ra

e@———® Chydorus sphaericus
@----© Cyclops bicuspidatus thomasi
@----© Simocephalus sp.

‘—'—" Ceriodaphnia pulchella

LITER

NUMBER

A 1 pe,
t—t+—
t—+—+t lo? tt OT oat to
WEEKS
Figure 2. Changes in population density (No./liter) in a mixed

zooplankton culture containing Chydorus sphaericus.


III. Methods for Cyclops bicuspidatus thomasi.

1.

Brood stock - C. bicuspidatus thomasi adults can usually be
obtained from field populations from temperate lakes. Since
this species is quite adaptable to a variety of environmental
conditions, it may be found at varying periods during the
year. We collected most of the brood stock for our experiments
during the winter and spring.

Cultures should initially be stocked with 50 mature C.
bicuspidatus thomasi per liter (approximate male-female ratio

1). ese plankton are relatively easy to handle and may
be transferred from one container to another with a pipette.
Food - Cultures of Paramecium multimicronucleatum should be
used as the food source for the C. bicuspidatus thomasi
cultures. Each of our C. bicuspidatus thomasi was fed three
times a week at a rate of 1,000 P. multimicronucleatum per
liter per week.

The protozoan cultures can be grown in 1 or 2 liters of
synthetic medium containing Cerophyl] (1 gram per liter)
inoculated with Enterobacter aerogenes. These cultures should
be maintained in subdued Tight at 25° C. To ensure that
abundant supplies of protozoans are available, new cultures
should be started periodically.

Since densities of protozoan cultures (under these
conditions) are quite variable, the quantity needed to feed
the desired number of organisms into the cyclopoid cultures
should be determined at each feeding. This may be done by
counting a 1 ml aliquot of the protozoan culture and
calculating the volume needed. duri iments
Temperature - Maximum standing crops during our experimen
were produced at 15° C (41° C).

33


LITERATURE CITED

Andrews, T.F. 1953. Seasonal variations in relative abundance of Cyclops
vernalis Fischer, Cyclops bicuspidatus Claus, and Mesocyclops
Teuckarti (claus) in western Lake Erie, from July 1946 to May 1948.
Ohio J. Sci. 53:91-100.

Balazs, G.H., £. Ross, and C.C, Brooks. 1973. Preliminary studies

on the preparation and feeding of crustacean diets. Aquaculture
2:369-377.

Burns, Carolyn W. 1968. The relationship between body size of filter-
feeding Cladocera and the maximum size of particle ingested.
Limnol. Oceanog. 13:675-678.

Coker, R.E. 1934. Reaction of some freshwater Copepods to high
temperatures. J. Elisha Mitchel] Sci. Soc. 50:143-159.

D'Agostino, A.S. and L. Provasoli. 1970. Dixenic culture of Daphnia
magna, Straus. Biol. Bull. 139:485-494.

Ewers, L.A. 1930. The larval development of freshwater Copepoda. Ohio
St. Univ. Press, Columbus. 43 pp.

1936. Propagation and rate of reproduction of some fresh-
water Copepoda. Trans. Amer. Micros. Soc. 55:230-238.

Gold, K. 1972. Growth characteristics of Tintinnida in continuous
culture. Preprint. 17th Int. Congr. Zoology, Monaco. 13 pp.

Keen, R. 1973. A probabilistic approach to the dynamics of natural
populations of the Chydoridae (Cladocera, Crustacea). Ecol.
54:524-534.

Lewis, B.G., S. Luff, and J.W. Whitehouse. 1971. Laboratory culture of
Cyclops abyssorum Sars, 1863 (Copepoda, Cyclopoida). Crustaceana
ABIES

McQueen, D.J. 1969. Reduction of zooplankton standing stocks by _
predaceous Cyclops bicuspidatus thomasi in Marion Lake, British
Columbia. J. Fish. Res. Board Canada 26:1605-1618.

Murphy, J.S. 1970. A general method for the monoxenic cultivation of
the Daphnidae. Biol. Bull. 139:321-332.

Provasoli, L., D.E. Conklin and A.S. D'Agostina. 1970, Factors inducing

fertility in aseptic Crustacea. Helgolander wiss. Meeresunters.
20:443-454.

29


Allen-Bradley “Sonic-Sifter": less than 37 microns; 37-53
microns; 53-75 microns; 75-90 microns; and 90-105 microns.

Stock solutions containing each size of each food were
prepared by adding known quantities to distilled water and were
kept frozen when not in use. Feeding solutions were prepared
fresh every week from stock solutions.

h) LIV (Farm and Wildlife Products, Inc.)

i) Trout Starter (Glencoe Mills Co.)

