TOTAL NUMBERS OF NAUPLIAR SIX (NVI), NAUPLIAR FIVE (NV), NAUPLIAR

APPENDIX E-7

FOUR (NIV), AND NAUPLIAR ONE THROUGH THREE (NI-NIII)
ON EACH COLLECTING DATE.

Date NVI NV NIV NI-NII
11-1971
9 0 86,965 347,862 1,739,310
21 0 1,017,589 740, 065 1,942, 671
23 367,076 367,076 367,076 458,845
25 0 94,355 471,778 1,604,046
27 0 377,422 1,037,912 3,868, 582
11-1971
1 0 466,236 932,473 2,237,935
3 93,247 93,247 279,741 2,331 , 182
5 0 0 45,330 158, 656
7 0 0 45,330 1,613,761
9 44,960 112,402 629, 452 1,528,670
u 449,609 359,687 1,079,061 4,586,009
13 1,528, 669 809,295 1,618,592 5,754,992
15 989,139 899,217 2,967,418 6,834, 053
17 1,978,278 1,348,826 1,618,591 4,316,244
19 1,162,982 629, 452 989,139 3,417,026
21 1,592,451 921,945 2,346,770 3,687,782
23 615,071 527,203 1,581,611 3,778,294
25 869,627 347,851 1,304,441 2,608,883
27 346,343 865,858 1,125,616 2,510,990
29 862,089 1,206, 925 1,120,716 2,327,642
31 772,713 858,570 772,713 2,232, 282
IV-1971
2 680,827 1,191,447 1,872,275 2, 638, 206
4 506,248 590, 623 928,122 2,278,118
6 83,620 250,862 668,967 2,675,871
8 416,346 333,076 416,346 1,332,307
10 330,061 330,061 247,546 1,072,700
12 407,049 569,869 488,459 2,605,115
14 240,912 562,130 401,521 1,124,260
16 0 160, 608 562,130 562,130
18 401,521 240,912 240,912 481 825
20 490,720 245,360 81,786 817,867

-218-


and oviposition at the two-thirds point in time between the two observations; e.g.,
if a female had a clutch at 12:00 noon and at 6:00 p.m. was found to have a new

clutch, hatching was estimated to have occurred at 2:00 p.m. and the oviposition

at 4:00 p.m.
All statistical analyses and tests follow the procedures of Sokal & Rohlf (1969).

FEMALE LIFE SPANS AFTER SEXUAL MATURITY

Generally, it was possible to recognize dying females by several indicators, al-
though some individuals, especially at the higher temperatures, died without ex~
hibiting prior indications. The most frequently observed indications of approach-
ing death were a noticeable slowing of the animal's feeding movements and less
active swimming. In most cases at the two lower temperatures, the females grew

_ progressively weaker, with the first indications begining as much as 10 days or more
before death. These females swam slower and often settled to the bottom for vari-
able lengths of time. On the other hand, lying motionless on the bottom was not
invariably associated with old age or approaching death. At 14, some females
ultimately became so feeble that they were unable to rise from the bottom of the beaker
except fo respond spastically after being touched. Females sometimes remained
alive in this condition for several days, their feeding movements reduced to an oc-
casfonal fanning motion and the hemocoele pumping very slowly and irregularly.
Undoubtedly, in a natural population, aging animals would be easy prey for pre-
dators; and it is unlikely any would survive long after the initial slowing processes
began. Certainly, none would be expected to live for days after becoming foo
feeble to swim.

Other signs of the female's approaching death were detritus accumulating on the
antennules or caudal setae and a growth, apparently of bacteria, over-her

body. It is not known whether the bacterial infection was the cause of death, or


PROPORTION:
OF

FEMALES
CARRYING
EGGS

~tll-

1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1

0.0

i 1 L 1 1 1 1

1 1 2 4 19 6 3
iT} tl IV Vv ViVi VIE 1X
DATE

Figure 11. Progortion of Adult Females Carrying Eggs on Each Collecting Date.
The Vertical Line Between 18 April and 24 April Shows a Change in
Scale from 2~Day to Approximately 14-Day Intervals.



separate series of six 350-ml containers in each of 3 temperature rooms with illumina-

tion and water temperature conditions as described above.

Table 4 shows the mortality over the 40-day span of the experiments among the ani-
mals cultured in each of the three media, The animals cultured in the conditioned
water medium had an average mortality rate of 72.9%, those cultured in distilled
water 65.6%, and those cultured in natural pond water only 16.3%. Statistically,
an aresine transformation (Sokal and Rohlf, 1969) was used to run t-tests to test for
equality between pairs of percentages. The results of those tests revealed that the
average mortality rate in the pond water differed significantly (p< 0.001) from that
of either of the other media, but the averages did not differ significantly between
conditioned water and distilled. These results should be valid for making a compari-
son between distilled water and pond water, but the comparisons between either of
these media and conditioned water are less reliable since the experiment with the
latter medium was conducted at a different time and with animals caught at a dif-

ferent season than for the experiment using distilled and pond water.

The reproduction data collected during these experiments did not show any large dif-
ferences for the results for the three media (Table 5). However, there were some indi-
cations of differences in mean clutch size. Surprisingly, the averages in distilled water
were larger than those for pond water in all three rooms, The conditioned water av-
erages were lower than those for either of the other media in all rooms, No explana-
tion for the apparent superiority of distilled water for production of large clutches can
be offered. It may be that a more complete study would not support this preliminary

finding as the differences reported here are not statistically significant.

The averages for the number of days the clutches were carried in each medium do not
show any substantial differences. However, they do show sizeable differences among
the different rooms, with the time being shortest in Room C and longest in Room A.

No definitive explanation can be provided for this observation. However, the rooms

-14-


to assure that individuals of C. vernalis cultured by the methods employed in our
work could be used in continuous flow bioassays, an attempt has been made to
culture them in a continuous flow system, This section describes the system used

and reports on the attempts to culture C. vernalis in this system.

The Continuous Flow System

Basically the continuous flow system consisted of a reservoir of culture medium (fil-
tered pond water) which was caused to flow through a culture container by the action
of an electrolysis pump. The pump consisted of a flask containing dilute H,SO, 4 in
which was immersed two platinum wire poles. A transformer connected to the elect-
rodes reduced the voltage from a wall plug to a level of 3 to 6 volts. The oO, and
Hy produced in the electrolysis flask flowed through glass tubing into the gas space
of a large,sealed container which contained 10 liters of culture medium. The water
from this reservoir flowed out through a glass tube which was immersed almost to the
bottom of the container. This tube terminated just inside the top of a sealed 1-liter
culture flask where the fresh medium dripped into the culture. The added water
caused some of the medium in the culture to flow out another piece of tubing which
was immersed in the culture almost to the bottom as was the tubing in the reservoir
container, This outflow tubing was covered with #25 plankton netting. The flow

through this system was approximately 2 liters per day.

The Culturing Results

A culture of C. vernalis was initiated in the continuous flow system by adding 6
adult males and 4 gravid females to the culture container. Two milliliters of the
fish food-alfalfa mixture were added to this culture every second day. Table 52
shows the number of adults, females, young, and total individuals found in this
culture after 4 weeks. It will be noted that the culture increased, Reproduction

obviously occurred and some of the young had matured by the time of sampling,
Thus, it is concluded that this species can be cultured in a continuous flow system.

-143-


~8€l-

Table 49, A COMPARISON OF THE RANKS OF TOTAL NUMBERS OF CYCLOPS VERNALIS (AS SHOWN
IN TABLE 48) KEPT {N SIX DIFFERENT TYPES OF WATER, THE SIX

WATERS WERE RANKED FROM HIGHEST TOTAL NUMBER OF

COPEPODS (RANK 1) TO LOWEST (RANK 6) SEPARATELY
FOR EACH WEEK OF SAMPLING

. Type of water
Week Noble Pond Tap Distilled 1/10 Noble | 1/5 Tap
9/10 Dist. 4/5 Dist.
1 6 1 5 2 4 3
2 5 1 2 3 6 4
3 5 1 6 2 4 3
4 4 1 6 2 3 5
5 3 1 4 2 6 5
é 3 1 5 2 4 6
7 4 2 5 1 3 6
8 4 2 5 3 1 6
Total of ranks 34 10 38 W 3} 38
App. TDS 1000 110 500 25 125 110



RESEARCH REPORTING SERIES

Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:

1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research

4. Environmental Monitoring

5. Socioeconomic Environmental Studies

This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
on the effects of pollution on humans, plant and
animal species, and materials. Problems are
assessed for their long- and short-term
influences. Investigations inelude formaticn,
transport, and pathway studies to determine the
fate of pollutants and their effects. This work
provides the technical basis for setting standards
to minimize undesirable changes in living
organisms in the aquatic, terrestrial and
atmospheric environments.

EPA REVIEW NOTICE

This report has been reviewed by the Office of Research and
Development ,EPA, and approved for publication. Approval does
not signify that the contents necessarily reflect the views
and pdlicies of the Environmental Protection Agency, nor does
mention of trade names or commercial products constitute
endorsement or recommendation for use.


12

1 4 1 4 1

19
ul

Figure 10. Specific Birth Rates (m

1 2
iit IV

i
4 1 9 6 3 14
Vv vio VUE VUE IX xX

,,) as Calculated for Each Collecting Date and the Weighted Mean m, Value (2.78) for

the Entire Reproductive Year, The Vertical Line Between 18 April and 24 April Shows a Change in Scale from

2-Day to Approximately 14-Day Intervals.


data at each incubation temperature (F=3.5; P<0.05), In the latter case a SNK
test shows no significant differences among means for 14°, 21°, and 27° incuba-
tion; or between the means for 14° and 31° incubation. The mean total number of
clutches per female decreases in the order 27°, 14°, 27°, and 31° for “native”
females, The order for the combined data at each incubation temperature is 21°,
7, 4, and 31°, which is almost the same order as that for mean clutch size,

only 21° and 27° being reversed.

Combining the effects of clutch size and total production, we can now look at
total lifetime egg production, At two incubation temperatures (21° and 31°), the
highest mean lifetime total number of eggs isreportedfora "non-native" female. As
was true for the mean number of clutches produced, the mean total egg production
is maximized at 21° incubation, regardless of hatching temperature (Figure 3 and
Table 18), Analysis of variance is presented in-Table 18, comparing only those
females “native” to each incubation temperature (F=2.0; ns) and again using data
-for all females at each incubation temperature (F=3.7; P<0:025). As for the mean
-total number of clutches, the overall mean total number of eggs produced per fe-
male decreases in the order 2v, 27, 14, and 31°. The results of a SNK test are
“also the same, the data for 14°, 21°, and 27°; and for 14° and 31° being homo-

geneous sets,

The means of all first clutches, second clutches, etc. were computed for the data

“at each incubation temperature, To avoid excessive variability, these mean se~
quential clutch sizes were computed only so long as there were at least five females
producing clutches. Analysis of variance was carried out for each incubation tem-
perature (Appendices A~1 through A-4) and in no case was there significant varia
tion. Combining all data regardless of incubation temperature (Table 19), no sig-
nificant difference (F=0.73; ns) is found among the sequential clutches for the over-
all data. Thus, it is demonstrated that mean clutch size does not vary with the

number of clutches the female has produced.

-57-


APPENDIX H-1
FECUNDITY IN EGGS PER CLUTCH OF FEMALE CYCLOPS VERNALIS AT 14°C,

Clutch Female No.
No. 1 2 3 4 5 6
1 21 61 25 67 39 24
2 32 75 22* 56 él 12
3 49 63 51 53
4 28 54 51 29
5 22 24 AA 28
6 W 41 24
ve 30277 47, 310284 36
No. of
clutches 6 5 2 6 6 2
per female

*Only one of two possible egg sacs present.

-223-


Table 51, PERCENTAGE OF NAUPLII OF CYCLOPS VERNALIS
SURVIVING 24 AND 48 HOURS AFTER HATCHING WITH AND
WITHOUT THE PRESENCE OF THE MOTHER.

Percentage of nauplii surviving

24 hours 48 hours

Mother Present

Mean 53.9 41.8
SES £4,95 £5.13
min. 30.0 12.0
max. 83.0 63.0
Mother Removed
Mean 98.4 96.2
SES 40.56 41.18
min. 96.0 88.0
max. | 100.0 100.0

-141-


Table 23, COMPARISON OF MEAN EGG PRODUCTION CYCLE FROM
OVIPOSITION TO OVIPOSITION AT FOUR INCU BATION TEMPERATURES

Incubation Mean 95%
temperature (hours) confidence limits
(degrees C)
14 166.7 147.5 - 188.3
21 63.7* 60.0 = 67.7*
27 47.3 36.9 - 60.7
31 55.2 48.4- 63.0

*Cycles which included a clutch of resting eggs excluded.

An Fingx7test indicates that variances of the data for the four incubation temperatures
are heterogeneous, A logarithmic transformation was made and an Anova carried out

with the transformed data.

Anova table: Cycles by incubation temperature. Data for cycles which included a
clutch of resting eggs are excluded from the 21° data.

Source of variation df MS F
Among incubation temperatures 3 2.1762 113.3437 P<0.001
Within incubation temperatures 217 0.0192
Total 220



Methods and Materials

As specified for each experiment, the animals used in this work were obtained either
from laboratory stock cultures or directly from the field. The laboratory stock cul-
tures were initiated with animals obtained from a farm pond in Range 3 west, Town-
ship 9 north, Section 11, southeast quadrant, in Cleveland County, Oklahoma, near
the city of Norman, Animals from this pond were separated from all other macro-
plankters and placed in large containers of conditioned water. The young produced
during experiments were also usually added to these cultures. The animals removed
from these stock cultures for experiments were thus a combination of individuals added
from field collections and from laboratory experiments as well as individuals produced

within the culture.

Periodically a few milliliters from a food culture were added to each stock culture.
The cultures were generally maintained at a temperature range of 24 to 26° C and

under moderate illumination, although other conditions were used early in the work,

Where animals directly from the field were used in experiments, these animals were
obtained mainly from the above pond, Unfortunately, this pond dried up during
early April, 1970, and did not refill until the middle of May, 1970. During the in-
terim, field animals were obtained from a farm pond located in Range 1 west, Town-
ship 8 north, Section 13, southwest quadrant, in Cleveland County. ‘For any given
experiments using animals from the field, care was taken to insure that all the in-

dividuals were obtained from the same population.

Field collections of copepods were made with a fine (No. 25 mesh), 10-inch diameter
plankton net. The net was thrown out into the body of water and pulled to shore
several times; or, if conditions permitted, it was towed through the water by a person
wearing chest waders, The zooplankton was concentrated in an 8 dram vial attached

to the smaller end of the net. All the organisms collected were transferred to a quart


APPENDIX E-7 (Cont.)

TOTAL NUMBERS OF NAUPLIAR SIX (NVI), NAUPLIAR FIVE (NV), NAUPLIAR
FOUR (NIV), AND NAUPLIAR ONE THROUGH THREE (NI-NIII)
ON EACH COLLECTING DATE.

Date NVI NV NIV NI-Nill
V-1971
4 523,302 320,205 465,211 887,493
18 321,217 160,608 321,217 883,347
28 0 0 80,304 160,608
Vt-1971
if 647,433 1,109,886 1,387, 358 1,757,320
25 190,910 0 95,455 286,366
VI-1971
9 0 362,625 271, 968 815,906
23 517,253 258, 626 603,462 2,069,015
Vill-1971
6 421,873 337,499 590, 623 1,181,246
20 569,869 651,278 895,508 3,419,214
IX-1971
3 242,043 161,362 80,695 806,812
17 0 0 0 0
X-1971
14 0 81,409 162,819 407,049
21 0 0 0 170,206
29 0 0 85,103 0

-219-


APPENDIX B-6 (Cont.)
21° INCUBATION: INTERVAL (HOURS) BETWEEN CLUTCHES

Intervals between successive clutches are listed from top fo botfom in the order of oc-
currence but are not necessarily consecutive, some intervals having been omitted for
various reasons as discussed in the text.

Variances of the data for the three hatching temperatures are heterogeneous. A logorith-
mic transformation was made and Anovas carried out with the transformed data.

Anova table: intervals following clutches of resting eggs (mean = 9.7) vs intervals fol-
lowing clutches of non-resting eggs (mean = 10.6).

Source of Variation df MS F
Among interval types 1 0.0284 0.1762 ns
Within interval types 107 0.1611
Total 108

Anova table: comparison among individuals of intervals between clutches, excluding
those intervals marked with an asterisk above which followed a clutch

of resting eggs.

Source of Variation df MS F
Among individuals 9 0.0969 0.5601 ns
Within individuals 74 0.1730
Total 83

-186-


Table 41, ANOVA TABLE TESTING THE DIFFERENCES AMONG THE MEAN
NUMBERS OF EGGS PER CLUTCH DURING THE VARIOUS SEASONS.

Source of variation df SS MS F
Among seasons 3 1779 9860 593.3286 8.7990*
Within seasons 43 2899 5354 67.4310

Total 46 4679 5214

*P<0.01

Table 42, A POSTERIORI COMPARISON OF THE SEASONAL MEAN NUMBERS
OF EGGS PER CLUTCH USING THE STUDENT-NEWMANG-KEULS TEST.

Summer Fall Spring Winter
Y 15,25 15,33 24.43 30.53
n 9 3 14 21
2 23.68 225.33 69.30 67.93

Yon

Su 15.25 9 -
F 15.33 3 7.5610 -
Sp 24,43 14 5.8300* 7.2155* --
W 30.53 21 5.9875* 8.4225* | 3.9130* --
*P< 0.05

-17-


~eZl-

Y
EGGS
PER
CLUTCH

40

30 Fo

12,

LL
AB

od A

1 3 10 15

Y, ADULTS PER LITER
Figure 16. Mean Clutch Size Plotted Against Density of Adults,

20


bal :7 4

APPENDIX A-~4
31° INCUBATION: NUMBER OF EGGS PER CLUTCH IN THE SEQUENCE PRODUCED

Female's hatching
temperature 2ie_| 27° 31° Standard

Individual De) DS Dx} DIT DI6 DI7 DI? D20 Total Mean error

Clutch No.
1 uu 30 20 2 27 24 25 7 183 22.9 2.29
2 12 4 (17) 20 33 27 21 164 23.4 3,11
3 3 % 2 a 2% 22 13 150 21.4 2.40
4 18 r:.) u 4 % 20 12 154 22.0 3.89
5 14 28 (25) 4 12 93 18.6 3.28
6 23 23 13
7 13 10
8 3a 10
9 9

Total 68116 69 147 225 2% 131 108

Mean . 3.6 29.0 17.20 29.4 25.0 24.0 21.8 13.5

Standard error 1.21 191 2.25 3.36 1.99 1.85 1.32

Parentheses indicate a clutch which was not counted. Number in parentheses Is an average value for that female.

Anove table: mean clutch size Anova table: comparison of sequential clutches

Source of variation df MS F Source of variation df MS F
Among individuals 7 220.7370 =8.8714 = P< 0.001 Among sequential clutches 4 20.0064 0.3238 ns
31° hatch vs other 1 5.5873 0.2246 ns Within sequential clutches 29 61.7759
D8 vs DS 1 527.0222 21.1810 P<0.001 Total 33
D3 vs Dilvs...D20 5 202.5100 8.1389 P<0.001
Within individuals 34 24.8818

Total 4



Comita, G.W. Oxygen Consumption in Diaptomus. Limnol. Oceanog. 13:51-57,
1968.

and G.C. Anderson. The Seasonal Development of a Population of Diopto-
mus aslandi Marsh, and Related Phytoplankton Cycles in Lake Washington. Limnol.
Oceanog. 4:37-52, 1959.

and J.J. Comita. The Infernal Distribution Patterns of a Calanoid Cope-
ped Population, and a Description of a Modified Clarke-~Bumpus Plankton Sompler.
Limnol. Oceanog. 2:321-332, 1957.

Conover, R.J. The Feeding Behavior and Respiration of some Marine Planktonic Crus~
tacea. Biol. Bull. 119:399-415, 1960.

. Reproductive Cycle, Early Development and Fecundity in Laboratory
Populations of the Copepod Calanus hyperboreus. Crustaceana. 13:61-72, 1967.

Corkett, C.J. Technique for Rearing Marine Calanoid Copepods in Laboratory Con-
ditions. Nature. 216:58-59, 1967.

and 1.A. McLaren. Egg Production and Oil Storage by the Copepod
Pseudocalanus in the Laboratory. J. Exp. Mar. Biol. Ecol. 3:90-105, 1969.

and D.L. Urry. Observations on the Keeping of Adult Female Pseudo-
calanus elongatus under Laboratory Conditions. J. Mar. Biol. Assoc. U.K. 48:97~
105, 1968.

Cummins, K.W., R.R. Costa, R.E. Rowe, G.A. Moshiri, R.M. Scanlon and R.K.
Zajdel. Ecological Energetics of a Natural Population of the Predaceous Zooplankter
Leptodora kindtii Focke (Cladocera). Oikos. 20:189-223, 1969.

Deevey, E.S. Life Tables for Natural Populations of Animals. Quart. Rev. Biol.
22:283-314, 1947.

Deevey, G.B. Annual Variations in Length of Copepods in the Sargasso Sea off
Bermuda. J. Mar. Biol. Assoc. U.K. 44:589-600, 1964.

Edmondson, W.T. Reproductive Rates of Rotifers in Natural Populations. Mem. Ist.
Ital. Idrobiol. 12:21-77, 1960.

Elbourn, C.A. The Life Cycle of Cyclops strenuus strenuus Fischer in a Smal! Pond.
J. Anim. Ecol. 35:333-347, 1966.

-167-


involved (Table 20). The mean time clutches of resting eggs were carried was sig-
nificantly shorter (F=15.3; P<0.001) than the mean carrying time of subitaneous
eggs. Since the mean intervals following clutches of the two egg types were not
significantly different (F=0.18), the significant difference (F=8.5; P<0.005) be-
tween the two complete cycles can be attributed to the shorter carrying time of rest-
ing egg clutches. Since both the mean carrying times and mean lengths of a com-
plete cycle were significantly shorter for clutches of resting eggs than for clutches
of subitaneous eggs, data for these intervals were excluded from the 21° data for
comparison with other incubation temperatures. For consistency, although the

mean interval between clutches was not significantly different, intervals following

resting clutches were also excluded from the 21° data used.

The data from each incubation temperature for all three time intervals were com~
pared for variability among individuals (Anova fables are shown with Appendices
B-1 through B-12). Significance (F=4.0; P<0.010) was indicated only for mean
carrying times of individuals incubated at 27° (Appendix B-3). An orthogonal set
of a priori comparisons was carried out for these data (Appendix B-3), with the re-
sult that the variability among individuals cannot be attributed to hatching tem-
peratures as was variability in clutch size (see preceding section). Clutches ap-
parently were produced at rates determined by the incubation temperatures alone,
Therefore, all data from each incubation temperature were used for Tables 21,22,

and 23, regardless of hatching temperatures.

When computing the mean clutch carrying times (Table 21) at the four incubation
temperatures, data for clutches of resting eggs were excluded from the 21° data.
However, sterile clutches could not be identified unequivocally and so could not
be excluded. If the carrying time of sterile clutches differs, the effect of their
inclusion in the data probably is slight, since the incidence of sterile clutches, as
determined in the next section, appears to be low. A highly significant (F=261.0;

P<0.001) inverse relationship between temperature and development time is apparent


-L@-

Table 7, A COMPARISON OF THE AVERAGE VALUES (SE) FOR CERTAIN REPRODUCTIVE AND
DEVELOPMENTAL PARAMETERS FOR D. CLAVIPES INDIVIDUALS AT
FOUR DIFFERENT TEMPERATURES .

Number Average Number Average Average Average Days | Average Days
Average of of Clutches Clutch Percent Clutch Development
Temperature | Clutches Per Container Size Hatch Carried To Adult
17.2 6 1.0 14,1 70.9 3.8 31.6
(£0.4) (£2.5) (£7.0) (£0.3) (€4.1)
21.9 4 0.7 9.8 75.1 2.3 23.6
(0.2) (£1.5) (£12.4) (£0.5) (£2.0)
26.4 i 1.8 10.5 65.6 1.5 31.8
0.4) @1.1) €£9.8) (40.2) ($4.2)
31.3 3 0.5 13.7 W.1 1.5 23
0.2) 6.1) 11.1) 0.5) (-)



APPENDIX E~4 (Cont.)

TOTAL NUMBER OF COPEPODITE THREE (CII!) IN EACH REGION OF THE POND
AND IN THE TOTAL POND ON EACH COLLECTING DATE, THE TOTAL
NUMBER WAS DETERMINED BY MULTIPLYING THE Y (MEAN) NUMBER
OF CII PER QUADRAT IN EACH REGION BY THE TOTAL NUMBER OF
QUADRATS IN THAT REGION AND SUMMING THE PRODUCTS
FOR EACH DATE.

Date Open water Rooted aquatics Total pond
region region
IV-1971
12 651,909 0 651,909
14 221,240 0 221,240
16 245,324 0 245,324
18 174,918 39,238 214,156
20 517,706 0 517,706
V-1971
4 175 , 282 59,521 234,804
18 220,214 348,974 « 569, 189
28 47,474 28,283 75,758
VI-1971
VW 1,265,551 39,473 1,305,024
25 586, 167 0 586, 167
VIN-1971
9 296,957 0 2% ,957
23 84,717 20,172 104,889
VIN-1971
6 33,886 0 33,886
20 40,048 146,700 186 , 748
IX-1971
3 26,031 207,632 233 ,634
W7 67,320 35,750 103,071
30 24,480 168,535 193,015
X-1971
14 36,720 180,574 217,294
21 7,314 0 7,314
29 7,344 61,073 68,417

-213-


Table 28. MEANS (Y) AND STANDARD ERRORS (SY) FOR THE TIMES EGGS
WERE CARRIED BY FEMALES IN SIMULATED WEED ENVIRONMENTS,
WEED-FREE ENVIRONMENTS, AND ENVIRONMENTS IN WHICH
THE SIMULATED WEEDS WERE RELEGATED TO THE
PERIMETER OF THE BEAKER.

Environment n Y (days) sy P
Simulated-weed 18 4.00 0.52

< 0.01
Weed-free 18 2.22 0.22

* Simulated weeds
relegated to the

perimeter of the
beaker 18 2.01 0.25

Table 29. MEANS (Y) AND STANDARD ERRORS (SY) FOR THE
TIMES EGGS WERE CARRIED BY FEMALES IN
SIMULATED WEED ENVIRONMENTS AND
WEED-FREE ENVIRONMENTS AFTER THE
ENVIRONMENTS OF THE ANIMALS
WERE REVERSED.

Environment n Y (days) Sy P
Simulated-weed 7 3.71 0.42

< 0.01
Weed-free 7 1.86 0.14

-92-


Table 10. A COMPARISON ON A WEEKLY BASIS OF THE AVERAGE

NUMBERS OF ADULT D. CLAVIPES (4SE) IN
POPULATIONS FED FROM CULTURES
OF A MIXED FOOD, OF YEAST
(ADDED TO BOTH LIGHT AND
DARK CONTAINERS), AND

OF CHLAMYDOMONAS.
Mixed Yeast in. Yeast in
Date food! light dark Chlamydomonas
17/X11/70 5.5 3.8 --- 3.2
(£0.5) (£0.9) (£0. 6)
23/Xi1/70 AY 4.2 — 2.6
(£0.6) (£0.64) 0.9)
29/X11/70 4.5 2.8 4.8 2.2
(£0.5) (0.9) (40.8) ¢1.0)
8/\/71 2.7 3.6 2.0 2.4
(40.7) (40.9) (0.8) (£0.2)
16/1/71 2.1 1.8 2.0 2.0
(40.6) (t0.9) (t0.6) (40.6)
2/7 0.7 2.8 0.6 1.8
(40.3) 1.1) (£0.2) (40.9)
28/1/71 0.9 4.0 2.0 2.2
(40.4) 1.5) (41.3) (¢t0.7)
4 3.0 6 1.6 5.2
(40.9) (£1.6) (t0.8) 1.4)
LPAI ZA) 2.8 6.0 1.4 6.2
(40.9) 2.0) (£0.7) (41.6)
18/11/71 1.6 3.6 1.2 5.4
¢0.7) 1.0) (0.6) (1.4)
25/1/71 0.9 4,2 1.0 5.4
¢to.6) 1.1) (t0.4) (41.5)

-28-


10.62 days at 14°C, Clutches were produced every 3.65 and 7.68 days at 26° and
21°C, respectively. A significant difference among the means at the four tempera~
tures was found at P < 0,001 using an anova test, Previous estimates of the time
period between successive clutches are shorter than those reported here. Andrews
(1953) found that C, vernalis, collected from Lake Erie and maintained in the lab-
oratory at 20°C, produced new egg clutches every third day. Ewers (1936) observed
that C, vernalis at room temperature produced eggs within 5 days following the adult
molt and successive clutches could be produced at 1.5-day intervals. Again, it may
be that a primarily vegetative diet slowed down the reproductive processes of the

animals in the present study.

The data suggest that the period between the production of the last egg clutch and
the death of the female is only slightly influenced by temperature (Table 53, Figure
17). Females died 1 to 37 days after the production of their last egg clutch (Appen-
dix G-1). The longest mean interval (10,5 days) was observed at the lowest temper-
ature of 14°C, and the shortest mean interval (6.3 days) was observed at 26°C. The
interval at 21°C, i.e. 9.9 days, was only slightly longer than at 26°C, Analysis of
variance showed no significant difference (P > 0.05) among these intervals at the

four temperatures.
FECUNDITY

The reproductive histories of Cyclops vernalis females at 14, 21°, 26°, and 31°C
are shown in Appendices H-1 through H-4, These data are summarized in Table 54,

Clutch Size
Each C. vernalis female observed produced at least one egg sac. The clutches that
were produced varied in size from 3 to 102 eggs. Both extremes were produced at

21°C. The variances for the mean clutch sizes at the four temperatures were signif-

icantly heterogeneous at P < 0,05 (Fmax test; Sokal and Rohlf, 1969), indicating that

-153-


Table 16. MEAN NUMBER OF EGGS PER CLUTCH IN RELATION
TO INCUBATION AND HATCHING TEMPERATURES

Female's
Incubation hatching
temperature Mean temperature “Mean
(degrees C) + SE (degrees C) ESE
4 26.8 = 1.14*
4 21.5% 0.85 a vee
31 20.2 + 1.05
14 erase
+ 21 28.0= 0.80
21 24.52 0.77 27 21.34 1,76*
31 15.9 = 1.20
14
+ 21 20.4 = 1.00*
27 28.8= 1.07 27 30.01.16
31 25.9 £ 3.65*
4 pO
+ 2 13.64 1.21*
31 21.1* 1.18 27 29.04 1.91*
31 21.3 1.30

*Mean clutch size ofa single female.

Anova table: Comparison of those females which were incubated at the same
temperature as that at which they hatched.

Source of variation df MS F
Among incubation temperatures 3 568.5867 8.5327 P<0.001
Within incubation temperatures 195 66.6358
Total 198

Anova table: Comparison of all females at each incubation temperature.

Source of variation df MS F
Among incubation temperatures 3 780.8564 10.7858 P<0.001
Within incubation temperatures 201 72,3966
Total 294

-53-


APPENDIX B-11

27° INCUBATION
EGG PRODUCTION CYCLE
Time (hours) from oviposition to succeeding oviposition

Female's
hatching
temperature | 21° 27° 31°
Individual co C7 c8 C13 C14 C15 Cl6 ci

57.0 50.0 48.0 51.0 49.5 48.5 50.0 35.0
39.5 | 45.5 | 36.5 | 39.0 | 40.5 | 39.5 | 39.0 | 41.5
30.0 | 51.0 | 38.0 | 57.0 | 57.0 | 56.0 | 22.0 | 58.5
49.5 ; 48.0 | 37.5 | 36.0 | 36.5 | 49.0 |178.0 | 48.0
42.5 44.0 55.0 39.0 38.5 36.0 47.0
37.5 51.0 48.0 46.5 36.0 44.0 48.5

25.0 48.5 48.5 43.0 55.0

48.0 49.0 46.5 51.5

49.5 47.5 49.0 45.5

45.0 73.0

38.0 43.0

60.5 47.5

48.0 28.0

61.5 | 65.5

121.0

{190.5

Lengths of cycles are listed from top to bottom in the order of occurrence but are not
necessarily consecutive, some cycles having been omitted for various reasons as dis~

cussed in the text.

Variances of the data for the three hatching temperatures are heterogeneous. A logorith-
mic transformation was made and Anovas carried out with the transformed data.

Anova table: comparison among individuals of complete egg production cycles.

Source of Variation df MS F
Among individuals 7 0.0141 0.6588 ns
Within individuals 63 0.0214
Total 70

-192-


Table 48 (Cont.)

-£01-

Type of water
Week Distilled 1/10 Noble, 9/10 Dist. 1/5 Tap, 4/5 Dist.
(TDS app. 25) (TDS app. 125) (TDS app. 110)

A G Y T A G Y T A G Y T
0 6.0 0.0 0.0 6.0 6.0 0.0 0.0 6.0 6.0 0.0 0.0 6.0
1 74 0.6 146.6 154.0 6.0 0.6 99.0 105.0 4,2 0.2 113.4 117.6

(1.0) (0.4) (14.3) (15.1) (0.6) (0.6) (34.7) (34.8) (0.6) (0.2) (29.8) (29.5)
2 6.8 1,2 112.4 W9.2 6.6 1.0 48.8 55.4 3.4 0.8 98.6 102.0

(1.4) 0.46) (17.9) (19.1) (1.2) (0.3) (12.0) (12.6) (0.5) (0.4) (25.8) (25.6)
3 6.6 0.6 101.2 107.8 9.6 0.8 77.8 87.4 3.0 0.0 87.4 90.4

(1.6) (0.2) (25.3) (26.0) (2.1) (0.5) (24.7) (24.9) (1.0) (-) (17.9) (17.5)
4 6.8 1.0 63.4 70.2 9.4 0.6 56.8 66.2 5.6 0.4 34.6 40.2

(0.9) .5) (12.8) (12.8) (2.3) (0.6) (19.3) (17.5) (1.7) (0.4) (7.6) (8.2)
5 9.0 1.2 100.8 109.8 8.8 0.2 29.2 38.0 9.2 0.2 31.4 40,6

(1.2) (0.5) (25.8) (25.7) (3.0) (0.2) (13.8) (12,5) (3.0) 0.2) (17.1) (17.3)
6 13.6 1.0 W114 125,0 W.4 0.2 33.2 44,6 10,2 0,0 26.8 37.0

(1.3) (0.6) (32.6) (32.1) (1.4) (0.2) (11.1) (12.4) .1) (-) (10.2) (10.5)
7 9.6 1.0 88.0 97.6 11.2 1.0 82,8 94.0 1.4 0.2 38.4 49.8

(1.3) 0.4) (14,8) (16.0) (0.5) (0.4) (30.7) (30.9) (4.0) 0.2) (13.7) (17.0)
8 10.8 1.0 AV.B 52.6 10.4 2.2 77.6 88.0 7.2 0.8 17.6 24.8

(1.4) 0.5) (13.0) (14,2) (1.2) (0.7) (23.9) (24.4) (2.3) (0.4) 6.9) (77.0)



reduced at this temperature, and it is likely that this is approaching the upper thermal!
limit for the species, (Preliminary results, not included in this report, for a study on
the upper thermal limit for this species seem to confirm this.) In general, the tempera~-

ture of 26.4°C appears to be the most favorable of those studied in this experiment.

From these studies it was concluded that a range of temperatures from about 30° to
20° C allows substantial reproduction and development, It may be that the optimum
temperature is just under 30°C. However, it is recommended that a somewhat lower
value, around 25° C, be used for culturing. Although a few degrees warmer might
increase culturing rate toa limited extent, these temperatures approach the upper
thermal limit, and it seems prudent to avoid this range. The detailed studies on tem=

perature reported in the next chapter fortify this recommendation.

Food

In the work described so far the food consisted of small volumes taken from a mixed
culture grown in the laboratory. As indicated in the previous subsections, questions
arose concerning the use of this type of food, Questions also arose concerning the
quantity of food that should be added. The present subsection presents the results
from a series of experiments designed to learn more about the effects of food on cul~

turing and to aid in the selection of the feeding conditions to be used for culturing.

An experiment was conducted to compare the suitability of the mixed culture food

with that of several other types, Each of 25 beakers was filled with 1000 ml of filtered
pond water and had 8 adult D, clavipes, four of each sex, added to it, Feeding took
place every second day, at which time the containers each received 2 ml of a food
suspension. While the volumes of the food additions were equal, the type of material
added varied; for 10 of the containers 2 ml of the mixed culture was added, for 10
others 2 ml from a yeast culture was added, and for the remaining five 2 ml from a

culture of Chlamydomonas sp. acted as the food. Inan attempt to make the quantity

-24-


We a 14° HATCH
& 21° HATCH
OF. o 27°HATCH ,
9 e 31° HATCH
rt 1
gE J
RANKED 7r 4
MEAN
NUMBER rae 4
OF
CLUTCHES =,
4b 4
3k 4
2+ 4

14 21 27 31
Incubation Temperatures
(degrees)

Figure 2. Mean Total Number of Clutches of Groups (Right Column of Table 17)
are Ranked from Lowest to Highest (I to 12) and Plotted Against the
Temperature at which the Females Were Incubated.


APPENDIX B-1
14° INCUBATION: TIME (HOURS) CLUTCHES WERE CARRIED

Female's hatching
temperature 44° 21° 31°

Individual A7 Aé A8 Ag Al0
107.5 120.0 119.5 115.5 115.5
114.0 116.5 104.5 121.0 96.5
127.0 114.5 97.5 114.5 107.5
132.5 123.0 152.0 1.0 124.5
119.5 106.5 117.0 113.5
119.5 109.5 97.5 120.5
120.0 114.0
107.0 118.5
112.0 65.0
113.0

Clutch carrying times are listed from top to bottom in the order of occurrence but are
not necessarily consecutive, some carrying times having been omitted for various
reasons as discussed in the text.

Variances of the data for the three hatching temperatures are marginally homogeneous.
A logarithmic transformation was made and Anovas carried out with the transformed
data.

Anova table: comparison among individuals of carrying times.

Source of Variation df MS F
Among individuals 4 0.0014 0.4242 ns
Within individuals 30 0.0033
Total 34

-179-


APPENDIX B-6
21° INCUBATION: INTERVAL (HOURS) BETWEEN CLUTCHES

BIS

moeneeonoononnon
QaTGninadiaysnsta

37°

B13

27°

B16

NEG SaFACHOGN

21°

B20

mooooomnooonn

SGHINGAR ORG ee
yon

BIg

B18

BI7

Bl4

BI2

OHSHSEEHSHESEHNDOESHHH_EW

BHLGAGATHHHOHYQVAOIONNaGY

BIT

eoneonnnnoonooonec’s4
Gant nr agd@uadynavta

Female's hatching

temperature
Individual”

-185-

*Indicates an interval following a ciutch of resting eggs.


the length of time eggs are carried by the females thus suggesting that the effect of
rooted aquatics on the copepods is partially due to the actual physical presence of
the plants, The cause or causes of this developmental retardation and the pathway

by which it is implemented are not known and deserve further investigation.

A comparison of the mean clutch carrying times for animals kept in restricted Potamo-
geton sp. environments and nylon-netting environments (Table 30) revealed no signi-

ficant difference,

Table 30. MEANS (Y) AND STANDARD ERRORS (Sy) FOR THE TIMES
EGGS WERE CARRIED BY FEMALES IN ENVIRONMENTS
IN WHICH POTAMOGETON SP. WAS RESTRICTED TO
A SMALL REGION BY NYLON NETTING AND
IN NYLON-NETTING ENVIRONMENTS.

Environment n ¥ (days) sy P
Weed-restricted 6 2.66 1.09

0.90
Nylonnetting 6 3.00 0.45

Field data revealed a heterogeneous distribution of D, clavipes during the entire study
period apparently related to the occurrence of rooted vegetation. Laboratory data
showed that rooted aquatics could cause the death of the copepods and that even the
presence of simulated weeds caused retardation of development and hence presumably
a decrease in reproductive rate. Apparently the rooted aquatics are an effective fac-
tor in determining the regions of a body of water in which this species can live and

reproduce.
In the past, investigations of zooplankton in small bodies of water have commonly

utilized one or two tows of a plankton net to determine species composition and abun-

dance. In using such data to draw conclusions concerning the populations in the pond,

-93-


that show that the body size of C. vernalis is dependent on the temperature of de-
velopment such that larger animals develop at lower temperatures. The fecundity
data reported in this paper is for C, vernalis females which had developed at the
temperature at which their egg production was observed. The variation of clutch
size at the different temperatures may be the indirect result of the effect of tempera-
ture on body size. However, the data obtained are insufficient to verify this hy-
pothesis. :

Eggs produced by cyclopoid copepods are normally carried in paired egg sacs (Ewers,
1936; Wilson and Yeatman, 1959). However, during our experiments several females
were observed carrying single egg sacs. One female (No. 11, Appendix H-3) was
observed to carry only one egg sac’in the majority of her clutches. If this female is
excluded, most of the clutches consisting of only 1 egg sac were either the first, the
last or the only egg clutch produced by that female. Although it may be that some of
the single egg sac clutches were the result of one of the egg sacs being accidentally
knocked off the female before the daily observations were made, most of the single
€gg sac clutches were probably due to a lower fecundity of the female at either the
beginning or the end of her reproductive life,

To determine the effect that age has on fecundity at different temperatures, a series
of comparisons were made using the Student's t-test. Tests were conducted to de-
termine whether significant differences existed between the mean sizes of: (1) the
first and last egg clutches, (2) the first clutches and all clutches and (3) the last
clutches and all clutches, The mean size of the first egg clutches produced at a tem-
perature was calculated by averaging the egg counts from the first and second clutches
for all females at that temperature which produced at least four clutches during their
lifetimes. The mean size for the last clutches was determined for each temperature by
averaging the eggs counts from the lost and next to last clutches for females which pro-
duced at least four egg clutches. The results (Table 55) show that clutch size was af-
fected by the age of the female as well as by temperature. The mean size of the first

-156~


“Glt-

Y,

EGGS
PER

CLUTCH

10 -

0 i _ 1 es eee 1!
19 1 2 4 1 9 6 3 14
u ty IV Vv Vi VE Vit 1X xX

DATE

Figure 13, Mean Number of Eggs Per Clutch on Each Collecting Date. The Vertical Line Between
18 April and 24 April Shows a Ghange in Scale from 2-Day to Approximately 14-Day
Intervals.



By the end of a month only three animals were left in the containers that received

no food, and no egg production or other signs of reproduction had been observed in
these containers. Thus, there was little evidence that the copepods could be sup-
ported by endogenous growth if food was not added.

The containers that received 1 ml of food per day initially showed a decline from
the 8 animals added originally. However, females carrying eggs were often present
in these containers and nauplii and young copepodites were commonly observed.
These cultures showed every indication of developing self-propagating populations,
and it was concluded that the use of the fish food solution as food showed promise.
However, the results of this work pointed out that the amount of food added is of

critical importance to culturing success and should be studied further.

To do this an experiment similar to the previous one but with lesser amounts of food
added was carried out. Sets of replicate cultures were initiated in this experiment
as in the previous one with the exception that only three sets of three replicates
each were used, The environmental factors were also the same as in the previous
work except for the feeding conditions. In the present experiment three containers
received 0.1 ml, three 0.5 ml, and three 1 ml of the food solution once every other
day.

The results from this experiment are presented in Table 12. The populations did quite
well in all the containers fed 0.1 or 0.5 ml of the fish food mixture. For the cul-
tures fed 1.0 ml, however, one container had lost all its copepods and another had
only one copepodite by the end of the experiment. It seemed that even 1 ml of this
food every other day was too much. Both of the other volumes of food addition seemed’
to be satisfactory.

A further study was carried out to explore the effects on D. clavipes of varying the
volumes of fish food-solution added as food. This work was conducted in quite a


The reduction in development time with increased temperature is greatest in the
lower range with differences lessening at the higher temperatures. Similarly,
Burgis (1970) reports, for Thermocyclops neglectus, a decrease in the effect of
temperature changes with rise in temperature and summarizes similar reports by
various authors for other species. An optimal temperature, above which develop-
ment time is increased with increasing temperature, was not observed in the pre~
sent experiments, although Burgis (1970) reports such an optimum for T. neglectus

and other species.

While an optimal temperature for development time was not observed, the length
of the complete egg production cycle does increase when the temperature exceeds
an optimum level. The time from oviposition to oviposition is inversely related to
temperature up to 27° incubation, but the cycle is longer at 31° incubation. Al-
though embryonic development within the egg is not inhibited at 31°, some tem-
perature-related maximum in the physiological process of egg production has been

exceeded, with the result of an increased interval between clutches.

Aycock (1942) reported 48.5% of Cyclops vernalis nauplii incubated at 28. r
reached maturity, while only 7.84% reached maturity when incubated at 7P.

In contrast, this poper reports an essentially inverse relationship for D. clavipes
"native" to their incubation temperature between hatching success and temperature
(the mean for WY is stightly lower than the mean for 21°). Although Aycock's
temperatures are quite different than ours, the data suggest the need for additional
experiments to determine whether the two species differ with respect to the rela-
tionship between temperature and survival, or whether higher hatching success at

the lower temperatures is balanced by higher mortality among nauplii.
Despite the fact that a 100% hatch is reported here rather infrequently (Appendices

C-1, -2, -3, ~4), it nevertheless seemed, as discussed previously, that in most

cases either all eggs hatched or none at all did. Due to the experimental design,

-76-


28.

29.

30.

31.
32.
33.
34,

35.

36,
37.
38.

39.

Page

Means and Standard Errors for the Times Eggs Were Carried by Females

in Simulated Weed Environments, Weed-free Environments, and Environ~
ments in Which the Simulated Weeds Were Relegated to the Parimeter

of the Beaker... eee eee cee cere reece eee ence ences eevee 92

Means and Standard Errors for the Times Eggs Were Carried by Females
in Simulated Weed Environments and Weed-free Environments After the
Environments of the Animals Were Reversed... e+ seeeeecvcccceee W2

Means and Standard Errors for the Times Eggs Were Carried by Females
in Environments in which Potamogeton sp. Was Restricted to a Small
Region by Nylon Netting and in Nylon Netting Environments........ 93

Durations of the Various Instars as Determined in the Field and Com-
puted from a Composite of the Laboratory Data... .. ee eee eee ae es

Life Table for the First Generation Using the First Occurrence to Deter-
mine the Durations of the Various Stages ........--- eee ee cc eee

Life Table for the Complete Year Study Using the Composite Labora-
tory Data to Determine the Durations of the Various Stages... ....26.- 99

Life Table for Laboratory Animals Using Complete Data From All Ani-
mals at All Temperatures... 0. sere cece cece ese cree cece 100

Calculation of the Intrinsic Rate of Natural Increase Using the Adult
Survivorship From the Full Year Study, a 40-Day Duration for the
Adult Stage, and the Average m, Value for the Year... .. eee ee ee 104

Anova Table Testing the Differences Among the m, Values of the
Various Seasons .--se cee c cece cece eer eee cee ence rece ee WO

A posteriori Comparison of the Mean Seasonal m, Values Using the Stu-
dent-Newman-Keuls Test... 2+ ses cceeccer er ecescocrerees WO

Anova Table Comparing the Mean Percentages of Females Carrying
Eggs During the Various Seasons... ... eee eee e errr ee creecee WT

Anova Table Comparing the Mean Ratios of Males to Females During
the Various Seasons. .2seeeeseescceccererecceevecccceee 16


CONTENTS
Page

Abstract ..... meee eee senncncocaccoeasevocecsseres bee eeeeecneeeeves ii
List of Figures ......0.ceeeceeceeceeee deve eeecneeeeees seeeeeceeeeeee VE
List of Tables ........64. ponsececne occ c cece nee e eens ees enscenerceres viii
Acknowledgments ......-.0s00 ee vcccceceres cece ee ceeeeeecuevecees xiii

Chapters

LT. Introduction... ccc cece cence cence nec eec ccc crettcereeeetserseeers J

2. The Culturing of a Calanoid ..........ee scenes dececceescesereavees 9

Methods and Materials .....cesscccsccceecerececccsceeceercseese 4

Temperature «2. eee eceeece eee ceeccceees
Food .....25+4-- be eeeeeeee bene eceeee eee a ne eceencneseee
Disturbance of Animals ..........- se eeeeeee eer reer

Culturing Success ..seccseccececsccnceecereeteereccccsercoscees Al
SUMMATY 2... c eee e eset eect nese ee nesses rsscecerscesserennes AZ
3. The Effects of Temperature on the Reproduction of Diaptomus clavipes .. 43
Methods and Materials .........ccecceeceecerecerecececeeeeeneee 43
Female Life Spans after Sexual Maturity ....-++.e+s seecccevenecees 48
Clutch Size and Total Egg Production cccecuacereeseseseesevstens 49
Rate of Clutch Production ........ececcceececsereenessccscecsens 62
Hatching Success .----- ee eeeeeee rer rrer ters ete eceeneeee 65

Discussion .......ccccccceecccccvcccsseccscecessocecersncercese JB


-6€l-

Table 50, A COMPARISON OF THE RANKS OF THE NUMBERS OF ADULT CYCLOPS VERNALIS (AS SHOWN
IN TABLE 48) KEPT IN SIX DIFFERENT TYPES OF WATER, THE SIX WATERS
WERE RANKED FROM HIGHEST NUMBER OF ADULTS (RANK 1) TO
LOWEST (RANK 6) SEPARATELY FOR EACH

WEEK OF SAMPLING

Type of water
1/10 Noble V/5 Tap
Week Noble Pond Tap Distilled 9/10 Dist. 4/5 Dist.

1 4.5 1.5 1.5 3 45 6
2 5 1 2 3.5 3.5 6
3 1 2 4.5 4.5 3 6
4 2 1 3.5 5 3.5 6
5 2 1 3 5 5 5
6 2 1 3 4 5 6
7 2 1 3 6 45 45
8 2 1 3 4 5 6
Total of ranks 20.5 9.5 23.5 35 wu 45.5
App. TDS 1000 110 500 25 125 110



The foregoing experiments in this subsection together with our general experience in
other experiments pointed out several problems with the use for food of material taken
from mixed laboratory cultures of algae and other microorganisms. The suitability of
this material fer culturing D, clavipes varied greatly with time. This was only to be
expected as the types and numbers of the organisms in these cultures undoubtedly
fluctuated greatly. Also, it was very difficult with this type of food to determine and
control the exact amounts of material being added. The food culture medium was di-
luted to a constant light transparency at each food addition in an attempt to standar~
dize the quantity of food added. However, as the shapes and sizes of the food particles
as well as their quantity are known to affect light transparency, this procedure could

only have been partially successful.

The problems with the use of the mixed culture material for food necessitated a search
for a more suitable material. Yeast and Chlamydomonas were used in the preceding
experiment partially to test their suitability. Both seemed adequate for some purposes,
but they still posed problems of exactly controlling the amounts added and both re~
quired a fair amount of effort to maintain the fresh cultures required to provide good
food material, Also, the favorable results obtained by culturing with yeast were
found to be due, quite largely, to the endogenous cultures growing within the cope~
pod culture beakers rather than to the yeast directly. This fact, which very possibly
is also true for Chlamydomonas, further complicates the standardization of food avail-
ability for copepod populations maintained in separate containers in the light.

Toward the end of our work in developing methods for culturing, a food material which
had been used successfully with Daphnia came to our attention. This material is basi-
cally a blend of commercially prepared fish food with dried grass. It was produced by
blending 10 g of Purine trout food, 0.5 g dried alfalfa grass, and 250 ml of filtered
pond water for 5 minutes at top speed ina Waring Blender. The blender was washed
with an additional 50 ml of pond water, and the entire mixture strained through #20

bolting cloth screen, The material retained on the screen was discarded and the filtrate

-31-


eggs from the female and to allow them to hatch while separated from her. Females
carrying egg sacs. were collected from the cultures at 14°, 2 °, 26°, and 31°C and
from the vials at 5°C. The egg sacs were removed from each female, the eggs were
counted, and then the sacs were placed in a Scm (diameter) Petri dish containing
5 ml of filtered pond water. The eggs were checked at intervals of several hours,

and the nauplii were counted as soon as possible after the eggs hatched.
DURATION OF THE FEMALE ADULT STAGE

The mean durations of the adult stage of Cyclops vernalis females were found to vary
inversely with temperature (Table 53, Figure 17). The longest period that a female
lived after its adult molt wos 84 days and the shortest period was 20 days (Appendix
G-1). The mean durations ranged from 26.2 days at 31°C to 74.2 days at 14°C,

A significant difference (P < 0.001) was found among these means by analysis of
variance. The homogeneity of the means was then tested by a posteriori comparisons
(Student-Newman-Keuls test; Sokal and Rohlf, 1969), The results indicated that the
duration of the adult stage was inversely related to temperature; the duration at each
temperature being significantly longer (P < 0,05) than that observed at any of the
higher temperatures and significantly shorter than that observed at any of the lower

temperatures.

The mean time intervals between the adult molt and the production of the first egg sacs
for females.at the four temperatures were also compared (Table 53, Figure 17, Apperi-
dix G-1). When tested by analysis of variance, a significant difference (P < 0.001)
was found among the mean intervals at the four temperatures. The time from the adult
molt to the production of the first egg sacs was longest at 14°C, i.e. 27.3 days, and
decreased with increasing temperatures; i.e. 23.9, 12.9,and 8.3 days at 21°, 26°
and 31°C, respectively. From Figure 17 it can be seen that approximately one-third
of the adult stage at each temperature was spent before egg production was initiated.

-150-


RATE OF CLUTCH PRODUCTION

Although listed in chronological order, the data on the rate of clutch production
presented in Appendices B-1 through B-i2 are disjunct, data for some clutches
being unobtainable for various reasons. For example, clutches were rather fre-
quently knocked off prematurely, usually by the experimenter, but on occasion by
the copepods themselves.. In these instances no data on clutch carrying time or in-
terval until the next clutch are reported for that clutch, although a value for the
time from oviposition to oviposition is recorded. Early in the study, there were sev-
eral instances in which a new clutch was not recognized as such until it was im-
possible to make a reasonable estimate of the time of oviposition, so that only the

interval following such a clutch could be determined.

Since D, clavipes females carry clutches until the eggs hatch, the cycle of egg pro-
duction is naturally divided into two parts: the time the female is carrying develop-
ing eggs and the time between clutches during which there are no eggs. If these
two parts of the cycle were to vary independently with temperature, it would be
possible for the length of the overall cycles at different temperatures to be iden-
tical, despite having the two component parts quite different. Therefore, all three
times (the length of time clutches were carried by the female, the Jength of the in-
terval between successive clutches, and the length of the complete cycle from ovi-
position to oviposition) are reported here. Since the data are incomplete, the re-
ported mean clutch carrying times plus mean intervals between clutches do nof ex-
actly equal the mean complete cycle times reported for each incubation tempera-

ture.

Three of the females incubated at 21° produced clutches of resting eggs. Since
this was a completely different egg type and the resting eggs obviously were not
carried until they hatched, the three time intervals when resting eggs were involved

were compared to the three time intervals when clutches of subitaneous eggs were

-62-


means presented in Table 24), the mean hatching success is highest for those females
which hatched and lived their entire lifetimes at a single temperature except at
21° incubation. However, orthogonal sets of a priori comparisons (shown with the

and 27°

‘ data and indicate significant variability even among the females “native” to each

Anova tables in Appendices C-1,-2, and -3) were made for the 14°, 21°

,

of these incubation temperatures.

Whether all data at each incubation temperature are used or only the data for fe-
males “native” to each incubation temperature, mean hatching success is highest
at 21° and is progressively lower at both higher and lower temperatures (Table 24),
Considering only those females "native" to each incubation temperature and there-
by eliminating any possible effects due to the individual's hatching temperature,
analysis of variance indicates that mean hatching success varies significantly (F=
4.4; P<O.010) with temperature (Table 24). An a posteriori SNK test, however,
shows no significant differences among the means for 4, 27°, and 31°, or be~
tween those for 14° and 21°. If all data, regardless of the female's hatching tem-
perature, for each incubation temperature are used, significance is even greater
(F=9.0; P<O.001), and the SNK test indicates that the mean for 21° is significantly
higher than the means for the other three incubation temperatures, which do not

significantly differ from one another.

‘Occasionally, a female lost her clutch before the eggs hatched. This happened
especially during the counting of the eggs in a new clutch. Until they hatched,
loose clutches were isolated in a small Petri dish containing water,and the resulting
data are included here. For these loose clutches, it was usually possible to locate
empty egg membranes after hatching, especially when they were all held together
by the egg sac membrane, as was often the case. Since the water volume was small,
about 20 ml, it was very easy to count all nauplii present. When the apparent
hatching success was less than 100% and even when it apparently was zero, it was

quite unusual to find either unhatched eggs or dead nauplii. In one case a nauplius

-71-


EPA-660/3-74-006
APRIL 1974

Ecological Research Series



Table 31. DURATIONS OF THE VARIOUS INSTARS AS
DETERMINED IN THE FIELD AND COMPUTED FROM
A COMPOSITE OF THE LABORATORY DATA
(approximately 20° to 25°C; Samples, 1972).

Duration
First
Stage Occurrence Laboratory
Egg-NIII 2 1.8
NIV 2 1.4
Nv 2 1.2
NVI 2 1.2
cl 2 1.5
cil 2 1.9
cil 6 3.5
CIV 6 4,4
cv 4 4.6
Composite
through CV 28 21.5
cvi 40 40.00
(used field duration
after comparison
with Table 15)
Total life cycle 68 days 61.5 days

-97-


Yeatman, H.C. American Cyclopoid Copepods of the viridis-vernalis Group (In-
cluding a description of Cyclops carolinianus n.sp.). Amer. Midland Nat. 32:1~-
90, 1944.

Zillioux, E.J. and D.F. Wilson. Culture of a Planktonic Calanoid Copepod through
Multiple Generations. Science. 151:996-998, 1966,

-171-


TEMP.

-0dl-

26

n n n n 1
4 Ww 9 6 -3
v vi VEE VEE 1X

n
14
x

Figure 14.

DATE

Mean Temperature and Range on Each Collecting Date. The Vertical Line Between 18 April
and 24 April Shows a Change in Scale from 2-Day to Approximately 14-Day Intervals.



from a dense population contained ina freshwater aquarium maintained in the Zoo-
logy Building on the University of Oklahoma campus, Norman, Oklahoma, The
source of these copepods could not be determined; however, the species C. vernalis
is known to be a common one in central Oklahoma (Kingsbury, 1968). A fifth cul-
ture was attempted at 5° C but was discontinued due to the low number of eggs pro~
duced and to the very slow rate of development of the immature stages. All animals
used in experiments at 5°C were reared at 21°C and then transferred to the 5°C room
at least 2 weeks before their use. The temperatures of 5, 14, and 21°C. were main-
tained in constant temperature rooms, while the temperatures of 26 and 31°C were
maintained in constant temperature chambers, Temperature was measured daily in
one container at each temperature throughout the experiments and wos never found
to vary more than 1°C in any chamber or room,. The culture medium consisted of
pond water which had been filtered through a double layer of #12 bolting cloth (125
threads per inch), Each temperature room and chamber was set for alternating per-
iods of 12-hours light and 12-hours dark, The cultures were fed the mixture of trout
food and dried alfalfa whose preparation is described in Chapter 2, With this mix-
ture a food source of constant ‘quality could be provided at each temperature. The
1-liter cultures were fed 0.5 ml of food mixture twice each week. Observations

of the copepods were made through a binocular microscope with magnifications of

7 to 30 times, Individual copepods were transferred using a micro-pipetie,

Experimental Procedures

Late-stage immature female copepodites were selected from the stock cultures at 14,
21°, 26°, and 31°C and placed singly in 4-dram shell vials, each of which contained
10 ml of filtered pond water, Two mature males were introduced and maintained in
each vial from the time the female matured (adult molt) until her death. These males
were always from the same stock culture as the female in whose vial they were placed.
Each female was examined daily from the time she was mated until her death. The

number of egg clutches produced by. each female and the number of eggs in each clutch
were recorded,

~148-


ABSTRACT

This report presents the results of studies undertaken to develop a method of maintain-

ing healthy, self-propagating, laboratory cultures of the freshwater calanoid copepod,
Diaptomus clavipes. Recommendations are given as to the conditions of container size,
type of culture medium, light conditions, temperature conditions, food type and quan-

tity, frequency of replacement medium, and amount of disturbance suggested for culturing.

The results of a study dealing with effects of temperature on certain reproductive at-
tributes of this species are presented. Temperature is shown to affect the longevity of
the adult females as well as the size, carrying time, and probably total lifetime pro-
duction of clutches. The results of this study indicate that certain of the reproductive
attributes of the females are affected by the temperature of early life os well as the

acclimation temperature.

The report includes the results from a study on the dynamics of a field population of
D. clavipes. The durations of the various life history stages were estimated both from
laboratory and field data. Life tables were constructed for the spring generation of
this population as well as all generations in a reproductive year combined. The stages

of greatest relative mortality were identified.

The report also presents recommendations for culturing the cyclopoid copepod, Cyclops
vernalis, and the results of studies concerning the effects of temperature on certain re~
productive attributes of this species. Temperature is shown to affect longevity of the

adult female, egg carrying duration, clutch size, and egg development rate.

This report was submitted in fulfillment of Project Number 158-250, Grant Number
18050 ELT by the Office of Research Administration, University of Oklahoma, under
the (partial) sponsorship of the Environmental Protection Agency. Work was completed
as of June 1973.


APPENDIX B-2
21° INCUBATION: TIME (HOURS) CLUTCHES WERE CARRIED

-08I-

Female's hatching
temperature 21° 27° 31°
Individual BIT B12 BI4 BI7 B18 B19 B20 BI6é B13 BIS

53.5 46.5 49.0 49.0 52.5 55.5 47.0 53.0 48.0 24.0
47.0 47.0 49.5 |. 49.0 49.5 45.0 |-49.5 49.0 44.0 44.5
56.5 38.0* | 60.0 54.0 54.0 46.5 52.5 57.5 10.0 47.0
55,5 36.0* 53.5 50.5 52.5 50.5 46.0 58.0 56.0
42.5 43.0* 46.0 41.0 54.0 53.5 51.0 43.5* | 44.5
54.5 45.5* 54,5 31.0* | 50.0 48.0 51.0 47,0* | 57.5
60.0 31.5* 3) .5* 55.5 48.0 49.0* | 46.5
39.0 43.0* 52.0 53.5 4L.5 40.0% | 45,0
43.5 46.5* 54.5 54.5 44.0* | 39.0
44.5 45.0* 48.5 47.0 63.5* | 72.0
45.5 | 38.5* 52.8 | 56.0 | 44.5* | 52.5
68.5 40.5* | 56.5 51.0 36.5* | 71.5
23.5 24.0% 50.5 | -48.0 45.0
52.0 43.0 | 52.0
43.0 45.0 65.0
64.5 24.0*

36.5 47 .5*

22.5 39.0*

*Indicates carrying time of a clutch of resting eggs.


APPENDIX D-1

MORPHOMETRIC MAP OF THE POND SHOWING DEPTH CONTOURS
IN CENTIMETERS. THE SCALE fS 1:200.

~200-


Table 32.

DETERMINE THE DURATIONS OF THE VARIOUS STAGES. *

LIFE TABLE FOR THE FIRST GENERATION USING THE FIRST OCCURRENCE TO

Stage Number living Number dying Mortality rate Mean number | Total life Mean lifetime
at beginning of In interval per 1000 alive alive expectancy | remaining for
age interval dy at beginning of ly Tx those attaining
k age interval age interval
qx ey
Egg~NIII 1000.00 836.62 836.62 581.69 3119.02 3.12
NIV 163.38 17,22 105.39 154.77 1955.64 11.97
NV UNABLE TO DETERMINE FROM DATA.
NVI 146.16 89.73 613.87 101 .30 1336.56 9.14
cl 56.44 1.05 18.61 55.92 1133.96 20.09
Cll 55.39 15.79 285.07 47.49 1022.12 18.45
cil 39.60 ‘ 16.74 422.73 31.58 927.14 23.41
CIV 22.86 0.57 24.93 22,58 739.76 32.36
cv 22.29 3.16 “141.63 23.87 604.28 27.11
cv 25.44 25,44 1000.00 12.72 508.80 20.00

Original 1,, equals 10.454 x 10°,


~6l-

Table 6. A COMPARISON OF THE AVERAGE VALUES (4SE) FOR CERTAIN REPRODUCTIVE AND
DEVELOPMENTAL PARAMETERS FOR D. CLAVIPES INDIVIDUALS AT

THREE DIFFERENT LIGHT INTENSITIES.

Light Total Average Number Average Average Average Days} Average Days
Intensity Number of of Clutches Clutch Percent Clutch For Development

(foocandles) | Clutches Per Container Size Hatch Carried To Adult

200-210 10 1.3 11.2 82.9 2.4 34.3
(£0.5) 2.4) (£4.5) (£0.5) (£3.3)

125-140 é 0.8 11.5 59.1 2.6 21.7
(€0.3) 0.9) (£8.3) (40.4) (£1.8)

0 8 1.0 12,2 43.0 1.8 27.0
(40.2) (42.6) 14.1) (0.5) (£1.8)



APPENDIX D-3

NUMBER OF QUADRATS IN EACH REGION OF THE POND AND IN THE TOTAL
POND ON EACH COLLECTING DATE, THE SURFACE AREA CAN BE
COMPUTED BY MULTIPLYING THE NUMBER OF QUADRATS

BY 45.54 CM?
Date Open water Rooted aquatic Total Pond
region region
"-1971
19 86,965 86,965 173,931
21 92,508 92,508 185,016
23 91,769 91,769 183,538
25 94,355 94,355 188,711
27 94,355 94,355 188,711
U-1971
1 93,247 93,247 186,494
3 93,247 93,247 186,494
5 90,660 90,660 181,321
7 90,660 90,660 181,321
9 89,921 89,921 179,843
ih] 89,921 89,921 179, 843
13 89,921 89,921 179, 843
15 89, 182 89, 182 179,364
W7 89, 182 89, 182 179,364
19 89, 182 89, 182 179,364
21 88,813 88,813 177,626
23 87,867 87,867 175,734
25 86,962 86,962 173,925
27 86,585 86,585 173,171
29 86,208 86,208 172,417
31 85,857 85,857 171,714
V-1971
2 115,099 55,107 170,206
4 109,198 59,551 168,749
6 99,363 67,878 167,241
8 95,429 71,108 166,538
10 91,495 73,534 165,030
12 81,409 81,409 162,819
14 73,746 86,862 160,608

~202-


Table 25, SUMMARIZED RESULTS

Incubation temperatures 14 21 27 31
(degrees C)
Adult longevity (days) 84.6 43.2 27.0 19.2
*Mean clutch size 26.8 28.0 30.0 21.3
*Mean total number of
eggs per female 322.0 343.9 340.0 117.3
Mean rate of clutch
production (hours) 166.7 63.7 47,3 55.2
Hatching success (%) 82.3 85.8 71.7 60.6
*Mean for females "native" toeach incubation temperature.
Table 26, ESTIMATED PRODUCTION OF NAUPLII
Incubation temperatures 4 21 27 31
:(degrees C)
A. Total nauplii per
female 272.4 | 295.1 243.8 71.1
B, Daily rate of pro-
duction of nauplii
averaged over adult
lifespan 3.13 6.83 9.03 - 3.70
C. Daily rate of produc-
tion of nauplii while
female is actively :
breeding 4,54 9.05 10.91 5.61



APPENDIX B-8
31° INCUBATION:. INTERVAL (HOURS) BETWEEN CLUTCHES

Female's
hatching
temperature | 21° 27° 31°
Individual D8 D5 D3 DI DI6é DI7 DI9 D20
22.0 | 24.0 | 191.5 | 10.5 | 17.5 | ----- W.0 | 49.5
13.5 24.5 12.0 9.5 19.0 14.0 18.0
13.0 | 24.0 81.5 33.0 9.0
61.0 24,0 24.0 | 22.0
21.5 4.5 | 38.0
96.0 31.0
22.5

Intervals between successive clutches are listed from top to bottom in the order of oc-
currence but are not necessarily consecutive, some having been omitted for various
reasons as discussed in the text.

Variances of the data for the three hatching temperatures are heterogeneous. A logarith—
mic transformation was made and Anovas carried out with the transformed data.

Anova table: comparison among individuals of intervals between clutches.

Source of Variation ; df MS F
Among individuals 6 0.1420 1.3172 ns
Within individuals 22 0.1078
Total 28

-188-


only half emerged from the egg was found dead. The impression, overall, was that
in almost all cases all eggs hatch, but that some nauplii die very shortly thereafter
and quickly decompose. In view of the usual delay between the time when the

nauplii hatched and the time when they were removed from the culture and counted,

the data reported here might more accurately be considered the percent hatching

and surviving for approximately 12 hours thereafter.

DISCUSSION

The inverse relationship between adult longevity and temperature reported in this
chapier is as expected for poikilotherms. The mean lifespans reported are quite
similar to adult lifespans previously reported for other species cultured in the lab-
oratory, Wilson & Parrish (1971) reported that females had a mean adult lifespan
of 37.7 days when the marine calanoid copepod Acartia tonsa was incubated at
17.5°C. Smyly (1970) reported that females of the predaceous cyclopoid Acantho-
cyclops viridis incubated at 16-18°C had a mean adult longevity of from 30.8 to
57.3 days, depending upon the type of food.

Incubation temperature at or below 27° apparently has little effect on clutch size,
at least among “native” females. This is in keeping with reports by other investi-
gators. Considerable work has been done, particularly with various marine species,
in an effort to discover what factors determine clutch size. The consensus is that
clutch size is determined by the female's body size, not directly by temperature or
food concentration (Corkett & McLaren, 1969; Smyly, 1968; Ravera & Tonolli,
1956), although it is conceded that under conditions of severe food shortage clutch
sizes will decrease (Corkett & McLaren, 1969). Body size has normally been found
inversely related to temperature (Coker, 1934a; Lock & McLaren, 1970; Smyly,
1970), but there have been reports both from the laboratory (Mullin & Brooks, 1970)
and from the field (Deevey, 1964) of adult size being directly related to the food

supply available to the immature stages. Obviously, the factors contributing to

-73-


CHAPTER 1
INTRODUCTION

Calanoid copepods, especially those from the genus Diaptomus, play a very impor-
tant role in the ecosystems of lakes and ponds. In most of the larger bodies of water
and many of the smaller ones, the diaptomids are very abundant, often the most a=
bundant, microcrustaceans. Their role in these ecosystems seems to be primarily
that of a herbivore. They, in turn, are fed on by carnivorous zooplankters and by
fish, especially larval and juvenile stages. Thus, they are of vital importance as
intermediate links in the food chain of our lakes and ponds and can be of significance
in determining types and amounts of fish, Their vital role not withstanding, how~
ever, our knowledge concerning the general biology, environmental requirements,
and interrelations within the ecosystem of this group of organisms is woefully inade-
quate. We are in no position to state what effect past changes in water quality
have had on these organisms nor to predict consequences of future environmental

alterations.

One of the major reasons, if not the main one, for our inadequate knowledge of

this group is our inability to maintain self-propagating cultures under controlled con=
ditions, This has acted as a major obstacle to efforts to ascertain environmental re-
quirements and to determine the consequences of changes in water quality for popu-

lations of these animals.

Thus, in order to increase our knowledge of this group, a research program has been
carried out which had the following objectives:
1. To learn how to keep healthy, self-propagating cultures of a diap-
tomid in the laboratory.

2. To study in controlled experiments the effects of environmental
factors, especially temperature, on diaptomids.


APPENDIX C-1
14° INCUBATION: PERCENT HATCH

Female's hatching
temperature 14° 21° 31°
Individual A7 Aé Ae A? Ald
100.0% 45.8% 15.0% 87.5% 100.0%
100.0 61.1 4.4 100.0 100.0
82.6 78.9 0.0 64.3 95.5
45.5 73.3 22.7 56.2 81.0
100.0 94.4 46.2 50.0 95.8
74.2 57.9 0.0 27.8 70.8
85.7 33.3 0.0 81.8
93.3 66.7
83.3 86.7
61.8
85.7
7.4
Mean % 82.3% 68.0% 9.2% 55.7% 92.9%

An arcsine transformation of the data above was made, the means above were com-

puted, and an Anova carried out:

Source of Variation df MS F
Among individuals 4 31.46.0922 8.2802 P<0.001
A7 vs Others 1 1797 1592 47.3521 P<0,001
Aé vs A8, 9 & 10 1 325.3057 0.8562 ns
A8 vs A9 vs A10 2 5230.9521 13.7674 P<0,001
Within individuals 36 379.9531
Total 40

194=


CHAPTER 3
THE EFFECTS OF TEMPERATURE ON THE REPRODUCTION
OF DIAPTOMUS.CLAVIPES

Relatively few controlled laboratory studies on the effects of temperature on fresh=
water copepods have been reported, although Coker (1933, 1934a, 1934b, 1934c)
carried out a series of significant experiments with cyclopoids in the early thirties.
Recently, other workers, notably Comita (1965, 1968), and Sfefken and Armitage
(1968) with diaptomids and Smyly (1970) with a cyclopoid, have conducted lab-

boratory studies that included some consideration of temperature effects on certain

physiological properties.

The development of a method for culturing Diaptomus clavipes, as reported in the
preceding chapter, opened up the possibility of studying the effects of temperature
on the reproduction of this species. The present. chapter presents the results from
such a study. This work was carried out to increase our understanding and predic-
tive capabilities concerning the consequences of temperature fluctuations on the
population dynamics of this species in particular and diaptomids in general, Ad--
mittedly, there is no guarantee that laboratory data accurately reflect what actu-
ally happens ir a natural population. For that reason, predictions based on labo~
ratory data alone can only be used with great caution. However, the laboratory
results can provide a quantitative basis upon which to design and intrepret field

studies.
METHODS AND MATERIALS

Stock cultures of Diaptomus clavipes were established using animals obtained on
May 19 and 21, 1971 from a farm pond near Norman, Cleveland County, Oklahoma.


Table 56. DEVELOPMENT TIMES OF CYCLOPS VERNALIS EGG CLUTCHES
AT DIFFERENT TEMPERA TURES.
Development Time (hours)
7
Clutch
No. aC 14°C arc 2c | 31°C
1 13 (ays) 64 40 24 24
2 13 (days) 72 40 24 24
3 14 (days) 80 40 24 24
4 80 40 24 24
5 80 40 32 24
6 40 32 24
7 40 32 24
8 48 32 24
9 48 32 24
10 48 32 24
VW 48 32 24
12 32 24
13 32 24
14 32 24
15 32 24
16 32 32
17 32 32
18 32 32
19 32 32
20 32 32
21 32 32
22 32 32
23 32 32
24 32 32
25 32 32
26 32 32
27 32 32
28 32 32
29 32 32
30 32 32
31 32 32
32 32 32
33 40 32
Mean 13.3 75.2 42.9 31.3 28.4
SEX +320 41.22 t0.54 £0.70
no. 3 5 11 33 33

-160-


the underside of the last body region, or urosome, until they hatch into nauplii.
We have found (see Chapter 3) that temperature affects the length of time the eggs
are carried by the female of this species. A series of experiments was designed to
determine if rooted aquatics also can affect the length of time eggs are carried and

something of the nature of any effect that was found.

Five sets of 10 replicates each were used in the study. One set of beakers (desig-
nated weed-restricted) had pieces of Potamogeton sp. restricted to a small region
with nylon netting, while in a second set only nylon netting was added. A third
group of containers had 15 pieces of polyethylene tubing of approximately the

same cross-sectional diameter as the Potamogeton sp. suspended vertically and ran-
domly in each container. Suspension of the polyethylene tubing in the containers
was accomplished by fastening a section of nylon netting over the top of the beaker
to act as a guide for the sections of tubing. The polyethylene tubing was soaked in
distilled water for 96 hours prior to the onset of the experiment, A fourth set of
beakers also included polyethylene tubing, but it was restricted to the perimeter of

the beaker, The final set of beakers, the controls, had nothing added.

In an attempt to keep conditions constant, animals were transferred weekly to a
clean beaker containing fresh water. At this time young were removed and discard-
ed. The beakers were checked at 24-hour intervals for animals carrying eggs and
for nauplii, Checking for nauplii was necessary to ensure that a clutch had not been
produced and then hatched during the preceding 24~hour period.

HETEROGENEITY OF DISTRIBUTION

Field Data

At the onset of the study the concentration of adults did not vary greatly between the

two regions of the study pond-~a higher concentration was noted in the open water on

“85+


a 74

21° INCUBATION: NUMBER OF EGGS PER CLUTCH IN THE SEQUENCE PRODUCED

APPENDIX A-2

Female's hatching]
temperature 21° 2? 31° +Standard

iia 8 81 a re nS Total Mean error

Clutch No.
1 2 4270 220 2h 8k | 8 | 8 225 22.5 1,46
2 29 2 1% 30 22 38 (28)| 20 | 24° 12 247 24.7 2.39
3 42 19 1 7 30 %6 361] 23}]o N 224 22.4 3.72
4 45 32 2 2% 35 2 | 18 | 15 12 230 25.6 3.47
5 a 31 42 40 2 2% | 32 | 19 27 263 29.2 2.62
6 22«7 17 4 33 «43 | 14] 2 24 244 27.1 3.51
7 28 9 «62535 «|| «17 «| 2718 195 24.4 2.15
8 28 (33 22 ~ | 3] 2 on 185 22.1 3.15
9 2 = 30 28 | 24 | 19 «17 143 23.8 2.05
10 2 35 27 | 2 | 4 13 135 22.5 3.40
W 2 42 32 | 15 | 14 oO 133 22.2 5AM
12 27-32 27 | 17 | 16. 08 127 21.2 3.66
13 16 37 os | 28 05 84 16.8 4.73
14 22028 34 12
15 30-26 14
16 23 34 24
17 1% (44 17
18 20 (34 13
yD 20%
20 2% «35
2 2835
22 40 34
23 3230
24 16 24
25 25

Total 629. 774 +56 157 229 204 358 298 213 265

Mean 26.2 31.0 18.7 26.2 28.6 2%.1 27.5 21.3 17.8 14.7

Standard error 1,58 1.11 1.76 3,68 3,08 2.30 2,33 1.76 2.13 1.38

Parentheses indicate a clutch which was not counted, Value is an average for that female.


177.8 eggs at 14°C and 180.9 eggs at 26°C. Total egg production by females at
21°C was found by a posteriori comparison to be significantly greater than that by
females at 31°C, All other paired comparisons of mean total egg production at the
different temperatures proved to be not significant. The extremes for individual life-
time egg production ranged from 430 eggs produced by a female at 26°C toa low of
3 eggs contained ina single egg sac and produced by a female at 21°,

DEVELOPMENT. TIME OF EGGS

Cumminset. al., (1969) considered the development period of copepod eggs as the
period from the first appearance of eggs in the egg sacs fo the time of hatching. In
our experiments with Cyclops vernalis, the egg development rate was determined

similarly.

The times for development of C, vernalis eggs in relation to temperature are shown
in Table 56. For these data each clutch at 5 and 14°C was produced by a different
female, whereas as many as 3 clutches may have been produced by a single female
at the temperatures of ar, 26°, and 31°C, The results indicate that the develop-
ment times of eggs increase as temperature decreases, The mean development times,
ranging from 28.4 hours at 31°C to 13.3 days at 5°C, were shown by analysis of
variance to differ significantly (P > 0.001). A posteriori comparisons among the mean
times at all the temperatures except 5°C indicated that the only two means not sig-
nificantly different (P_ 0,05) were those at 26 and 31°C, The development times
at these temperatures were 31.3 hours and 28.4 hours, respectively. As the mean
development time at 5°C was obviously much longer than the times at the higher

temperatures, it was not included in the comparisons.

The rates of development of eggs, expressed as the reciprocal of mean development
time, have been plotted against temperature (Figure 18). This plot shows a steady

increase in the rate with temperature up to 26°C, From 26°C to 31°C the rate of
increase of the development rate slows.

-159-


Table 20, COMPARISON OF TIME INTERVALS OF RESTING AND
SUBITANEOUS EGGS PRODUCED AT 21° INCUBATION

Resting Subitaneous Analysis of
eggs eggs variance results
Mean carrying time
(hours) 39.0 48.2 F=15.3; P<0,001
Mean interval
following clutches
(hours) 9.7 10.6 F=0.18; ns
Mean complete cycle
which included clutches
(hours) 53.2 63.7 F=8.5; P<0.005

~64-


-922-

E-T OFE/S1E-99S FL6T:39IEIO ONILNILd ANZWNYZAOD Se

APPENDIX H-4
FECUNDITY IN EGGS PER CLUTCH OF FEMALE CYCLOPS VERNALIS AT 31°C

Clutch Female No.
No. | 1 2 3 4 5 é 7 8 9 0 3 12° #13 4 15 %6 #17) Ww #9
1 | 31 440 48 50 ATK * 3 6 2 DB 4 8 3 62 5
2 a 970 42ST iC 3305 6 Dp 3 8
3 {19 52 2% 15 2 2 2 17 2% 7 a 4 3% 38 32
4 3100 32C—iaR nm 2 18 472538
5 33° (34) 14 % 20 50
ra 4 35 51
7 15 %
8 12
9 16

Total

rer 42 208 204 145 12% 106 65 82 OI7 2 7 % «107 54 4 242 1430 «191 356
female

No, of

a

female

*Only one of two possible egg sacs present.