5) Salmon Starter (A preparation acquired from the
Gavins Point National Fish Hatchery).

LIV, Trout Starter, and Salmon Starter were ground into
powder in the "micro-mill"; but they could not be separated
into size categories in the "Sonic-Sifter" because of their
tendency to lump together. Instead, the powder was first added
to distilled water and the resulting mixture was passed through
a series of standard soil testing seives (W. S. Tyler, Inc.).
The same particle size ranges as those described earlier were
obtained. The quantity of food in each stock solution was
determined by filtering a sample onto a glass-fiber filter and
drying to a constant weight at 100° C. Feeding solutions were
then prepared fresh each week by diluting stock solutions
appropriately. The latter were stored frozen.

k) Bacterized - Cerophyll. This was prepared by boiling
1 gram of Cerophyl] powder for about 1 hr in 1 liter of distilled
water. After the solution had cooled to room temperature, it
was inoculated with Enterobacter aerogenes. This was the same
food that was used to culture Paramecium multimicronucleatum
that in turn was used for food for Cyclops bicuspidatus thomasi.
(See later section).

Each of these foods (a-k) were used separately, in various concentrations,
and in several combinations.

4. Medium - Hatchery pond water from which the organisms were
collected, and Missouri River water. The water was filtered
through a Millipore HA membrane (0.45-micron pore size).

Organisms could not be obtained from the field between October 1972 and
May 1973, and the experiments just summarized depleted the populations
in all but one of the mixed stock cultures. In June 1973 the decision
was made to terminate efforts for culturing B. longirostris. The
remaining culture containing B. longirostris was maintained until
November 1973. Zooplankton populations In this culture had been sampled
biweekly (between August 1972 and November 1973). Filtered Missouri
River water was added to maintain the culture volume at 2,000 ml.


ganisms expend more energy at 20° C, the 9 mg/wk of food was not sufficient ©
in these cultures. The uncertainty in interpretation of these results
points up the necessity of further experiments involving the interaction

of food supply and temperature. When these relationships are known

optimum conditions for mass culturing this organism will be more nearly
defined.

In spite of the obvious need for more work, a simple, reproducible, and
relatively efficient method for producing large numbers of C. sphaericus
has been found. The small size of the organisms make them appear desir-
able as larval fish food. Furthermore, repeated handling in the laboratory
did not result in any significant mortality of individuals. In contrast
to our experience with B. longirostris, air-locked C. sphaericus were
rarely observed. The low mortalities observed in laboratory cultures

under controlled conditions make this species appear particularly

desirable for bioassay test purposes.

16


TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)

T. REPORT NO. z [3. RECIPIENT'S ACCESSION NO.
EPA-660/3-75-010
4. TITLE AND SUBTITLE iS. REPORT DATE
Studies to Determine Methods for Culturing Three 2/75 (date of approval)

5. Pi
Freshwater Zooplankton Species 6. PERFORMING ORGANIZATION CODE

7. AUTHOR(S) 8. PERFORMING ORGANIZATION REPORT NO.
Dan B. Martin and Jerry F. Novotny

9. PERFORMING ORGANIZATION NAME AND AODRESS 10. PROGRAM ELEMENT NO.
North Central Reservoir Investigations 1BA021
U. S. Fish and Wildlife Service 11, CONTRACT/GRANT NO.

Yankton, South Dakota 57078 EPA-1AG-0152(D)

12. SPONSORING AGENCY NAME AND ADDRESS 13, TYPE OF REPORT AND PERIOD COVERED
. Final
National Water Quality Laboratory

6201 Congdon Boulevard 14, SPONSORING AGENCY CODE
Duluth, Minnesota 55804

18. SUPPLEMENTARY NOTES.

Te. ABSTRACT Studies to determine laboratory methods for culturing unispecific
populations of Bosmina longirostris, Chydorus sphaericus and Cyclops bicuspidatus
thomasi were carried out. These cultures are to provide a source of animals to be
used as live food for fish and as bioassay test organisms. B. longirostris was not
successfully cultured. High mortalities, apparently associated with the phenomenon
of "air-locking," always occurred during handling in the laboratory. C. sphaericus
was successfully maintained in relatively dense cultures (approximately 1,000 per
liter) using a mixture of dried foods, less than 37 microns in size. One-fourth
of the standing crop was harvested each week without apparently reducing the
production in the culture. C. bicuspidatus thomasi could be grown using both dried
food and live Paramecium multimicronucleatum as an energy source. However, the
latter resulted in higher standing crops. Total standing crop as well as the
proportion of each life stage in the population fluctuated greatly in the C.
bicuspidatus thomasi cultures. Both C. bicuspidatus thomasi and C. sphaericus were
grown at 15° C, at a light:dark cycle of 12:12 hours, and in a synthetic medium of
known chemical composition. C. sphaericus was recommended as being best suited

for live fish food and as a bioassay test animal.