() egg count not available, number ts average clutch size for that female.


APPENDIX E-1 (Cont.)

TOTAL NUMBER OF ADULTS (CVI) IN EACH REGION OF THE POND AND IN
THE TOTAL POND ON EACH COLLECTING DATE, THE TOTAL NUMBER
WAS DETERMINED BY MULTIPLYING THE Y (MEAN) NUMBER OF

ADULTS PER QUADRAT IN EACH REGION BY THE TOTAL

NUMBER OF QUADRATS IN THAT REGION AND
SUMMING THE PRODUCTS FOR EACH DATE.

Date Open water Rooted aquatic Total pond
region region
V-1971
12 1,641,783 54,006 1,695,789
14 1,908,190 21,715 1,929,906
16 “1,690,980 101,830 1,792,811
18 643,697 104,634 748 ,332
20 1,886,343 41,056 1,927,399
24 1,497,714 26,408 1,524,122
V-1971 .
4 2,572,700 178,565 2,751,264
18 911,433 781,697 1,693, 130
28 344,186 14,141 358,327
VI-1971
YW 1,813,607 118,418 1,932,025
25 4,782,553 0 4,782,553
VII-1971
9 1,994,547 106,288 2,100,835
23 4,181,914. 388, 330 4,570,244
VIN-1971
6 1,827,506 957 ,939 2,785,445
20. 1,521,805 1,173,600 2,695,405
1X-1971 :
3 1,085 , 287 2,579,838 3,665,125
7 940,950 858,012 1,798,962
30 348,840 1,137,614 1,486,454
X-1971
4 218,025 415,319 633,344
21 17,600 255,784 273 , 384
29 25,704 346,081 371,785

-207-


of food material of these different types that was added somewhat equal, the suspen-
sions were all diluted with distilled water to a common spectrophotometric transparency

value of 70% at 420 millimicrons.

All but five of the beakers were exposed to a 12-hour~on 12~hour-off cycle of mo-
derate illumination, and efforts were made to equalize the distance of the beakers
from the fluorescent light source. Five of the beakers that received yeast as food
were not exposed to any light--except for a very small time during the addition of
food, These beakers were completely covered with aluminum foil and were added
to the experiment 3 weeks after the other beakers were initiated. The experimental

containers were all kept in a constant temperature room at approximately a°c,

The total number of copepodites in each container was estimated on a weekly basis
for the duration of this experiment. These estimates were based on counts from three
subsamples which were obtained by using glass tubing as described previously. Also,
after the first few weeks, separate estimates were made of the number of adults in
each of these containers. The results from the counts are presented in Tables 9 and

10 respectively.

The immediate impression gathered from these tables is that the mixed culture furnished
the poorest of the three types of food. All 10 containers to which this food was added
during the experiment were devoid of copepodites by the end of the work, This re-
sult is attributed primarily to the quality, as a food for D. clavipes, of the material
added from the mixed culture. The production of individuals in the copepod stock
cultures while this experiment was in progress was very poor relative to the production
at other periods, A microscopic examination of the mixed culture material showed
very few living cells, and even the color of the material was noticeably paler than

usual,

A contributing factor to the extinction of the populations in all the mixed culture

containers was the relatively small volumes used. With food of poor quality, only a

~25-


Ron, U. On the Number of Eggs in some Free-living Freshwater Copepods. Verh,
Internat. Verein. Limnol. 12:447-454, 1955.

Samples, C.R. Laboratory Culturing of the Colanoid Copepod Diaptomus (Aglaodiap-
tomus) clavipes Schacht 1897. Unpublished M.S. Thesis, Univ. of Okla. 60 Per
1972,

Siefken, M. and K.B. Armitage. Seasonal Variation in Metabolism and Organic

Nutrients in Three Diaptomus (Crustacea: Copepoda). Comp. Biochem. Physiol.
24:591-609, 1968,

Small, L.F. An Optical Method of Measuring Phytoplankton Standing Crop. lowa
State J. Sci. 35:343-354, 1961.

Smyly, W.J.P. Number of Eggs and Body Size inthe Freshwater Copepod Diaptomus
gracilis Sars in the English Lake District. Oikos. 19:323-338, 1968.

« Observations on Rate of Development, Longevity and Fecundity of
Acanthocyclops viridis (Jurine) (Copepoda, Cyclopoida) in Relation to Type of Prey.
Crustaceana, 18:21-36, 1970. ,

Sokal, R.R. and F.J. Rohif. Biometry. W.H. Freeman & Co, (San Francisco). 776 p.,
1969.

Taub, F.B. and A.M. Dollar, The Nutritional Inadequacy of Chlorella and Chla-
mydomonas as Food for Daphnia pulex. Limnol. Oceanog. 13:607-617, 1968.

Tonolli, V. Studio sulla Dinamica de! Popolamento di un Copepode (Eudiaptomus
vulgaris Schmeil), Mem. Ist. Ita. Idrobiol. 13:179=202, 1961.

. Biometry as a Means of Evaluating Demographic Changes in Natural
Populations of Copepods, Verh, Internat, Verein, Limnol. 15:752-757, 1964.

Walter, E. Uber die Lebensdaver der freilebenden Susswasser-Cyclopiden und andere
Fragen ihrere Biologie, Zool. Jb. (Syst.). 44:375-420, 1922.

Welch, P.S. Limnological Methods. McGraw-Hill Book Co. (New York). 381 p.,
1948,

Wilson, D.F. and K.K. Parrish. Remating ina Planktonic Marine Calanoid Copepod.
Mar, Biol. 9:202-204, 1971.

Wilson, M.S. and H.C. Yeatman. Free-living Copepoda. In: Fresh-water Biology.
Emondson, W.T., (ed.), 2nd ed., New York City, John Wiley & Sons. p. 735-861,
1959.

-170-


APPENDIX E-2 (Cont.)

TOTAL NUMBER OF COPEPODITE FIVE (CV) IN EACH REGION OF THE POND
AND IN THE TOTAL POND ON EACH COLLECTING DATE, THE TOTAL
NUMBER WAS DETERMINED BY MULTIPLYING THE Y (MEAN)
NUMBER OF CV PER QUADRAT IN EACH REGION BY
THE TOTAL NUMBER OF QUADRATS IN THAT
REGION AND SUMMING THE PRODUCTS
FOR EACH DATE,

|
Date Open water Rooted aquatics Total pond
: region region
V-1971
12 1,037,973 30,529 1,068,502
4 719,028 0 719,028
16 718,448 33,944 752,391
18 279,868 13,079 292,948
20 635 ,542 0 635,542
V-1971
4 327,948 89,282 417,230
18 183,512 125,630 309, 143
28 89,015 0 89,015
VI-1971
W 1,863,449 26,315 1,889,764
25 1,372, 163 21,083 1,393,247
VU-1971
9 148,478 (¢) 148,478
23 396, 630 40,345 436,976
VIN-1971 .
6 221,805 93,916 . 315,721
20 104,125 55,012 159, 137
IX-1971
3 68,081 218,014 286 ,096
7 119,341 53,625 172,967
30 6,845 144,459 151,344
X-1971
4 44,589 72,229 116,818
21 8,000 196,758 204,758
29 11,934 81,431 93,365

-209-


Table 13. A COMPARISON OF MORTALITY AND REPRODUCTIVE SUCCESS
(tSE) FOR D. CLAVIPES FEMALES KEPT UNDER THREE
DIFFERENT FOOD CONDITIONS. (EACH
VALUE IS THE MEAN FOR THE RESULTS
FROM SIX INDIVIDUALS.)

Experiment Food added every third day (ml)
No food 0.4 0.8
Number of 1 0 0 3
animals
dying 2 0 0 3
during
experiment 3 - 2 5
1 0.11 0.41 0.47
Number of (40.0) 40.02) (40.04)
Cte er 2 0.11 0.39 0.39
nimal per (40.0) (40.02 (40.03)
“ey 3 - 0.21 0.23
- (40.06) (40.06)
Me 1 18.3 24,5 26.9
number of (41.3) (22.2) (33.4)
ease 2 19.0 18.0 24.8
tlutch ‘ (42.5) (41.4) 4.9)
3 - 15.2 16.5
- (41.7) (41.9)



-901-

1000 ne |
LABORATORY DATA
\\
NN
100 -
8 9]
[4
wi 0. \ -
2
@)
2
z TOTAL YEAR
> 10- Xs \
a NY
0 [a oe oe es oe ee
EGG 4 5 6 1 2 3.4 5 6
N Cc 40 DAYS

+ 1
Figure 9. Survivorship Curves for the First Generation (g1), Laboratory Population, and
Lonwplete Year's Data.


SUMMARY

A study has been conducted with the primary objective of developing a reproducible
method of maintaining self~propagating cultures of the calanoid copepod D. clavipes
in the laboratory. Based on the results of this study the following culturing conditions
are recommended:

1. Containers--Wide-mouth glass vessels over one-half full and con-
Jaining at least one liter of culturing medium. For most purposes
a volume of medium in the range of 2 to 5 liters is to be preferred.

2. Culture medium--Water from an environment that naturally con-
tains D. clavipes. This water should be filtered several times
through fine mesh netting to remove all multicellular animals and
the larger algal cells.

3. Light--Moderate illumination (50 to 150 foot-candles at the water
sorface) on a cycle of 12 hours on and 12 hours off,

4, Temperature--Within a couple of degrees of 25°C.

5. Food typer-An aqueous mixture of commercial fish food and dried
a grass prepared by blending these materials as described in
the report and then filtering the resulting material. Other food
materials were found to be satisfactory for some purposes but the
fish food solution is preferable in genera! because it is easy to pro-
duce with a reproducible composition.

6. Food quantity “Approximately 0.1 ml of the fish food solution

once every 3 days. Both the volume and the frequency of feeding
can be altered somewhat as long as the total amount of material
added is kept low.

7. Changing medium-~Once every week or two.

8. Disturbance of animals--Should be minimized.

Culturing success has been tested by determining how long several females and their
progeny could be maintained in the laboratory. This test was carried out before all
the above recommendations had been arrived at, however, and so the conditions
chosen were not as suitable as those specified above. Even so, the animals were

cultured through six complete generations and into the seventh when the test was

voluntarily terminated.

-42-


APPENDIX B-12

31° INCUBATION
EGG PRODUCTION CYCLE
Time (hours) from oviposition to succeeding oviposition

Female's
hatching
temperature | 21° 27° 31°

Individual D8 D5 D3 DI D6 DI7 DI? D20

47.0 46.5 36.5 48.0 48.0 | ----- 40.5 79.5

48.5 | 48.5 24.5 | 97.5 51.0 | 40.5

47.0 | 48.0 46.5 | 47.5 68.5 | 47.5

97.0 96.0 | 116.0 55.0 50.0

76.0 30.0 70.5

47.5 63.5

49.5 57.5

119.0

Lengths of cycles are listed from top to bottom in the order of occurrence but are not
necessarily consecutive, some cycles having been omitted for various reasons as dis-
cussed in the text.

Variances of the data for the three hatching temperatures are heterogeneous. A logorith-
mic transformation was made and Anovas carried out with the transformed dato.

Anova table: comparison among individuals of complete egg production cycle.

Source of Variation df MS F
Among individuals 6 0.0276 1.1219 ns
Within individuals 25 0.0246

Total 31

-193-


METHODS AND MATERIALS

The methods used in the experiments reported in this chapter were similiar, in mosi

respects, to the methods used in the more extensive study on the effects of tempera-
ture on reproduction for C, vernalis reported in the next chapter (Chapter 6). Thus,
this section will be kept brief and the Methods and Materials section in that chapter

may be referred to if more details are desired.

Our stock cultures of C. vernalis were begun with animals from a dense population
of this species found in a freshwater aquarium in the Zoology Building on the Uni-
versity of Oklahoma campus, Norman, Oklahoma, All experiments were conducted
at approximately 21°C and under alternating periods of 12-hours light (moderate
intensity) and 12-hours dark, The measurements of total dissolved solids (TDS) were
made with a Myron L DS Meter (Model 532T 1).

The food used was the trout food-alfalfa mixture whose preparation has been descri-
bed in Chapter 2, All experimental cultures were fed a predetermined volume of
this misture at 2-day intervals.. The population in each experimental container
was censused every 7 days, and the water in each container was changed at the
same time. Unless specified otherwise, pond water filtered through a double layer

of #12 bolting silk was used as the culturing medium.

The counting method entailed reducing the volume of water in each experimental
container to about 10 ml. The excess water was drawn out of each container by the
use of a piece of glass tubing covered at one end by a double layer of #25 bolting
cloth and containing a suction bulb at the other end, After the animals had been
concentrated in this manner, small volumes of the remaining 10 ml were separately
pipetted into a small Petri dish, and the animals present in each volume counted
with the aid of a binocular dissecting microscope. The numbers of adults, gravid
(egg carrying) females, and young (copepodites plus nauplii) were recorded for each

experimental culture at each census time.

-127-


Mortality was substantially greater in the containers receiving 0.8 ml of food every
third day than in the other containers. Probably this amount of food was still too

great to avoid deterioration of the quality of the medium.

On the other hand, clutch production per animal per day was as great in the containers
receiving 0.8 ml as in those receiving 0.4 ml but, as expected, was reduced in the
containers to which no food was added. In these latter containers each animal pro-
duced one clutch and no more. Thus, if these initial clutches, which were presumably
partially formed when the experiments were started, are excluded, the rate of clutch
production under conditions of no food additions fs zero. Mean numbers of eggs per

clutch did not vary greatly among the different food conditions.

As an interesting sidelight of these experiments, it will be noted that mortality tended
to be higher and both rate of clutch production and size of clutch tended to be lower
in the third experiment. The explanation for these differences is not apparent, but
their existence does interject a note of warning against automatically assuming repli-

catibility of experiments using even the supposedly standardized fish food solution.

As an adjunct to the second and third experiments of the preceding work, one extra
set of containers was initiated and maintained during each experiment in exactly the
same way as the regular sets of containers. The animals in these extra containers were
fed 0.4 ml of fish food solution every third day. The only difference between the
treatment of the individuals in these containers and those that received 0.4 ml in the
regular experiments was that the fish food solution added to the extra containers had
been made up 2 weeks before the start of the experiment instead of at the experi-
ment's start as was the food added to the regular containers. The food for all con-

tainers was held ina refrigerator at all times when not in use.

The mortality and reproduction for the females in these extra containers are summarized

in Table 14. The results for the comparable animals from the regular experiments are


APPENDIX D4
VOLUME IN LITERS OF EACH REGION OF THE POND AND IN THE

TOTAL POND ON EACH COLLECTING DATE

Date Open water Rooted aquatic Total pond
tegion region
11-1971
19 429,690 150,739 580,429
21 504,605 201,313 705,919
23 496,246 192,942 689, 188
25 532, 242 215,506 747 ,748
27 532,242 215,506 747,748
-1971 ,
1 485,087 237 ,562 722,649
3 485,087 237,562 722,649
5 477,640 186,448 664,089
7 477,640 186,448 664,089
9 469,500 177,857 647,358
W 469,500 177,857 647,358
13 469,500 177,857 647,358
5 457,366 173,261 630,627
Ww 457,366 173,261 630,627
19 457,366 173,261 630,627
21 451,349 170,910 622,259
23 438,341 167, 187 605,528
25 429,676 150,753 580,429
27. 423,790 148,277 572,067
29 417,938 145,760 563,699
31 412,247 143,089 555,336
IV-1971
2 469,895 68,705 538,600
4 444,849 77,020 521,869
6 425,731 79,407 505 , 138
8 408, 138 88,632 496,770
10 390,545 89,494 480,039
12 358,480 96,459 454,940
14 332,976 105,233 438 , 209
16 320,128 109,718 429,847
18 284,478 145,369 499, 847

-204-


APPENDIX C-4
31° INCUBATION: PERCENT HATCH

Female's
hatching :
temperature | 21° 27° 31°
Individual D8 DS D3 DN DIé DI7 DI? D20
0.0% | 0.0% |----- 17.2% | 44.4% | 58.3% | 60.0% | 64.7%
25.0 | 41.2 45.0 |100.0 65.0 | 66.7
84.6 | 65.4 4.4 0.0 42.9 | 23.1
61.1 42.3 82.9 | 53.8 87.0 }100.0
21.4 100.0 | 88.0 25.0
69.2 61.5
90.3 90.0
52.6 0.0
Mean % 33.2% | 30.7% | ----- 62.8% | 63.6% | 58.3% | 64.7% | 54.4%

An aresine transformation of the above data was made, the means above were computed,
and an Anova carried out:

Source of Variation df MS F
Among individuals 6 337.7368 0.5347 ns
Within individuals 28 631.5237
Total 34

-198-


APPENDIX D-4 (Cont.)
VOLUME IN LITERS OF EACH REGION OF THE POND AND IN THE

TOTAL POND ON EACH COLLECTING DATE

Date Open water Rooted aquatic Total pond
region region
V-1971
20 - 281,736 181,572 463,308
24 285,380 152,829 438,209
V-1971
4 230,704 240,972 471,677
18 257,514 172,332 429,847
28 247,147 182,699 429,847
VI-1971
i 462,438 243,481 705,919
25 593,881 178,966 772,847
Vil-1971
9 266,846 397,243 664,089
23 207,631 356,067 563,699
VIN-1971
6 137,205 384,664 521,869
20 115,993 338,947 454,940
IX-1971
3 114,534 323,675 438,209
7 127,927 310,282 438,209
30 129,599 325,341 454,940
X-1971
14 129,599 325,341 454,940
21 102,011 436,589 538,600
29 61,873 476,726 538,600

-205-


Table 22, COMPARISON OF MEAN INTERVAL BETWEEN
CLUTCHES AT FOUR INCUBATION TEMPERATURES

Incubation Mean 95%
temperature (hours) confidence limits
(degrees C)
14 35.4 23.3 - 54,0
21 10.7* 8.7 - 13,1*
27 11.5 9.2 - 14.3
31 22.9 17.0 - 30.8

* Intervals following clutches of resting eggs excluded.

An FmaxTest indicates that the variances of the data for the four incubation fempera-
tures are heterogeneous, A logarithmic transformation was made and an Anova carried

out with the transformed data.

Anova table: Intervals by incubation temperature. Datg for intervals following
clutches of resting eggs excluded from 21° data.

Source of variation df MS F
Among incubation temperatures 3 2.4857 15,6826 P<0,001
Within incubation temperatures 199 0.1585
Total 202

-67-


it was not possible to determine more accurately if hatching successes actually were
greater than reported. However, Taub and Dollar (1968) found that in cultures of
Daphnia pulex the frequency with which entire broods failed was greater than the
frequency with which only a fraction of a brood failed. This is in agreement with
what is suspected in the present study, although Taub and Dollar attribute the fail-

ures to inadequate food and this condition is not suspected in our cultures.

Using the results of this study (summarized in part in Table 25), the daily recruit-
ment of nauplii per female at each temperature can be estimated. Multiplying
hatching success by mean total number of eggs produced at each temperature shows
(row A, Table 26) a decrease in total nauplii in the order 21°, a, 27°, 31°.
This value, divided by the mean adult longevity of females at each temperature
yields (row B, Table 26) the daily rate at which nauplii are added to the popula~
tion at each temperature averaged over the adult lifetime of the individual. Row
C, Table 26 (computed by multiplying mean clutch size by hatching success, then
dividing by the rate of clutch production and converting from hours to days) lists
the mean rate of addition to the population during the time that females are breed:
ing. Both of these rates are highest at 27°, despite the reduced hatching success
at that incubation temperature, and the maximum rate of recruitment appears to
occur between 21° and 27°.


served as the feeding solution. This material was made up fresh every two weeks or so
and kept in the refrigerator when not in use so that bacterial growth and other changes

were retarded,

This food has the great advantage that it can be made up to the same specifications
each time it is produced. This permits contro! of the relative amounts of food added

to different cultures at one time or to one culture at various times.

Besides changing the type of food added, it was also decided at this point to change
the frequency at which the water in the experimental containers was changed. Rather
than maintaining the same medium throughout an experiment as had been done up to
this point, the water was changed periodically during an experiment in the rest of

our work, This procedure was initiated in order to cut down growth of food organ-
isms within the cultures and thus to aid in minimizing the availability of food from
sources other than the known quantifies that had been added.

A preliminary study was conducted to determine whether the fish food solution would
support D. clavipes populations at all and thus whether it deserved further investiga-
tion. Twenty cultures were initiated in 1-1 containers by adding 4 pairs of adult cope~
pods to each, These cultures were divided into 4 sets of 5 replicates each, and a dif~
ferent amount of food was added fo each set. Five of the containers received 50 ml

of the fish food solution per day, five received 10 ml per day, five received 1 ml per
day, and five received no food at all, These cultures were maintained in a constant
temperature room at approximately 21° C and under moderate light conditions of 12

hours on and 12 hours off, The water in each container was changed once a week.

The results from this experiment are presented in Table 11. It will be noted that in
less than two weeks all animals in the containers receiving either 10 or 50 ml per day
were dead. The water in these containers turned cloudy and gave off a bad smell.
It seemed obvious that the animals died out because so much food material was added

that the water became anoxic and putrid conditions developed.

=32-


per clutch appears to be the most important of the three factors studied in determin-

ing the m, values.

In an attempt to investigate the cause of the variations in the mean number of eggs

per clutch, correlation coefficients were calculated between this parameter and each
of three environmental factors; i.e. chlorophyll a content in the water, water tempera-
ture, and density of adults (Table 43). Significant inverse correlations were found

with density and temperature but not with chlorophyll a.

‘Table 43, CORRELATION COEFFICIENTS FOUND BETWEEN THE MEAN NUMBEP
OF EGGS PER CLUTCH AND CHLOROPHYLL CONTENT, TEMPERATURE,
AND DENSITY OF ADULTS DURING THE STUDY YEAR.

Parameter n Correlation Coefficient P
Chlorophy!I 18 --0.3650 nonsignif.
Temperature 47 ~0.6003 <0.01
Adult densities 47 ~0.4176 <0.01

Chlorophyll content has been taken as a quantitative measure of the total food avail-
able for grazing in several studies (e.g. Hall, 1964). Ina predator-prey (D. clavipes-
algae) food relation, an inverse correlation would be expected when the density of
the predator increased to the level where the prey were harvested faster than they
could reproduce. On the other hand, a positive correlation would be expected when
the prey was not limited by the predator, but rather the reproductive intensity of the
predator was determined by the amount of prey available. Although an insignificant
correlation was found between the mean number of eggs per clutch and chlorophyll
content during the full study period, analysis of data during the early spring season
(19 February to 31 March) showed a highly significant positive correlation, while a

similar analysis for the data from the remainder of the year revealed a significant

-118-


Except for the adult instar which, of course, had a mortality rate ,. ) of 100%, the
egg to NIII stage had the highest mortality rate, The second highest « mortality rate
occurred for stage NVI with 613.87 out of every 1,000 individuals dying during this
interval. The CIll stage had the third highest rate, 422.73 per 1,000. The observa~

_ tion of an apparently negative mortality rate for stage CV is due to the inaccuracies
of our population estimates. An overestimate of the numbers of CVI, an underestimate
of the numbers of CV, or some combination of these two would lead to such a result.

The apparently negative rate does suggest that mortality was low for stage CV.

The shortest life expectancy during g, was in the egg to NIII stage, with the remain~
der of the naupliar stages also having lower e values than any of the copepodite
stages (Table 32). The longest life expectancy in this generation (32.36 days) was
found in the CIV, although in all copepodite stages except Cll (e, of 18.45 days)
the life expectancy was greater than 20 days. The life expectancies of the six cope-
podite stages contrast with the shorter expectancies of 3.12 days, 11.97 days, and
9.14 days found for the egg to NIII, NIV, and NVI stages, respectively.

The survivorship, mortality, and longevity rates for the complete study (Table 33)
reveal similar trends as those for a° The highest mortality rate, 881.40 per 1,000
individuals, occurred in the egg to NIII stage. This was followed by a mortality rate
of 469.42 per 1,000 for the NVI stage. The greatest life expectancy (22.13 days)
was found in the Clll stage. Although life expectancies for the various stages of the
complete year's data were not as long as those for the first generation, they followed
a similar trend with all the copepodite stages having longer life expectancies than
any of the naupliar stages. The lowest life expectancy was again in the egg to NIII
stage, 1.69 days, while NIV, NV, and NVI had life expectancies of 5.73 days,
8.09 days, and 9.05 days, respectively. The lowest life expectancy among the cope-
podite stages was found for Cll (14.46 days), with each of the other copepodite in-

stars having a life expectancy of greater than 15 days.

-102-


Three factors interacted to produce the specific m values observed; i.e., the per-
cent of adult females carrying eggs, the ratio of males to females in the population,
and the mean number of eggs per clutch on each date. As indicated in Figure 11,
at least 50% of the adult females were carrying eggs during the greater part of this
study. The only extended periods when less than 50% of the females were carrying
eggs occurred from mid-April to mid-May and from mid-October until the culmina-
tion of the study on 29 October. The latter period coincided with a steady decrease

both in water temperature and total adult population.

Although the average percent of the females carrying eggs appeared to differ among
the four seasons, the variance was so great within the groups that analysis of variance
showed no significant differences among the seasonal means (Table 38), It appears ,
therefore, that variation in the percentage of females carrying eggs was not a major
determining factor for the seasonal variations of the m,. values that were noted in

this study.

Table 38. ANOVA, TABLE COMPARING THE MEAN PERCENTAGES
OF FEMALES CARRYING EGGS DURING THE VARIOUS SEASONS.

Source of variation | df ss MS F
Among seasons 3 74.1164 24,7054 2.3181*
Within seasons 43 458.2621 10,6572

Total 42 532.3785

*P <0.10

Several investigators, including Chapman (1969), have suggested that, for certain co-
pepods, males have a shorter life span than females. Elgmork (1959) found evidence

to suggest that the males develop somewhat more quickly in Cyclops strennus strennus

-11i-


APPENDIX D

All measurements pertaining to the morphometry of the pond were determined follow~-
ing procedures outlined by Welch (1948). These include construction of the morpho~
metric map (Appendix D-1), determination of the surface area and volume for each
depth stratum (Appendix D~-2), and the determination of surface area (Appendix D~-3)
and volume (Appendix D-4) of each region of the pond on each collecting date. The
two regions of the pond are described in the Methods and Materials section of Chapter
4. On each collecting date the depth of the pond and the depth of the water at the
junction of the open water region and the area of rooted aquatics was measured. By
subtracting the depth of the water at the junction of the two regions from the total

depth of the pond the appropriate dimensions of the open water area could be calcu-
lated.

-199-


Table 39. ANOVA TABLE COMPARING. THE MEAN RATIOS
OF MALES TO FEMALES DURING THE VARIOUS SEASONS.

Source of variation df ss MS F
Among seasons 3 8.2784 2.7594 28.8037*
Within seasons 42 4.0268 0.0958

Total 45 12.3052

*P< 0.01

Table 40, A POSTERIORI COMPARISON OF THE SEASONAL MEANS OF THE
RATIOS OF MALES TO FEMALES USING THE STUDENT-NEWMAN-KEULS TEST

Summer Winter Spring Fall
¥ 0.9278 | 0.9938 | 1.2436 | 2.7367
no 9 21 “4 3
3 0.0654 | 0.0779 | 0.1489 | 0.0901
Yon
Su 0.9278 9 -

W 0.9938 21 0.2515 od
Sp 1.2436 14 0.3248 0.2178 =

F 2.7367 3 0.5583* 0.4692* | 0.4016* -

*P< 0.05

-116-


APPENDIX G-1

DURATIONS IN DAYS OF THE ADULT STAGE, THE PERIODS FROM MATURATION TO THE
PRODUCTION OF THE FIRST EGG CLUTCH, THE PERIODS FROM THE PRODUCTION
OF THE LAST EGG CLUTCH TO DEATH AND THE PERIODS BETWEEN THE
PRODUCTION OF SUCCESSIVE EGG CLUTCHES FOR CYCLOPS
VERNALIS FEMALES AT TEMPERATURES OF 14,

21, 26, and 31°C,

Total Before After Between Total Before After Between
adult eggs eggs clutches adult eggs eggs clutches
4 21
7 7 12 1.6 52 15 12 6.3
74 33 5 9.5 37 W 4 7.7
id 38 20 10.0 31 7 10 6.8
58 a] 9 7.6 4B 6 37 -—
84 18 7 11.0 63 30 9 6.0
83 57 10 14.0 64 37 13 14.0
59 6 10 6.1
55 31 3 10.5
76 27 6 9.0
57 26 10 6.4
59 31 3 6.0
67 3 8 5.3
60 51 4 8.0
Mean 38.5 12.9 6.3 3.65 26.2 8.3 6.9 3.62
SE 2.49 1.97 1.31 0.268 0.72 1.06 0.88 0.335
n W an W W Ww WwW Ww 7
26 31
39 2% 1 4.0 26 4 4 4.0
39 22 7 3.0 25 2 1 44
32 10 2 48 26 5 5 3.2
37 3 3 2.6 30 2 é 5.5
59 16 é 3.5 27 10 5 4.0
40 9 1 3.6 22 8 uv 45
37 13 12 3.0 24 7 7 5.0
30 7 W 2.4 29 1 4 6.7
28 12 W 5.0 25 9 6 3.3
38 3 u 4.7 25 16 7 2.0
a uN 4 3.6 27 13 8 3.0
20 8 W _
28 13 8 1.8
22 1% 4 2.0
23 10 12 _
29 7 5 2.8
32 8 6 4S
28 4 18 2.0
30 4 4 2.8
Mean 74,2 27.3 10.5 10.62 57.2 23.9 9.9 7,68
SE- 43.9% £7.73 £2.14 20.881 *2.80 £3.85 22.45 20.717
n* 6 6 6 6 3 13 13 13

-222-


APPENDIX E~3 (Cont.)

TOTAL NUMBER OF COPEPODITE FOUR (C1V) IN EACH REGION OF THE POND
AND IN THE TOTAL POND ON EACH COLLECTING DATE, THE TOTAL
NUMBER WAS DETERMINED BY MULTIPLYING THE Y (MEAN)

NUMBER OF CIV PER QUADRAT IN EACH REGION BY
THE TOTAL NUMBER OF QUADRATS IN THAT
REGION AND SUMMING THE PRODUCTS
FOR EACH DATE.

Date Open water Rooted aquatics Total pond
region region
V-1971
10 823,463 0 823,463
12 783,568 10,176 793,744
14 516,226 0 516,226
16 464,363 0 464,363
18 118, 944 26,159 145,103
20 610,429 13,686 624,115
V-1971
4 197,900 74,401 272,301
18 336,439 348,974 685,414 -
28 41,540 0 41,540
VI-1971
WW 2,072,713 78,946 2,151,659
25 785,996 0 785 ,996
VII-1971
9 287,058 : 17,714 304,773
23 134,777 0 134,777
VIN-1971
6 95,499 56,349 151,848
20 50,060 48,899 98,959
IX-1971
3 40,048 124,579 164,627
17 58,140 115,285 173,426
30 0 41,273 41,273
X-1971
14 34,425 126,402 160, 827
21 5,485 118,054 123,540
29 4,590 61,073 65,663

-211-


with TDS readings close to those of the pond water showed no better results than
the Noble water witha TDS of 1000 or the so-called distilled water with a TDS of
25. :

Cannibalism

Cannibalism, especially by the mother on her newly hatched offspring, was observed
several times during our research and has been reported in cyclopoids by several
other authors, Fryer (1957) reported C, vernalis as a carnivorous copepod, having
found cyclopoid remains in the guts of individuals of this species. Khan (1965) and
Smyly (1970) both noted high mortality of nauplii when left in the presence of adult
Acanthocyclops viridis, but neither reported the magnitude of this mortality.

To determine the extent of the mortality of newly hatched C, vernalis nauplii when
in the presence of their mother, the following experiment was carried out. Twenty
gravid females were placed in separate 5-cm Petri dishes, each containing 10 ml of
filtered pond water and maintained at 21°C. Within 2 hours after the clutch carried
by each female had hatched, the nauplii were counted, Also at that time, the fe-
males were removed from ten of the dishes. The nauplii in each dish were counted
again 24 and 48 hours after the first count (Appendix F-1).

At both 24 and 48 hours after the first count, the mean percentage of nauplii sur-
viving in dishes with females was approximately one-half that of the nauplii which
were in dishes from which the females had been removed (Table 51). Student's
t-test comparing the percentages with and without the mother present at both 24
and 48 hours showed differences between the two groups to be highly significant

at both times (P< 0.001). Further, using the same test it was found that for both
the dishes with adult copepods and those without, the mean percentage of nauplii
surviving for 48 hours did not differ significantly (P> 0,05) from the mean percent-

age surviving for 24 hours,

-140-


Table 52, NUMBERS OF CYCLOPS VERNALIS AFTER 4 WEEKS IN
A CONTINUOUS FLOW CULTURING SYSTEM,

Week ‘Numbers of ‘individuals
Males Females Immature copepodites Nauplii
0 6 4 0 0
4 8 22 8 48

Several problems were encountered in the continuous flow experiment, however.
Fine mesh bolting cloth (#25) was placed over the end of the culture container
outlet tube in an attempt to prevent the escape of young nauplii, Even so, many
of the smallest nauplii were noted to pass through this netting. To prevent this
escape the layer of netting over the end of the tube was doubled. This seemed to
prevent the escape of most of the nauplii but led to other problems.

The double-thickness netting became clogged quite quickly with food particles which
greatly impeded the flow through the system. This problem was partially overcome
by connecting a small inverted funnel to the end of the outlet tube so that the water
from the culture entered the tubing through the wide end of the funnel. This out-
let was then covered with a double thickness of #25 netting, The increased area of
this outlet decreased the clogging problem while still providing the safeguard of a
double thickness of netting to retain the nauplii. Another aid to reduce clogging
was provided by filtering the food mixture through #25 bolting silk while it was being
produced, rather than through the #20 silk usually used.

Even with the refinements mentioned qbove, clogging of the netting was some pro-
blem. Overall it seems, at least at present, that culturing C, vernalis ina con-
tinuous flow system is more difficult and time-consuming than culturing the species
in a standing water system. If bioassays concerning the toxic effects of certain ma-
terials on adults of this species are to be conducted, it may well be advisable to

-144-


EPA~660/3-74~006
April 1974

CULTURING AND ECOLOGY OF DIAPTOMUS CLAVIPES

AND CYCLOPS VERNALIS

By

Andrew Robertson
Carl W. Gehrs
Bryan D. Hardin
Gary W. Hunt
Department of Zoology
University of Oklahoma
Norman, Oklahoma 73069

Grant No. 18050 ELT
Program Element 1BA021

Project Officer

Dr. Kenneth Biesinger
National Water Quality Laboratory
6201 Congdon Boulevard

Duluth, Minnesota 55804

Prepared for

OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460

For sale by the Superintendent of Documents, U.8. Government Printing Office, Washington, D.C, 20402 - Price $2.55°


~vel-

Table 47, A COMPARISON OF THE MEAN NUMBERS OF CYCLOPS VERNALIS FOUND UNDER THREE
DIFFERENT RATES OF FOOD ADDITIONS ON A SERIES OF SAMPLING
DATES. EACH VALUE IS THE MEAN OF THREE REPLICATES, THE
STANDARD ERRORS OF THE MEANS ARE INCLUDED IN
PARENTHESES, (A = ADULTS, G = GRAVID FEMALES,
Y = IMMATURE COPEPODITES PLUS NAUPLII,
T = TOTAL NUMBERS.)

Rate of food addition every second day

0.4 mi/Iiter 1.0 ml/liter 2.0 mi/liter
— Week A S Na I A SG x T A G Y T

0 8.0 4.0 0.0 8.0 8.0 4.0 0.0 8.0 8.0 4.0 0.0 8.0
6.7 2.7 147.7 154.3 7.3 3.3 169.7 177.0 5.7 1.3 82.0. 87.7

0.3) 0.9) (14.2) (14,3) (0.3) @.7) (19.1) (19.1) (1.2) (0.3) (24.6) (24.8)

2 13.7 2.0 196.3 210.0 17.7 0.7 183.3 201.0 17.0 2.3 198.0 215.0
(1.3) (1.0) (50,2). (51.4) 5.8 (0.3) (1541) (20.9) (7.6) (1.3) (83.5) (91.1)

3 11.0 0.3 88.0 99.0 13.0 0.7 103.7 116.7 16.7 0.0 93.7 107.3
(0.02) = @.3) 7) (3.0) (0.7) (35.6) (98.4) 4.9) (-) (25.2) (26.4)

4 21.3 1.3 55.0 > 76,3 25.7 1.0 43.0 68.7 24,7 4,0 100.3 125.0
0.9) = 0.9) (2,9) 3,3) (2.4) (0.6) (22.2) (24,3) (2.4) (142) (2743) (27.5)

5 17.7 0.7 59.0 78,7 22.0 3.7, 145.0 167.0 23.7 5.3 273,0 296.7
(2.3) @.3) (17.8) (19,7) (4.0) (1.3) (93.0) (94.5) (7.2) (1,5) (97,8) (103.0)

6 19.7 3.3. 146.0 165.7 21,7 3.0 334,3 356.0 38,0 3.7, 191.0 229.0
(2.0) (1.7) (54.0) (56.1) (5) (2.9) (149.3) (150.7) (6.4) (2.7) (30.1)-—(36.3)

7 19.7 3.3 121.3 141.0 26.7 2.7 206.0 232.7 AL. 2.7 217.7 259.3
2.9) (1.2) (52.1) (49.2) (7.7) (7) (53.4) (60.1) (8.8) (1.5) (89.6) (80.8)

8 23.7 3.7 149.7 ° 173.3 37.3 1.30 61.7 99.0 54.3 3.7. 7.7 «172.0
(0.3) (1.2) (45.8) (45.9) (20.9) (0.3) (18.3) (26.1) (9.7) (0.9) (54.6) (56.8)

9 22.3 1.0 106.3 128.7 33.3 0.7 70.7 104.0 44,7 6.3 144.3 189.0
(1.2) (0.6) (26.5) (26,0) (10.5) (0.7) (34.3) (44.5) (7.0) (4.4) (56.5) (51.8)

10 23.7 13° 134.3 158.0 35.3 3.0 108.7 144.0 52.3 7.7 168.0 220.3
(1.2) (1.3) (41.4) (42.5) (13.5) (1.5) (37.0) (34.9) (8.9) (2.0) (50.9) (53.0)

v 18.7 1,3 68.7 87.3 38.0 5.0 112.0 150.0 “49.0 6.3 147.7 196.7
(1.9) 0.9) (38.8) (40.3) (12.2) (1.5) (67.6) (61.5) (8,7) (2.4) 51.4) (60,0)



-S6-

TOTAL
ADULTS

(x 10°)

1 1 1 L 1 1 1
2 4 VW 9 6 3 14
IV Vv Vi VU VEE IX x

DATE
Figure 8. Size of the Adult Population from 2 April to 29 October. The Vertical Line
Between 18 April and 24 April Shows a Change in Scale From Two Day to Approxi=
mately Fourteen Day Intervals.



Table 17. LIFETIME TOTAL NUMBER OF CLUTCHES PER FEMALE IN
RELATION TO HATCHING AND INCUBATION TEMPERATURES

Female's
Incubation hatching
temperature Mean temperature Mean
(degrees C) *SE (degrees C) ESE
14 12*
21 9*
+
14 8.24 1.07 nn
31 6.7 £0.33
14 aren
21 12.3 = 3.35
+ . .
21 13.0 = 2,36 7 14%
31 15.0 3,00
14 oe
21 7*
£
27 10.2% 1.69 27 11.3 £ 2.09
31 7*
14° Seared
+ 21 5*
31 5.2 = 0.88 7 4
31 5.541,18

*Lifetime total of a single female.

Anova table: Comparison of those females which were incubated at the same
temperature as that at which they hatched.

Source of variation df MS F
Among incubation temperatures 3 57.5626 1.4295 ns
Within incubation temperatures 16 40,2663
Total 19

Anova table: Comparison of all females at each incubation temperature,

Source of variation df MS F

Among incubation temperatures 3 93.3140 3.4617 PKO.05
Within incubation temperatures 27 26,9555
Total 30

~55-


CULTURING SUCCESS

While the techniques for culturing that have been described in this chapter were being
worked out, a study was initiated to determine if the use of the techniques as then
available would lead to self-propagating cultures. The animals used to initiate the
study were collected from a field population in November 1969. Adults from this
collection were maintained in several 350-ml glass containers at a density of 1 fe~
male and 2 males per 250- to 300-ml volume of natural pond water. Each newly
gravid female was transferred to an individual 100-m! glass beaker containing 70 ml
of natural pond water, These females remained in these smal! beakers until their
clutches hatched and then were transferred back into their previous 350-ml beakers,
The immatures remained in and were allowed to mature in the 100-ml beakers. At
maturity, some of the new adults were transferred into 350-ml beakers. The same den-
sity of animals and the same type and volume of medium as stated above were used to
culture these adults, This procedure was repeated for each new generation, The
animals were fed a couple of milliliters of the mixed culture food every 2 to 3 days.
Water temperatures in the 24 to 26°C range in combination with moderate light in-

tensities were used.

This study extended over an 8-month period (November 1969 to July 1970) and was
terminated voluntarily. The cultures passed through six and were in the seventh filial
generation at termination, Thus, it was concluded that the techniques employed were
suitable for the development of self-propagating cultures. As our work has ascertained
that the conditions for some of the factors can be improved over those used for this
culturing, it is assumed that the employment of the full procedures developed in our
work and summarized in the next section of this chapter will allow easy culturing and
experimentation with D. clavipes. In fact, this has been borne out by further work

on this species as reported in the next chapter of this report.

-4]-


Several authors, Elbourn (1966), Khan (1965), and Cummins etal, (1969), have
observed,an increase in development rate of cyclopoid eggs with increasing temper-
ature. On the other hand, Burgis (1970) reported that the development rate of the
eggs of the tropical cyclopoid Thermocyclops neglectus was retarded at temperatures
above 32,5°C, That the development rate of Cyclops vernalis eggs also would pro-
bably be retarded at higher temperatures is suggested by the decrease in the rate of
increase of the development rate that was observed between 26 and 3i°c,

A correlation between egg development rate and the interval between successive
clutches at different temperatures was noted for C, strenuus strenuus by Elgmork
(1959). Similar results have been found for C. vernalis. Both the development

rate of the eggs and the period between successive egg clutches are inversely re-
lated to temperature, The duration that egg sacs are carried by female C. vernalis

is dependent'on the development rate of the eggs (the sacs are normally carried until
the eggs hatch). Possibly the carrying of the egg sacs inhibits further egg production.
If this is true, the development rate of eggs, as determined by temperature, would

to some extent determine the rate at which successive egg clutches are produced,

HATCHING SUCCESS

The percentage of the eggs that hatched in the clutches examined ranged from 75.4%
to 100% (Table 57). Over 90% hatching success. was quite common, The mean per-
centages of hatching at the five temperatures ranged from 88.1% at 5°C to 96.0%
at 26°C, An analysis of variance showed no significant differences (P > 0.05) among
the mean percentages of hatching at the different temperatures. The method used in
removing the eggs from the females and in counting the eggs may have resulted in in-
jury toa few eggs, If this is true, the percentage of hatching under natural condi-
tions may be even closer to 100% than is indicated in this study.

-162-


population began then. In approximately 75% of the collections during early spring
and 88% of the collections during the spring-summer period, the proportion of the
total adult population located in the open water region was greater than the propor-
tion of the total water volume located in this region, These values are quite differ-
ent from expected and are further evidence that the adult population is not evenly
distributed between the two regions.

Although the Student's t-test showed a detectable difference in the concentration
of adults in the two regions of the pond during most of the sampling dates during the
spring-summer period, it did not show a similar pattern during the early spring period.
Another method of testing for differences between the two regions in the early spring
would be to run an F-test comparing all the data from the entire period in one test.
That is determining whether there were significant differences in the early spring be-
tween the average number of adults per liter in the open water region and the average
number per liter in the area in which rooted aquatics were found. When such a test
was conducted, the F value was found to be significant at the 0.05 level (Table 27).
The results of this test plus those of the t-tests described earlier show that significant
differences existed between the concentrations of adults in the two regions of the pond
throughout much if not all of the collecting period. In the early spring the differences
were less pronounced and could be demonstrated for the entire period only, while in
the spring-summer period they were stronger and were demonstrated for most of the in-
dividual sampling dates.

Table 27. AN ANOVA TEST COMPARING THE MEAN NUMBER OF ADULTS PER

LITER IN THE OPEN WATER REGION AND THE MEAN NUMBER IN THE

REGION IN WHICH ROOTED AQUATICS WERE FOUND DURING THE
EARLY SPRING (19 February to 4 April, 1971).

Source of variation | df DD MS F
Among regions 1 2.2751 2.2751 7.1454*
Within regions 44 14,0138 0.3184

Total 45 16.2889

*P< 0.05.

-90-


-SI-

Table 4, MORTALITY OCCURRING AMONG ADULT D, CLAVIPES CULTURED IN
3 TYPES OF CULTURE MEDIA OVER A PERIOD OF 40 DAYS.

Culture
Media

No.. Animals Used

Mortality (Numbers)

% Mortality

Females Males Total

Females Males Total

Females Males Average

Conditioned Water

Distilled Water

Pond Water

58 75 133

35 60 90

21 22 43

40 57 97

17 42 59

69.0

48.6

14,3

76.0 72.9
70.0 65.6
18,2 16.3



AND IN THE TOTAL POND ON EACH COLLECTING DATE.

APPENDIX E-é (Cont.)
TOTAL NUMBER OF COPEPODITE ONE (I) IN EACH REGION OF THE POND

THE TOTAL

NUMBER WAS DETERMINED BY MULTIPLYING THE Y (MEAN)
NUMBER OF Cl PER QUADRAT IN EACH REGION BY

THE TOTAL NUMBER OF QUADRATS IN THAT
REGION AND SUMMING THE PRODUCTS

FOR EACH DATE,

Date Open water Rooted aquatics Total pond
region region
IV-1971
14 92,183 10,858 103,041
16 480,634 38,793 519,426
18 118,944 134,530 253,474
20 494,525 0 494,525
V-1971
4 390,145 0 390,145
18 220,214 181,466 401,681
28 41,540 0 41,540
VI-1971
a 966,601 26,315 992,917
25 213,151 0 213,151
VII-1971
9 79 ,188 (0) 79,188
23 88,017 0 88,017
VIN-1971
6 80,096 18,783 98,879
20 74,089 48,899 122,988
IX-1971
3 30,036 0 30,036
17 30,600 17,875 48,475
30 146,882 24,075 170,957
X-1971
14 16,065 0 16,065
21 3,657 19,675 23,332
29 0 0 0

-217-


APPENDIX H-3

FECUNDITY IN EGGS PER CLUTCH OF FEMALE CYCLOPS VERNALIS AT 26°C,

Clutch Female No.

No. 1 2 3 4 5 6 7 8 9 10 nN
1 37, 42 «+49 «56 448) 22% 34. ««332:—6* 54 14
2 35 31 70 62 51 30 31 28 «19 47 13*
3 (28) 17 66 63 45 10 25 18 35 20*
4 13° 19 «461 «6 «((37)—«*18 7 32 15 17*
5 23 48 «44 21 26 21 16*
6 (48) 30 16 26 13*
7 35-32 5* 10*
8 3828 5* 15
9 14* 23* 13 6*

Total eggs | 473 109 269 430 338 140 133 158 25 IST 124

per female

No. of —

clutches 4 4 5 9 9 9 5 6 2 4 9

*Only one of two possible egg sacs present.

() Egg count not available, number is average clutch size for that female.

~225-


Table 2, ADULT POPULATION SIZE (TOTAL NUMBER AND NUMBER PER LITER) ON A SERIES OF
DATES FOR D. CLAVIPES CULTURES MAINTAINED IN CONTAINERS OF 5

DIFI

OF THREE REPLICATES.)

FERENT VOLUMES. (EACH VOLUME !S THE AVERAGE

Container Volume (ml)

Sampling 50 250 1,000 3,000 20,000
Date Total No. | No./} | Total No. | No./I | Total No. | No.1 | Total No. | No./l | Total No. | No./I
(1970)
24-V1 10.0 200.0 10.0 40,0 10.0 10.0 10.0 3.3 10,0 0.5
27-VI 8.3 166.0 8.7 34,8 10,0 10.0 9.0 3.0 7.7 0.4
Vl 0.7 14.0 8.0 32.0 8,0 8.0 8.8 2.9 91 0.5
3-Vil 0.3 6.0 6.3 25.2 11.6 11.6 8.6 2.9 9.2 0.5
7Nu 0.0 0.0 5.0 20.0 10.5 10.5 6.8 2.3 - -
15-VII* - - 6.5 6.5 19.3 6.4 70.3 3.5
20-VIl 8.7 34.8 30.0 30.0 28.1 9.4 189,0 9.5
27-Vil 6.0 24,0 41,4 Al.4 43.1 14,4 136.4 6.8
i-vill 3.7 14,8 26,7 26.7 37.2 12.4 98.9 49
31-Vill 0.0 0.0 21.3 21.3 30.8 10.3 81.1 4.1

* Subsampling initiated in 1,000-, 3,000-, and 20,000-ml cultures


it has usually been assumed that a relatively homogeneous environment existed in the
pond such that the species present were either randomly or uniformly distributed.
The data from the current study demonstrate that this assumption is not necessarily cor-

rect, at least if macrophytes are present near the surface.
LIFE TABLE APPROACH TO POPULATION DYNAMICS

During February and early March 1971, false starts in the population development
occurred during periods of relatively warm weather (surface water temperature be-
tween 6° and 93°C). Eggs were produced and hatched, and the nauplii developed to
naupliar stage IV at which point development ceased and the individuals eventually
died. Data from these individuals were not used in determining the durations of the
various instars. Since only adults and a few early nauplii were found at the start of
this investigation, the duration of each immature instar was estimated as the length
of time between the first observation of the instar in the population and the first ob-
servation of the succeeding instar. This procedure could not be used in estimating
the duration of the adult instar, however, since at no time were adults absent from
the population. Instead, the duration of the copepodite VI stage was estimated by
measuring the length of time between successive minima in the numbers of adults in
the population. The minima in adult numbers were assumed to result from periods
high mortality in this instar group. A minimum occurred on 18 April 1971 with an-
other one occurring on 28 May 1971 (Figure 8). The period of time between these
two dates, 40 days, was taken as an estimate of duration of CVI. This agrees closely
with the estimate of 43.2 days obtained for the duration of the adult female life span
in the laboratory at 21°C (Table 15). The mean water temperature in the field dur-
ing this period was between 15° and 20°C. The value of the mean temperature in
this context is debatable, however, since vertical migration of the individuals and
the differing temperature patterns at the various water depths would enable the or-

ganisms to live ina variety of temperature situations. The exact correlation of instar

-94-


Table 53. MEAN DURATIONS OF THE ADULT STAGE, THE MEAN TIME
INTERVALS FROM THE ADULT MOLT TO THE PRODUCTION OF THE
FIRST EGG CLUTCH, THE MEAN TIMES FROM THE PRODUCTION
OF THE LAST EGG CLUTCH TO DEATH, AND THE MEAN TIME
INTERVALS BETWEEN SUCCESSIVE EGG CLUTCHES
FOR CYCLOPS VERNALIS FEMALES AT

FOUR DIFFERENT TEMPERATURES

Times in days
Temperature | Adult Before After Between
(°C) stage sey Ist clutch SES last clutch SES clutches SEy
14 74.2 |3.96| 27.3 7.73 10.5 2.14] 10.62 | 0.881
21 57.2 | 2.80] 23.9 3.85 99 2.45 7.68 | 0.717
26 38.5 | 2.49} 12.9 1.97 6.3 1.31 3.65 | 0.268
31 26.2 | 0.72 8.3 1.06 6.9 0.88) 3.62 | 0.335

Coker (1934b) observed egg production beginning much earlier in the adult stage of
Cyclops vernalis than reported in this paper. He found this period ranged from 7 days
at 7 to 10°C to 3 days at 20 to 23°C. He also observed an increase in this interval
at the highest temperature he studied, an increase to 7 days at 28 to 30°C. A similar
result was not observed for the animals in the present study. Smyly (1970), ina study
of the effect of diet on longevity of Acanthocyclops viridis, found that the interval
from the adult molt to the initial egg clutch was 1 to 2 weeks for animals maintained
on an animal diet and 4 weeks for animals fed algae only. The C., vernalis observed
by Coker were fed protozoan infusions, whereas the animals observed for this paper
were maintained on a diet which was composed mainly of plant material. The dif-
ferences in diet composition may explain the discrepancy between the intervals pre-

ceding the production of the first egg clutch observed by Coker and those recorded

in this paper.

The mean time between egg clutches decreased as temperature increased (Table 53,

Figure 17, Appendix G-17). The mean intervals ranged from 3.62 days at 31 °C to

-151-


Table 54. SUMMARY OF STATISTICAL DATA FOR CLUTCH SIZE, THE
NUMBER OF CLUTCHES PRODUCED PER FEMALE, AND THE TOTAL
EGG PRODUCTION PER FEMALE AT THE TEMPERATURES
14, 21, 26, AND 31°C,

Temperature (°C) 14 21 26 31
Clutch size (antilogs of
log values)
Mean 35.2 39.7 25.2 27.5
95% Confidence limits
L 28.7 33.7 21.5 24.1
by 4.1 46.9 29.5 31.2
n 27 65 66 7%
Min. W 3 5 4
Max, 75 102 70 62
Number of clutches per female
Mean 45 5.0 6.0 4.0
SE, 10.81 40.63 40.77 40.47
n 6 13 WV 19
Min. 2 1 2
Max. 6 8 9 9
Total egg production per female
Mean 177.8 233.9 180.9 123.9
SE, 47,58 430.44 «435.32, H19.94
n 1 6 13 W 19
Min. 36 3 25 4
Max. 310 388 430 356

-154-


clutches was found to be significantly different (14°C, P< 0,05; 21°C, P< 0.01;

26 and 31° Cc, P< 0,001) than the mean size of the last clutches at all temperatures,
with the first clutches always being larger. At the higher temperatures, i.e. 26°

and 31°C, the mean size of the first clutches was significantly different (26°C, P<
0.05; 31°C, P< 0.01) from the mean size for all clutches, again the first clutches
were larger, At 21°C and 31°C the mean for the last clutches was different 21%,
P<0,05; 31°C, P< 0,01) than the mean size for all clutches. Smyly (1970) observed
that age affected clutch size in Acanthocyclops viridis, i.e. diminishing size with
increasing age, and that diet affected the rate of this diminution. The experiments

with C. vernalis also suggest that fecundity decreases as age increases.

Number of Clutches Per Female

Previous observations of C, vernalis indicated that females could produce a maximum
of 12 egg clutches per lifetime (Ewers, 1936). Females in the experiments reported

in this paper produced from a minimum of 1 toa maximum of 9 clutches per lifetime.
The mean number of.clutches produced per female varied little among the four temper-
atures studied (Table 54), The largest mean number of clutches (6.0) was produced

by females at 26°C, and the smallest mean number of clutches (4.0) was produced by
females at 31°C. No significant difference (P > 0.05) was found among the mean
numbers of clutches produced at the four temperatures.

Total Egg Production

An anova showed the mean total number of eggs produced by a female during her
lifetime voried among the four temperatures (P< 0.05). This would be expected since
clutch size was found to vary with temperature, even though the number of clutches
in a lifetime did not, Mean total egg production was highest at 21°C, ine. 233.9
eggs, and lowest at 31°C, i.e. 123.9 eggs (Table 54), Means of egg production by

° . .
females at 14° and 26 C were intermediate between the extremes and were very similar,

-158-


~€Z-

Table 8, A COMPARISON OF THE AVERAGE VALUES (4SE) FOR CERTAIN REPRODUCTIVE AND
DEVELOPMENTAL PARAMETERS FOR D. CLAVIPES INDIVIDUALS AT
FOUR DIFFERENT TEMPERATURES.

Average Number Average Average Average Days | Average Days
Average Number of | of Clutches Clutch Percent Clutch Development
Temperature | Clutches | Per Container Size Hatch Carried To Adult
9.7 0 0.0 -—- 7 -- —
(-)

22.0 17 2.8 1.1 51.8 3.7 20.2
41.1) (€0.8) (£21.7) (£0.8) (£1.8)
26.2 20 3.3 10.5 68.3 2.0 25.7
1.1) 41.2) 13.6) 0.4) (6.9)
29.6 39 6.5 16.5 74.5 1.8 18.1
(£0.4) (2.0) 9.9) 0.1) (1.5)



The results in the present study agree generally with the findings of Walter (1922)

in work with Cyclops viridis and Elbourn (1966) in work with C, strenuus strenuus,
both of whom also found that the hatching success of cyclopoid eggs was not af-
fected by temperature. However, Elbourn reported hatching success of only 60 to
80% at all temperatures, whereas Walter reported approximately 80 to 90% success,
Khan (1965) reported decreasing hatching success of eggs of Acanthocyclops viridis:
at temperatures above 26°C but did not elaborate on the extent of the egg mortality.
Burgis (1970) observed that all eggs of the tropical cyclopoid Thermocyclops neg-
lectus failed to hatch at temperatures above 35°C,

SUMMARY

The effects of temperature on certain of the reproductive attributes of the cyclopoid
copepod Cyclops vernalis have been studied. Four temperatures, 14, 21°, 26°,
and.31°C, were used in all experiments and a fifth, 5°C, in some. The following

conclusions were reached:

1. The mean duration of the adult stage of the females is in-
versely related to temperature.

2. The mean time interval from the adult molt to the start of
egg laying and the time between successive clutches are
both also inversely related to temperature. The time from
the last clutch to death did not seem to be significantly af-
fected by temperature, however.

3. Clutch size was found to be significantly different among
femperatures, with a tendency for the animals at 21 and
14°C to have larger clutch sizes than the animals at 26
and 31°C,

4, Temperature was not, however, related to the number of
clutches produced by a female during her lifetime.

5, As expected from the combined effects of temperature on
clutch size and number, a significant inverse relation was
found between total lifetime production of eggs and temper-
ature.

164


APPENDIX E-8

TOTAL NUMBER OF EGGS IN THE POND ON EACH COLLECTING DATE. THE
TOTAL NUMBER OF EGGS WAS DETERMINED BY MULTIPLYING THE Y
(MEAN) NUMBER OF EGGS PER ADULT BY THE TOTAL NUMBER

OF ADULTS IN THE POPULATION ON EACH DATE.

Date Number $f eggs Date Number of eggs
(x 10”) (x 103)
W-1971 IV-1971
19 7,631 20 5,905
2 1,359 24 6,028
23 6,561 V-1971
25 6,955 4 11,220
27 9,892 18 4,505
HI-1971 28 1,511
1 10,385 VI-1971
3 17,134 n 7,162
5 15,646 25 27,339
7 10,454 VII-1971
9 11,060 9 11,845
YW 8,295 23 29,489
13 16,186 VIN-1971
15 18,687 6 17,844
17 15,644 20 9,560
19 19,048 1X-1971
2 20,557 3 21,832
23 16,277 17 11,689
25 23,890 30 5,310
27 35,243 X-1971
29 18,793 14 433
31 18,602 21 0
IV-1971 29 744
2 19,444
4 10,565
6 12,650
8 16,674
10 11,849
12 4,753
14 2,570
16 2,229
18 3,416

~220-


Table 35, CALCULATION OF THE INTRINSIC RATE OF NATURAL INCREASE
(r) USING THE ADULT SURVIVORSHIP FROM THE FULL YEAR STUDY,
A 40-DAY DURATION FOR THE ADULT STAGE, AND
THE AVERAGE m,. VALUE FOR THE YEAR.

Stage | Duration 1 m i) xl om
x x xX xx

Egg | 0-1.81 | 1.00000 | 00.00
cv | 21.46
cvi | 25.46 | 0.00878 | 2.78339 | 0.02443 | 0.62980
cvi | 29.46 | 0.00878 | 2.78339 | 0.02443 | 0.71970
cvi | 33.46 | 0.00878 | 2.78339 | 0.02443 | 0.81742
cvi | 37.46 | 0.00878 | 2.78339 | 0.02443 | 0.91514
CVI | 41.46 | 0.00878 | 2.78339 | 0.02443 | 1.01286
cv | 45.46 | 0.00878 | 2.78339 | 0.02443 | 1.11058
cvi | 49.46 | 0.00878 | 2.78339 | 0.02443 | 1.20830
cvi | 53.46 | 0.00878 | 2.78339 | 0.02443 | 1.30602
“cvl | 57.46 | 0.00878 | 2.78339 | 0.02443 | 0.40374

cvl 61.46 0.00878 2.78339 | 0.02443 1.50146

Total 0.24430 | 10.61720

T (generation time) = 39.45968
RS (net reproductive rate) = 0.24430; In = -1.4094
r= -0.03572

-104-


APPENDIX B-4
31° INCUBATION: TIME (HOURS) CLUTCHES WERE CARRIED

Female's
hatching
temperature 21° 27° 31°
Individual D8 |. D5 D3 DI DI6é DI7 Di? D20
25.0 | 22.5 24.5 | 37.5} 30.5 | 23.5 29.5 | 30.0
35.0] 24.0 37.0 | 28.5 37.0 | 22.5
34.0 | 24.0 34.5 35.5 | 38.5
36.0 | 35.0 23.5 31.0 | 28.0
35.5 28.0 25.5 | 32.5
23.0 29.0 | 32.0
29.5 35.0
30.0

Clutch carrying times are listed from top to bottom in the order of occurrence but are
not necessarily consecutive, some carrying times having been omitted for various reasons
as discussed in the text.

Variances of the data for the three hatching temperatures are marginally homogeneous.
A logarithmic transformation was made and Anovas carried out with the transformed data.

Anova table: comparison among individuals of carrying times.

Source of Variation df MS F
Among individuals 7 0.0097 2.2045 ns
Within individuals 26 0.0044
Total 33

-183-


APPENDIX E-5

TOTAL NUMBER OF COPEPODITE TWO (II) IN EACH REGION OF THE POND
AND IN THE TOTAL POND ON EACH COLLECTING DATE. THE TOTAL
NUMBER WAS DETERMINED BY MULTIPLYING THE Y- (MEAN)
NUMBER OF CII PER QUADRAT IN EACH REGION BY
THE TOTAL NUMBER. OF QUADRATS IN THAT
REGION AND SUMMING THE PRODUCTS
FOR EACH DATE,

Date Open water Rooted aquatics Total pond
region region:
11-1971
19 0 0 0
21 0 0 0
23 (0) 0 0
25 0 0 0
27 0 0 0
11-1971
1 0 0 0
3 0 0 0
5 0 0 0
7 0 0 0
9 0 0 0
n 0 0 0
13 0 0 )
15 38,221 0 38,221 -
7 568,540 11,148 579,688
19 925,271 78,035 1,003,306
21 » 810,421 253,752 1,064,173
23 263,602 219,668 : 483,270
25 293,499 43,481 336,981
27 606,101 272,126 878, 228
29 495,702 886,721 1,382,422
31 331,163 85,857 417,021
IV-1971
2 1,107,831 158,434 1,266,265
4 709,790 44,663 754,454
6 335,353 25,454 360,807
8 477,149 8,889 486,038
10 194,429 9,192 203 , 621

-214-


Life tables were constructed for the first generation, the total year,
and the laboratory animals. These tables agree in indicating the

highest mortality rates occurred in the egg to NII] and the NVI to
Cl stages.

The laboratory population showed substantially greater survival than
the field population,

The indications are that a physiological survivorship curve character~
ized the adults of both the field and the laboratory population.

Reproduction in the population was analyzed by determining the
specific birth rates in the various seasons. Analysis of three fac~
tors=-male to female ratio, percent of females carrying eggs, and
the mean number of eggs per clutch--suggested that the mean num=
ber of eggs per clutch was the most important factor in determining
the specific birth rates.

Chlorophyll a (food), water temperature, and adult densities were
studied to evaluate their effects on clutch size. Although signifi-
cant correlations between the mean number of eggs per clutch and
both temperature and adult densities existed, analysis of field and
laboratory data suggested that density may be the regulating factor
in controlling clutch size and hence reproduction, as long as the
temperature is within the range necessary for reproduction in this
population.

-125-


REFERENCES

Andrews, T.F. Seasonal Variations in Relative Abundance of Cyclops vernalis Fischer,
Cyclops bicuspidatus Claus, and Mesocyclops leuckarti (Claus) in Western Lake Erie
from July, 1946 to May, 1948. Ohio J. Sci. 53:91-100, 1953.

Armitage, K.B., and M. Davis. Population Structure of Some Pond Microcrustacea.
Hydrobiol. 29:205-225, 1967.

and J.C, Tash. The Life Cycle of Cyclops bicuspidatus thomasi S.A.
Forbes in Leavenworth County State Lake, Kansas, U.S.A. (Copepoda). Crustaceana.
13:94-102, 1967.

Aycock, D. Influence of Temperature on Size and Form of Cyclops vernalis Fischer.
J. Elisha Mitchell Sci. Soc. 58:84-93, 1942.

Birch, L.C. The Intrinsic Rate of Natural Increase of an Insect Population. J. Anim.
Ecol. 17:15-26, 1948,

Burgis, M.J. The Effect of Temperature on the Development Time of Eggs of Thermo-
cyclops sp., a Tropical Cyclopoid Copepod from Lake George, Uganda, Limnol.
Oceanog. 15:742-747, 1970.

Chapman, A. The Bionomics of Diaptomus gracilis Sars (Copepoda:Calanoida) in Loch
Lomond, Scotland, J. Anim, Ecol. 38:257-283, 1969.

Coker, R.E. Influence of Temperature on Size of Freshwater Copepoda (Cyc lops).
Internat. Rev. Ges. Hydrobiol. Hydrog. 29:406-436, 1933,

+ Some Aspects of the Influence of Temperature on Copepoda. Science,

79;323-324, 1934a.

- Reaction of Some Freshwater Copepods to High Temperatures. J. Elisha
Mitchell Sci. Soc. 50:143-159, 1934b.

+ Influence of Temperature on Form of the Freshwater Copepod, Cyclops
vernalis Fischer. Internat. Rev. Ges. Hydrobiol. Hydrog. 30:411-427, 1934c.

Comita, G.W. A Study ofa CalanoidCopepod Population in an Arctic Lake. Ecology.
37:576-591 , 1956.

+ Oxygen Uptake in Diaptomus siciloides Lilljeborg. Limnol. Oceanog.
10:466-468, 1965.

-166-


of nauplii. At other times, observations were made with the naked eye simply by
looking into the beaker and recording the presence or absence of a clutch, al-

though it was often possible also to observe and record the condition of the ovaries.

When the observations with the microscope were made, the eggs in each new clutch
were counted and the nauplii were removed from any beakers in which eggs had
hatched. Nauplii were removed from the beakers with a pipette, leaving the
adults behind, and were discarded or put into a stock culture after they had been
counted. Water removed from the experimental beakers while removing nauplii

was returned, minus nauplii, to the same beaker, thereby maintaining the water

level without loss or dilution of the food.

To count eggs, females were isolated, as described above, in a small volume of
water which was gently poured into a Petri dish. Because these animals swim con-
stantly, it was necessary to arrest their movement. Simply pipetting all water from
the Petri dish did not permit counting of eggs, since the females reacted with vio-
lent swimming motions when out of water. Therefore, after all water was removed,
a drop of methyl! cellulose (about 4% solution) was put on the female. The animal
was almost totally immobilized and eggs could be counted with ease. Using insect
pins, it was possible to rotate the female for both dorsal and ventral views of the
clutch. After the eggs were counted, filtered pond water was added from a plastic
rinse bottle to wash the. methy! cellulose away, Females were rinsed two or three
times, as necessary, to restore free movement and normal swimming. After the
rinse water was pipetted from the Petri dish, the edge was dipped into the female's

culture beaker, allowing the animal to swim away.

When a female put on eggs, or when a clutch hatched between two observations,
the time midway between was used as an estimate of the time the event occurred.
Often, an old clutch hatched and a new clutch was put on between two observa-

tons. In such a case, hatching was estimated as occurring at the one-third point

~47-


one stage to the next. In 91 the number of individuals reaching CVI was estimated
by multiplying the ratio of new adults to total adults by the total adult population on
31 March, the first date new adults were observed in the population. The cH adults
were distinguished from older animals by their more opaque appearance and slightly
smaller size. Prior to 31 March none of the adults collected were opaque. This
opaque appearance was possibly related to the new carapace (following the molt from
CV to CVI) not yet having hardened, although the cause was not determined. The
total number of individuals entering each instar during the different generations or

periods was determined by using the following formula:

nek jot tt Me
pre, 2 D,
x=] i

where: 1 refers to the number of individuals alive;
i refers to the instar designation;
x refers to the collection designation;
j refers to the first collection prior to the appearance of instar i;

k refers to the collection following the last collection in which
instar i appears;

D refers to the duration in days of instar i;

W, refers to the interval in days between collection x and collection
x1;

N, refers to the number of individuals of instar i produced in the in-
terval x to x + 1; it also refers to the number of individuals of
instar i produced in a particular generation.

Table 32 shows the life table for 9° The 1 and d columns of this life table reveal
over 80% mortality occurred within the egg to NI! stage, with 836.62 out of every
1,000 individuals that entered the egg stage dying before reaching NIV. (Technical~
ly some animals undoubtedly reached NIV but died before being counted. To simplify
the presentation in this section all the mortality recorded for a stage will be assumed

to have occurred between the time of counting and the transformation to the next stage.)

-101-


in Table 21. The mean for 31° is only slightly lower than the mean for 27°, and an

SNK test indicates the two are not significantly different.

The relationship between temperature and the interval between successive clutches
(Table 22) is not so straightforward. The longest interval is at 14° as was the long-
est development time. However, for the remaining three incubation temperatures,
the relationship is direct. Differences in the means are highly significant (F=15.7;
P<0,001), but a SNK test indicates that neither the means for 21° and 27° nor the
means for 14° and 31° are significantly different, Therefore, the data indicate a
fairly narrow range of intermediate temperatures in which the mean interval be-
tween clutches is minimized, with the interval increasing markedly at both higher

and lower temperatures.

Similarly, the length of the complete cycle from oviposition to oviposition (Table
23) is minimal at the intermediate temperatures and higher at the extremes. The
minimum is shifted toward the higher temperatures by the strong inverse relation-
ship between development time and temperature, but the interval between clutches
is sufficiently long at the upper extreme to make the complete cycle longer at 31°
than at 27° despite the slightly shorter mean development time at 31°, Analysis of
variance indicates highly significant (F=113.3; P<0.001) differences among the
means of the four incubation temperatures. Comparison of these means by a SNK
test indicates that the mean for each incubation temperature differs significantly

from the meansfor all other incubation temperatures.
HATCHING SUCCESS

Data on hatching success are not presented for every clutch produced by the ex~
perimental females. Some of the clutches consisted of resting eggs, a few of which
hatched up to two and one-half months after oviposition. No data were obtained

on the hatching success of these clutches. Of those resting eggs that did not hatch

-65-


varied toa certain extent in temperature and to a considerable extent in light intensity ,
and duration. Room C had continual light of moderately low intensity, and these con-
ditions may have encouraged algal growth in this room. This could then have led to
increased food supply, and thus shorter average times for the carrying of the clutches.
The results obtained in this work suggested that the influence of light conditions should

be explored further. This was done and the results are considered in the next subsection.

The results presented here indicate that, in general, pond water is to be preferred for
culturing because a lower mortality rate was observed in this medium than in the other
two. Although no controlled experiments were conducted, the superior qualities of
pond water for culturing were also indicated by certain observations on development
of the young in the different types of media. In an informal comparison of develop
ment in distilled and pond water, approximately 500 nauplii were kept in distilled
water, while approximately 2000 nauplii were simultaneously maintained in pond
water under the same conditions, None of the nauplii in the distilled water lived to
maturity, while over one-quarter of those in pond water did. General impressions on
development in conditioned water indicated only poor development, although some
individuals did mature in this medium. The general conclusion arising from our con-
sideration of the three types of media is that pond water should be used for culturing
unless an alternate medium has been shown to be as good or better. Obviously, how-
ever, the pond water should be from a pond where the species to be cultured occurs,

as chemical conditions vary greatly from pond to pond.
Light

The influence of light on culturing has been explored by measuring simultaneously
under three light intensities certain reproductive and developmental attributes. The
experiment was designed to measure the effects of temperature (as described in the
next subsection) as well as light, and so four temperatures regimes were used, . How-

ever, animals exposed to each light intensity were represented only once in each

-17-


over, the tubes were removed from the water bath; the dissolved materials were trans—
ferred to centrifuge tubes; and these were centrifuged at high speed for 1 minute.
The supernate was then transferred to a cuvette, and, using the tap water extract
to zero the spectrocolorimeter (B&L Spectronic 20), the percent absorbance of the
two extracts at a wavelength of 665 millimicrons was determined. The average of

two readings was recorded as a measure of the chlorophyll a present.
Field Data

To determine the dispersion pattern of the population, it was necessary to obtain data
appropriate for statistical testing. During the preliminary period of sampling, it was
apparent that most individuals of this species were located in open water rather than
where rooted aquatics were growing. For this reason,the pond was divided horizon-
tally into two regions or strata, the area where rooted aquatics came within 40 cm

of the surface and the open water area. Eight samples were taken from each region
of each date with the sites to be sampled selected by means of a random numbers
table. The number of adults (copepodite VI) per liter in each sample was determined

by dividing the total number of adults by the number of liters sampled.

The numbers of adult and copepodite V individuals of each sex, the number of females
carrying eggs, and the numbers of the other copepodite stages were determined by
complete census of each sample. The numbers of the various naupliar stages were de-
termined after combining the samples into two groups, one from each stratum. Each
pooled sample was then divided several times through use of a plankton splitter, and
the nauplii in one of the resulting subsamples were counted. The mean number of
eggs per clutch was determined at the same time. The number of splits to be carried
out was chosen fo ensure the census of at least 20 clutches. The amount of time an

individual spends in naupliar stages | fo III is relatively small (apparently less than

24 hours); and, for this reason, these stages were uncommon in the samples, and so

-83-


No.

40.

4.

42.

45.

46.

47.

49,

50.

Sl.

52.

53.

Page
A posteriori Comparison of the Seasonal Means of the Ratios of Males
to Females Using the Student-Newman~-Keuls Test,...... ce eeee 16
Anova Table Testing the Differences Among the Mean Numbers of
Eggs Per Clutch During the Various Seasons, ..... 0. eee eee cues 7
A posteriori Comparison of the Seasonal Mean Numbers of Eggs Per
Clutch Using the Student-Newman-Keuls Test. ......0..2000ceee 7
Correlation Coefficients Found Between the Mean Number of Eggs Per
Clutch and Chlorophy!| Content, Temperature, and Density of Adults
During the Study Year... se. eee eee eee eee see cence renee HB

A Comparison of the Mean Numbers of Cyclops vernalis That Were
Present in Four Culture Volumes on a Series of Sampling Times ,...,.. 129

A Comparison of the Mean Densities of Cyclops vernalis That Were
Present in Four Culture Volumes on a Series of Sampling Times .......
A Comparison of the Mean Numbers of Cyclops vernalis Found Under

Three Different Rates of Food Additions on a Series of Sampling Dates. . - 132

A Comparison of the Mean Numbers of Cyclops vernalis Found Under
Three Different Rates of Food Additions on a Series of Sampling Dates. . - 134

. A Comparison of the Mean Numbers of Cyclops vernalis Present on a

Series of Sampling Dates in Six Different Types of Culturing Water... .. 136

A Comparison of the Ranks of Total Numbers of Cyclops vernalis Kept
in Six Different Types of Water «+ +++ eeeee eect ewer rerence 138

A Comparison of the Ranks of the Numbers of Adult Cyclops vernalis
Kept in Six Different Types of Water... . ce cee cee eee eee eee
Percentage of Nauplii of Cyclops vernalis Surviving 24 and 48 Hours
After Hatching With and Without the Presence of the Mother.........
Numbers of Cyclops vernalis after 4 Weeks in « Continuous Flow Cul-
turing System... eee cee eee ee eee ences eter

144

Mean Durations of the Adult Stage, the Mean Time Intervals From the
Adult Molt to the Production of the First Egg Clutch, the Mean Times


Table 21, COMPARISON OF MEAN CLUTCH CARRYING
TIME AT FOUR INCUBATION TEMPERATURES

Incubation Mean 95%
temperature (hours) confidence limits
(degrees: ©)
14 113.1 108,2 - 118.2
21 48,,2* 45.7 - 50.9%
27 32.7 30.6 ~ 34.9
31 29.8 28.0- 31.6

*Carrying times of clutches of resting eggs excluded.

An Fmax7Test indicates that variances of the data for the four incubation temperatures
are heterogeneous. A logarithmic transformation was made and an Anova carried out
with the transformed data.

Anova table: Carrying times by incubation temperature. Data for clutches of
resting eggs excluded from 21° data.

Source of variation df MS F
Among incubation temperatures 3 2.6887 261.0388 P<0.001
Within incubation temperatures 220 0.0103
Total 223 :



Table 24. HATCHING SUCCESS

Female's
Incubation Overall hatching
temperature mean temperature Mean
(degrees C) (%) (degrees C) (%)
14 82.3*
2 68.0*
14 66.8 7 —
31 55.8
14 wenn
al 85.8
a 85.9 27 96.2%
31 76.9
. 4 -———
21 51,.7*
7 66.6 7 7.7
31 23.7*
14 oon
21 33.2*
3 53.3 27 30.7*
31 60.6

*Mean hatching success of a single female.

An Anova was carried out using the Arcsine transformation of the data for those females
"native" to each incubation temperature. Data were not used for those females which
were not incubated at the same temperature as that at which they hatched.

Source of variation df MS F
Among incubation temperatures 3 2065 .1329 4.3675 P< 0.010
Within incubation temperatures 161 472.8348
Total 164

-72-


APPENDIX E~-6

TOTAL NUMBER OF COPEPODITE ONE (I) IN EACH REGION OF THE POND
AND IN THE TOTAL POND ON EACH COLLECTING DATE, THE TOTAL

FOR EACH DATE.

NUMBER WAS DETERMINED BY MULTIPLYING THE Y (MEAN)
NUMBER OF Cl PER QUADRAT IN EACH REGION BY
THE TOTAL NUMBER OF QUADRATS IN THAT
REGION AND SUMMING THE PRODUCTS

Date Open water Rooted aquatics Total pond
region region
N-1971
19 0 0 0
21 0 0 0
23 0 0 0
25 (9) 0 0
27 0 0 0
H-1971
1 0 0 0
3 0 0 0
5 0 0 0)
7 0 0 0
9 0 0 0
" (0) 0 i)
13 67,441 11,240 78,682
15 535,097 55,739 590,836
7 624,279 78,035 702,314
19 590,836 122,626 713,462
21 588,388 177,626 766,014
23 637, 038 208, 685 845,723
25 271,759 10,870 282,629
27 284,496 86,586 371 ,082
29 775,881 812,827 1,588,708
31 760,449 57,238 817,687
IV-1971
2 776,921 110,215 887,136
4 696,141 7,444 703,584
6 273,250 25,454 298,705
8 262,432 17,777 280,209
10 194,429 0 194,429
12 437,577 10,176 447,753

-216-


No. Page

15. Mean Number of Adults Per Liter in the Open Water Region on Each
Collecting Date... .- ae rs 072

16. Mean Clutch Size Plotted Against Density of Adults . , 123

17. Comparison of the Mean Durations of the Adult Stage, the Mean Time
From Maturation to the Production of the First Egg Clutch, and the
Mean Time From the Production of the Last Egg Clutch to Death For C.
vernalis Females at Different Temperatures: 1.2. e cece eeeeeeree 152

18. The Rate of Development of Eggs of C. vernalis in Relation to Tempera-

FUE we ec eer e eee were rere ere t seer reer ete rsrceceee 16)

vii


-00l-

Table 34.