17. KEY WORDS AND DOCUMENT ANALYSIS

ja. DESCRIPTORS b.IDENTIFIERS/OPEN ENDED TERMS |c. COSATI Field/Group

*Cladocera, *Copepods, *Invertebrates, Bosmina longirostris,

*Fish Food Organisms, Zooplankton, Cultures, Chydorus sphaericus,

Bioassay, Fresh Water Cyclops bicuspidatus 6
thomasi,

Method development,

Test animal

TS, DISTRIBUTION STATEMENT 19, SECURITY CLASS (This Report) 21. NO. OF PAGES
41 p.
20. SEGURITY CLASS (This page) 22, PRICE
—

EPA Form 2220-1 (9-73) # U.S. GOVERNMENT PRINTING OFFICE: 1975~698- 446/138 REGION 10


Abstract... 1... eee eee

List of Figures... .......0.

CONTENTS

List of Tables... 2... .....0.2.
Acknowledgments. ...... we ee
1. Introduction... ....... .

2. General Methods... ........
3. Bosmina longirostris........
4, Chydorus sphaericus. .... see
5. Cyclops bicuspidatus thomasi....
6. General Discussion and Recommendation
7. Literature Cited... ... .

8. Appendix. ..........0.4-


SECTION VI
GENERAL DISCUSSION AND RECOMMENDATION

An outline of the proposed methods for culturing C. sphaericus, and C
bicuspidatus thomasi will be given in this section along with some

other general observations relative to the use of these methods in future
studies. The methods outlined are not intended to be interpreted as
"optimum" culture conditions. Rather, these methods represent a degree

of success in attaining the most efficient means of culturing these species.

We recommend C. sphaericus as a live food for larval fish and as a test
organism in bioassay experiments. Large populations of this species can
be maintained on a relatively simple diet, under easily controlled
environmental conditions. Once established the cultures produce a fairly
stable standing crop. Test populations can be handled without significant
mortalities. In contrast to C. bicuspidatus thomasi, C. sphaericus

seems to have a rather limited range of tolerance to certain environmental

factors (e.g., temperature). This makes the latter species a more
sensitive experimental animal.

To culture C. sphaericus, one should use glass containers with a volume
of at least 2 Titers. The effects of container size were not tested in
our experiments but 2 liters is sufficiently large. A temperature of
15° C, a light:dark cycle of 12:12 hours, and the medium described by
Sheer and Armitage (1973) should be used. Food consisting of equal
proportions of LIV, brewer's yeast, and Cerophyl] (less than 37 microns)
should be added three times a week in a concentration equivalent to 4
or 5 mg/liter per week. One-fourth of the culture volume (along with
the organisms) should be removed each week and fresh medium added to
restore the original volume. These methods resulted in populations in
which all size classes (age groups) were constantly evident.

C. bicuspidatus thomasi was best cultured using live Paramecium sp.
cultures as food. About 1,000 of the protozoans/liter per week seemed

to produce maximum standing crops at 15° C. A light:dark cycle of 12:12
hours was satisfactory. Cultures should be maintained in glass
containers of at least 2-liter volume, in the synthetic medium of Sheer
and Armitage, 1973. One-fourth of the culture medium (and organisms)

was removed each week and fresh medium was added. Under these conditions,
the number of organisms produced will be more variable and less predictable
than those found in the C. sphaericus cultures. The proportion of each
life stage in the total Fopitation may also fluctuate greatly. These
factors plus the present inability to efficiently separate the various
life stages make this species less desirable as live fish food or for
bioassay test purposes.

In considering future work with zooplankton cultures, one of two directions
seems inevitable. The first involves the use of small cultures containing

27


containing 2 liters of filtered Missouri River water at 15°C. One
milligram each of LIV, Cerophyll, and brewer's yeast (all less than 37
microns particle size) was added to each culture three times a week.

The cultures were sampled once per week and fresh medium was added to the
container to maintain the 2 liter volume.