LIFE TABLE FOR LABORATORY ANIMALS USING COMPLETE DATA FROM ALL ANIMALS AT

ALL TEMPERATURES.
Stage Number living Number dying Mortality rate Mean number | Total life Mean lifetime
at beginning of In Interval per 1000 alive alive expectancy | remaining for
age Interval dq, at beginning of ky Ty those attaining
ly age interval age interval
qx &x

Egg-Nill 1000.00 281 .51 281 .51 859.29 1291772 12.92
NIV 718.49 115.55 160.82 660.71 11362.49 15.81
NV 602.94 71.48 118.47 567.23 -10444,11 17.32
NVI 531.51 151.26 284.58 455.88 9763.43 18.37
fof 380.25 18.91 49.72 370.80 9198.14 24.19
cil 361.34 35.71 98.84 343.49 8653 .06 23.95
Ci - 325.63 27.31 83.87 311.97 8003 .87 24.58
CIV 298.32 18.91 63.38 288 .87 6924.45 23.21
cv 279,41 54.62 195.49 252.10 5667.87 20.29
cvi 224.79 224.79 1000.00 112.39 4495.60 20.00

Original 1, equals 1.302 x 10°,


-0L-

12
W

10

RANKED

MEAN é
PERCENT

HATCH 5

0 14° HATCH
4 21° HATCH 4

o o 27°HATCH
e 31° HATCH

14 21 27 31

Incubation Temperatures
(degrees)
Figure 4,. Mean Percent Hatch of Groups of Females (Right Column of Table 24)
are Ranked from Lowest to Highest (1 to 12) and Plotted Against the
Temperature at which the Females Were Incubated.


APPENDIX A-3 (Cont.)
27° INCUBATION: ANALYSES OF VARIANCE

Anova table: mean clutch sizes

Source of variation df MS F
Among individuals 7 517.4556 97579 P< 0.001
27° hatch vs other 1 545.8955 10.2962 P< 0.005
21° hatch vs 31° hatch = 1 103.1429 1.9454 ns
Among 27° hatch 5 594.6302 11.2154 P< 0.005
Within individuals 74 53.0191
Total 81

Anova table: comparison of sequential clutches

Source of variation df MS F

Among sequential clutches 7 123.4961 1.2403 ns
Within sequential clutches 51 99.562 *
Total 58

477-


durations with temperature is further complicated by food, since both quantity and
quality of food apparently affect the developmental rates of copepods (Coker, 1933;
Smyly, 1970).

Table 31 shows the durations of the various instars as determined in the field and also
as computed from a composite of laboratory data. The laboratory data are from Sam=
ples (1972) and were obtained within a temperature range of approximately 20°C to
25°C. Several! studies have shown an inverse relationship between temperature and
the developmental time of the various instars, for example McLaren (1965) found a
longer developmental time in the Arctic calanoid Pseudocalanus at lower temperatures
than at higher ones. When the field durations (obtained at lower temperatures) are
compared to the laboratory durations, similar results are noted for D, clavipes with

a total developmental time to CVI of 28 days in the field and 21.5 days in the labora—
tory.

Life tables were constructed for the first generation (g,) (Table 32), total year (Table
33), and laboratory animals (Table 34). In computing the life tables, the durations
based on the laboratory results were used except for the first generation (g,). This
procedure was justified for the field population on the basis that during the reproduc
tive part of the year, except during O° the pond temperatures were generally within the
range 15° to 25°C (see Figure 14, page!20), and the animals probably migrated with-
in the pond in order to avoid higher temperatures when they did occur, Thus, the
durations from the laboratory for temperatures in the range 20° to 25°C were assumed
to represent a reasonable first approximation for the instar durations during the genera
tions after 9° Since the field durations were directly determined for the first genera~

tion,these were used for developing the life table for this generation,
The number of individuals entering each of the various stages for the a and labora-

tory study were determined by following the survival of a cohort of individuals at close

intervals. This was accomplished by computing the loss of individuals in passing from

~96-


METHODS AND MATERIALS

The study pond is located in Section 11, Township 9N, Range 3W of Cleveland
County, Oklahoma. It is approximately 13.1 km SSE of Norman, Oklahoma. It
is a man-made impoundment receiving runoff from the surrounding prairie at its

east and southwest margins. There are no fish in the pond, The morphometry of
the pond (Appendices D-1 and D~2) was determined following procedures outlined
by Welch (1948). Pennak (1957) suggested that if one wants a reliable estimate of
species composition and relative abundance of each species in an aquatic environ-
ment sampling from top to bottom is imperative. The same reasoning applies when
determining the absolute numbers of a given species. In the present study sampling
was carried out by means of a semi=pliable, 6,5-cm diameter, wire-embedded poly-
ethylene tubing, suitably calibrated for depth measurements. A rope was attached
to one end of the tube. This end was lowered perpendicular to the surfaceuntil it
reached the bottom of the pond. After attachinga #20 plankton net over the op-
posite end of the tube, the lowered end was raised in a manner similar to that de-
scribed by Pennak (1962), thereby causing a vertical column of water to be filtered

through the plankton net.

Although sampling was carried out from May, 1970 until October, 1971, only data
from 19 February through 29 October, 1971 are considered in this paper. These

dates encompass the period of the year during which successful reproduction occurred
in this population. On 19 February almost the entire population was in the adult

stage and reproductive activity had begun, as evidenced by the high number of females
carrying eggs. By the termination date, 29 October, although the temperature was
still well within the range necessary for successful reproduction (approximately 10°

to 30°C), successful reproduction was greatly curtailed. This was evidenced by the
fact that only low numbers of immature copepodites were collected on the last three

dates. Collections were taken every other day from 19 February to 20 April and

-81-


Kingsbury, P.J. Plankton Production and Species Distribution in the Limnological _
Provinces of Oklahoma. Univ. of Okla., Ph.D. Dissertation. 84 p. 1968.

Lewis, A.G. An Enrichment Solution for Culturing the Early Developmental Stages
on the Planktonic Marine Copepod Euchaeta japonica Marukawa. Limnol. Qceanog.
12:147-148, 1967.

Lock, A.R. and 1.A. Mclaren, The Effect of Varying and Constant Temperatures
on the Size of a Marine Copepod. Limnol. Oceanog. 15:638-640, 1970.

Margalef, R. Temperature, Dimensions y Evolucion. Proc. Inst. Biol. Apl., Barce-
lona. °19:13-94, 1955.

Marshall, S.M, and A.P. Orr, On the Biology of Calanus finmarchicus. VII. Fac-
tors Affecting Egg Production. J. Mar. Biol. Assoc. U.K. 30:527-547, 1952,

McLaren, !1.A. Some Relationships between Temperature and Egg Size, Body Size,
Development Rate, and Fecundity of the Copepod Pseudocalanus. Limnol. Oceanog.
10;528-538, 1965. ere

Mullin, M.M. Some Factors Affecting the Feeding of Marine Copepods of the Genus
Calanus. Limnol, Oceanog. 8:239-250, 1963.

and E.R. Brooks, Laboratory Culture, Growth Rate, and Feeding Behavior
ofa Planktonic Marine Copepod. Limnol. Oceanog. 12:657-666, 1967.

and . The Effect of Concentration of Food and Body Weight,
Cumulative Ingestion; and Rate of Growth of the Marine Copepod Calanus helgo-
landicus. Limno!. Oceanog. 15:748-755, 1970.

Pennak, R.W. Species Composition of Limnetic Zooplankton Communities. Limnol.
Oceanog. 2:222-232, 1957. ‘

+ Quantitative Zooplankton Sampling in Littoral Vegetation Areas.
Limnol, Oceanog. 7:487-489, 1962.

Ravera, O. La Struttura Demografica dei Copepodi del Lago Maggiore. Mem. Ist.
Ital. Idrobiol. 8:109-150, 1954.

. Seasonal Variation of the Reproductive Rate in Pelagic Copepods of
Lake Maggiore. Internat. Verein. Limnol. 12:436-446, 1955.

and V. Tonolli. Body Size and Number of Eggs in Diaptomids, as Re-
Tated to Water Renewal in Mountain Lakes. Limnol. Oceanog. 1:118-122, 1956,

-169-


LIST OF ARTICLES SUBMITTED FOR PUBLICATION
AND LIST OF DISSERTATIONS

Gehrs, C.W. Aspects of the Population Dynamics of the Calanoid Copepod, Diap-
tomus clavipes Schacht. Univ. of Okla., Ph.D. Dissertation. 111 p., 1972.

. Low Temperature: A Potential Aid in Analyzing the Reproductive Char~
acteristics of Calanoid Copepod Populations. Prog. Fish Culturist. (Accepted for
publication).

. Horizontal Distribution and Abundance of Diaptomus clavipes Schacht
Related fo the Occurrence of Rooted Aquatic Vegetation. Gebait ted to Limnol.
Cceanog. ).

and B.D. Hardin. : Production of Resting Eggs by Diaptomus clavipes
Schacht (Copepoda, Calanoida). (Submitted to Amer. Midland Nat.) :

Hardin, B.D. Temperature and other Factors Affecting the Reproduction of Diaptomus
clavipes (Crustacea, Copepoda, Calanoida). Univ. of Okla., M.S. Thesis, 75 p.,
1972.

Hunt, G.W. The Influence of Temperature on Reproduction of Cyclops vernalis
Fischer (Copepoda). Univ. of Okla., M.S. Thesis, 52 p., 1972.

Samples, C.R. Laboratory Culturing of the Calanoid Copepod Diaptomus (Aglaodiap-
tomus) clavipes Schacht 1897. Univ. of Okla., M.S. Thesis. 60 p., 1972.

-172-


only occurred on animals already weak and dying, but in any event death invari-
ably followed within days after this growth was first observed. Once begun, the
bacterial growth quickly covered the female, giving a velvety appearance to those
that lived more than a day or two. Other workers (Corkett & Urry, 1968) have
made similar observations and found that addition of antibiotics to the cultures ex-
tended the longevity of the copepods. No antibiotics were used in the experiments

reported here.

Table 15 presents the values observed for female life span after maturity at the dif-
ferent incubation and hatching temperatures as well as the mean for each tempera-
ture. The samples in the present study were too small to permit statistical analysis
in terms of the female's hatching temperatures, but, by inspection of Table 15, it
appears that adult life span is determined by the incubation temperature, with no
obvious influences attributable to hatching temperature. Thus, analysis of vari-
ance was carried out using all data at each incubation temperature. Not unex-
pectedly, mean adult longevity was found to decrease significantly (F=23.7;
P<0.001) with increasing temperature. Since the females apparently lived to phy-
siological old age, the values reported represent estimates of the longest expected
adult life spans and may not be an accurate indication of adult life spans in a natu-
ral population. The conclusion that physiological longevity is inversely related to

incubation temperature, however, is no less valid.
CLUTCH SIZE AND TOTAL EGG PRODUCTION

The data for egg production at the four different temperatures are presented in Ap-
pendices A-1 through A~4. In three cases (second clutch of B20, Appendix A-2;
second clutch of D3; and fifth clutch of D16, both Appendix A-4), the eggs ina
clutch were not counted. In those three cases, the average value for that female is re-
ported. In all other cases, the values reported are numbers of eggs actually counted.

Accuracy of the counts probably declines slightly with increasing clutch size. The

~49~


males, The water level was maintained by additions as necessary, but since the
beakers were covered with a cellophane kitchen wrap, addition of water was sel-

dom necessary.

When copepodites were needed for isolation, a stock culture was filtered, result-
ing in the collection of many more copepodites than were necessary for isolation
at any one temperature. Other workers (Marshall & Orr, 1952; Corkett, 1967;
Mullin & Brooks, 1957; Katona & Moodie, 1969) have emphasized that successful
culturing of planktonic marine copepods requires that disturbances of cultures be
minimized, Therefore, when copepodites were isolated from one stock culture,
some were transferred to a different temperature chamber where they matured and
lived out their lives. This provided considerable acclimation time, especially in
relation to that allowed by some others (e.g., Comita, 1965; 1968) for laboratory
experiments on animals taken in plankton samples. These individuals, which
hatched and developed through all naupliar and some early copepodite stages at a
temperature other than that at which they were kept as late copepodites and adults,

are identified in the discussions which follow.

The introduction of copepodites from a warmer temperature was necessary ot 14 to
have experimental animals maturing at that temperature. Despite the continuous
presence of egg-bearing females in the stock cultures at 14° and the regular addi-
tion to these cultures of nauplii produced by experimental animals at 14°, very few
copepodites were ever found in these cultures. Of the few found and isolated,
only one female (A7) reached maturity. (One or two males also reached maturity.)
Aycock (1942), working with Cyclops vernalis, reported both higher mortality at
lower temperatures and development time inversely related to temperature. If both
conditions are true for D. clavipes (the inverse relationship between development
time and temperature was confirmed in this study), they would account, in part at

least,. for the absence of copepodites in the 14° stock cultures.

-45-


and temperature ona 24-hour basis. Light intensities were recorded with a Weston
Light Meter, while all water temperatures were measured with a thermometer graded
in degrees Celcius and correct within +1 .0°C. Air temperatures in the constant
temperature rooms and chambers were usually within +1 .5°C of those temperatures
set on the controls. The temperatures of the waters in the culture vessels were nor

mally within £1,.0° C of the desired temperature.

Selection of Culturing Conditions

This section is structured in a series of subsections, each of which deals with a dif-
ferent factor of importance in culturing. Such structuring was deemed desirable since
the individual experiments often produced data that could be applied to the study of

more than one of the culturing factors.

The experiments presented in the following subsections are much too limited to pro-
vide statistically defensible results concerning the effects of the culturing factors.
However, it should be borne in mind that the objective of this research was to de-
velop a method for culturing D. clavipes. Thus, the work undertaken was restricted

to that judged necessary to develop such a method. In the next chapter a more rigorous

consideration of the effects of temperature will be presented.
Container Characteristics

It was felt that the culture vessels should satisfy the following requirements: 1) the
containers should not appreciably affect the composition of the culturing medium; 2)
the containers should facilitate observations on and manipulation of the animals; and,
most importantly, 3) the animals should exhibit reasonable survival and reproductive
rates in the containers. To satisfy the first requirement glass containers were used.
The second requirement was met by using only wide-mouth containers made of clear
glass. With these restrictions set up, the only other question was what the volume

of the cultures should be to ensure good survival and reproduction,


~vee-

APPENDIX H-2

FECUNDITY IN EGGS PER CLUTCH OF FEMALE CYCLOPS VERNALIS AT 21°C,

Clutch Female No.
No. 1 2 3 4 #5 & 7 8 9 -0. 12. 43
1 101 5B 69 3* 40S si (as sia
2 60 5455 50 74 62 102, 81 10 29 43. 7
3 81 575] 36 6 51 68 13 3% 47. 50
4 7 6* 50 28 52 62 13" 44 56
5 7 48 33 64 12 3% 29h
6 23 43 27* 27 4829
7 29 12* 34g
8 15 %

Total eggs 350-175 «296 3ss1B7)s«129,—-38BeSC«ACsC3s]s263 3481985

per female

No. of

clutches 5 4 61 5 2 8 3 7 6 7 8 3

per female

*Only one of two possible egg sacs present.


different manner than the three previous experiments, however. In this work the

mortality and reproductive success were monitored for individual females.

To initiate the work a number of females that had been raised in stock cultures at

21° C were placed in separate 100-ml beakers each containing 80 ml of filtered

pond water. Before being added to the beakers, these females had been examined
for the condition of their ovaries. The appearance of the ovaries was judged to fall
into one of three categories—-clear, somewhat opaque, or very opaque. Very opaque
ovaries signaled an animal that was almost ready for oviposition; after oviposition the
ovaries were clear, Only animals with opaque or very opaque ovaries were used to
initiate this study.

Two adult males were also added to each beaker, and these were replaced during the
experiment if any died. The females were not replaced, however. The beakers were
maintained ina 21° C constant temperature room under light conditions of moderate

iumination and 12-hours light and 12-hours dark.

From the beakers set up in this manner, three groups of six were selected using a ran~
dom number table. One group received 0.8 ml of fish food solution every third day,
one group 0.4 ml every third day, and one group no food at all. This experiment was
repeated using all three food conditions and then repeated a second time with the
omission of the beakers to which no food was added.

Each experiment lasted 9 days and during this time each beaker was examined once

a day. At examination it was determined for each container: 1) whether the female
was still alive, 2) if so whether she was carrying an egg clutch, and 3) if so how many
eggs were in the clutch. The results from these experiments are summarized in Table

13.


included for comparative purposes. No substantial differences can be noted between
the results obtained for the animals fed the two kinds of food. This work gives no indi-
cation that the older fish food solution was any different in its ability to serve as food
than the fresh material. Thus, it seems reasonable to suggest that the practice of
making up new food only every 2 weeks rather than more often probably has little in-
fluence on the results obtained.

Overall, it is concluded that the fish food solution provides an excellent food for cul-
turing and experimentation with D, clavipes. However, the volume and frequency

of food additions are of crucial importance. Too much food leads to high mortalities
probably caused by deterioration of the quality of the medium. Food additions as
small as a few tenths of a milliliter every third day seem quite adequate to support
strong reproduction and growth. Changing the water every few days even in stock
cultures is also recommended with this food to avoid build-up of nutrients and the

deterioration of water quality.
Disturbance of Animals

No controlled experiments were conducted to study the effects of disturbance of the
animals on culturing success. However, an attempt was made throughout the work to
keep the disturbance as little as possible. Minimal disturbance of the immatures and
adult males was accomplished by making observations without removing them from their
containers. It was necessary to disturb the adult females on numerous occasions in
order to isolate gravid individuals. Yet, the females appeared to be remarkably toler-
ant to both the frequent handling required to make microscopic counts of the eggs in
their clutches and to the methy! cellulose used to immobilize them during the process.
Individual females were frequently isolated two or three times, a few as many as eight

times, for this purpose.


during the course of this study, none decomposed, so it is possible that these might

have hatched had the study continued longer,

Pipetting the individual nauplii from the culture was not a completely satisfactory
method to count nauplii; and, as a result, the data for some clutches were so con~
fused that no estimate of hatching was obtained. However, the pipetting was the
only method that did not traumatize the females and thus did not invalidate the
data on other factors being studied. Normally, the nauplii were slightly pigmented
and were easily visible immediately upon hatching. Occasionally, nauplii were
produced which totally lacked pigment and therefore were almost invisible until
they reached the third or fourth naupliar stage. Additionally, at 27° and 31°C,
the water became quite cloudy with suspended material and algal growth during the
last three or four days before the regular water change. On occasion, the water
was cloudy enough to moke finding nauplii extremely difficult even if they were
distinctly pigmented. As a result, not all nauplii could be found and pipetted out
at one time, and it usually required two or more observation periods for all nauplii
to be removed. In this time, some no doubt died. Others may have been missed
several times and then removed and counted with nauplii from a later clutch. In
the latter case, a difference in naupliar stage sometimes distinguished the older
nauplii from the newly hatched ones, but it was not always possible to determine
positively whether the older nauplii came from the previous clutch or from an even
earlier one. Ina few cases, overlapping between two clutches was so great that no

data are reported for either,

The hatching successes of individual females were tested by analysis of variance for
differences among the individuals at each incubation temperature (Appendices C-1
through C-4). Differences among individuals were highly significant at three in-
cubation temperatures (P<0.001 at 14° and 21°, P<0.010 at 27°). Only at 31° in-
cubation, where the hatching successes were uniformly low, were there no signi-

ficant differences among individuals, As shown in Figure 4 (constructed using the

-69~


This work indicates that culture volume over the range studied in this experiment
does not greatly affect culturing success. More animals developed in the larger
volumes but the highest densities were noted in the smallest volume. These differ-
ences are probably related to food availability in the different volumes. These
differences aside, however, it was generally observed that the cultures at all four
volumes were very successful, There seems little indication of culturing becoming

more difficult at low volumes as was observed for Diaptomus clavipes.
Food Concentration

Two separate tests were conducted to determine the effects on culturing success of
varying food concentration. In the first test, 9 separate cultures were initiated in
J-liter beakers by adding 8 adults, 4 males and 4 gravid females, to each beaker.
These cultures were divided into three sets of three replicates each. One set of re-

plicates received 0.1 ml of food solution every second day, one set 0,5 ml, and

one set 1.0 ml.

The results from this experiment are presented in Table 46. It is immediately obvious
that vigorous cultures developed at all three feeding rates. There is some indica-
tion that the population density increased with the amount of food added. This is
quite evident for the adults but not so clear for the total numbers, where the dens~
ities in the cultures receiving 0.5 ml seem to have been at least as great as those in

the cultures receiving 1.0 ml.

A second test was run in much the same way as the one just described. However,
4-inch finger bowls each containing 250 ml of culture water were used instead of 1-
liter beakers, and the food volumes added were somewhat different. One set of
three replicates recieved food at the rate of 0.4 ml per liter of culture water ever
second day, in other words 0.1 ml were added to the 250-ml cultures each time.

The second set received 1.0 ml per liter every second day, and the third set received

2.0 ml per liter every second day.

~131-


being inversely correlated with water temperature during development. Other in-
vestigators have found a correlation between the number of eggs per clutch and food.
Similar correlations were found between both temperature and food and the mean
number of eggs per clutch in the population we studied. Our laboratory data suggest
that the significant correlation observed between temperature and clutch size does

not necessarily indicate a cause and effect relationship. Interpretation of these data
suggest, therefore, that the density of adults may be the regulating mechanism for
determining the number of eggs per clutch produced by the females. Selection for a
self-regulating mechanism would be expected in a species whose environment is limit-

ed and whose individual members are unable to migrate.
SUMMARY

A study dealing with certain aspects of the population dynamics of a field population
of Diaptomus clavipes has been carried out, The following results were obtained:

1. When samples were collected every two days from a pond population
from 19 February to 20 April and every 14 days from 24 April to 29
October 1971, the adult population was found to be distributed un-
evenly in the pond. ©. During the entire period but especially in
the summer and fall, greater concentrations of copepods occurred
in the open water regions than in areas in which rooted aquatics
grew close to the surface.

2. Analysis of data from laboratory experiments revealed that, when
concentrations of rooted aquatics were present in culture containers,
adult animals were unable to survive.

3. Simulating the physical presence of the weeds with pieces of poly-
ethylene tubing caused a retardation in the developmental rate of
the egg to NII stage.

4. Durations of the various instar stages were determined through data
obtained during the intensive (every second day) collection periodand
also through analysis of laboratory data. The durations obtained in
the two different ways show quite good agreement,

-124-


3. To follow and analyze a field population of a diaptomid with special
attention to the relation between population dynamics and environ=
mental conditions.

This project was supported by a grant (USDI Grant 18050 ELT) from the Federal Water
Pollution Control Administration (now part of the Environmental Protection Agency)
of the U.S, Department of Interior to the University of Oklahoma Research Institute.
This is the final report for this grant.

The grant was awarded for one year, extended for six months, and then continued
for a subsequent year plus a six month extension. During the continuation period,
the grant work was expanded to include a certain amount of research on a cyclopoid
copepod. This entailed adding two objectives to the study. These were:
4 To develop dependable, reproducible methods for culturing a cyclo-~
poid.

5, To explore the effects of certain environmental factors, especially
temperature, on the cyclopoid.

The research on cyclopoids is also covered in this final report.
In order to simplify and clarify the presentation, this report is divided into five

sections. Each section reports the results that correspond to one of the five objec~

tives outlined above.


CHAPTER 5
THE CULTURING OF A CYCLOPOID

In general, freshwater cyclopoid copepods are easier to maintain and culture under
laboratory conditions than are calanoids, Cyclops vernalis Fischer, the species used
in our work, has been cultured previously and used in experimental studies (e.g.
Coker, 1933, 1934b, 1934c; Aycock, 1942). Thus, there was no need to develop
culturing methods in order to make this form available for use in bioassays or other

experimental work.

However, to use a species for such studies, it is very desirable to have some under~
standing of the relations between culturing conditions and success in order to assure
that the methods used are dependable and easily reproducible. This guarantees that
experimental animals can always be obtained in the quantities and at the times need-
ed. Also, if the relations between culturing conditions and culturing success are
understood, care can be taken so ‘that, when the influence of a certain environmental
factor is being studied,:'the influences of other factors are minimized or controlled.
Thus, for example, size of container can be kept lorge enough so that it does not
unduly affect the results obtained in an experiment studying the effects of varying
temperature.

The work reported in this chapter had as its objective the development of a depend-
able, reproducible method for culturing a cyclopoid, specifically Cyclops vernalis.
Also, this work was intended to clarify the relation between culturing success and

certain of the factors influencing it. Asan aid in the use of these animals for bio~
assays, a short section is included on their maintenance and culture in a continuous

flow system.

~126-


The pond is located on the west side of Flood Street, 2.7 miles north of Robinson
Street (Section 11, Township 10 N, Range 3 W). In the laboratory D. clavipes
was seporated from D. siciloides, which is also found in the pond, on the basis of
the larger size and red antennules of D. clavipes. Between May 19 and 28, 1971,
stock cultures were established at 14°, 21°, 27°, and 31°C in constant tempera-
ture chambers set for 12-hour alternating periods of light and dark. In the 4
and 21° chambers, water temperatures were quite stable, never fluctuating more
than 40.5°C. In the other two chambers, water temperatures routinely fluctuated
41.0°C and occasionally £2.0°C for short periods.

The pond from which the animals were originally obtained was highly turbid, so
water for culturing was taken from a pond 3.5 miles east and 2.8 miles south of the
intersection in Norman of Lindsey and Classen Streets (Section 13, Township 8 N,
Range 2 W). Water was collected in 5-gallon plastic bottles as needed, approxi-
mately every 7 to 14 days. In the laboratory, the wafer was twice poured from one
bottle to another, both times with a single thickness of No. 20 bolting cloth cover-
ing the mouth of each bottle. Beakers of filtered water were maintained on two
occasions for periods of 14 and 16 days, respectively, and were "fed" on the same
schedule as used for experimental cultures. The only contaminating microcrus~
tacean was a small cyclopoid copepod which probably passed through the bolting
cloth as‘eggs or early nauplii. In addition to protozoans and algae, several small
rotifers and rhabdocoel flatworms were observed.

Beginning on June 14, 1971, immature copepodites from stock cultures were isolated
as needed for experimental purposes in 250-ml beakers containing 150 to 175 ml of
water. No effort was made to determine the stage of the copepodites, since no ex~
perimental observations were made until after the animals matured. The isolated
copepodites were observed once daily until they reached sexual maturity. When

an individual was found to have molted to an adult female, it was transferred to a
400-ml beaker containing 250 to 275 ml of filtered pond water and one or two adult


SUMMATY cose cece cere ec ee een n eee sean seer cere essessnenesee o
4. Aspects of the Dynamics of a Natural Population of Diaptomus clavipes .

Methods and Materials ........... dee eereeceveene bee ceeeeneesee

Field Data ....sesesscecssseeees
Laboratory Data .....sseeessceves

Oem e eee eee ceereteeee

Heterogeneity of Distribution ........cccccccceccccvcrscscceccce

Field Data ..... cece cece nce ee sc ccecceeeesssenvcresceees ee
Laboratory Data ....scceceececcecccccvcerencestcceceesereceees

Life Table Approach to Population Dynamics .......csececceeceeees
Reproduction ..sececcevccsceccccceccncccseccreescerecceoeeres
Summary’. .sceccsscccccccccecccencscesscrsencrscscrcessssecece
5. The Culturing of a Cyclopoid 10... .cccecccccsecctsececeececceness
Methods and Materials .....ccesscecccccccecensscercerssteeeees

A Dependable, Reproducible Culturing Method .......csseeceeresce

Culture Volume ....... eee e eens seer ne cen seer ceeeeeeneeeeeeees
Food Concentration...

soccer eseccees eee en i ca

Continuous Flow Culture ...ecccccceccccceceserstveteccccesereee

The Continuous Flow System .....cccccceccsesccccessoeeseseceee
The Culturing Results .....ccccccccccccccccescrcecessererceeeue

SUMMOTY 2... cece cececeeceeceseceeceensnseerereeeesesteeeeete
6. The Influence of Temperature on the Reproduction of Cyclops vernalis ,

Methods and Materials .......cccevccsccssccceseccssserccacencs

Stock Cultures ......escceccsscceceececenectsscencneeesterenee
Experimental Procedures ..scecsececcccsccenceensccecncessecees

Duration of the Female Adult Stage ....sccseuccescescecerenecees

Page

79
80

81

83
84

85

85
n

94
107
124
126
127

128
128
131
133
140
142
142
143
143
145
147

147
147

150


-e91-

Table 57, SUCCESS OF HATCHING FOR EGG CLUTCHES PRODUCED BY CYCLOPS VERNALIS
IN RELATION TO TEMPERATURE,

Exe Ke 21°C 26°C 31°C

Clutch | Eggs Nouplii % Eggs Nouplii % Eggs Noauplii % Eggs Nouplii % Eggs Navuplii %
No, hatch hatch hatch hatch hatch

1 59 «52S BY 4 43 (95.5 44 40 (90.0 59 5B 983 78 729243

2 47 4h 9386. 60 56 (93.3 60 58986 8 84 698.8 7l 7098.6

3 80 66 = 82.5 63 «5587.3 70 62 8B 6 63 95.5 67 65 «97.0

4 59 5186.4 8 875 (84.3 68 6595.6 56 54964

5 72 69 = (95.8 60 50 = 83,3 48 45 O95=B 67 6292.5

6 7 72 93,5 82 71-866 7 69 = 945 7 60 7.9

7 42 4095.2 6 66 100.0 82 7 92.7 70 6288.6

8 4b 3892.7 63 6095.2 320 31 959 51 5098.0

9 “a 3 75 102 «97—S«95.1 42, 40 (95,2
10 34 34 100.0 60 60 100.0 82 78 95.1
u 67 62925 124 7 94.3 65 60 (9243
12 55 50 90.9 110,105 95.5 75 65 86.7
13 43 3888.4 3% «= 32—s« 88 78 74949
14 33 (3090.9 140 124 88.6

15 72 68 94.4 80.78 (97.5

16 114 86 75.4 “ag 43 100.0

W7 90 69 98.8 107 9 92.5

18 87 87_—s:*100.0 63 «59 «93,7

9 70 65 «= 92.9 74 6689.2

20 34 30 88,2 54 50 92.6

2 97 88 90.7

22 68 67 98.5

Mean 68.1 916 92.8 96.0 92.7
SE, 43.20 41.39 £1.06 40.70 41.57
No. 3 20 22 8 13



27° INCUBATION: INTERVAL (HOURS) BETWEEN CLUTCHES

APPENDIX B-7

Female's
hatching
temperature {| 21° 27° 31°
individual co C7 c8 CI3 Cci4 C15 C6 cil
11.5 8.0 | 12.5] 11.0 9.0 4.0 | 24.0 10,0
45 8.5 | 25.0 4.0 3.5 18.0 | 11.0 3.5
4.5 8.0 | 17.0 | 22.5 | 22.5 18.5 4.5 | 22.5
12.0 11.0 4.5 8.5 9.0 9.5 | 168.0 | 12.5
4.5 12.5 4.0 3.5 3.5 19,5
12.0 | 12.5 4.5 4.0 10.0
5.5 | 12.5 10.0 8.0
25.5 | 12.0 7.0
12.5 | 24.0 5.5
37.5 | 24.0
97.0 | 39.0
172.0 | 12.5
24.0
7.5
24.5

Intervals between successive clutches are listed from top to bottom in the order of oc
currence but are not necessarily consecutive, some intervals having been omitted for
various reasons as discussed in the text.

Variances of the data for the three hatching temperatures are heterogeneous, A logarith-
mic transformation was made and Anovas carried out with the transformed data.

Anova table: comparison among individuals of intervals between clutches.

Source of Variation df MS F
Among individuals 7 0.2682 2.1370 ns
Within individuals 54 0.1255
Total 61

-187-


APPENDIX E-3

TOTAL NUMBER OF COPEPODITE FOUR (CIV) IN EACH REGION OF THE POND
AND IN THE TOTAL POND ON EACH COLLECTING DATE, THE TOTAL
NUMBER WAS DETERMINED BY MULTIPLYING THE Y (MEAN)

NUMBER OF CIV PER QUADRAT IN EACH REGION BY
THE TOTAL.NUMBER OF QUADRATS IN THAT
REGION AND SUMMING THE PRODUCTS
FOR EACH DATE.

Date Open water Rooted aquatics Total pond
region region
M-1971
19 0 0 0
21 0 0 0
23 0 0 0
25 0 0 0
27 0 0 0
WI-1971
1 0 ) 0
3 0 0 0
5 0 0 0
7 0 0 0
9 0 0 0
i 0 0 0
13 0 0 0
15 0 0 0
17 0 0 0
9 0 0 0
21 0 0 0
23 0 21,967 21,967
25 239, 148 0 239, 148
27 507,146 284,496 791,642
29 290,955 2,142,909 2,433,864
31 723,654 85,857 809,511
V-1971
2
4 327,596 141,434 469,029
6 621,023 398,784 1,019,808
8 858,869 8,889 867,757

-210-


-tl-

90
80
70
60
50

40

Time (days)

30

20

10

Duration of the adult stage
Adult molt to the production of the first egg sac

COD Production of the last egg sac to death 4

\N

WM

A

AA

UMM

A\\

30 34

1

oa

18 20 22 24
Temperature (°C)

Figure 17. Comparison of the Mean Durations of the Adult Stage, the Mean Times From Maturation to the Pro=-

duction of the First Egg Clutch, and'the Mean Times From the Production of the Last Egg Clutch to
Death for C. vernalis Females at Different Temperatures.


Table 19. COMPARISON OF MEAN SEQUENTIAL CLUTCH SIZES

Standard

Clutch no. No. Mean error
1 31 22.6 1.19
2 30 24.6 1.55
3 30 24.2 1.80
4 29 24.0 1.66
5 27 25.3 1.80
6 24 26.9 1.81
7 21 23.5 1.99
8 16 26.6 . 2.81
9 13 23.5 1.52
10 YW 24.7 2.26
11 9 25.4 3.74
12 9 26.9 4.07
13 7 17.4 3.32
14 6 25.8 3.39
15 5 25.6 3.12
16 5 28.6 2.99
17 5 28.4 6.61

Anova table: Comparison of sequential clutches using all data, regardless of hatching
or incubation temperature.

Source of variation df MS F

vw Among sequential clutches 16 62.3168 0.7330 ns
Within sequential clutches 261 85.0152
Total 277

-6l-


-91-

Table 5. REPRODUCTION OCCURRING OVER A SPAN OF 40 DAYS AMONG F, GENERATION D.
CLAVIPES CULTURED IN CONDITIONED WATER, DISTILLED WATER ~~
AND NATURAL POND WATER MEDIA.

Range of
Total Range of Mean Time Mean Time
Culture Number Clutch Clutch Clutch Carried | Clutch Carried
Room Medium Clutches Size Size (Days) (Days)
A Conditioned Water 18 6-28 13.50 1-11 3.61
A Pond Water 18 7-27 15.83 1-8 3.39
A Distilled Water 7 11-28 19,71 2-10 3.86
B Conditioned Water 3 1-8 5.00 1-3 2.33
B Pond Water 9 8-15 10,44 1-6 2.78
B Distilled Water 7 8-21 14,00 2-4 2.71
c Conditioned Water 4 4-12 7.75 1-2 1.25
Cc Pond Water 10 7-19 10.50 1-3 1,70
Cc Distilled Water 13 5-26 15.08 1-5 1.92



Table 18.

LIFETIME TOTAL NUMBER OF EGGS PER FEMALE

Female's
Incubation hatching
temperature Mean with 95% temperature Mean with 95%
(degrees C) confidence limits (degrees C) confidence limits
14 322*
14 164.0 2) 158*
98.69 ~ 272.4 27 atatead
31 132.6 81.51 ~ 215.6
14 ated
21 255.9 21 255.8 112.2 ~ 582.8
151.6 - 431.8 27 298*
31 237.6 59.31 ~ 951.7
14 one
27 234.9 21 143*
123.7 - 446.0 27 266.4 107.9 - 657.9
31 181*
14 coee
31 94.03 21 638*
53.43 - 165.5 27 116*
31 95.84 42,23 - 217.5

*Lifetime total of a single female.

An Fmax~test indicates that the variances of the groups are heterogeneous. Therefore,
a logarithmic transformation was made. The means and confidence limits above are
the antilogarithms of the means and confidence limits of the transformed data. Using
the transformed data, the following Analyses of Variance were carried out:

Anova table: Comparison of those females which were incubated at the same tem-
perature as that at which they hatched--"native" females only.

Source of variation df MS F
Among incubation temperatures 3 0.2752 2.0294 ns
Within incubation temperatures 16 0.1356
Total 19

~59=


at the University of Oklahoma (TDS approximately 500 ppm), this water orginated
from wells on the University campus; 3) pond water--from a stock pond near Noble
(TDS approximately 110 ppm); and 4) distilled-water--low quality distilled water
produced by a general purpose still at the University (TDS approximately 25 ppm).
Also two mixtures were made up with resultant TDS values similar to those for the
pond water. One mixture was composed of I-part Noble water to every 9 parts of
distilled water, resulting ina TDS of approximately 125 ppm. The second mixture
was composed of 1 part tap water for every 4 parts distilled water; this had on ap-
proximate TDS value of 110 ppm.

Five replicate cultures were monitored for each type of water making a total of 30
cultures. The results from this experiment (expressed as means) are presented in Table
48, Successful cultures developed in all the types of water tested, However, there

were differences among the population densities that developed in the different water

types.

To look at these differences more closely, the numbers for total individuals for the
six types of water have been ranked from highest (rank 1) tolowest (ronk 6) separately
for each week (Table 49). The same procedure has been followed for the numbers of
adults (Table 50), {t may be noted in both tables that the pond water had the high-
est or almost the highest numbers all the time. The other water types were roughly
similar in the results obtained, Certainly there is no evidence that the two mixtures
which had TDS values similar to those for the pond water produced more animals than

the waters with higher or lower TDS readings.

From this work it is concluded that the pond water was somewhat better than the other
waters tested for culturing. Perhaps this superiority was due to nothing more than
the small particles or organic matter that were not removed from the water by the #12
silk used for filtering, providing an extra source of food, Whatever caused the pond

water to be somewhat superior, it certainly was not related to TDS value as the waters

-135-


-Zg-

RANKED
MEAN
CLUTCH
SIZE

12

WW

10

0 14° HATCH
“& 21° HATCH
© 27° HATCH
© 31° HATCH
a
1 il 1 L
14 21 27 31
Incubation Temperatures
(degrees)

Figure 1. Mean Elutch Sizes of Groups of Females (Right Column of Table 16)
are Ranked from Lowest to Highest (1 to 12) and Plotted Against the
Temperature at which the Females Were incubated.



five subsamples were taken from the 1000-, 3000-, and 20,000-ml containers respec-
tively. The locations on the surface of the cultures where the tubing was inserted
were chosen with the aid of a random numbers table so that the subsampling was car-
ried out randomly. Tables 1, 2, and 3 present respectively the average numbers of

copepodites, of adults, and of egg clutches ona series of dates for each culture size.

The 50-m! cultures were very unsatisfactory. No egg clutches were ever noted, and

after two weeks no copepods were left in the containers at all,

For all the other culture sizes some reproduction was noted. However, the 250-ml
containers were also unsatisfactory. A few clutches were noted on the first two dates
the cultures were examined. After this 1 however, no eggs were ever found, The
cultures increased in size somewhat when the nauplii from the early egg clutches grew
to where they were counted as copepodites, but the last time the containers were ex-
amined all three cultures had gone to extinction. It was obvious that the conditions
in these containers were not satisfactory for the development of self-propagating cul-

tures.

The three larger culture sizes all seemed to be satisfactory for culturing. While they
exhibited marked fluctuations in numbers of copepodites and of clutches, the popula-
tion levels stayed at high enough levels to indicate that satisfactory culturing condi-

tions were present,

The largest numbers of individuals were produced in the 20,000-ml cultures. However,
the highest densities of individuals seemed to develop in the 1000-m! cultures. As

the same amount of food was added to each culture, it was not surprising that the 1000-ml
cultures had generally higher densities than found in the larger cultures. The fact that
more individuals were produced in the larger containers than in the 1000-ml ones shows,
however, that even with the same amount of food added the carrying capacities were

higher in these containers.