A summary of C. sphaericus produced in the stock cultures is found in
Table 1, Culture No. 1 was sacrificed to supply stock for another
experiment (to be discussed later). Culture No. 2 was discontinued in
order to supply stock for cultures at the National Water Quality
Laboratory (EPA), Duluth, Minnesota. The remaining two cultures were
maintained for 225 days. The average standing crop in the stock cultures
ranged from 1,624 to 2,268/liter. One-fourth of the standing crop was
removed each week, and it appeared that production was fairly well balanced
with the number harvested, since there were not large fluctuations in
standing crop after the populations became established. It can be
concluded that 200-300 individuals per liter per day can be produced in
large cultures under the conditions described for these stock cultures.
Time did not allow for additional experiments to determine how closely

the present values approach maximum production. Different rates of
feeding and harvesting, as well as different temperatures must be inves-
tigated to determine conditions for maximum yield.

Table 1. Summary of stock cultures of Chydorus sphaericus.

Average Average
harvested standing
Total (Number crop
Culture Duration number per liter (Number
number (Days) harvested per day) per liter) _
1 49 6,713 274 1,918
2 78 9,048 232 1,624
3 225 36,354 324 2,268
4 225 33,685 300 2,100

As soon as it became apparent that C. sphaericus could be grown in a
medium of Missouri River water, another experiment was started to determine
if a synthetic medium, of defined chemical composition, could be used in
place of the natural water. Synthetic water would have wide applicability
and may reduce variability in future experiments. The filtered Missouri
River water was compared with the synthetic medium described by Sheer

and Armitage, 1973. Treatments were set up in the following manner: (a)
Treatments 1A and 1B used filtered Missouri River water, while 2A and

2B used synthetic medium; (b) 1A and 2A both received food in the same

13


and (c) 2,000 Paramecium sp. per liter per week. The cultures were
sampled weekly. Fresh Missouri River water was added each week to main-
tain the volume at 2 liters.

At the end of 25 weeks no difference in the standing crops of C. bicuspidatus
thomasi could be shown among any of the three food treatments (Table a

Standing crops and composition for each life stage were comparable to
those found in the 10-liter stock cultures, and the large variations within
each 2-liter culture were also comparable to the larger cultures. No
conclusions could be drawn as to the precise quantity of food necessary

to produce maximum population densities. However, it was apparent that
1,000 to 1,500 Paramecium sp. per liter per week was sufficient under
these culture conditions to maintain maximum populations, and it is not

likely that greater quantities of food would result in greater standing
crops.

Table 5. Average standing crop and average percentage composition
for each life stage of Cyclops bicuspidatus thomasi in 2-liter cultures
fed three different quantities of Paramecium sp. for 176 days at 15° C.

“Culture A Culture B Culture C
Number Percent Number Percent Number = Percent
per of per of per of

Life stage liter total liter total liter total
Adults 112 34.2 84 22.7 90 26.7
Copepodites 160 48.8 168 45.4 150 44.5
Nauplii 56 7.1 118 31.9 97 28.8
Total 328 100.1 370 100.0 337 100.0

Culture A - 1,000 Paramecium sp. per liter per week
Culture B - 1,500 Paramecium sp. per liter per week
Culture C - 2,000 Paramecium sp. per liter per week

Another approach was also used in an effort to determine the amount of
food necessary for maximum production. Individual adults were placed in
a series of 50-ml containers with about 30 ml of filtered Missouri River
water. Different numbers of Paramecium sp. (from 10 to 100) were then
placed with each adults. It was assumed that the number of Paramecium
sp. needed for mass culture could be determined by knowing the number

Of Paramecium sp. consumed by an individual, on a daily basis. The
same food levels were set up at 10° and 20° C as it was further assumed
that food consumption would be greater at increased temperatures.
Observations were made every 4 hr on each culture, and the number of
Paramecium sp. remaining were counted.

19


Temperature had a marked effect on standing crop and population fecundity
(Table 3). There was at least one order of magnitude difference in both
the average number of individuals per liter and the average number
harvested at the two temperatures. The number of eggs produced by the
Populations followed the same trends. Temperature did not have any
apparent affect on the population age structure. The percent of the
total population with eggs was relatively constant at both temperatures,
indicating about the same proportion of juveniles and adults in all
cultures. The lower temperature did not seemingly impair the ability
of the individual animal to produce eggs. Both the average clutch size
and the ratio of eggs per animal appeared independent of temperature.

Table 3. The effects of two temperatures (10° and 20° C) on population

density and reproduction of Chydorus sphaericus. All cultures 87
days duration.