~9-


Table 9. A COMPARISON ON A WEEKLY BASIS OF THE AVERAGE NUMBERS

OF TOTAL COPEPODITES OF D. CLAVIPES (4SE) IN
POPULATIONS FED FROM A MIXED FOOD
CULTURE, FROM A YEAST CULTURE
(ADDED TO BOTH LIGHT AND
DARK CONTAINERS), AND

FROM A CHLAMYDOMO-
NAS CULTURE
Date Mixed Yeast in Yeast in | Chlamydomonas
food light dark
20/X1/70 (Start) 8.0 8.0 -- 8.0
27/XV/70 7.8 9.9 - 7.6
¢1.3) 2.8) 3.2)
4/X1V/70 7.5 WW ~~ 7.0
1.9) 4.3) 3.6)
17/11/70 14.0 7.8 8.0 9.4
2.3) (43.5) (start) (4.0)
23/X11/70 11.6 6.6 17.4 7.8
(41.6) (42.5) (2.2) (41.8)
29/X1/70 14.4 10.8 7.2 6.2
(2.4) (2.2) (41.8) (42.6)
B/7i 0.2 9.6 4A 8.2
on (41.3) (41.6) (41.9)
1/71 8.7 7.8 4.8 9.8
(22.4) (41,8) (41.4) 2.4
2/7 6.2 6.8 2.2 5.8
€1.9 (2.2) 41.1) &1.6)
28/1/71 5.7 6.6 3.8 6.2
(4.8) 1.1) ¢£1.8) 1.7)
4/7 3.6 5.6 3.4 5.8
€1.1) €1.7) 1.5) 41.6)

-26-


that developed in the mixtures were no greater than those
in the waters from which they were prepared. Thus, the
total dissolved solids value can not be detected as having
an effect on:culturing success.

6. Removal of adults--A comparison of the amounts of mor-
Jallty for newly hatched nauplii with their mothers present
and removed showed much higher mortalities with the mothers
present,suggesting cannibalism. Thus, to maximize culturing
success, egg-carrying females should be placed in separate
containers and then removed as soon as their eggs hatch.

7. Temperature--No experiments concerning the effects of
venving this factor were conducted [n the work reported In
this chapter. However, based on the results from the studies
tn the next chapter, a temperature In the range 20 to 25°C
is recommended for culturing.

An experiment was conducted to determine if C, vernalis can be cultured In a con-
tinuous flow system. The system employed is described in the text, and its flow was
driven by an electrolysis pump. Although the experiment only ran for 4 weeks, a
population which appeared to be self-sustaining developed in the system. Nauplii
escaped with the outflow water In this system unless fine netting was used to cover
the outlet. Unfortunately, the netting tended to clog with food particles thus imped-
ing flow. This problem could be corrected, to some:extent, by increasing the area
of the outflow, but it made continuous flow culturing with the system used less satis-
factory than culturing In a standing water system.

-146-


For this work, the cultures were fed known volumes of the blend of commercial
trout food and alfalfa whose preparation was described in Chapter 2. This food
was stored in a refrigerator fo retard bacterial growth and was freshly prepared
every 10 to 15 days as needed. . Each experimental culture received 0.4 ml of this
preparation every third day. In order to avoid great differences in the algal and
bacterial cultures growing in the experimental beakers and to prevent an accumu-
lation of detritus in these containers, the animals were transferred to clean beakers

containing fresh water every ninth day.

In the preliminary stages of the study, the contents of the beakers were filtered
through a small hand net when it was necessary to isolate the females for egg count-
ing, to transfer the animals to a fresh beaker, or to reduce the water volume to
count nauplii. It soon became apparent that this almost daily (and in some cases
twice daily) filtering was not satisfactory for a number of reasons. In addition to
causing frequent stress on the animals, individuals were occasionally lost or killed.
Therefore, to minimize the chances of error introduced by handling, the females
used in the experiments reported here were removed from the water only for egg
counting and for transferring to a fresh beaker each ninth day. In both of these
procedures, females were isolated by pouring them out of the cultures into a second
beaker. Thereafter, water wos poured off, leaving the female behind in progres-
sively smaller volumes of water. In this way, trauma and possible physical damage
were minimized. As described below, nauplii were removed from the. cultures with-

out disturbing the experimental animals.

From maturity to death, females were observed at least orice daily and, as possible,
two or more times each day. At least one, but often more, of the observations

each day was made by placing the beaker under a microscope and determining the
following: 1) whether or not the female was carrying eggs; 2) the condition of the
female's ovaries (prior to oviposition, the ovaries become progressively more opaque,

thus providing a warning that oviposition is imminent); and 3) the presence or absence


-Zl-

Table 3. THE NUMBER OF CLUTCHES (TOTAL NUMBER AND NUMBER PER ADULT) BEING CARRIED ON
A SERIES OF DATES IN D, CLAVIPES CULTURES MAINTAINED IN CONTAINERS
OF 5 DIFFERENT VOLUMES. (EACH VALUE IS THE AVERAGE
OF THREE REPLICATES.)

Container Volume (ml)

Sompling 50 250 1,000 3,000 20,500
art) Total No. | No./adult | Total No. | No./adult | Total No. | No./adult | Total No. | No. /adult ] Total No. | No. /adult
24-V1 - - - - - - - - - -
27-1 0.0 0,0 3 0.345 5 0,500 4 0,444 3 0,390
1Vil 0.0 0.0 1 0,125 4 0,500 5 0.568 4 0,440
3-Vil 0.0 0.0 0 0,0 6 0,517 5 0,581 3 0,326
7-VIl 0.0 0,0 0 0.0 2 0,190 5 0.735 uN -
15-V |" - - - - - a - -
20-V Il 0 0.0 0 0,000 tt) 0,000 68 0,360
27-Vil - - 4 0.097 15 0.348 27 0,198
l-ViIL - - 9 0,337 3 0.081 33 0.334
3t-Vill to) 0.0 4 0.188 13 0.422 Vv) 0,000

* Subsampling initiated In 1,000-, 3000-, and 20,000-ml cultures.


The results from this second experiment are presented in Table 47. They agree with
the preceding work in showing successful cultures at all three feeding levels. These
results also show increases in density associated with increasing amounts of food
added. In this experiment the increase in density with food seemed to hold for the

numbers of total individuals as well as the numbers of adults.

This work indicates that, over the range of food concentrations studied, successful
cultures are produced irregardless of feeding level. There are no indications of the
culturing failures noted for Diaptomus clavipes at rates of food addition above 0.1
or 0.2 ml every second or third day. In fact, the indications are that the culture
densities for C. vernalis increase with increasing food at least up to the highest

level attempted, 2.0 ml per liter every second day.

Water Quality

An attempt was made to learn something concerning the effects of the chemical com-
position of the water used for culturing on culfuring success. Asa simple first step

in this type of study, the effects of culturing in waters from four different sources
were monitored, As an overall indication of the quality of these waters, the concen-
trations of total dissolved solids (TDS) were measured, Obviously, TDS is only one
chemical property of many that may vary among various waters and thus affect their
suitability for raising copepods. However, it was considered as good an indicator

as any of general chemical conditions and thus was considered an appropriate pro-

perty to measure in an initial study.

Populations were initiated in 100 ml beakers, each containing approximately 60 ml
of pond water, by adding 6 adults, 3 of each sex. Waters of four fypes were
tested, The sources of these four wafers were: 1) Noble woter--from wells on the
floodplain of the South Canadian River near the town of Noble, Cleveland County,
Oklahoma (TDS approximately 1,000 ppm); 2) tap water--the water from the faps

-133-


SUMMARY

The effects of temperature and certain other factors on a number of the reproduc~

tive attributes of Diaptomus clavipes have been evaluated. The following con-

clusions have been reached:

1. The physiological longevity of adult females is inversely related
to temperature.

2. There are some indications that the temperature at which a fe~
male hatches and spends her early immature stages is related to
the size of the clutches she produces. It appears that, except
near the upper thermal limit, the largest clutches are produced
py females that have been at a constant temperature all their

ives.

3. The temperature at which the female is kept also affects clutch
size, This effect was noted primarily, however, with regard to
the average clutch size being reduced at the highest tempera-
ture. The differences in average clutch size among females
from 27, 21, and 14°C were relatively minor.

4, The total number of clutches produced by a female during her
lifetime showed some indications of being related to tempera-
ture.

5. The number of clutches a female has produced previously does
not have any large effect on clutch size.

6. Based on results from only three females, resting eggs are car-
ried by the females for a shorter time than are subitaneous eggs,
but the time after a clutch of resting eggs until a new clutch is
produced is not different between the two types of eggs.

7. The temperature of hatching and early life does not seem to have
any substantial effect on rate of clutch production.

8. The amount of time females carry their clutches is inversely re-
lated to temperature.

9, The interval between successive clutches is also related to tem-
perature but with the shortest time between 21 and 2c.

10. Hatching success is related to temperature with a maximum in
the vicinity of 21-C.

-79-,


Reciprocal of the time (in hours) of development

0.04 T T T T T

0.03

0.02

0.01

0 N 1 \ mn i
5 10 15 20 25 30 35

Temperature (°C)
Figure 18. The Rate of Development (Expressed as the Reciprocal

of Development Time in Hours) of Eggs of Cyclops
vernalis in Relation to Temperature.

-161-


Table 11.

A COMPARISON OF THE NUMBERS OF D, CLAVIPES

COPEPODITES PER LITER (SE) ON A SERTES
OF DATES IN CONTAINERS RECEIVING

DIFFERENT AMOUNTS OF FOOD.
(EACH VALUE IS THE MEAN

OF FIVE REPLICATES.)

‘Date Volume of food per day (ml)

(1971) 0 1 10 50

12/1N (Start) 8.0 8.0 8.0 8.0

23/IV 2.0 3.2 0.0 0.0
¢¢0.7) (0.4) (----) (----)

29/1V 0.6 4.0 0.0 0.0
¢£0.4) 1.5) (----) (--=-)

pv 0.6 3.6 0.0 0.0
40.4) 1.7) (--=-) (----)



clutch size are multiple. Inheritance is probably also important and could
cause the frequently significant variability reported here among females with com-

mon hatching and incubation temperatures (Appendices A-1, -2, -3, and ~4).

As noted above, the literature generally indicates that clutch size is directly re-
lated to the body size of the female which in turn is partially determined by tem-
perature (to which body size is inversely related). Accordingly, those females
which hatched and underwent most of their development at a temperature lower than
that at which incubated as late copepodites and adults would be expected to produce
larger clutches than did females that lived exclusively at the higher temperature.
Such is not the case with the data reported here, the data for females which hatched
at 21° being particularly divergent from expectations (Figure 1). Unfortunately,
samples are small and. the females used were not measured. If the data are valid,
reproductive potential is reduced unless the ambient temperature remains relatively
constant throughout the life of the reproducing females. This, of course, is not the
case in natural populations and such reduced reproductive potential in an environ-
ment of changing temperature appears to lack selective value. Therefore, these re-
sults should be verified by further experimentation before final conclusions are
reached.

At the three lower incubation temperatures, the meon clutch size, the total number
of clutches, and the total egg production seem to be unaffected by temperature, at
least for females living their lives at a single constant temperature. This implies
that, under these conditions, the reproductive potential of an individual is approxi-
mately the same, about 340 eggs, regardless of the temperature at which incubated.
However, the potential for females in a natural population may differ considerably.
Conover (1967) reported that female Calanus hyperboreus from laboratory molts were
less fecund than were females taken from plankton samples. On the average, life-
time egg production by laboratory females was less than half that of females from
the plankton. Similarly, Mullin & Brooks (1967) reported lower fecundity among
laboratory females of two species of marine calanoids. In any event, for laboratory

-74-


6. A female's clutch size is related to her age. The earlier
clutches laid by a female tended to be larger than the
later ones.

7. Egg development rate is inversely related to temperature.

8. No significant differences could be detected in the per-
cent of the eggs that hatched at the various temperatures.

~165-


APPENDIX D-2
SURFACE AREA AND VOLUME OF THE VARIOUS DEPTH STRATA OF THE POND

Depth stratum Surface area Volume
(em) (cm?) ()

0- 68 9,536,250 981,996
68- 88 7,247,250 13,116
88-108 5,689,500 284 ,058
108-128 3,898,000 188,746
128-148 2,827,750 121,774
148-168 2,162,000 72,025
168-228 1,683,500 : 33,670

~201-


APPENDIX B-10 (Cont.)

21° INCUBATION
EGG PRODUCTION CYCLE
Time (hours) from oviposition to succeeding oviposition

Lengths of cycles are listed from top to bottom in the order of occurrence but are not

necessarily consecutive, some cycles having been omitted for various reasons as dis~
cussed in the text,

Variances of the data for the three hatching temperatures are heterogeneous. A logarith~
mic transformation was made and Anovas carried out with the transformed data.

Anova table: cycles including clutches of resting eggs (mean = 53.2) vs cycles including
clutches of non-testing eggs (mean = 63.7).

Source of Variation df MS F
Among cycle types 1 0,1248 8.5479 PK0.005
Within cycle types 112 0.0146
Total 113 ;

Anova table: comparison among individuals of complete egg production cycles excluding
those cycles marked with an asterisk above which included a clutch of
resting eggs.

Source of Variation df MS F
Among individuals 9 0.0196 1.2980 ns
Within individuals 78 0.0151
Total 87

“191-


eggs in larger clutches were very tightly packed, often with more than two layers
of eggs, making large clutches difficult to count accurately, Clutches in which
eggs were irregularly arranged within the clutch also were difficult to count ac-
curately. When a clutch was accidentally dislodged from a female during count-
ing, the clutch was broken up and the eggs counted individually as a check on ac-
curacy. In almost all cases, the number counted in the intact clutch was correct.
Occasionally, with larger clutches or those in which eggs were irregularly arrange:
the count made of the intact clutch was short by as many as two eggs. All errors
discovered in this manner were low counts, so that the data reported, if biased, ar

slightly conservative.

With each of the appendices is an Anova table for comparison of mean clutch size
among all the individuals incubated at the temperature considered in that appendix.
An a priori orthogonal set of comparisons also was made, grouping individuals by
hatching temperatures. Only at an incubation temperature of 21° (Appendix A-2)
did mean clutch size not vary significantly among individuals with a common hatch~
_ ing temperature. At the remaining three incubation temperatures (Appendices A-1,
"3, and -4) variation among individuals with a common hatching temperature tended
to be as great or greater than that among groups of individuals with different hatch-

ing temperatures.

Table 2 summarizes the mean clutch sizes found at the various hatching and incuba-
tion temperatures. Figure 1, using the data from Table 16, reveals two interest-
ing features of the mean clutch size of "native" females (those incubated at the
same temperature as that at which they hatched) compared to "non-native" females
(those incubated at a temperature other than their hatching temperature). At every
incubation temperature except 31°, "native" females have the largest mean clutch
size. Futhermore, for every hatching temperature except 31°, mean clutch size is
maximized when the incubation temperature corresponds. Although the evidence

is not conclusive, the female's hatching temperature apparently can affect mean

-51-


culturing, incubation temperature is important only as it affects survivorship to

adulthood. On the other hand, while temperature over a certain range apparently
does not greatly affect reproductive potential, it does affect the rate at which that
potential is realized. If predation or other factors in a natural population operate
to reduce the adult population before physiological old age, then temperature be-

comes an important factor as it affects the reproductive rate.

The question of whether females require more than one copulation to produce fer-

tile eggs throughout their adult lifespans has been debated in the literature for many
years (Ewers, 1936; Hill & Coker, 1930). Most marine calanoids apparently require
only one copulation, although Wilson and Parrish (1971) have reported that Acartia

tonsa will mate more than once in the laboratory. The intensity and duration of

breeding activity among freshwater copepodshave been estimated by counting the
number of females carrying spermatophores (Comita, 1956; Comita & Anderson,

1959). The transfer of spermatophores was observed to last up to six months in some
populations, which seems to imply repeated matings. D. clavipes, at least in the
laboratory, is obliged to copulate each time a clutch fs produced. Failure to copu-
late results in the extrusion of the egg material into a spherical sac without forma-
tion of individual eggs. This sac soon bursts and the egg material is dispersed. This
would result in unacceptable waste of energy if it occurred with regularity in a
natural population, but D. clavipes is largely restricted to small bodies of fresh water

in which it is unlikely that any female would fail to encounter a suitable male.

Future studies of egg production by D. clavipes need not be concerned with the age
of females with respect to clutch size, since this study has shown that clutch size
does not change with the age of the female. This is not a safe general assumption
for other copepod species, however. Smyly (1970) has reported not only that the
number of eggs laid in successive broods by Acanthocyclops viridis diminishes with
age, but also that the rate of diminution in clutch size is affected by the type of
food.

-75-


Table 36. ANOVA TABLE TESTING THE DIFFERENCES AMONG
THE m VALUES OF THE VARIOUS SEASONS.

Source of variation | df ss MS F

—S__
Among seasons 3 109.6651 36,5534 8.7624*
Within seasons 42 175.3545 4.1701
Total 45 285.0196
*P< 0.01

Table 37, A POSTERIORI COMPARISON OF THE MEAN SEASONAL
m, VALUES USING THE STUDENT-NEWMANCKEULS TEST.

Fall Summer Spring Winter
Y 0.4472 2.5779 | 3.0306 5.3306
n 3 9 14 21
s? 0.2583 | 2.7774 | 5.3713 | 5.3967
Y oon
F 0.4472 3 --

Su 2.5779 9 2.1307* -
Sp 3.0316 14 2.5844* 0.4537 --

W 5.3306 21 4.8834* 2.7527* | 2.2990* eal

*P< 0.05

-110-


APPENDIX B-5
14° INCUBATION: INTERVAL (HOURS) BETWEEN CLUTCHES

Female's hatching
temperature 14° 21° 31°
Individual A7 Aé A8 Ag AlO
13.0 60.5 48.0 24.0 25.0
5.5 8.0 25.0 47.5 8.0
24.0 20.5 24.5 8.5 93.0
151.0 60.0 19.5 185.5
49.5 59.0
14.0 170.0
39.5 9.0
352.0 99.5
96.0

Intervals between successive clutches are listed from top to bottom in the order of
occurrence but are not necessarily consecutive, some intervals having been omitted
for various reasons as discussed in the text.

Variances of the data for the three hatching temperatures are marginally homogeneous.

A logarithmic transformation was made and Anovas carried out with the transformed
data. :

Anova table: comparison among individuals of intervals between clutches.

Source of Variation df MS F
Among individuals 4 0.0404 0.1596 ns
Within individuals 23 0.2530
Total 27

-184-


thm the females, and thus males predominated during the early stages of several in-
creases in the numbers of adults in his populations. Interpretation of the ratio of
males to females on the various collecting dates in our study (Figure 12) suggests
that males appeared before females in the various generations of this population also,
This conclusion was reached from an examination of the ratio of males to females
during the low periods of adult population numbers. On both 18 April and 28 May
when the numbers in the total population were low (Figure 8), the ratio of males to
females was significantly above the yearly average of 1.28 (tested by a modification
of the Student's t-test designed to test the difference between a single observation
and the mean of a group; Sokal and Rohlf, 1969), Previously it was indicated that
these dates were taken as the die-off dates for the previous generations (winter and

ye respectively).

An analysis of variance performed on the values for the mean ratios of males to fe-
males during the four seasons (Table 39) revealed a significant difference among the
seasonal means. However, when an SNK test was run on these data (Table 40), the
only seasonal differences detected were between the fall mean and those for each of
the remaining three seasons. Since the ratio of males to females in the fall would
have no bearing on the m,. values obtained during the preceding reproductive year,
the ratio of males to females was assumed to be unimportant in determining the m

values obtained in this study.

The final factor studied was the mean number of eggs per clutch (Figure 13), Analy-
sis of variance showed a significant difference among the means for the four seasons
(Table 41). The lowest mean number of eggs per clutch (15.25) occurred in the sum-
mer period,with the means for fall, spring, and winter following in ascending order
(Table 42). The mean value for winter was twice that for summer, with 30,53 eggs
per clutch occurring. A detectable difference occurred between the members of

each pair of means except the summer-fall pair (Table 42), The mean number of eggs

-113-


level, probably because the food was of rather poor quality at that time. (This prob-
lem will be discussed further in the subsequent section on food.) Thus, only large

differences in the influence of light intensity could have been detected.

No very large differences in the effects of the different intensities are evident from
the results. There is some indication that the percent hatch increases with intensity
and also that development time may be lowest at the medium intensity. However,
there are too few data to establish these trends statistically, and overall it seems pru-~
dent to state that there is little indication that light intensity has a major effect on
culturing success. From this work it was concluded that the use of a moderate to low
light intensity with a period of 12-hours light and 12-hours dark should be satisfactory

for culturing.

Temperature

The effects of temperature on the reproduction of D. clavipes will be considered at

some length in the next chapter. However, before such detailed work could be car-
ried out, culturing techniques including the determination of a temperature for cul-
turing had to be determined. Thus, a certain amount of preliminary experimentation

on the effects of temperature was conducted and is reported in this subsection.

Two experiments were carried out to study the effects of temperature. One of these
was an examination of the combined effects of light and temperature. This is the
same experiment as that described in the preceding subsection on light intensity.
However, rather than averaging the data with regard to light as was done there, in
this subsection the data on developmental and reproductive attributes from that ex-
periment have been averaged for each temperature (Table 1. As there were two con-
tainers at each of three light intensities in each temperature room, averaging the

data this way balances out the first order effects of light.

-20-


culture the animals in standing water systems and then transfer them to continuous

flow ones for the measurement of toxic effects.
SUMMARY

_ An investigation has been conducted with the objective of specifying a set of con-
ditions that will allow dependable, reproducible culturing of Cyclops vernalis.

Based on this work the following conditions are recommended for culturing:

1. Culture volume--Comparing the size and density of the popu-
lations that developed in volumes of 20, 80, 250 and 1000 ml
showed that this factor had little effect on the culturing success
for C, vernalis, at least in the range from 20 to 1000 ml. Cul-
tures with volumes of about 100 mi should be convenient for
many experimental purposes.

2. Food type--Only one type of food was used during this work.
This food, a fish food-alfalfa mixture, proved quite satis~
factory and is recommended.

3, Food quantity~-Successful cultures developed at rates of food
addition from 0.1 to 2.0 ml of food mixture per liter of cul-
ture medium every second day. The densities that developed
in the cultures seemed to be directly related to quantity of
food addition, although not ona linear basis. Food additions
anywhere in the above range are recommended with the exact
amount determined by the density desired.

4, Culture medium~-Culturing was attempted with four types of
water, i.e. Noble water (well water), tap water, pond water,
and low quality distilled water. Successful populations de-
veloped in all four water types. However, pond water is sug-
gested for culturing because the densities were higher in it
than in the other types. The other three types developed
approximately equal densities.

5. Total dissolved solids--Two mixtures, one of Noble water
‘DS = 1000 ppm) plus distilled water (TDS = 25 ppm) and one
of the tap water (TDS = 500 ppm) plus distilled water, were
prepared so that both had a TDS value approaching that of
the pond water (IDS = 110 ppm). The population densities

-145-


Elgmork, K. Seasonal Occurrence of Cyclops strenuus strenuus. Folia Limnol.
Scanad, No. 11, 196p., 1959.

Elster, H.-J. Uberdie Populations-dynamik von Eudiaptomus gracilis Sars und Het-
erocope borealis Fischer im Bodensee-Obersee. Arch. Hydrobiol. Supp!. 20:546-
614, 1954,

Ewers, L.A. Propagation and Rate of Reproduction of some Freshwater Copepoda.
Trans. Amer. Micros. Soc. 55:230-238, 1936.

Fassett, N.C. A Manual of Aquatic Plants. Univ. Wisc. Press, Madison, Wisconsin,
1957.

Fryer, G. The Food of some Freshwater Cyclopoid Copepods and its Ecological Sig-
nificance. J. Anim. Ecol. 26:263-286, 1957.

Hall, D.J. An Experimental Approach to the Dynamics of a Natural Population of
Daphnia galeata mendotae. Ecology. 45:94-112, 1964.

Healey, M.C, The Seasonal and Diel Changes in Distribution of Diaptomus leptopus.
ina small eutrophic lake, Limnol. Oceanog., 12:34-39, 1967.

Heinle, D.R. Culture of Calanoid Copepods in Synthetic Seawater. J. Fish. Res.
Bd. Canada. 26:150-153, 1969.

Hill, L.1. and R.E. Coker. Observations on Mating Habits of Cyclops. J. Elisha
Mitchell Sei. Soc, 45:206-220, 1930.

Jacobs, J. Laboratory Cultivation of the Marine Copepod Pseudodiaptomus corona~
tus Williams. Limnol. Oceanog. 6:443-446, 1961.

Kamal, A. and K.B. Armitage. External Morphology of Adult and Copepodid Stages
of Diaptomus clavipes Schacht 1897. Univ. Kansas Sci. Bull. 47:559-573, 1967.

Katona, $.K. andC.F. Moodie. Breeding of Pseudocalanus elongatus in the Labora—
tory. J. Mar. Biol. Assoc. U.K. 49:743-747, 1969.

Khan, M.F. The Effect of Constant and Varying Temperatures on the Development
of Acanthocyclops viridis Jurine. Proc. Roy. Irish Acad., Sect, B. 64:117-130,
1969.

Kibby, H.V. Energetics and Population Dynamics of Diaptomus gracilis. Ecol. Mono-
graphs. 41:311-327, 1971.

-168-


No.

12.

13,

14,

15.

16.

7.

18.
19.
20.

21.

22.

24.
25.
26.

27.

Page
A Comparison of the Numbers of D. clavipes Copepodites Per Liter on
a Series of Dates in Containers Receiving Different Amounts of Food... . 35

A Comparison of Mortality and Reproductive Success for D. clavipes
Females Kept Under Three Different Food Conditions. ..+e+eeeeeeee 37

A Comparison of Mortality and Reproductive Success for D. clavipes
Fed 0.4 ml of Either Fresh or Old Fish Food Solution... .e+eeeeree se 3

Female Life Spans Afer Sexual Maturity in Relation to Incubation and
Hatching Temperatures... cece eee eee ere reece eere ee 50

Mean Number of Eggs Per Clutch in Relation to Incubation and Hatch-
ing Temperatures... 6 eee cece ee eee eee w eee e sees eee eet H3

Lifetime Total Number of Clutches Per Female in Relation to Hatching
and Incubation Temperatures .... cece ec eee reer er cree ees Od

Lifetime Total Number of Eggs Per Female .......2 20 ee ce eee eee SD
Comparison of Mean Sequential Clutch Sizes ete e ee neee ee eee el

Comparison of Time Intervals of Resting and Subitaneous Eggs Produced
at 219 Incubation... ec cee ee cece ee eee eee erence nt 4

Comparison of Mean Clutch Carrying Time at Four Incubation Tempera-

TUES. oe ewww eee ewer essere ecrcseescosrecserec see 6 6G

Comparison of Mean Interval between Clutches at Four Incubation
Temperatures... cece eee c cece cree ees e rest ereerecees OF

Comparison of Mean Egg Production Cycle from Oviposition to Ovi-
position at Four Incubation Temperatures. 6... eee see e cere eee s 68

Hatching Success. +--+ + +e ec cece eee eee e eet e reste renee 72
Summarized Results... 2.0 c cece cre eer reese reece cesses eB
Estimated Production of Nauplii . 0c se eee eee eee e eee eee e ee JB
An Anova Test Comparing the Mean Number of Adults Per Liter in the

Open Water Region and the Mean Number in the Region in Which Rooted
Aquatics Were Found During the Early Spring... esses eee reese ee A

ix


“G6l-

21° INCUBATION: PERCENT HATCH

APPENDIX C-2

Female's hatching

temperature 21° 97° 31°
Individual Bll BI2 BI4 BI7 B18 BID B20 Blé BI3 BIS
24.0% | 74.1%| 81.8%} 91.7%| 92.2% | 86.4%] 96.3%| 100.0% | 14.3% | 100.0%
100.0 {100.0 | 37.5 | 83.3 | 59.1 86.8 | 88.6 | 85.0 | 70.8 25.0
95.2 |100.0 0.0 {100.0 | 73.3 | 88.5 95.7 | 91.3 0.0 | 54.5
46.7 | 65.4 95.2 96.0 | 71.4 |100.0 | 77.8 | 60.0 | 75.0
23.8 | 20.6 88.2 | 22.5 | 96.0 | 79.1 {100.0 85.2
63.6 | 60.0 72.7. | 66.7 | 94.3 | 92.9 100.0
100.0 100.0 | 68.4 | 88.2 100.0
89.3 89.3 92.3 81.8
100.0 100.0 | 87.5 94.1
42.9 93.8 | 92.0 53.8
100.0 96.3 | 100.0 100.0
37.0 100.0 {100.0 3.8
100.0 100.0 100.0
95.5 100.0 75.0
70.0 92.9
100.0 95.8
87.5 76.5
100.0 100.0
90.0
100.0
100.0
Mean % 88.1% | 77.7% | 31.6% | 93.5% | 72.2% | 88.0% | 94.5% | 96.2% | 29.0% | 85.5%



A DEPENDABLE, REPRODUCIBLE CULTURING METHOD

The conditions developed for culturing a diaptomid (see Chapter 2) were used as the
starting point for culturing Cyclops vernalis, Healthy self-sustaining cultures of the
cyclopoid were easily obtained under these conditions, However, in order to gain
a greater understanding of the factors affecting culturing success for this species,
several sets of experiments considering the effects of varying certain culture con-:
ditions were conducted, The results of these experiments are presented in this sec~

tion,
Culture Volume

The effects of variations in culture volume on culturing success were studied by
comparing the size and density of the populations that developed in four different
volumes, The culture containers used were 2-inch diameter Petri dishes, 100-ml
beakers, 4-inch diameter finger bowls, and I-liter beakers. These containers were
filled with 20 ml, 80 ml, 250 ml, and 1000 ml of water, respectively. Three re-
plicates of each culture volume were used, The volume of food added was adjusted
for the different containers so that the rate of addition for each container was 1 ml
of food per liter of culture water every second day. Each culture was initiated with

8 adults, 4 males and 4 gravid females.

The average counts obtained during this experiment are presented in Table 44, It
will be noted that the greatest numbers of individuals developed in the largest
culture volume. This pattem was especially evident with regard to the numbers of
adults,

An opposite pattern is noted, however, if one compares the densities rather than
the total numbers that developed in each culture volume (Table 45). Here the

smallest volume tends to have the greatest density and again the Pattern i is clearly
exhibited by the adults.

-128-


Table 48, A COMPARISON OF THE MEAN NUMBERS OF CYCLOPS VERNALIS PRESENT ON A SERIES OF
]ATER. EACH VALUE IS THE MEAN

SAMPLING DATES IN SIX DIFFERENT TYPES OF CUL!

OF FIVE REPLICATES. THE STANDARD ERRORS OF THE MEANS ARE INCLUDED IN
PARENTHESES. (A = ADULTS, G = GRAVID FEMALES, Y = IMMATURE
COPEPODITES PLUS NAUPLII, T = TOTAL NUMBERS.)

“9El-

Type of water
Noble Pond
(TDS app. 1000) (TDS app. 110) (TDS app. 500)
A G Y T A Ss Y T A Y

6.0 0.0 0.0 6.0 6.0 0.0 0.0 6.0 6.0 0.0 0.0

6.6 0.6 17.6 24.2 9.0 1,8 155.8 164.8 7.2 0.6 98.8
(0.4) 0.4) (11.3) (11.1) (1.7) (0.5) (28.3) (29.5) (0.4) (0.4) (26.7) -

4.0 0.6 74,4 78,4 10.6 2.0 142.4 153.0 8.0 0.6 115.6
0.3) 0.2) 13.3) (13.2) (1.7) 0.9) (24,1) (24.5) 1.5) (0.4) (26,2)
12,0 0.6 46,0 58,0 11.2 0.6 151.0 162.2 6.6 0.4 44
@.5) (0.2) (6.2) (8.2) (1.2) (0.2) (28.3) (29.4) (1.0) (0.2) 77.6)
10,2 0.2 50.6 60.8 13.2 0.4 135.8 149.0 8.8 0.2 37,4
1.3) 0.2) (11.0) (10,3) (0.6) (0.4) 28.7) (29.0) 1.2) 0.2) (9.2)
17.0- 0.0 39.4 56,4 17.8 0.6 100.6 118.4 12.4 0.2 35.2
(2.2) ¢) (8.7) 8.7) (1.6) (0.4) (12.9) (12.8) (1.8) 0.2) (14.4)
21.4 0.4 42.6 64,0 24.6 0.2 103.6 128.2 15,2 0.2 28.8
0.8) 0.2). 6.9) (7.2) @.4) (0.2) @0.1) (19.1) a.9) (0.2) (8.0)
22,2 1.4 34,2 56,4 27.7 0.5 67.2 95.0 12.8 0.0 38.6
(1.0) 0.5) (4.0) (3.8) (4.2) (0.3) 03.6) (16.1) 0.9) ) (16,7)
19.0 0.8 22.0 41,0 26.2 1,0 44,2 70.5 11.8 0.6 20.2
@.2) 0.4) (4.8) (5.6) (2.2) (0.6) (1.2) 1.1) @.2) (0.6) (8.8)



These results indicate that a high mortality among newly hatched C, vernalis
nauplii can result when they are in the presence of an adult. Smyly (1970) stated
that such cannibalism depends directly on the frequency of encounter of the mother
with the nauplii. In our results mortality of approximately 50% occurred in the
first 24-hour period, whereas only about 20% mortality of the remaining animals
occurred during the second 24-hour period. Possibly an increase in the size of the
nauplii or an increase in their mobility may have caused this decrease in mortulity
during the second day after hatching.

Temperature

A study specifically designed to investigate the relation between temperature and
certain reproductive attributes for C. vernalis is reported in the next chapter (Chapter
6), Because that study provided a good deal of information on the influence of
temperature on populations of C, vernalis, no experiments in which temperature was
varied were conducted during the research on culturing reported in the present chap-
ter. :

When the results from Chapter 6 are examined, it is clear that the animals repro-
duced quite well anywhere within a range from about 14 to 31°C. The rate of re~
production was retarded somewhat in the lower part of this range, however, and the
number of eggs produced by a female during her life was reduced at the upper temper~
ature. With these results in mind it seems prudent to suggest a temperature between
20° and 25° C for culturing of this species.

CONTINUOUS FLOW CULTURE

lt has been noted that many organisms are more sensitive to a toxic material when
they are exposed fo a certain concentration of it in a continuous flow system than

when they are exposed to the same concentration ina standing water system. Thus,

-142-


Table 12, A COMPARISON OF THE NUMBERS OF D. CLAVIPES

COPEPODITES PER LITER (4SE) ONA
SERIES OF DATES IN CONTAINERS
RECEIVING DIFFERENT AMOUNTS

OF FOOD. (EACH VALUE IS THE
MEAN OF THREE REPLICATES.)

Date Volume of food every other day (ml)
(1971)

0.1 0.5 1.0
8/V (Start) 8.0 8.0 8.0
14/Vv 11.0 9.0 6.7
(40.6) (42.1) (42.9)
20/V 22.0 26.3 3.7
(£2.1) (412.0) (£0.9)
27N 10.3 12.0 2.7
(2.4) 4.6) (1.5)
3VI 6.3 15.7 4.3
0.9) (46.6) (£3.0)
10/Vi 6.0 9.0 2.7
1.2) (46.5) (€2.2)

-35-


APPENDIX C-3
27° INCUBATION: PERCENT HATCH

Female's
hatching
temperature | 21° 27° 31°
Individual co C7 c8 C13 ci4 cis C16 cil
0.0%) 54.3% | 44.8%} 87.5%| 100.0%) 65.0%| 0.0% | 13.3%
86.7 {100.0 20.0 48.6 | 100.0 82.8 42.9 45.0
27.8 12.5 62.2 76.2 85.2 96.3 76.0 6.7
43.5 0.0 44,4 81.8 | 100.0 79.3 |100.0 61.1
100.0 |100.0 36.2 55.3 | 100.0 96.0 50.0
100.0 94.7 68.3 58.8 79.2 93.8 0.0
0.0 84.5 42.2 60.6 |100.0 |100.0
32.6 66.0 66.7 97.1 56.7
15.8 48.5 43.5 89.7
54.5 35.3 95.5 64.0
100.0 38.9
19.0 | 78.3
29.4 85.7
96.0 70.6
80.8 |100.0
90.0 | 19.4
75.9 42.2
61.9
Mean % 51.7%) 65.7% | 55.3% | 69.0%| 98.3%] 85.8% | 55.1% | 23.7%

An arcsine transformation of the data above was made, the means above were com-

puted, and an Anova carried out:

Source of Variation df MS F
Among individuals 7 1913.2415 3.9134 PK0.010
C7, 8, ... 16 vs Others 1 4214.2313 8.6199 P<0.010
C9 vs C11 1 912.0849 = 1.8656 ns
C7 vs C8 vs ... vs C16 5 1653.2749 3.3817 P<0.010
Within individuals 72 488.8929
Total 79

-197-


APPENDIX E-5 (Cont.)

TOTAL NUMBER OF COPEPODITE TWO (II) IN EACH REGION OF THE POND
AND IN THE TOTAL POND ON EACH COLLECTING DATE, THE TOTAL
NUMBER WAS DETERMINED BY MULTIPLYING THE Y (MEAN)

NUMBER OF CII PER QUADRAT IN EACH REGION BY
THE TOTAL NUMBER OF QUADRATS IN THAT
REGION AND SUMMING THE PRODUCTS

FOR EACH DATE,
Date Open water Rooted aquatics Total pond
region region
IV-1971
12 325,639 10,176 335,815
14. 73,747 0 73,747
16 330,434 12,930 343,366
18 188,911 254,113 443,024
20 664,518 13,686 678, 204
V-1971
4 378, 837 0° 378,837
18 250,800 265,22! 516,021
28 23,737 . 0 23,737
VI-1971
i 1,145,971 78,946 1,224,917
25 306,405 10,541 316,947
VII-1971
9 113,833 0 113,833
23 83,616 0 83,616
VIN-1971
6 43,128 0 43,128
20 64,077 122,249 186,326
1X-1971
3 12,114 62,289 74,304
17 39,780 71,501 111,281
30 104,041 385,224 489,265
X-1971
14 32,130 18,057 50,187
21 3,657 59,027 62,684
29 1,836 0 1,836

-215-


TABLES

1, Total Copepodite Population Size on a Series of Mates for D. Clavipes
Cultures Maintained in Containers of Five Different Volumes......... 10

2. Adult Population Size on a Series of Dates for D. clavipes Cultures
Maintained in Containers of Five Different Volumes ..... seeteeces TT

3. The Number of Clutches Being Carried on a-Series of Dates in D.
clavipes Cultures Maintained in Containers of Five Different Volumes... 12

4. Mortality Occurring Among Adult D. clavipes Cultured in Three Types
of Culture Media Over'a Period of 40 Days... se ee ee eee ee eevee V5

5. Reproduction Occurring Over a Span of 40 Days Among Fg Generation
D. clavipes Cultured in Conditioned Water, Distilled Water, and
Natural Pond Water Media 6.6... sees eee eee cere nee eree 16

6. A Comparison of the Average Values for Certain Reproductive and
Developmental Parameters for D. clavipes Individuals at Three Dif-
ferent Light Intensities... 0... eee e eect eee reece tenet cee WY

7. A Comparison of the Average Values for Certain Reproductive and
Developmental Parameters for D. clavipes Individuals at Four Dif-
ferent Temperatures... cc cece eet ee eee eee e ee reereee Bf

8. A Comparison of the Average Values for Certain Reproductive and
Developmental Parameters for D. clavipes Individuals at Four Dif-
ferent Temperatures... cece cee eset rere reece etreerene 2B

9. A Comparison on a Weekly Basis of the Average Number of Total
Copepodites of D. clavipes in Populations Fed From a Mixed Food
Culture, From a Yeast Culture (Added to Both Light and Dark Con-
tainers), and From a Chlamydomonas Culture... 6+ + teen eeneee BH

10. A Comparison on a Weekly Basis of the Average Numbers of Adult
D. clavipes in Populations Fed From Cultures of a Mixed Food, of
Yeast (Added to Both Light and Dark Containers), and of Chlamy~

domonas .. cc eee e cece eee e centre cece eres eeteceese BB

11. A Comparison of the Numbers of D. clavipes Copepodites Per Liter on
Several Dates in Containers Receiving Different Amounts of Food. ..--- 33


Table 10 (Continued)

Mixed Yeast in Yeast in
Date food light dark Chlamydomonas
3/ll/71 0.7 4.6 1.2 6.0
(40,3) (41,3) (£0.6) (41.3)
VALI Y ZAI 0.7 3.8 1,2 5.8
(£0.3) 1.1) (£0.6) (41.1)
18/71 0.4 4.6 0. 5.6
¢t0.2) 1.2) (t0.6) (41.0)
25/1/71 0.3 3.6 0.6 5.6
(£0.2) (£1.0) (t0.6) (t0.9)
ANZA! 0.2 2.6 0.2 5.2
¢t0.1) (£1.3) (40.2) (¢1.2)
8/V/71 0.2 2.4 0.4 44
0.1) 1.3) (40.4) 0.7)
19/\V/71 0.0 3.0 0.4 5.2
(--) (41.5) (40.4) (£1.7)

-29~


weighted mean, Starting with 21 February and continuing until 27 March, the m,.
values tended to rise, peaking at a value over 9. After this date the m, values
steadily decreased but remained above the mean until 10 April, From this date until
the culmination of the study, the m,. values rose above the mean only during the period
of 9 July to 6 August and for a short time in September. The mean water tempera~
ture (see Figure 14, page 120) tended to rise during the period from 27 March until

3 September and was above 20°C for much of the summer. Our results in Chapter 3
indicated that reproductive rate increases with temperature to 25°C or somewhat
higher. Thus, the seasonal variations in m, values did not show the pattern expected

from the temperature variations.