20° C 10° ¢
Replicate Replicate
A B A B
Total number harvested 3,784 2,530 234 262
Average harvested (Number
per liter per day) 86 58 6 6
Average standing crop
(Number per liter) 602 406 42 42
Percent of total population
with eggs 19.7 21.6 23.1 18.3
Average clutch size 2.1 2.1 1.6 2.2
Average standing crop eggs
(Number per liter) 250 187 14 7
Average eggs per animal 0.4 0.5 0.4 0.4
Total number eggs harvested 1,552 1,162 88 104

These results indicate that the lower temperature reduced the population
density by decreasing growth rates and increasing the time for develop-
ment of each stage in the life cycle. No evidence was found that the

lower temperature was detrimental to the individual animal. Keen (1973)
reported an egg duration time of about 60 hr at 20° C, and about 300 hr

at 10° C for Chydorus sp.

The average standing crop and the number of animals produced per day were
less for the 20° C treatments of this experiment than those observed in
the previous experiments at 15° C under similar feeding, conditions. It
is possible that 15° C is more near optimum, and 20° C is too warm for
maximum production. It is also possible that since the individual or-

15


one to several individuals. The second would make use of mass culture
techniques in which large containers would be used to produce thousands
of organisms, There is no doubt that the small cultures, containing a
single specimen, have yielded valuable information for a number of
species. These small cultures, where individuals are subjected to direct
observation, are particularly well suited for gaining certain kinds of
data, (e.g., the number of eggs produced by a single female). However,
after using these techniques over an extended period, this author
questions the application of such results to entire populations inhabiting
large and complex habitats. It seems-more desirable, where possible, to
use large cultures to test the effects of environmental variables (or
pollutants) on populations rather than on individuals. This approach

in culturing populations has worked particularly well with planktonic
algae, and it seems equally well suited to zooplankton.

28


for survival of this organism. Information on food supply is very
limited because there. was no way of determining exactly what the B. long-
irostris were utilizing in the mixed cultures, and there was no survival
in any of the experiments using defined food. It seems unlikely that
either quantity or quality of food was the major reason for mortality.
The concentration, size, and nutritional diversity of the food used in
these experiments covered a wide range of conditions, and seemingly
included those considerations reportedly important for other cladocerans
(Murphy, 1970; D'Agostino and Provasoli, 1970; Burns, 1968).

In spite of the difficulties encountered in the present study and the
reports received from the aforementioned investigators, there are still a
few reports in the literature of apparent success with respect to culturing
B. longirostris. Zhdanova (1969) reported several characteristics of
growth and development for B. longirostris and 8. coregoni in culture.
Semenova (1968) studied the effect of temperature on various life stages
of B. coregoni, and Burns (1968) included B. longirostris in her study of
the relation between food particle size ingestion and body size for
several cladocerans. These studies all had one thing in common. The
methods of obtaining and manipulating individuals as well as the actual
conditions of the culturing were poorly described. All the studies used
local natural water which contained a portion of the original particulate
matter (living and nonliving). The two Russian investigators used mixed
algal cultures (predominately Chlorella vulgaris) as food, and culture
vessels with a volume of 50 to 100 ml. Burns used 250 ml experimental
cultures and reported that a high percentage of the test animals failed
to ingest any of the experimental food. Careful study of these papers
failed to reveal any factors not thoroughly explored or considered in

the present experiments. However, long-term maintenance of individuals
under defined conditions was not the objective of any of these studies,
but they do suggest some degree of success at short-term maintenance.

On the basis of our experience it does not appear as if B. longiros-

tris is a suitable organism to use as live fish food orfor bioassay test
purposes. The requirements for large-scale culturing are unknown but it
now appears that these requirements, when known, will be more complex than
those for other common zooplankters. Unless the problems of air-locking
are solved, there will be large mortalities when individuals or entire
populations are manipulated experimentally. This factor greatly reduces
the desirability of the species, since high mortalities in the control
treatments of a bioassay tend to obscure the effects of the variable

being tested. These variable and extreme characteristics of mortality
would also complicate any attempts to regulate a constant and/or uniform
food supply to fish cultures. In the following section, a method is
described for maintaining relatively dense cultures of Chydorus sphaericus,
a similar organism.

10


The following steps were considered necessary to accomplish these objectives:

1. A method that can be considered “adequate" for maintenance of
a limited population must be developed.

2. The method must be simplified and improved, and the important
variables defined.

3. The optimum conditions for production must be determined.

Once suitable culture methods became available, they could also be used
to study the effects of certain environmental variables on the life
history and ecology of the organisms. Such studies were the secondary
objective of this project, however, most of the effort was spent on
evaluation of mass-culture methods. The degree of progress varied with
each of the three species, and additional data are needed in several
areas. It is hoped that the information in this report will serve as a
starting point for future efforts in culturing zooplankton.