To determine whether the apparent differences in m values during the various periods
of the year were statistically different, the data were divided into four groups by sea-
son. The first group of data, called the winter data, included the m values obtain-
ed from samples collected from the onset of the study, 19 February, until 31 March,
when the first new individuals reached maturity. The second or spring group of m
values were those from samples collected between 31 March and 28 May. This latter
date was chosen because it was assumed to be the final die-off date for the first gen-
eration. The summer group included the values obtained for samples from 28 May
until 30 September, while the fall group included values obtained from 30 September
until 29 October. The fall season was characterized by decreasing temperatures,

although they were well above the lower limit for reproduction.

Analysis of variance of the four group (Table 36) showed a significant difference
among the means of the various seasons. To determine which pairs of means were
significantly different, a Student-Newman-Keuls (SNK) test (Sokal and Rohlf, 1969)
was used. The lowest mean m,. value was that for the fall season, followed in in-
creasing order by the summer, spring, and then winter means (Table 37), The SNK
test detected a significant difference between the members of all pairs of means

except the spring-summer one (Table 37).

-109-


APPENDIX D-3 (Cont.)

NUMBER OF QUADRATS IN EACH REGION OF THE POND AND IN THE TOTAL
POND ON EACH COLLECTING DATE, THE SURFACE AREA CAN BE
COMPUTED BY MULTIPLYING THE NUMBER OF QUADRATS

BY 45.54 CM2
Date Open water Rooted aquatic Total pond
region region
V-1971
16 70,092 90,516 160,608
18 55,973 104,634 160,608
20 54,088 109,494 163,573
24 55,728 105,633 161,362
V-1971
4 45,234 119,042 164,277
18 48,936 111,671 160,608
28 47,474 113,133 160,608
VI-1971 .
VW 79,719 105,261 184,981
25 106,575 84,335 190,910
VU-1971
9 39,594 141,718 181,312
23 30,806 141,211 172,417
VIN-1971 :
6 18,483 150,265 168,749
20 16,019 146,700 162,819
IX-1971
3 16,019 145,343 161,362
7 18,360 143 ,002 161,362
30 18,360 144,459 162,819
X-1971
14 18,360 . 144,459 162,819
21 12,800 157,406 170,206
29 7,344 162,862 170,206

-203-


To study this question these properties were measured for animals maintained in five
different volumes of culture media, i.e., 50, 250, 1000, 3000, and 20,000 ml.
Containers were selected so that the depth of water was approximately the same in
the different cultures. This meant that the ratio of volume to surface area was ap-
proximately constant for all cultures, and so the exchange with the atmosphere and
the concentrations of the dissolved gases should not have varied greatly among the

cultures.

The cultures were maintained in the laboratory at a temperature of 23 +3° Cand
with a light cycle of approximately 12-hours light and 12-hours dark. They were
fed 4 ml from the mixed algal cultures at intervals of 2 to 3 days. In the containers
of the three larger sizes, small quantities of distilled water were added as needed to
keep the volumes stable. In the containers of the two smaller sizes, it was necessary

to remove small quantities of the media occasionally to keep the volumes constant.

Three replicate cultures were maintained for each volume, so there were 15 cultures
in total. Each culture was initiated with the addition of 5 adult males and 5 adult
females of D, clavipes. Survival and reproduction were determined by periodically
counting the number of adults, of total copepodites, and of egg clutches in each con-

tainer.

In the 50- and 250-ml containers these counts were always made by direct examination.
However, in the larger containers the numbers became too great for this approach to

be practical. Thus, after the first four examinations, the counts for these larger cul-
tures were estimated through the use of a subsampling technique. This consisted of
obtaining a subsample by inserting a piece of clear plastic tubing, having an inside
diameter of 3 cm, to the bottom of each culture container. The tube was then stop-
pered on top and sealed, by means of a plastic Petri dish, at the bottom. The sub~
sample was withdrawn from the culture and the contained animals and clutches counted.

After counting the entire subsample was returned to its container. Three, four, and


CHAPTER 2

THE CULTURING OF A CALANOID

Until quite recently very little success had been obtained in culturing calanoid cope-
pods in the laboratory. Within the last decade or so, however, methods for culturing
have been developed for a number of marine and estuarine species (see for example
Conover, 1960; Jacobs, 1961; Mullin, 1963; Zillioux and Wilson, 1966; Corkett,
1967; Lewis, 1967; Mullin and Brooks, 1967; Corkett and Urry, 1968; Heinle, 1969;
and Katona and Moodie, 1969). As far as can be determined, no comparable studies
reporting on methods for culturing freshwater calanoids have been published.

, The objective of the research reported in this chapter was to develop a reproducible
method for maintaining healthy, self-propagating cultures of a diaptomid in the labo-
ratory. Favorable conditions for culturing have been determined by varying the factors
thought most likely to affect the culturing success and determining the effects of these

variations.

The diaptomid selected for study was the species, Diaptomus (Aglaodiaptomus) clavipes

Schacht. The reasons for this choice were the following:

1. This species is relatively large for a diaptomid. The large size facili-
tated counting and handling.

2. This species is found in small bodies of water such as ponds, as well as
in large reservoirs. It was assumed that a form that inhabits small bodies
of water would be easier to culture in the rather restricted volumes avail~
able in experimental containers than would more strictly limnetic forms.

3. This species is rather important in the ecology of the lakes and ponds
of central North America. It is one of the three or four most com-
monly found diaptomids in the Plains States and the Southwest.

4, This species is found in many of the farm ponds in the vicinity of
Norman, Oklahoma, and thus was readily available to the investi-
gators.


Page

Fecundity ..ccecceseccescrsecevencscecetcsrresrererecsssecces 153

Clutch Size ..... ccc cece cece eee c eee cee enc encncee sevcsceeees 153
Number of Clutches Per Female ......-ceceeseeecccercrececereee 158
Total Egg Production ......cccccccececcececscccteceseseceesees 158
Development Time of Eggs ......seeeeeecrceee secccecccecreescee 159
Hatching Success .......c cece cece ccecntcccecsscssscecsctsseres 162
SUMMAOTY cccecceecsenecercrencsrcrecncrercstcsessecsersertces 164
7. References ...ccseseceeccecees sec cecseccccertessccesvectoreces 166

8. List of Articles Submitted for Publication and List of Dissertations ..... 172

9. Appendices 173

Se ee


Although the correlation we observed between temperature and mean number of eggs
per clutch agrees with the findings of Chapman (1969), Corkett and McLaren (1969),
and Comita and Anderson (1959), the data do not, in themselves, show a cause and
effect relationship. In Chapter 3 when we compared the mean number of eggs per
clutch at various temperatures, using only data from animals incubated at the same
temperature at which they developed, no significant difference was found among the
means for the various temperatures, except at 31°C where the mean number of eggs
per clutch was lower, These findings suggest that other factors may be more impor-

tant than temperature in controlling the number of eggs per clutch in the. field.

The final factor studied in relation to egg production was adult density (Figure 15).
Only the densities in the open water were considered. Concentrations of adults
ranged from less than 0.5 to greater than 17 adults per liter during the reproductive
year, the higher values were found later in the year. Although the concentration

of adults had a 30-fold range during the study period, the total number of adults did
not fluctuate to this extent, The greater variation in concentration than in popula-
tion resulted primarily from the decrease in open water volume due to the encroachment

of rooted aquatics.

As with temperature an inverse correlation was found between density and the mean
number of eggs per clutch. Figure 16 is a plot of clutch size versus density. 1t shows
a wide range of clutch sizes were observed when densities were below 3 adults per
liter, thereby indicating little effect of densities on clutch size at these levels. When
the density was greater than 3 adults per liter, however, there are indications that an
inverse relationship between the mean number of eggs per clutch and adult density

prevailed.
In this study it was found that the major factor affecting the reproductive rate was

the mean number of eggs per clutch during the various seasons. Several investigators

have correlated the number of eggs per clutch with the size of the female; the size

-121-


~66-

Table 33,

DATA TO DETERMINE THE DURATIONS OF THE VARIOUS STAGES,

LIFE TABLE FOR THE COMPLETE YEAR STUDY USING THE COMPOSITE LABORATORY

Stage Number living Number dying Mortality rate Mean number | Total life Mean lifetime
at beginning of in interval per 1000 alive alive expectancy | remaining for
age interval dy at beginning of L, TI, those attaining
I age interval age interval
a ex
Egg-NIII 1000.00 881 .40 881.40 559.30 1691.90 1.69
NIV 118.60 50.62 426.79 93.29 679.57 5.73
NV 67.98 15,24 224.15 60.36 549,89 8.09
NVI 52.74 24.76 469.42 40.36 477 46 9.05
ci 27.98 1.22 43.54 27.38 427.41 15.28
cil 26.77 11.09 414.21 21.22 387.17 14.46
Cui 15,68 0.85 53.93 15.26 347.06 22.13
CIV 14,83 0.18 11.80 14.75 294.26 19.84
cv 14.66 5.88 401 .01 11.72 230.10 15.70
CVI 8.78 8.78 1000.00 4.39 175.60 20.00



-9g-

We 14° HATCH

& 21° HATCH

27° HATCH
ob e 31° HATCH

oO

10+

°

RANKED
MEAN 6b
PERCENT
HATCH

14 21 27 31

Incubation Temperatures
(degrees)
Figure 3. Mean Total Number of Eggs Produced by the Groups of Females
(Right Column of Table 18)are Ranked from Lowest to Highest
(I to 12) and Plotted Against the Temperature at which the Fe-
males Were Incubated.


APPENDIX C-2 (Cont.)
21° INCUBATION: PERCENT HATCH

An arcsine transformation of the data on the previous page was made, the means on the
previous page were computed, and an Anova carried out:

Source of Variation . df MS F

Among individuals 9 1345.5168 3.6499  PX0.001
BI1, B12, ..., B20 vs Others 1 1.2514 ' 0.0034 ns
B16 vs BI3 & BIS 1 26279419 7.1287 PX0.010
"B13 vs B15 1 4027.4391 10.9251 — PX0.010
BI] vs B12 vs .... vs B20 6 908 .8366. 2.4654 — PK0.050

Within individuals 86 368.6425

Total 95

=196=


It would seem that it can be concluded from this work that large containers are better
for culturing than smaller ones and that, if the containers are too small, culturing will
not be possible at all. The choice of container size will depend partly on the goals

of the work being undertaken. It has been found that the large containers are too
difficult to manipulate, clean, etc. to be used in experiments that require a lot of
examination and care of the animals, and containers of the 1000-ml range are advised
for such experimental studies. However, for general culturing to provide stock animals
where less care is needed, the large containers are convenient, and 5- to 10-gallon

aquaria have proven quite satisfactory for this type of use.
Culture Medium

The selection of a culture medium was made, in part, on the basis of a comparison of
data collected by daily monitoring of the mortality and egg production that occurred
among field-collected (designated generation Fy) animals maintained in the three
different water described in the Methods and Materials section, i.e., distilled water,
conditioned tap water, and pond water. An experiment comparing distilled and pond
water was conducted using animals collected during November 1969. Separate series
of six 350-ml containers were placed in each of three temperature rooms. Three of the
containers in each room held distilled water, while the other three contained pond
water. Three adults,.1 female and 2 males, were placed in each of the containers,
and this number was maintained throughout the experiment by replacing all dead in-
dividuals. The containers were kept under moderate to low illumination. Photoperiod
was adjusted to be approximately 12-hours light and 12 dark in two of the rooms
(Rooms A and B). In the third room (Room C) the light was left on all the time as this
condition was required for other work being conducted in that room, A water tem~
perature of approximately 25° C was used in two of the rooms (Rooms A and C), while
21° C was used in the third (Room B). Mortality and reproduction among these animals
were monitored for 40 days. Data for mortality and reproduction in conditioned water
were obtained from a previous experiment that covered a 40-day period and used ani-

mals collected during the summer months of 1969. The latter experiment employed

-13-


~wl~

Table 45, A COMPARISON OF THE MEAN NUMBERS OF CYCLOPS VERNALIS FOUND UNDER THREE

DIFFERENT RATES OF FOOD ADDITIONS ON A SERIES OF SAMPLING
DATES. EACH VALUE [S THE MEAN OF THREE REPLICATES, THE

STANDARD ERRORS OF THE MEANS ARE INCLUDED IN

PARENTHESES, (A = ADULTS, G = GRAVID FEMALES,

Y = IMMATURES PLUS NAUPLI!,
T = TOTAL NUMBERS.)

Rate of food addition every second day

0.1 mi/liter 0.5 mi/litter 1.0 mi/liter
Week A G Y T A G Y T A G aa T

0 8.0 4,0 0.0 8.0 8.0 4.0 0.0 8.0 8.0 4.0 0.0 8,0
1 8.0 3.0 118.7 126.7 7.0 2,0 27.0 34.0 4.0 0.7 29.0 33.0
(1.0) (0.6) (105.7) (106.2) (1.0) 0.0) (5,1) (4.2) (1.0) (0.3) (2.1) (2.1)

2 11.0 1.0 106.7 117.7 7 2.7 82.0 93.7 13.3 1.0 56.3 69.7
(1.7) (1.0) - (88.3) (40.0) (1.5) (0.9) (23.3) (22.0) (0.7) (1.0) (22.6) (23.2)

3 17.0 0.7 92.7 109.7 27.7 2.7 87.0 114.7 37.3 5.3 202.0 239.3
5.6) (0.3) (87.7) (4149) 6.9) (0.9) (3.2) (8.7) (10.2) (2.3) (79.3) (88.8)

4 27.3 0.0 78,3 105.7 38.0 7.7 151.7 189.7 63.3 3.7 95.0 158,3
(9.6) (-) (34.5) (44.1) (0.0) (2.4) (84.4) (84.4) (25.9) (1.2) 39.2) (16.6)

5 22.3 0.3 40.0 62.3 37.3 2.0 240.7 278.0 50.3 4.7 149.7 -200,0
4.1 (0.3) (9.5) (11.1) (5.2) (1.0) (148.5). (150.3) (10.3) (2.2) (61,8) (69.4)

é 13.7 1.0 48.7 62.3 29.7 4.3 151.0 180.7 36.0 8.0 37.7 73,7
(3.0) 0.6) (31.4) (82.5) (7.9) (47)—(97.6) (105.3) (10.1) (2.5) (9.3) (19.0)

7 18.0 1.0 79.7 97.7 33.3 2.0 320.3 353.7 41.3 9.0 306.0 347.3
(4.4) (0.6) (34.2) (38.3) (10.9) (1.5) (71.2) (70.3) (12.0) (4.6) (108.9) (117.1)

8 18.7 1.3 86.0 104.7 43.3 143° 225,0 268.3 55.0 8.0 183.3 238,3
4.9) (1.3) (12.9) (16.5) (2.4) (0.7) (74.5) (72.3) (9.0) (2.3) (68.4)_—(77.2)

9 18.0 1,0 313.5 331.5 45.5 3.0 208.5 254.0 67.0 7.7 260.7 327.7
(4.0) (1.0) (47.5) (51.5) (2.5) (1.0) + (3.5) (1.0) (12.7) (1.8) (54.1) (48.3)

10 28.5 2.0 308.5 337.0 48.0 4.0 477.0 525.0 53,3 16.0 539.3 592.7
(0.5) (0.0) (19.5) (20,0) (8.0) (2,0) (118.0) (140.0) (6.5) (4.9) (94.9) (94.3)



From the Production of the Last Egg Clutch to Death, and the Mean
Time Intervals Between Successive Egg Clutches for Cyclops vernalis
Females at Four Different Temperatures, , ....cccceccccevececs

Summary of Statistical Data for Clutch Size, the Number of Clutches
Produced Per Female, and the Total Egg Production Per Female at the
Temperatures 14°, 21°, 26°, and 31° Co. . cece cee cece eee eene
Clutch Size in Relation to Age and to Temperature ....ceee cn eeee

Development Times of Cyclops vernalis Egg Clutches at Different Tem-

PEFAEUTES » pe ee eee tere cere r eer e reer asec seerores

Success of Hatching for Egg Clutches Produced by Cyclops vernalis in
Relation to Temperature ... cece cece cree ee ste rnceerearas

xii

151

154
157

160

163


~0El-

Table 45. A COMPARISON OF THE MEAN DENSITIES OF CYCLOPS VERNALIS THAT. WERE PRESENT IN

FOUR CULTURE VOLUMES ON A SERIES OF SAMPLING TIMES. THESE
DENSITIES ARE BASED ON THE DATA IN TABLE 44, (A = ADULTS,
G = GRAVID FEMALES, Y = IMMATURE COPEPODITES
PLUS NAUPLII, T = TOTAL NUMBERS.)

Culture volume

20 mi 80 ml 250 mi 1000 mi
Week A_G ¥ T A_G Y T AG Y T AG Y T
oO 400 200 0 400 100 50 Oo 100 32.=«16 UY) 32 84 0 8
1 235° 35 «3.300 535 66 12 241 309 29 13° 679 =—708 41 2 33
2 235 O 2350 2585 162 21 1329 1491 71 3 = «=733—— (804 3 1 56 10
3 450 15 2265 2715 . 246 16 504 750 52 3 415 467 37, 5 202 239
4 235 0 1515 1750 204 12 «612 «816 1030-4 «61720275 63 4 95 158 -
5 385 0 1065 1450 246 25 1429 1675 88 15 580 668 50 5 150 200
6 300 © 5085 5385 250 16 1650 1900 87 12 1337 1424 3% 8 74
7 385 0 2600 2985 254 9 1775 2029 107 «1 824981 41 9 306 347
8 450 50 2865 3315 241 4 1359 1600 149 5 «247 396 55 8 183 238
9 450 50 3200 3650 246 9 1384 1629 13306-3283 416 67 8 261 328



APPENDIX A-1
14°C INCUBATION: NUMBER OF EGGS PER CLUTCH IN THE SEQUENCE PRODUCED

-ELl-

Female's hatching
temperature 14° 2° 31° +Standard
Individual a7 AS A8 Ag Al0 Total Mean error
Clutch No.
1 30 24 20 16 23 113 22.6 2.32
2 26 18 23 18 30 5 23.0 2,32
3 23 9 9 14 22 97 19.4 1.57
4 22 15 22 % 21 % 19.2 1.53
5 26 18 13 24 24 105 21.0 2.41
6 31 19 24 18 24 116 23.2 2.31
7 2 15 10 22
8 30 15
9 24 15
10 34
VW 28
12 27
Total 322 158 121 116 166
Mean 26.8 17.6 20.2 16.6 23,7
Standard error 1,14 1.00 1.62 . 1,62 1,13
Anova table: mean clutch size Anova table: comparison of sequential clutches
Source of variation = df. = MS F Source of varlation df MS F
Among Individuals 4 174.0824 12.8986 P< 0,001 Among sequential clutches 5 16,2400 0,7298 ns
A7 vs Others 1 475.9767 35.2675 P< 0.001 Within sequential clutches 24 22,2500
Ab vs 31° hatch 1 41.7795 3.0956 ons Total 29

AB vs AF vs AlO0 2 89,2869 6.6157 P< 0.050
Within individuals 36 13.4962
Total 40



temperature room, so that the first order effects of temperature were balanced out
under each light condition, and the results will be presented in this subsection as
simply testing the influence of light. The average temperatures of the temperature

rooms used were approximately 17.2°, 21 9, 26.4°, and 31,0° C.

Six 350-ml containers, each containing 1 adult female and 2 adult males in pond
water, were set up in each room. The animals used in these experiments were the

offspring of laboratory-cultured animals.

To achieve the desired illumination conditions, two containers in each room were
completely covered with aluminum foil providing conditions of complete darkness

for the animals in these containers. Two other containers in each room were covered
with aluminum foil except for the top, which was covered by a piece of wax-paper
resulting in a light intensity at the surface of the water of 125 to 140 foot-candles.
The final two containers were covered on the side and bottom with foil as with the
other containers, but the top was covered by a 4 1/2 x 4 1/2 x 1/8-inch piece of
plate glass. This provided light of 200 to 218 foot-candles at the surface of the
water, The light conditions were adjusted to approximately 12-hours light and 12
dark.

The reproduction of the adults was followed for approximately 30 days and the develop-
ment of the young until they died or matured. A summary of the results is presented

in Table 6. In order to minimize the influence of individual animals on the results, a
double-averaging procedure was followed in obtaining the data in this table, The re~
sults for a parameter were averaged for each container, and then these values were
averaged separately for each light intensity resulting in an overall mean at each in-

tensity for each parameter.

It is immediately apparent from the table that the results are based on the production

of only a few clutches, During the experiment reproductive activity was at a low

-18-


the data did not meet the requirements for the anova test. In order to do such a test
and ensuing a posteriori comparisons, the clutch size values were transformed to log-
arithms (base 10). All tests of significance were performed on the transformed data.
The values for the mean clutch sizes discussed below are the antilogs of the means of
the logarithmically transformed data. The greatest mean number of eggs per clutch
was 39.7 eggs produced at 21°C, with the clutch size at 14°C only slightly lessthan this
with 35,2 eggs. The least number of eggs per clutch was 25.2 eggs, produced at
26°C, The mean clutch size at 31°C was also quite low with only 27.5 eggs per
clutch. Ewers (1936) and Armitage and Davis (1967) observed C. vernalis producing
clutches of roughly the same size as those reported in this report (40 to 80 eggs per
clutch and 16 to 23 eggs per sac, respectively). However, Andrews (1953) reported
clutches ranging in size from 100 to 150 eggs.

Analysis of variance showed a significant difference (P < 0.001) among the means
for clutch size at the four temperatures. A posteriori comparisons indicated that the
"mean size of egg clutches produced at 21°C was significantly larger (P < 0.05) than
the means at either 26 or 31°C, Also, the mean size of the egg clutches produced

at 14°C was significantly larger (P< 0,05) than that at 26°C, These results give some
suggestion 07 an inverse relationship between clutch size and temperature, but the re-
lation is not well defined,by our data, Andrews (1953) reported a maximum rate of
egg production by C. vernalis in the laboratory at the temperature of 21°C,

Several authors have suggested that the size of the egg clutches produced by cyclo-
poids is only indirectly related to temperature and that the factor directly determin
ing clutch size is the female's body size. Margalef (1955) stated that the potential
fecundity of cyclopoids is a function of body size and therefore of the temperature
during development. Elbourn (1966) reported that the female's size and the tempera~ ,
fure are equally important as factors determining the number of eggs produced per
clutch by C, strenuus strenuus. The females’ body sizes were not measured in the

present experiments; however, Coker (1933) and Aycock (1942) published findings

-155-


APPENDIX A-2 (Cont.)
21° INCUBATION: ANALYSES OF VARIANCE

Anova table: mean clutch sizes

Source of variation df MS F

Among individuals 9 450.1521 9.1601 P< 0.001
21° hatch vs other 1 3119.2990 63.4747, P< 0.001
27° hatch vs 31° hatch 1 273.4580 5.5646 P< 0.050
BI3 vs BIS 1 66.0056 1.3431 ns
Among 21° hatch 6 98.7679 2.0098 ns

Within individuals 120 49 1424

Total 127

Anova table: comparison of sequential clutches

Source of variation df MS F

Among sequential clutches 12 (64.1855 0.8088 = ns
Within sequential clutches 88 = 79.3545
Total 100

-175-


1
foo}
~

t

NUMBER
OF

. ADULTS

PER
LITER

50
40
30

20

w BR UONDW0O

T

— oer

a

@ OPEN WATER AREA
© ROOTED AQUATIC AREA

1

1 L 1 1 1 inl Ll 1 y i 1 1

fj pitt

py iiriy

19
I

21

Figure 5. Mean Numbersof Adults Per Liter in the Two Regions of the Pond from 19 February to 4

23 2.

1 14 it
5 27 1 3 5 7 9 11 13 15 I7 19 21 23 2527 29 31 2 4
ul DATE IV

April, 1971. Enlarged Arabic Numerals show those Dates when there were Detectable
Differences (P<O.05) Between Concentrations of Adults in the Two Regions.


~62l~

Table 44, A COMPARISON OF THE MEAN NUMBERS OF CYCLOPS VERNALIS THAT WERE PRESENT IN.
FOUR CULTURE VOLUMES ON A SERIES OF

“TIMES.” EACH VALUE

1S THE MEAN OF THREE REPLICATES, THE STANDARD ERRORS OF THE

MEANS ARE INCLUDED IN PARENTHESES, (A = ADULTS,
G = GRAVID FEMALES, Y = IMMATURE COPEPODITES
PLUS NAUPLII, T = TOTAL NUMBERS.)

Culture volume

20 ml 80 mi 250 ml 1000 ml
Week A GY T A G Y T A G Y T A c Y T
0 8.0 4.0 0.0 8.0 8.0 4.0 0.0 8.0 8.0 40 0.0 8.0 8.0 4,0 0.0 8.0
1 4.7 0.7 6.0 10.7 5.3 1.0 19.3 24,7 7.3 3.3 169.7 177.0 4.0 0.7 29.0 33,0
(0.3) 0.7) (3.8) (3.5) (0.7) (1.0) (9.4) (9.5) | (0.3) (0.7) (19.1) (19.1) (1,0) (0.3) (2.1) (2.1)
2 4.7 0.0 47.0 51.7 13.0 1.7. 106.3. 119,3 17.7 0.7—-183.3 201.0 13.3 1.0 56,3 69,7
(0.9) (-) (14.5) 04.7) 6.7) (0.9) 1.9) (31.9) | (5.8) (0.3) (15.1) (20.9) 0.7) (1.0) (22.6) (23.2)
3 9.0 0.3 45,3 54.3 19.7 1.3 40.3 60.0 13,0 0,7 103.7 116.7 37.3 5.3 202.0 239.3
(3.0) (0.3) (2.6) (3.8) (8.7) (0.7) (7.4) (16.0) | (3.0) (0.7) (35.6) (38.6) | (10.2) (2.3) (79.3) (88.8)
4 4:7 0.0 30.3 35.0 16.3 1.0 49.0 65.3 25.7 1,0 43.0 68.7 63.3 3.7 95.0 158,3
(1.2) (-) (10.5) (11.7) | 45) 0.6) (13.3) (18.5) | (2.4) (0.6) (22.2) (24.3) | (25.9) (1.2) (39.2) (16.6)
5 7.7 0.0 21.3 29.0 19.7 2.0 114,3 134.0 | 22.0 3.7. 145.0 167.0 50.3 4.7 149.7 200.0
(1.2) () (20) (2.5) | (2.2) (0.6) (28.8) (30.7) | (4.0) (1.3) (93.0) (94.5) | (10.3) (2.2) (61.5) (69.4)
6 40 0,0 101.7 107.7 20.0 1.3 132.0 152.0 21.7 3.0 334.3 356.0 3.0 8.0 37.7 73.7
(5) (-) 45.9) (47.4) (7.5) (0.3) (37.9) (44.4) | (1.5) (2.1) (149.3)(150.7) | (10.1) (2.5) (9.3) (19.0)
7 7.7 0,0 52,0 59,7 20.3 0.7 142.0 162.3 26.7 2.7 206.0 232.7 41.3 9.0 306,0 347.3
(1.2) (+) (24.8) (26.0) (7.3) (0.3) (20.2). (14.6) | (7.7) (1.7) 63.4) (60.1) | (12.0) (4.6) (108.9) (117.1)
8 9.0 1.0 57,3 66.3 19.3 0.3 108.7 128.0 | 37.3 1.3 61.7 99.0 55.0 6.0 183.3 238.3
(0.6) (0.6) (6.1) (5.8) 3.3) (0.3) (7.6) (7.8) | (20.9) (0.3) (18.3) (26.1) (9.0) (2.3) (68.4) (77.2)
9 9.0 1,0 64,0 73.0 19.7 0.7. 110.7 130.3 | 33.3. 0.7 70.7 104.0 67.0 7.7 260.7 327.7
(1.5) 0.6) (22.1) (21.3) (2.3) (0.7) (44.4) (46.5) | (10.5) (0.7) (34.3) (44.5) | (12.7) (1.8) (64.1) (48.3)



CHAPTER 4
ASPECTS OF THE DYNAMICS OF A NATURAL POPULATION
OF DIAPTOMUS CLAVIPES

Even with the ability to culture and experiment with an organism in the laboratory,

it is still necessary to study naturally occurring populations. Controlled laboratory
experiments provide a means of separating the effects of the many factors that in-
fluence a population, and thus they furnish a basis for interpreting the usually rather
complicated population fluctuations found in nature. However, only from studies

on field populations is it possible to determine whether the relations found in the lab-
oratory hold in nature and, if so, which of the relations cre of importance in actual

environmental situations.

Thus, considering our overall goal of providing increased knowledge of the relation
of diaptomids to water quality, a study on certain aspects of the population dynamics
of a field population of Diaptomus clavipes was carried out. This work naturally
fell into three broad categories: 1) heterogeneity of distribution, 2) a life table ap-
proach to population dynamics, and 3) reproduction. Separate sections dealing with
the results and discussion for each of these aspects are included in this chapter

after the Methods and Materials section.

A number of studies have dealt with some aspects of the relations between a diaptomid
population and environmental factors. Such work was pioneered by G.W. Comita
and coworkers (Comita, 1956; Comita and Comita, 1956; and Comita and Anderson,
1959) and by Ravera and Tonolli (Ravera, 1954, 1955; Tonolli, 1964, 1961; and
Ravera and Tonolli, 1956). More recently,significant studies along these lines have
included the work of Elster (1964), Armitage and Davis (1967), Healy (1967), Smyly
(1968), Chapman (1969), and Kibby (1971). ,

~80-


APPENDIX E-2

TOTAL NUMBER OF COPEPODITE FIVE (CV) IN EACH REGION OF THE POND
AND IN THE TOTAL POND ON EACH COLLECTING DATE, THE TOTAL
NUMBER WAS DETERMINED BY MULTIPLYING THE Y (MEAN)
NUMBER OF CV PER QUADRAT IN EACH REGION BY
THE TOTAL NUMBER OF QUADRATS IN THAT
REGION AND SUMMING THE PRODUCTS
FOR EACH DATE.

Date Open water Rooted aquatics Total pond
region region
1-197]
9 0 0 0
21 0 0 0
23 ia) 0 0
25 0 0 0
27 (0) 0 0
IH-1971
1 0 0 0
3 0 0 0
5 0 0 0
7 0 0 0
9 0 0 0
i 0 0 0
13 0 0 0
15 0 0 0
7 0 0 0
21 0 0 0
23 0 0 0
25 0 0 0
27 0 0 0
29 21,552 49,262 70,814
31 233,041 71,547 304,588
IV-1971
2 766,921 41,331 818,251
4 286,646 126,546 413,192
6 633,444 263,028 894,472
8 966,227 8,889 975,116
10 972,144 9,192 981,336

-208-


APPENDIX B-3

27° INCUBATION: TIME (HOURS) CLUTCHES WERE CARRIED

Female's
hatching
temperature 2° 27° 3r°
Individual co C7 C8 C13 7 Cl4 cis Clé ci
45.5 | 41.5 35.5 | 40.0 | 40.5 | 44.5 26.0 | 25.0
25.5 | 43.0 19.5 | 35.5 | 37.0 | -38.0 28.0 | 38.0
45.0 | 40.0 33.5 | 34.5 | 34.5 | 30.5 17.5 | 36.0
30.5 | 37.0 33.5 | 27.5 | 27.5 | 34.5 10.0 | 34.5
33.0 | 37.0 42.5 | 35.5 | 35.0 | 32.0
35.0 | 33.0 35.5 | 42.0 | 32.0 | 35.0
32.5 36.5 | 38.5 | 35.0 | 39.0
35.0 25.0 | 39.5 | 44.0
35.5 23.5 | 43.5
24.0 34.0 | 44.0
24.0 30.5
18.5 23.5
24.0 20.5
41.0
52.5

Clutch carrying times are listed from top to bottom in the order of occurrence but are
not necessarily consecutive, some carrying times having been omitted for various rea~
sons as discussed in the text.

Variances of the data for the three hatching temperatures are marginally homogeneous.
A logarithmic transformation was made and Anovas carried out with the transformed data.

Anova table: comparison among individuals of carrying times.

Source of Variation df MS F
Among individuals 7 0.0427 3.9537 PK0.010
27° hatch vs other 1 0.0045 0.4170 ns
21° hatch vs 31° hatch 1 0.0016 0.1519 ns
C7 vs C8 vs ... vs C16 5 0.0587 5.4380 P<0.001
Within individuals 59 0.0108
Total 66

-182-


ACKNOWLEDGMENTS

The assistance of the Department of Zoology of the University of Oklahoma in making
facilities available in support of our studies is most gratefully acknowledged. A num~
ber of the members of that department provided advice and assistance during the work,
and we would like to thank especially Drs. Harley P. Brown, Howord Haines, Donald
Perkins, and Frank Sonleitner. A special word of gratitude is owed Dr. Haines who
assumed the position of Grant Director for this project part of the way through the study
and by his efforts assured ifs completion. We also acknowledge with our thanks the
assistance of Mr. Charles R. Samples and Mr. J.T. Yacovino whose efforts provided
much of the material in Chapter 2 and of Dr. Kenneth Biesinger of the National Water
Quality Laboratory, EPA, for his advice throughout this project and especially for sug-
gesting the food material which proved the most satisfactory for our cultures. Finally,
we owe our most special thanks to our wives and families who supported this work in

ways too numerous to mention, but especially with their patience.

xiii


one date and in the region of rooted aquatics on the next (Figure 5). Starting with
6 April, however, a definite pattern, with a higher concentration of adults in the
open water region than in the region of rooted aquatics, began (Figure 6). This pat-
tern prevailed until 21 October. The concentration of adults ranged from 0.00 per
liter in the area of rooted aquatics on 25 June to approximaiely 18 per liter in the

open water region on 23 July.

A Student's t-test was conducted separately on the data from each sampling date to
determine whether the concentration of adults varied significantly between the two
regions. A detectable difference was found on only 5 of 23 dates prior to 6
April, i.e.,. 23 February; 9, 17, and 25 March; and 2 April (Figure 5), but.
from 6 April until 20 August, a significant difference (P<0.25) between means
was found on all but 1 of 18 dates (Figure 6). This onset of relatively con-
tinuous stratification coincided with two observations, an increased rate of growth
of rooted aquatics and an addition to the population of new adults which developed
from the current year's reproductive activity (see Life Table Approach to Population

Dynamics).

More evidence of differential distribution between the two areas of the pond was ob-
tained when the proportion of the total water volume in the open water region and
the fraction of animals collected in open water were both graphed against time (Fi-
gure 7). If the animals were not ‘concentrating in one of the areas of the pond, the
probability of one proportion being larger than the other on any given date would be
0.5. However, if the animals were concentrating in one of the areas, one of the
proportions would be consistently larger than the other. Each data point was placed
in two of three groups on the basis of the time of year it was obtained. The group=
ings were total sampling period (19 February to 29 October), early spring (19 Feb~
ruary to 4 April), and spring-summer (6 April to 29 October). The early April date
was chosen as the dividing time because of the increased rate of growth observed in

the rooted aquatics from that date on and because the addition of new adults to the


~9Ll>

27° INCUBATION: NUMBER OF EGGS PER CLUTCH IN THE SEQUENCE PRODUCED

APPENDIX A-3

Female's hatching

temperature 21° 27° 31° Standard
individual co | C7 ce] cis 7 ci 7 cis 7 tie |—cn 7] Total Mean error
Clutch No,
1 19 35 2 32 7 20 04 15 181 22.6 3.60
2 18 3% 45 35 2 2 07 20 212 26.5 4,28
3 18 40 45 42 7 7 25 30 254 31.8 3.36
4 23 26 36 33 21 2» 13 36 217 27.1 2.84
5 20 29 47 38 22 2 ) 32 223 27.9 4,10
r 20 39 4 34 24 32 35 225 32,1 2.89
7 25 34 45 33 25 32 13 207 29.6 3.75
8 43 50 27 35 30 185 37.0 4.23
9 19 33 23 2a
10 22 34 22 2
" 32 36
12 42 4%
13 17 2
4 25 34
15 % 32
16 20 36
7 20 45
18 21
Total 143 «5505 67817 2088s 58 («181
Mean 20.4 2.7 37.6 31.9 25.4 27.9 11.6 25.9
Standard error 1.00 2.07 2.02 1,99 1.59 (1.14 3,66 (3.65



Laboratory Data

The field data revealed heterogeneity of distribution prevailed throughout the col-
lecting period and appeared to be correlated with the growth of higher aquatics.
The laboratory experiments discussed below showed that these differences in con=

centrations were, in fact, a result of the rooted aquatics.

In an experiment where 20 animals were forced to live in environments in which
pieces of Potamogeton sp. were floating freely, 18 died within 96 hours; whereas of
20 animals in a comparable environment without Potamogeton,only one died in the
same interval, This experiment clearly demonstrated that adult D, clavipes are un~

able to survive when forced to live in proximity to Potamogeton sp.

Comparison of the mean clutch carrying times for females in the simulated-weed and
weed-free environments revealed that those animals in simulated weeds carried their
eggs almost twice as long as did the animals in weed=free environments, 4.00 days

and 2,22 days respectively (Table 28), Asa check on these results, the environments

__ of the animals were reversed, with those animals previously in simulated-weed environ-
ments now in weed~free environments and those animals previously in weed-free en-
vironments now in simulated-weed environments. After the environments were switch-
ed, the mean clutch carrying times were 3.71 days inthe simulated-weed environment
and 1.86 days in the weed-free environment (Table 29), again almost twice as long

in the simulated weeds as in the weed-free environments. In both cases the results
were highly significant (P< 0.01) when the Student's t-test was employed. Compari-
son of the mean clutch carrying times for females in the weed~free environments and
the environments having polyethylene tubing around the perimeter of the beakers
(Table 28) showed little difference between the two. This indicated that the differ~
ence between the average clutch carrying time for animals in simulated-weed environ~
ments and that for animals in weed-free environments was not due to some chemical

found in the polyethylene tubing. This work showed that simulated weeds increase

-91-


APPENDIX E-4

TOTAL NUMBER OF COPEPODITE THREE (CII) IN EACH REGION OF THE POND
AND IN THE TOTAL POND ON EACH COLLECTING DATE, THE TOTAL
NUMBER WAS DETERMINED BY MULTIPLYING THE Y (MEAN) NUMBER

OF Cll! PER QUADRAT IN EACH REGION BY THE TOTAL NUMBER OF

FOR EACH DATE.

QUADRATS IN THAT REGION AND SUMMING THE PRODUCTS

Date Open water Rooted aquatics Total pond
region region
-1971
19 0 0 0
21 0 0 0
23 0 0) 0
25 0 0 0
27 0 0 0
W-1971
1 0 0 0
3 0 0 0
5 0 0 0
7 0 0 0
9 0 0 0
W 0 0 0
13 0 0 0
15 0 0 0
7 44,591 11,148 55,739
19 66,887 11, 148 78,035
21 388,558 25,375 413,933
23 285,572 329,502 615,074
25 793 , 535 32,611 826, 146
27 655,578 556,623 1,212,201
29 420,269 1,490, 183 1,910,452
31 1,018,021 157,405 1,175,426
IV-1971
2 .
4 368,545 37,219 405 , 764
6 285,671 93,333 379,003
8 846,940 8,889 855,828
10 651,909 0 651,909

-212-


-06I-

Time (hours) from oviposition to succeeding oviposition

APPENDIX B-10

21° INCUBATION

EGG PRODUCTION CYCLE

Female's hatching

temperature 21° 27° 31°
Individual BI] B12 B14 BI7 B18 BIg B20 | . B16 B13 BIS
73.5 | 61.5 | 59.0 | 72.0 | 61.0 | 63.0 | 56.5 | 59.0 | 53.5 | 60.5
50.5 | 50.5 | 73.0 | 72.5 | 63.5 } 57,0 | 54.5 | 60.5 | 166.0 | 59.5
47.0 | 55.5 136.5 ] 111.5 | 49.5 | 63.5 | 60.5 | 48.0 | 61.0
52.0 49.0% 56.5 56.0 | 110.5 57.0 61.0 61.0 61.5
60.0 | 39,5* 50.5 | 52.0 | 57.5 | 56.5 | 56.5 | 47.0*| 56.5
60.0 46.0* 78.0*} 71.5 56.0 59.0 50.0* {| 62.5
47.0 48 .5* 92.0% 153.0 75.5 57.0*} 65.0
57,0 | 36,0* 58.5 | 57.5 | 47.5*}| 53.0
71.5 | 48.0* 163.5 } 47.5 | 96.0*] 47.0
47.5 | 65.0% | 56.5 | 79.5 | 71,0*| 116.5
66.5 | 53.0* 104.0 | 55.0 | 68,5*| 71.0
52.5 | 46.5% 86.0 | 62.0 68.5
51.0 | 48.5* 58.5 79.0
40,0 | 48.5* 115.5
76.5 | 66.5 56.0
39.5 | 53.0 68.5
76.0 | 40,0*
50.5 55.5*
72.5 46.5*
40.5 | 50.0*
: 46.5%
50.0*

*Indicates a cycle which includes a clutch of resting eggs.


APPENDIX B-2 (Cont.)
21° INCUBATION: TIME (HOURS) CLUTCHES WERE CARRIED

Clutch carrying times are listed from top to bottom in the order of occurrence but are

not necessarily consecutive, some carrying times having been omitted for various reasons
as discussed in the text.

Variances of the data for the three hatching temperatures are heterogeneous. A logarith~
mic transformation was made and Anovos carried out with the transformed data.

Anova table: carrying times of resting eggs (mean = 39.0) vs carrying times of non-
resting eggs (mean = 48.2).

Source of Variation df MS F
Among egg types 1 0.1755 15.2608 P<0.001
Within egg types n3 0.0115
Total 114

Anova table: comparison among individuals of carrying times, excluding those times

marked with an asterisk above which are the carrying times of clutches
of resting eggs.

Source of Variation df MS F
Among individuals 9 0.0160 1.3793 ns
Within individuals 78 0.0116
Total 87

-181-


species of animal, the use of V curves gives the information necessary in determining
where to concentrate and what to study. In D. clavipes it is apparent that the cause
of mortality (what) is of importance primarily during the egg to NIII interval (where)
with the possible inclusion of the metamorphosis from NVI to Cl. Mortality seems
of little consequence in the adult instar, since a physiological 1 curve is apparently
present in this stage. Consequently with the adults, population studies should focus

on those factors which may affect the reproductive rate.
REPRODUCTION

Analysis of the survivorship curves suggested three points of the life cycle for special
consideration--egg production, survivorship from egg to NIII, and survivorship of
NVI to CI. In this section consideration will be given to egg production and its re-

lation to certain environmental factors.

Edmondson (1960) used as his reproductive index the ratio of eggs to animals in the
population (crude birth rate). Since with D, clavipes the eggs are carried by the
females and the adults are easily distinguished from the immature copepodite stages
(Kamal and Armitage, 1967), it was possible to determine the ratio of eggs to adults
in our work, Because the egg development time varies only slightly with temperature
(Table 21) except near the lower limit for reproduction, a 2-day duration for the

egg stage has been used to compute the specific birth rates. By dividing the total
number of eggs by the product of the total adult population and the duration of the

egg stage, a specific birth rate, or m,. value, can be calculated for any date.

An average (weighted for the differences in the sampling intervals) of 2.78 eggs per
adult per day was calculated for the entire study period (Figure 10). During the first
three collecting dates, when the temperature was well below the lower limit for repro-

ductive activity as determined in the laboratory, the m, values were below the yearly

-107-


clutch size, females producing larger clutches if they remain at the same constant
temperature throughout life, and the largest clutches at each temperature being
produced by these "native" females. Since the data are inconclusive, two analyses
of variance have been carried out to examine the effect of temperature on clutch
size (Table 16).

The first compares only those females "native" to each incubation temperature, all
"non-native" females being omitted. For these "native" females, mean clutch size
decreases in the order 27, ar, 1, and 31° incubation. Differences in means
are highly significant (F=8.5; P<0.001), but an a posteriori comparison of means by
a Student-Newman-Keuls (SNK) test reveals that the means of females "native" to
4° , 21°, ond 27° are not significantly different. Only the mean of females "na-
tive" to 31° differs significantly from the others.

The second analysis of variance in Table 16 includes data for all females at each
incubation temperature, regardless of their hatching temperature. These overall
means decrease in the same order as the means for "native" females and the dif-
ferences are also highly significant (F=10.8; P<0.001). Applying a SNK fest to
these combined data, the means of 1”, ar, and 31° are shown not to differ, only

the mean for 27° being significantly higher.

Table 17 summarizes the data from Appendices A-1 through A~4 in terms of the mean
total numbers of clutches produced during a lifetime at the various temperatures.
Except at 21°, “non-native” females produced fewer clutches than did "native"
females. However, the mean number of clutches wos maximized at 21° incubation,

regardless of the hatching temperature (Figure 2).
Analysis of variance (Table 17), comparing the mean total numbers of clutches pro-

duced at each incubation temperature, was first carried out using only data from

females "native" to each incubation temperature. (F=1.4;ns) and again using all

-54-


Table 18 (continued). LIFETIME TOTAL NUMBER OF EGGS PER FEMALE

Anova table: Comparison of all females at each incubation temperature.

Source of variation df MS F
Among incubation temperatures 3 0.3281 3.6741 P< 0.025
Within incubation temperatures | 27 0.0893
Total 30

~60-


APPENDIX B-9

14° INCUBATION
EGG PRODUCTION CYCLE
Time (hours) from oviposition to succeeding oviposition

Female's hatching
temperature 14° 21° 31°

Individual A7 A6 As Ag Ald
168.0 133.0 167.5 139.5 139.0
122.0 122.0 129.5 166.5 227.5
147.5 138.5 122.0 168.5 132.0
192.5 272.0 136.5 123.0 132.5
279.5 156.0 296.5 206.5
129.0 123.5
206.5 153.5
208.0 470.5

Lengths of cycles are listed from top to bottom in the order of occurrence but are not
necessarily consecutive, some cycles having been omitted for various reasons as dis~
cussed in the text.

Variances of the data for the three hatching temperatures are marginally homogeneous.

A logarithmic transformation was made and Anovas carried out with the transformed
data.

Anova table: comparison among individuals of complete egg production cycle.

Source of Variation df MS F
Among individuals 4 0.0087 0.3954 ns
.Within individuals 25 0.0220
Total 29

-189-


jar and transported to the laboratory where they were surveyed with a binocular dis-

secting microscope.

Unless otherwise specified, 350 ml glass containers (finger bowls with a 4 1/2-inch
diameter) were used for the culturing experiments, while the immatures were reared

in 100-ml glass beakers. Ina few initial experiments, the culture water was changed

at 7- to 10-day intervals, but in most experiments it was never totally changed, Evapo-
ration losses were reduced by placing a cover over the mouth of each container, but

it was still occastonally necessary to make up losses by adding distilled water.

Some consideration was given in this work to what type of water should be used for
culturing. Three types of culture media (i.e., conditioned water, distilled water,
and natural pond water) were tested, Conditioned water was prepared by placing
tap water in 3.8-1 glass containers and aging it for two weeks prior to use. The nat-
ural pond water was obtained from the pond in which the animals were collected and
was filtered twice through a double layer of No. 25 silk bolting cloth before being
placed in the culture containers. The distilled water was the product of a general-
use laboratory still and was of rather low quality. Except where specified otherwise,

conditioned tap water was used in this work,

During experiments unless otherwise specified, the animals in each container were
examined daily. Any individuals found dead were isolated on a depression slide and
examined microscopically to determine the sex. Each dead individual was replaced
by an individual of the same sex. Each newly gravid female was isolated ina drop
of water on a depression slide. A drop of methyl cellulose was added to immobilize
her, and a dissecting microscope was used to count the number of eggs in her clutch.
After the eggs had been counted, the females were either returned to their culture
containers or were placed in separate 100-ml beakers, containing 70 to 80 ml of pond
water, until their eggs hatched. After hatch, the females that had been placed in

100-m! beakers were returned to their original culture containers.


In order to reduce handling, egg counts were made with the egg sacs intact on the
female, Counting of the eggs was facilitated by isolating the females ina small
drop of water, thus restricting movement. No detrimental effect was noted for fe-
males handled by this method. Counting accuracy was checked occasionally by
counting, in the usual way, the eggs on a gravid (egg-carrying) female taken from
a stock culture; then removing the egg sacs and recounting after the egg sacs had
been teased apart and the eggs were lying more or less in one plane, Some error
was found in counts for the larger sacs of 30 or more eggs, but this error was small

(not more than + 3 eggs).

Nauplii were removed from a vial as soon as possible after a clutch of eggs hatched,
Two drops of the food mixture were added to each vial twice per week, and the cul-

ture medium in each vial was replaced once per week,

To determine the development time for eggs at 14, 21, 26, and 31°C, each female
in the egg production experiment, described above, was monitored at 8 hour inter-
vals during.a 4 week period. The development time for the eggs was estimated as

the time from the first observation of the egg sac to the first time when the sac was

no longer on the female. In any individual case this estimate may have been in
error by as much as 8 hours, but on the average the differences should have largely

balanced out and an unbiased mean value should have been obtained.

In addition to determining egg. development at the above temperatures, egg develop-
ment times were observed at 5°C, At this temperature several vials, each with
mature female and 2 mature males taken originally from the 21°C cultures as pre=
viously mentioned, were examined once each day for several weeks, always at
about the same time of day. The development times for egg sacs produced at this

temperature were thus found within +1 day.

Data were also gathered on the influence of temferature on hatching success. Be-

cause of cannibalism (see Chapter 5), it was necessary, for this work, to remove the

-149-


-1@z-

PERCENTAGE OF CYCLOPS VERNALIS NAUPLII SURVIVING 24 AND 48 HOURS AFTER

APPENDIX F-1

HATCHING WITH AND WITHOUT THE PRESENCE OF THE MOTHER.

Clutch Female present Clutch Female removed at time 0
No. Number of nauplii surviving No. Number of nauplii surviving
Ohr 24hr. % 48hr. % Ohr 24hr. % 48 hr. %
1 83 25 30 10 12 1 76 73 97 73 97
2 68 33 49 26 38 2 95 91 % 84 88
3 49 30 61 29 59 3 59 59 100 58 98
4 55 3360s; 4 52 52 100 51 98
5 32 19 59 15 47 5 24 24 100 22 92
6 65 54 83 4l 63 6 38 37 97 36 95
7 51 24 47 18 35 7 96 96 100 % 100
8 73 34 47 28 38 8 26 26 100 26 100
9 4 16 35 13 28 9 57 55 % 55 %
10 50 34 68 31 62 10 50 49 98 49 98
Mean 53.9 41.8 98.4 96.2
SE, 14,95 45,13 40.56 41.18
No. 10 10 10 10



50 oT TTT

40 4
30 b OPEN WATER AREA |
© ROOTED AQUATIC AREA
204 \ 4
1
1
1
19k \ 4
a ! 4
; E H | 1 4
5b | ]
4b \ J
NUMBER | } i
OF 3F ' 4
g ADULTS t '
? PER 2 | ' 7
LITER |
'
oly ' q
is !
0:6 ' J
0.5 1 4
0.4) 1 4
1
0.3 4
0.2 H
'
i
0.1 i 4
0 L L 1 1 1 1 1 H al L A 1 1 L L 1 1 L 1 1
68 10 l2 4 16 18 20244 18 2 ll 25 9 23.6 20 3 7 30 14 21 29
V oare VEO VE vi x x

Figure 6, Mean Number of Adults Per Liter in the TwoRegions of the Pond from 6 April to 29 October, 1971.
Enlarged Arabic Numerals Show those Dates when there were Detectable Differences (PX0,05) Between
Concentrations of Adults in the Two Regions. The Vertical Dashed Line Between 18 April and 24 April
Shows a Change in Scale from 2-Day to Approximately 14~Day Intervals.


-01-

Table 1. TOTAL COPEPODITE POPULATION SIZE (TOTAL NUMBER AND NUMBER PER LITER) ON A

OF THREE REPLICATES.)

SERIES CF DATES FOR D. CLAVIPES CULTURES MAINTAINED IN CONTAINERS
OF 5 DIFFERENT VOLUMES. (EACH VALUE IS THE AVERAGE

Container Volume (ml)

Sampling 2 1,000 3,000 20,000
aa) Total No. per Total No. p= Total No. | No./i | Total No. [ No./} | Total No. [ No./}
24-VE 10.0 200.0 10.0 40,0 10.0 10.0 10.0 3.3 10.0 0.5
27-V1 8.3 166.0 8.7 34,8 10.0 10.0 9.0 3.0 7.7 0.4
1-Vil 0.7 14.0 8.0 32.0 W7 W.7 W.7 39 V7 0.6
3-Vi 0.3 6.0 6.3 25.2 14,3 14,3 9.7 3.2 11.7 0.6
7VU 0.0 0.0 5.0 20.0 16.7 16.7 9.3 3.1 - -
15-V II* - - 24.0 24.0 58.7 19.6 331.7 16.6
20-VII 8.7 34.8 38.0 38.0 43.7 14.6 387.3 19.4
27-Vil 6.0 24.0 47,7 47.7 67.3 22.4 328.7 16.4
1-Vill 3.7 14.8 41.0 41.0 47.3 15.8 313.0 15.7
31-Vill 0.0 0.0 25.3 25.3 40.7 13.6 123.0 6.2

* Subsampling initialed In 1,000-, 3,000-,and 20,000-m! cultures


Table 14. ACOMPARISON OF MORTALITY AND REPRODUCTIVE SUCCESS
(ESE) FOR D. CLAVIPES FED 0.4 ML OF EITHER FRESH
OR OLD FISH FOOD SOLUTION. (EACH VALUE
IS THE MEAN FOR SIX INDIVIDUALS. )

Experiment Type of food
Fresh Old
Number of 2 0 0
animals dying
during experiment 3 2 2
No. of clutches 2 40102) nee
produced per . (70.02)
animal per day 3 0.21 0.21
(£0.06) (20.03)
2 18.0 20.1
Mean number 4 rate
of eggs (41.4) (*1.4)
per clutch 3 15.2 15.8
(41.7) (41.3)

~39-


they were lumped for counting. The results of all counts are presented in Appendices
E-1 through E-8.

Laboratory Data -

Animals were cultured in 100-ml beakers, each containing 80 ml of pond water which
had been filtered twice through #20 bolting cloth, The beakers were kept ina con=
stant temperature chamber at 21°C and under a daily cycle of 12 hours of light and
12 hours of dark. Twice weekly the animals were fed 0,1 ml of the aqueous mix-
ture of trout food and alfalfa whose preparation has been described in Chapter 2.

To initiate an experiment, a single pair of adult animals (one male and one female)
was added to each beaker. The animals to be used were taken from a stock culture
reared at the same temperature and light as used in the experiment and consisting

of individuals at least one generation removed from the field.

Two questions pertaining to the effect of rooted aquatics on D, clavipes were studied
in the laboratory. The first question concerned the ability of the adults to survive
when they were forced to live in an environment in which Potamogeton sp., a "nar-
row leafed species" (Fassett, 1957), was allowed to float free. Two sets of 10
replicates each were used in this study. One set of beakers had Potamogeton sp.
floating freely in the water, while the other set had no Potamogeton sp. added.
Toensure that the results obtained were due to the vegetation rather than to peri-
phyton, the Potamogeton sp, was soaked for 15 minutes in tap water and then for 15
minutes in distilled water. The vegetation was then washed with fast flowing tap
water for 5 minutes after which it was placed ina container of double filtered pond
water until it was used. After 4 days, during which time the beakers were censused

daily for mortality, the experiment was terminated.

The second question studied in the laboratory concerned the effect of higher aquatics

on reproduction. The female of D. clavipes generally carries her eggs ina sac on


SELECTED WATER Bierogne d= "
hi

RESOURCES. ABSTRACTS . Ww

INPUT TRANSACTION FORM

4. Tithe . ie Report Date
Culturing and Ecology of Diaptomus Clavipes and 7 [6 we
Cyclops Vernalis 8. Perfotming Organisation

Gather Report No.
Robertson, Andrew . 1. ees

” Offtce of Research Administration TE
University of Oklahoma | 18050 ELT
Norman, Oklahoma 7306' ; ct BBs deere mt

ee ae ee

Supplementary Notes i

Environmental Protection Agency report number,
EPA~660/3-74-006, April 1974,

16. Abstract : :

This report presents the results of studies undertaken to develop a method of maintaining health, self-
propagating, laboratory cultures of the freshwater calanold copepod, Diaptomus clavipes. Recom-
mendations are given as to the conditions of contalner size, type of culture medium, fight conditions|
temperature conditions, food type and quantity, frequency of replacement medium, and amount of
disturbance suggested for culturing. ve

The results of a study dealing with effects of temperature on certaln reproductive attributes of this
species are presented. Temperature Is shown to affect the longevity of the adult females as well as
the size, carrying time, and probably total IIfetime production of’clutches, The results of this study
Indicate that certain of the reproductive attributes of the females are affected by the temperature of
early life as well as the acclimation temperature. oe

The report Includes the results from a study on the dynamles of a fleld population of D. clavipes. The
durations of the various life history stages were estimated both from laboratory and fleld data, Life
tables were constructed for the spring generation of thls population as well as all generations Ina
reproductive year combined, The stages of greatest relative mortality were identified. The report

also presents recommendations for culturing the cyclopold copepod, Cyclops vernalis, and the re=
sults of studies conceming effects of tempetature on certaln reproductive attributes of this spectes.

174. Descriptors *Copepods, *Water temperature, *Aquatlc populations, *Bloassay, Crustaceans,
Animal ecology, Zooplankton, Food abundance, Oklahoma laboratory animals, Environmental
effects, Reproduction.

17b. Identitiers *Dlaptomus clavipes, “Cyclops vernalls, *Laboratory culturing, *Temperature relations,
*Food relations, lation dynamles, tie _

17ce. COWRR Field & Group

18, Avaitavility 49. Security Class. | 21. No.of | send To:

. (Repo: >~ . Pages So,
“1 Se vigyCt 5 | pmo | MATERAEROURGEBSRiTinc zonMarion cenren
[0 (Page) ~ WASHINGTON. ©. C. 30240

Abstractor Tastitution



The second experiment (Table 8) was carried out under the same conditions as the
first with three exceptions: 1) light was not varied but.was at moderate intensity
for 12 hours a day, 2) animals captured in the field were used rather than labora-

tory-cultured animals, and 3) the temperatures were somewhat different.

As can be seen from Table 8, no reproduction at all was observed for animals main-
tained at approximately 10° C, although substantial numbers of clutches were pro-
duced at the other three temperatures used in this experiment. Comparing the clutch
production at the three higher temperatures, there were many more clutches produced
at 29.6° than at 26.2° or 22.0°C, The average clutch size and average percent
hatch were also highest at this temperature, although the differences are not as sub-
stantial as with the numbers of clutches. Further, the average number of days the
clutches were carried and the average number of days for development to an adult
were lower at 29.6° C than at the other temperatures. Overall there seems substan-
tial indication that 29.6° C aided reproduction and maybe development and thus that
this temperature should be good for culturing.

A contrast to the foregoing results is evident in Table 7. The most obvious difference

is that, even though the two experiments were of approximately equal duration, many
fewer clutches were produced in the Table 7 experiment. The reason for this is not
immediately obvious, However, the two experiments were conducted at different times,
and it is suspected that differences in the quality and, toa lesser extent, quantity of
the food cultures at the different times may have played an important role in causing

the differences in reproductive rate.

In this latter experiment the lowest temperature is no longer 9.7°, but is 17.2°C.
Reproduction took place at this temperature, although the clutch carrying times show
indication of being longer at this temperature than at the higher ones. Also it will
be noted that the highest temperature is almost two degrees higher in this experiment

than in the other one. Clutch production and percent hatch show indications of being

-22—-


Table 55, CLUTCH SIZE IN RELATION TO AGE AND TO TEMPERATURE. THE VALUES FOR THE FIRST

CLUTCHES ARE BASED ON THE AVERAGE OF THE FIRST AND SECOND
CLUTCHES PRODUCED BY ALL FEMALES WHICH HAD FOUR OR MORE
CLUTCHES, VALUES FOR THE LAST CLUTCHES ARE BASED ON THE
AVERAGE OF THE NEXT TO LAST AND LAST CLUTCHES PRODUCED
BY ALL FEMALES WHICH HAD FOUR OR MORE CLUTCHES. THE

VALUES FOR ALL CLUTCHES ARE THE MEANS FOR ALL
FEMALES WHICH HAD AT LEAST ONE EGG CLUTCH.

Temperature °C Mean SE= on Mean SEZ on t,

First Clutches Lost Clutches

4 51.5 6.63 8 31,0 4,97 8 2,47*

21 55.8 4.61 18 32.5 3.74 18 3.95**

26 39.4 3.40 20 22.2 2.80 20 3.90***

31 39.3 2.34 22 22,5 1.91 22 5.55***
First Clutches All Clutches

4 51.5 6.63 8 39.5 3.47 27 T.64ns

21 55.8 4.61 18 46.8 2.75 65 1.57ns

26 39.4 3.40 20 30.1 2.08 66 2.18*

31 39.3 2.34 22 31.0 1.52 76 2.68**
Last Clutches All Clutches

4 31.0 4.97 8 39.5 3.46 27 1.23ns

21 32.5 3.74 18 46.8 2.75 65 2.55*

26 22,2 2.80 20 30.1 2.08 66 1.95ns

31 22.5 1.91 22 31.0 1.52 76 2.82**

ns Not significant at P < 0,05
* Significant at P< 0.05.

‘** Significant at P< 0.01.
*** Significant at P< 0.001,

-157-


¥,

NUMBER

ADULTS
PER
LITER

-ezl-

18

17+

16,
Ub

1

0
19
{

1
Mt

2
IV

4
DATE

19 63 #14
VoOVEVILVIIEXE X

Figure 15, Mean Number of Adults Per Liter In the Open Water Region on Each Collect=
ing Date. The Vertical Line Between 18 April and 24 April Shows a Change

in Scale from 2-Day to Approximately 14-Day Intervals.


No attempt was made to isolate the immature animals ona daily basis during any
experiment since the total number of young involved precluded such a procedure.
Instead, when information on the young was gathered at all, a total count as well

as an estimate of the developmental stage of each animal was made by observing the
animals in well-lighted surroundings. The estimates of developmental stage were
made on the basis of differences in total body size and appearance and not on micro-

scopic examination of specific morphological features.

The animals were fed from mixed laboratory cultures which had been seeded initially
with water and contained organisms from the pond located in Township 9 north. The
cultures were maintained in two 2-gallon aquaria. A small number of ammonium ni-
trate granules were added at infrequent intervals, These aquaria were maintained at
approximately 26° C and under moderate illumination. The liquid used for feeding
was obtained by removing a volume from one of these aquaria and filtering it twice
through a piece of No. 25 silk bolting cloth. Some consistency in the amount of
material being added at each feeding during an experiment was maintained by using
distilled water to adjust the concentrations of the feeding solutions to a constant light
transmittance (85%) in a Hach Photometric Colorimeter. No qualitative determina-
tions were made of the organisms present in the feeding liquid; but periodic, cursory
examinations revealed that there were several types of algae and protozoans present.
As the water in the experimental containers was usually not changed during the ex-
periments, food organisms obviously must have reproduced in the containers. Thus,
while approximately the same amount of food culture was added fo each container
during an experiment, different amounts of food may have been available in the dif-
ferent containers, especially later in an experiment, because the separate containers
had experienced differences in the growth and reproduction of their endogenous food

cultures.

The constant temperature rooms and chambers in which all of the experiments were

conducted had automatic controls that permitted independent regulation of both light


Table 15. FEMALE LIFE SPANS AFTER SEXUAL MATURITY IN
RELATION TOINCUBATION AND HATCHING TEMPERATURES

Female's
Incubation Mean hatching Mean Days survival
temperature (days) temperature (days) after sexual
(degrees C) ts... (degrees C) =S.E. Individual | maturity

14 103 A7 103

21 82 AG 82

14 84.6 27 —— = —

+ 7,84 AS 72

31 79.3 Ag 102

= 11.57 Alo 64

14 — — ---

BIT a

BI2 56

BI4 16

21 38.9 BI7 27

21 43.2 + 6.86 B18 38

+ 5,57 BI9 22

B20 49

27 51 BIS 51

31 54.5 BI3 41

= 13.50 BIS 68

14 — -- ---

21 17 co 17

C7 3T

27 27.0 ce 45

4,77 27 30.3 C13 21

# 5.79 cl4 16

C15 26

Clé 23

31 17 “Chl 17

14 — o— —

21 25 Ds [25

27 16 D5 16

31 49.2 D3 16

= 2.45 DI 13

31 21.5 DI6 31

+ 1,32 DI7 iW

DI9 17

D20 25



NUMBER OF QUADRATS IN THAT REGION AND
SUMMING THE PRODUCTS FOR EACH DATE,

APPENDIX E-1

TOTAL NUMBER OF ADULTS (CVI) IN EACH REGION OF THE POND AND IN
THE TOTAL POND ON EACH COLLECTING DATE. THE TOTAL NUMBER
WAS DETERMINED BY MULTIPLYING THE Y (MEAN) NUMBER OF

ADULTS PER QUADRAT IN EACH REGION BY THE TOTAL

Date Open water Rooted aquatics Total pond
region region
-1971
9 1,052,454 263,113 1,315,568
21 300,651 346,905 647,556
23 1,295,112 127,198 1,422,556
25 1,086,969 312,214 1,399, 183
27 778,387 247,668 1,026 ,056
W-1971
1 648,656 342,018 990,675
3 1,130,623 256,430 1,387,053
5 1,002,468 554,306 1,556,775
7 813,768 754,800 1,568,568
9 1,002,860 146,122 1,168,982
WV 802,295 281,005 1,090,301
13 1,067,820 438,368 1,506, 189
15 1,047,897 379,026 1,426,924
7 1,696,500 70,687 1,767, 187
9 1, 189,906 294,531 1,484,437
21 1,121,267 199,829 1,321,096
23 735,888 801,789 1,537,678
25 1,521,848 76,092 1,597,940
27 1,114,793 454,575 1,569, 369
29 366,308 1,009,878 1,376, 264
31 1,083,947 139,517 1,223,465
V-1971
2 1,251,705 13,887 1,265,593
4 668,839 89,326 758, 166
6 1,068, 160 161,210 1,229,371
8 1,526,877 26,665 1,553,543
10 1,749,859 32,682 1,782,542

-206-


inverse correlation, Apparently, quantity of food was not a limiting factor during

the early spring season, whereas it may have been during the remainder of the year.

Food quality (e.g. species of algae) present would be important in determining the
valve of chlorophyll as an indicator of available food. In the spring of the year
filamentous Spirogyra and colonial Volvox were the dominant genera numerically,

while in the summer Ankistrodesmus and Scenedesmus were the dominant algal genera.

If the filamentous and colonial algae were of such size that the filter-feeding diap-
tomids could not ingest them, they would be of no food value to these animals; even

though the measured quantity of chlorophyll might have been high.

A second problem relating to food was suggested by the laboratory study on hetero-
geneity of distribution where it appeared that materials such as detritus and protists
might be sources of food. Since the actual food source of D, clavipes is not known,

placing too great a value on chlorophyll content alone is tenuous.:

Several investigators, including Comita and Anderson (1959), Corkett and McLaren
(1969), and Chapman (1969), have suggested that the mean number of eggs per clutch
in copepods is correlated to the size of the female. They further state that, since the
size a female attains is inversely correlated to the temperature at which she develops,
temperature is an important indirect factor in determining the mean number of eggs
per clutch. In the current study a significant inverse correlation between tempera-

ture and mean number of eggs per clutch was also found.

Figure 14 shows the mean temperatures and the ranges on the various dates of this
study, At the onset of the study,the temperature was below the lower limit for suc~
cessful reproduction as determined in the laboratory (Chapter 3), The temperature
steadily increased, however, until 18 August when the yearly maximum of approxi-
mately 27°C was reached. After this date the temperature steadily decreased until

the termination of the study.

-119-


The life table from the full year furnishes further evidence that the duration estimated
for the adult stage is relatively accurate. A successful population having an annual
reproductive period, no emigration or immigration, and numbers that are in part de-
termined by the cycle of available food should have approximately the same number
of individuals alive at the beginning of the reproductive period in one year as the
next, provided that the available environment remains the same, The intrinsic rate
of natural increase (Birch, 1948), or r, for such a population would be expected to
approach 0 for the complete year. When r was calculated from the full year's data
using a 40-day duration for the adult stage and the average m, (number of eggs/adult/
day) value for the year (see Figure 10, page 108), the result obtained was -0.03
(Table 35), surprisingly close to 0 in view of the limitations of the method employed,

The life table prepared from laboratory data (Table 34) shows that the mortality rates
of the egg to NIII stage and the NVI stage were similar, with 281.51 and 284,58
deaths per 1,000 individuals, respectively. The mortality rates for the early stages
were higher than those for the copepodite stages except CV. The mean life expec-
fancies for the copepodite stages were all higher than those for the early stages, with
values ranging from 20.00 days for the CVI instar to 24.19 days for Cl and 24.58 days
for Cll. The life expectancies for the early stages ranged from a low of 12.92 days
for the egg to NIII stage to 18.37 days for the NVI instar.

Comparison of the three life tables reveals several interesting points. Although the
three tables have similar trends in their mortality rates with the egg NII and the
NVI stages having the highest mortalities, the table derived from laboratory data shows
almost the same mortality rate in the egg to NIll as in the NVI stage; whereas, in the
tables derived from field results, mortality is far greater in the egg to NIII stage than
in the NVI stage. Another difference between laboratory and field derived mortality
is in degree. Approximately 28% of the eggs perished in the laboratory, while over
84% of all the eggs produced in the field and almost 84% of the eggs produced by the

overwintering females failed to develop to NVI.

-103-


Figure 9 provides plots of the 1 curves for the first generation, laboratory popula-
tion, and complete year's data thus allowing visual comparison of survivorship for
the three groups. A definite similarity in form exists among the three curves with

the greatest survival occurring in the laboratory population, If the survivorship curve
of the laboratory population is considered to show the survival for the various stages
without the effects of a natural environment, then the differences between this curve
and the two field derived curves can be considered as rough indications of the ef-

fects of environmental factors.

The forms of the three survivorship curves range from a near diagonal curve for the
immature stages of the laboratory animals (death occurring independent of age) to
the positively skewed curve for the instars of the two field populations (high early
mortality). It appears that a negatively skewed curve characterizes the adult stage
of each group with those animals reaching adulthood surviving until they die of old
age. This idea is supported by three pieces of evidence:

1. The adult’ population underwent relatively sharp declines in numbers,
e.g., on 18 April and 28 May, which are assumed to have been the
result of more or less synchronous death of a large segment of the adult

population;

2. Ther value calculated for the entire year was found to be close to
0.0 when a 40-day duration was assumed for the adult instar (Table

35);

3. The laboratory results show a life span close to that calculated from
the field work (Table 15).

Life tables are valuable aids in determining general characteristics of animal popu-
lations. They also aid the investigator in determining those stages of the life cycle
which have the highest mortality rates. This is of value to the individual studying
pest species in that he can, through use of this approach, determine the weak points
of the life cycle; and he can concentrate on increasing the mortality rates in these

stages. If one is interested in studying the population dynamics or productivity of a

-105-


FIGURES
New Poge
1. Mean Clutch Sizes of Groups of Females Ranked From Lowest to High

est and Plotted Against the Temperature at Which the Females Were In-
cubated. cc eee cece tec cette erent ree tarecratcenes OZ

2. Mean Total Numbers of Clutches Ranked From Lowest to Highest and
Plotted Against the Temperature at Which the Females Were Incubated.. 56

3. Mean Total Numbers of Eggs Ranked From Lowest to Highest and Plotted
Against the Temperature at Which the Females Were Incubated....... 58

4. Mean Percent Hatch Ranked From Lowest to Highest and Plotted Against
the Temperature at Which the Females Were Incubated..+++-+eee++ 70

5. Mean Numbers of Adults Per Liter in the Two Regions of the Pond From
19 February to 4 April, 1971 2. ese c cere creer er eeeercceece 87

6. Mean Numbers of Adults Per Liter in the Two Regions of the Pond From
6 April fo 29 October, 1971... eee e cece cece eee e cee eceece BB

7. Proportion of Total Water Volume of the Pond Which Was in the Open
Water Area and Proportion of the Total Number of Adults Which Were
Collected in the Open Water Region on Each Collecting Date.....-. 89
8. Size of the Adult Population on Fach Collecting Date ...----+eeee 95

9. Survivorship Curves for the First Generation (g;), Laboratory Popula~
tion, and Complete Year's Data... esse cere ever ccc ecccccce 106

10. Specific Birth Rates (m,) as Calculated for Each Collecting Date and
the Weighted Mean m, Value for the Entire Reproductive Year......- 108

11. Proportion of Adult Females Carrying Eggs on Each Collecting Date.... 112

12. Ratio of Males to Females in the Adult Population on Each Collecting

Date... cece cece e erence cece eee ee eerererecsceee 4

13, Mean Number of Eggs Per Clutch on Each Collecting Date......... 115

14. Mean Temperature and Range on Each Collecting Date....-.-+.+.+- 120

vi


every two weeks from 24 April until the termination of the study. The intensive col-
lection period was instigated to determine the duration of the instars (Comita, 1956)
and to allow the development of a horizontal life table (Deevey, 1947) for the first

generation.

In analyzing reproduction it was the intent of this study, not only fo follow and
describe the various reproductive parameters, but also to relate variations in these
parameters to temperature and food. Thus, at the same time that samples of animals
were collected, the vertical temperature profile of the water was determined. Tem-
perature readings were taken at 0.5-m depth intervals from surface to bottom in the
open water region with a Whitney Underwater Thermistor.

Chlorophyll a concentrations were used as an index of food availability. Water for
chlorophyll a determinations was gathered at 2-week iritervals at the same time
animals were collected. Five hundred milliliters of water was collected from both
the surface and bottom of the open water region of the pond through use of a Kem-
merer water sampler, The two samples were combined, the water was returned to
the laboratory, and chlorophy!! a determinations were made following a modifica~
tion of the procedures outlined by Small (1961). After shaking the water sample to
mix it thoroughly two 200- or 300-m! subsamples were poured into separate 300-ml
beakers. Which of these two amounts was used was determined by the turbidity of
the water~- high turbidity, a.200-m! subsample, low turbidity, a 300-ml subsample.
Each of the subsamples was passed through a membrane filter apparatus which con-
tained a 0.8-micron (average pore size) filter to concentrate the phytoplankton.

The filter was then removed from the apparatus and placed in a 50-ml test tube.

The test tube was then capped. A duplicate procedure was follawed using tap water
rather than pond water in order to provide a control, Ten milliliters of 90% acetone
were added to each test tube. The tubes were capped and placed in a constant tem-
perature water bath (21°C) for 30 minutes. After 15 minutes the tubes were lightly

shaken to aid mixing of the materials. When the 30-minute incubation period was

-82=


TABLE 9 (Continued)

3

S

B | ad <@ ~F oF aF aF LF a a aq

me oR OF OTF Or Ber ORF CH NH —-H OM-

S 2° °E “HE “EH MWR Sh SH - £

5

=

eo)

c = =>

Se] sh ae ef aS aS eG +F oS xT KF

851-9 -9g -g -9 -9o K-F KF COP OF

ge) "2 "hf "EL "EE CH CH LX

e

se| 92 22 ST CF BR OF OR QT eH ag

a}; woo ine ne -_

o2| °@ PH WE PH “LE WH “HR PH ME OR

33] 33 25 33 33 58 38 38 a= ae eT

z NOS -—-9 Sf CH CH Cf SCP Cf Cf OC!

s2| “8 "HR CR CR CR CH CH MT CH OL
Ee & EE § FE FEE GE

els = § §$§ 2 2 @# $ $ 8

[>]

a > 8 F 5 fF &® # & § 8

-27-


CHAPTER 6

THE INFLUENCE OF TEMPERATURE ON THE REPRODUCTION
OF CYCLOPS VERNALIS

If we are to protect the quality of our aquatic environments, we should possess a
thorough understanding of the environmental relations of the common aquatic organisms.
The cyclopoid copepod Cyclops vernalis Fischer is one of the most aburdent and widely
distributed planktonic forms in North America, occurring over most of the continent
and ina variety of habitats (Yeaiman, 1944), Thus, it was deemed important to in=
crease our knowledge concerning this form. Such added knowledge should aid our
understanding of the ecological relations, not only of this species, but of planktonic
cyclopoid copepods in general.

The development of a dependable culturing method reported in the preceding chapter
was the first step in this work, Using these methods experimental studies on environ-

mental relations could be conducted.

A number of studies, including those of Ewers (1936), Andrews (1953), Roen (1955),
Elgmork (1959),and Armitage and Tash (1967), have suggested temperature as a very
important influence on cyclopoids, We have conducted a study whose primary ob-
jective was to investigate the influence of this factor on certain reproductive attributes

of C, vernalis. The results from this study are presented in this chapter.
METHODS AND MATERIALS
Stock Cultures

Stock cultures of Cyclops vernalis were set up in | -liter beakers at temperatures of
14°, 21°, 26°, and 31°C, All animals used to begin these cultures were collected

-147-


“vil-

M/F

aL 1 1 L

4 n
2 4 11 9 6 3 14
IV v vi ovil VEE IX x

DATE

Figure 12, Ratio of Males to Females in the Adult Population on certain Collecting
Date. The Vertical Dashed Line Between 18 April and 24 April Shows
a Change in Scale from 2-Day to Approximately 14-Day Intervals.



1.0 b \ 4
'
1
1
09 F ! 4
1
1
i
0.8 H 4
i)
1
i}
1 4
0.7 |
i]
1
0f6 + 4
NUMBER i
OF i
ADULTS 9.5 - \ 1
PER \
& 1
$ LITER |
0.45- f +
1
1
i
i}
0.3 - © RATIO OPEN WATER VOLUME \ 7
TO TOTAL VOLUME !
© RATIO ADULTS IN OPEN WATER !
0.2 + TO TOTAL ADULTS ! 4
1
1
Orb {
1
.
0 225 1 913 7 21, 2529.2 610, 14,1824 18? 6 FF

1
19 23°28 37 «W115 19 23 27 31 4 8 12 16 20 4 28°25 23 20 17 14 28
i Ty v VI VIL VIE IX x

DATE
Figure 7. Proportion of Total Water Volume of the Pond which was inthe OpenWater Area and Proportion of the Total Number
ofAdults which were Collected inthe Open Water Region on each Collecting Date. The Vertical Dashed Line Between
TA Faw

AN Anetl and OA Anetl Chain a Charman In Canta fone DF Revs ta Annvavinntaly 1 Mnbaes le


few individuals could be supported. What seemed to occur was that the populations
fluctuated to a certain extent, and the carrying capacity of these containers was so
low that these fluctuations often resulted in the populations being completely wiped
out. Obviously, after extinction the population could not recover even though the
food being added might actually be capable of supporting a few individuals on the
average. Thus, although it was concluded in a previous subsection dealing with con-
tainer characteristics that 1000-m! containers are satisfactory for culturing, this is

really only true when food conditions are favorable.

The populations supported by additions of yeast to containers kept in the light or of
Chlamydomonas did not all go to extinction during the experiment as did the mixed
culture ones. Both materials seemed to serve as quite good food sources, Chlamydomonas
may possibly have been somewhat superior as none of its populations had reached zero

by the end of the experiment, while two of the culfures supported on yeast in the light

had done so.

The relatively satisfactory culturing results with these two materials raised a question
as to how much the success was due to the material added serving directly as food and
how much the success was due to this material acting as a nutrient source for the growth
of mixed cultures of food organisms in the culture beakers themselves. The beakers
which received yeast as a food but were maintained in the dark were added to the ex-
periment in an attempt to gather some information concerning this question. It was
reasoned that with light excluded growth of food microorganisms within the cultures
would be greatly reduced, and so any reduction in copepod population development
in the dark containers compared with the results for the populations fed yeast and

kept in the light should indicate the effects of the in situ growth. Such a compari-
son (Tables 9 and 10) shows large differences, The populations kept in the dark but
fed the same food as the populations kept in the light did much poorer than the latter
populations. By the end of the experiment only one of the five dark populations still
had any copepodites. Thus, as expected, it appears that in situ growth plays a major

part in providing food in the containers kept in the light.