C, bicuspidatus thomasi are knownto be bisexual animals with no reports

of parthenogenesis. Copulation was observed several times in these studies.
In this process, the male grasped the female in the region of the 4th

or 5th metasomal segment with his antenule claspers. The joined pair
remained attached for 2 to 15 minutes. In our experiments, an unfertilized
female was placed in a small chamber with one mature male. After
copulation, the male was removed. On most occasions this resulted in
viable eggs within 1-3 days following copulation (temperature 15° C).
After the first clutch hatched, viable eggs continued to be produced until
4 to 7 clutches hatched. If no further contact with a male was permitted
the female would continue to produce unviable eggs. These eggs remained
with the female for varying lengths of time, but eventually deteriorated
and were dropped. As many as six unviable clutches were produced but

the number of eggs usually decreased each time. Ewers (1936) found 3

to 4 consecutive clutches of viable eggs were formed after contact with

a male and females would continue to produce ova in the absence of

sperm in the seminal receptacle.

Development time of each life stage was temperature dependent (Table 9).
The duration of the egg incubation varied from an average of 8.3 days

at 5° C to 2.1 days at 20° C. Development through the naupliar stages
averaged from 37.3 days at 5° C to 8.0 days at 25° C, and for copepodite
stages, 70.4 and 19.4 days at 5° C and 25° C, respectively. Development
of an egg to an adult averaged 29.6 days at 25° C, and 116.0 days at

5° C. These developmental periods are similar to those reported by Ewers
(1930) who found that 28 to 35 days were required for completion of the
life cycle of C. bicuspidatus thomasi at 20° C, and Andrews (1953) who
reported an average 0 lays for egg to adult in field populations at
about 15° C.

Table 9. Development time (days) for each major life stage of Cyclops
bicuspidatus thomasi at five experimental temperatures. (A) Average,
(B) Standard deviation, (C) Number of observations.

Life stage 5° C 10° € 15° C 20° C 25° C
E. (A) 8.3 5.9 3.6 2.1 2.2
"8 (B) 1.2 0.9 0.6 0.3 0.7
es ee)  ? A 2

Naupliar A 37.3 29.7 . . .
{3} 6.4 5.5 5.8 0.7 2.0
ty 0 3 3 1 26 6 18 4

Copepodite A 70.4 58.5 43. . :
pep (B) 14.1 16.9 12.2 4.2 3.2

(c) 2 3 9 6 9
Total (A) 116.0 94.1 65.5 38.8 29.6

25


Table 7. Average standing crop and average percentage composition for
each life stage of Cyclops bicuspidatus thomasi in 2-liter cultures

fed different combinations of dried food (particle size 37-90 microns)
for 254 days at 15° C.

Ration No. 1 Ration No. 2 Ration No. 3
Number Percent Number Percent Number Percent

per of per of per of

Life stage liter total liter total liter total
Adults 36 25.9 10 11.4 15 21.7
Copepodites 47 33.8 53 60.2 21 30.4
Nauplii 56 40.3 25 28.4 33 47.8
Total 139 100.0 88 100.0 69 99.9

Ration No. 1 - LIV, Brewer's Yeast, Cerophy!1
Ration No. 2 - Tetra, trout starter, salmon starter
Ration No. 3 - Brewer's Yeast, Cerophyl], Tetra, trout starter

All of the rations were added at the rate of 9 mg per week

All three combinations of dried food supported a population of C. bicuspi-
datus thomasi for over 36 weeks (Table 7). The proportion of each Tife
stage in the populations was about the same as that observed in the
Paramecium sp.-fed cultures, but the total standing crops were lower.
Direct observations on individual animals in small containers indicated
that adults and late stage copepodites readily devoured particles of dried
food. When the particles were placed in the small containers they

would sink to the bottom. Eventually, the animals would search the bottom
of the container and find the food. It was repeatedly noted that these
individuals seemed highly selective in regard to the size of particle
they would attack. Small particles were seldom taken. C. bicuspidatus
thomasi would select particles in a size range from about the maximum
size that they could ingest upwards to large particles. It was not
unusual to watch an individual spend several minutes struggling with a
particle that was several times larger than it could possibly ingest.

Another experiment was set up to demonstrate the effects of different
size food particles on standing crops of C. bicuspidatus thomasi cultures
(Table 8). Three 2-liter cultures were started with 100 adults each,

in filtered Missouri River water at 15° C. A combination of LIV,
brewer's yeast, and Cerophyl] (equal proportions of each) was added to
the cultures at the rate of 9 mg/week. Cultures were sampled once a week.

22


TABLES

Summary of stock cultures of Chydorus sphaericus

A comparison of Missouri River water (MRW) and synthetic

medium (SM) for culturing Chydorus sphaericus at two
levels of feeding

The effects of two temperatures (10° and 20° C) on population

density and reproduction of Chydorus sphaericus. All cultures
87 days duration

Average standing crop and average percentage composition for
each life stage of Cyclops bicuspidatus thomasi in 10-liter
stock cultures. Cultures were maintained in 15° C and were
fed 1,500 Paramecium sp. per liter per week

Average standing crop and average percentage composition for
each life stage of Cyclops bicuspidatus thomasi in 2-liter
cultures fed three aS forent quantities of Paramecium Sp.
for 176 days at 15° C

Average standing crop and average percentage composition for
each life stage of Cyclops bicuspidatus thomasi in 2-liter
cultures at three experimental temperatures. Feeding rate was
1,500 Paramecium sp. per liter per week and duration was 210 days

Average standing crop and average percentage composition for
each life stage of Cyclops bicuspidatus thomasi in 2-liter
cultures fed different combinations of dried food (particle
size 37-90 microns), for 254 days at 15° C

Average standing crop and average percentage composition for
each life stage of Cyclops bicuspidatus thomasi in 2-liter
cultures fed different size particles of LIV, brewer's yeast,
and Cerophyl1. Culture temperature was 15° C, duration was
191 days, and 9 mg of food were added per week

Development time (days) for each major life stage of Cyclops
bicuspidatus thomasi at five experimental temperatures. (A)
verage, (B) Standard deviation, (C) number of observations

14

15

18

19

20

22

23

25


FIGURES

CHANGES IN POPULATION DENSITY (NO./LITER) IN A MIXED
ZOOPLANKTON CULTURE CONTAINING BOSMINA LONGIROSTRIS

CHANGES IN POPULATION DENSITY (NO./LITER) IN A MIXED
ZOOPLANKTON CULTURE CONTAINING CHYDORUS SPHAERICUS

iv

PAGE

12


SECTION IV
CHYDORUS SPHAERICUS

During attempts to establish unispecific cultures of B. longirostris, a
procedure for maintaining relatively dense populations of th ‘dorus
sphaericus was discovered. Numerous collections of mixed zooplankton
populations were brought into the laboratory during the summer and autumn
of 1972. These mixed assemblages were placed in 2-liter containers and
subsequently treated in a variety of ways. These were simply trial and
error methods of looking for a combination of conditions that would be
suitable for B. longirostris culturing and were also an application of
"enrichment culture techniques". Initially, from 4 to 12 species would
be present in a typical culture. As time passed, most of the species
would be eliminated and 1 to 3 species would remain. Usually, at the
end, the dominant species would be one cladoceran and one copepod.

The history of one of the cultures just described is shown in Figure 2.
This culture was started in August 1972 and was maintained for 66 weeks.
Originally, the culture contained: (a) C. bicuspidatus thomasi, 148/liters
(b) Simocephalus sp., 31/liter; (c) C. pulchelTa, 5/liter; and (d) C.
sphaericus, 2/liter. Initially, the medium was unfiltered hatchery pond
water. The culture was sampled biweekly. Filtered Missouri River water
was added to maintain the volume of 2 liters. No food was added for 14
weeks. A mixture of C. reinhardtii, C. ellipsoidea, and £. aerogenes

was added to the culture once a week from week-14 to week-30. At week-30,
these food additions were discontinued, and a mixture of LIV, Cerophyll,

and brewer's yeast (all less than 37-microns particle size) was added
three times a week.

C. pulchella was found only in the initial sample and at week-2.
Simocephalus sp. increased during the first 4 weeks, then rapidly declined,
and was not found after week-6. C. bicuspidatus thomasi persisted
throughout the life of the culture, fluctuating between 15 and 404/liter.
C. sphaericus fluctuated between 2 and 49/liter during the first phase

of the culture (the period during which no food was added). No apparent
trend in population density occurred during the second phase (when algae
and bacteria were added as food); as numbers during this period ranged
between 2 and 24/liter. Following the change to LIV, Cerophy11,. and
brewer's yeast, the population density of C. s haericus began to increase.
The rise continued until shortly before the culture was terminated. The
maximum population density reached was 1,098/liter. Similar results

were noted in several other mixed cultures containing C. sphaericus, and
it became evident that perhaps a method had been found for maintenance

of relatively dense populations of this organism.

Four unispecific, stock cultures of C. sphaericus were started in October
1973. . Ten to twenty mature individuals were placed in 4-liter vessels

VW


SECTION I
INTRODUCTION

Laboratory culture of zooplankton is still in the early stages of
development. At present, it seems as if most efforts have proceeded along
two divergent lines of investigation. The first is exemplified by the
work of Murphy (1970), Provasoli et al. (1970), D'Agostino and Provasoli
(1970), or Taub and Dollar (1964), in which the zooplankters are grown

in bacteria-free systems, under rigorously defined chemical and physical
conditions. Such studies are valuable in determining nutritional require-
ments of the animals or in determining certain physiological responses to
changing environmental variables. However, monoxenic culturing methods
are often tedious and do not lend themselves to the production of large
numbers of organisms for routine use. Furthermore, the culture conditions
are highly artificial with respect to the natural environment. In contrast
to these rigidly controlled culture techniques, organisms are often
cultured under conditions where many of the important environmental
properties are poorly defined. Typically, the medium employed is "pond"
or "lake" water. Often, the food consists of mixed algae, bacteria,
protozoa or detritus (both living and non-living). These methods are
useful for holding organisms for short periods of time for a variety of
test purposes. Over long periods of time, populations in such cultures
tend to fluctuate in an unpredictable (and often inconvenient) manner.
These methods are often difficult to repeat in other laboratories.

The purpose of this study was to develop methods for culturing Cyclops
bicuspidatus thomasi, Bosmina longirostris, and Chydorus sphaericus.
These three species were chosen because they are known to be important
foods for fish at first feeding (Siefert, 1972).

Culture methods are needed to provide:

1. An adequate and natural food supply for larval and juvenile fish
being used in chronic toxicology testing programs.

2. A-reliable supply of these zooplankters to be used for the
determination of water quality criteria (bioassay).

Specifically, the culture methods would have to:

1. Be capable of supplying a constant (and predictable) number of
organisms. .

2. Produce organisms that were morphologically and physiologically
equivalent to individuals found in natural populations.

3. Be well enough defined so that they could be reproduced by any
investigator.


SECTION V
CYCLOPS BICUSPIDATUS THOMASI

Two 10-liter stock cultures of Cyclops bicuspidatus thomasi were begun
in November 1972 by isolating mature males and females with eggs from
collections obtained from fish ponds at the Gavins Point National Fish
Hatchery. The cultures were maintained until March 1973, using filtered
Missouri River water. In March 1973 all of the C. bicuspidatus thomasi
were removed from one of the cultures and placed in a similar 10-liter
culture containing filtered Lake Superior water. All other conditions,
including food supply remained the same in both cultures. These two
different stock cultures were maintained from March 1973 until November
1973. In November 1973 all C. bicuspidatus thomasi were removed from
both cultures and placed two 10-Titer cultures containing the synthetic
medium described by Sheer and Armitage (1973) and were maintained until

termination of the study in May 1974. Temperature was 15° C throughout
the study.

Previous work had shown that mixed protozoan populations, grown in water
enriched with sheep manure, could be used as food for culturing C.
bicuspidatus thomasi. Therefore, pure cultures of Paramecium sp. were
at a rate of 500/liter three times a week to the stock cultures.
This resulted in a total of 15,000 Paramecium sp. per culture per week.

Paramecium sp. were grown in 1- or 2-liter batch cultures, using Cerophyl}
gram/liter) that had been inoculated with E. aerogenes. Cultures were

grown in subdued light at 25° C. Initially, effort was spent in trying

to perfect methods of growing Paramecium sp. in continuous cultures

(chemostat-type). Several modifications of the techniques described by

Gold (1972) were tried, but sufficient numbers could not be obtained.

Eventually, batch culturing techniques were relied upon entirely. The

quantity of Paramecium sp. used as food was determined by direct count

of a 1-ml sample in all experiments, as well as the stock cultures. A

j-ml sample was removed from a well mixed batch culture and the total

number of Paramecium sp. in the sample was counted. This number was

then used to calculate the volume of culture needed to obtain the desired

number of Paramecium sp. for feeding. A significant portion of the bac-

terized Cerophyll always accompanied the Paramecium sp. into the C.

bicuspidatus thomasi cultures at each feeding. This was considered .

lesirable as this bacterial population was probably utilized by the nauplii.

The 10-liter stock cultures were sampled biweekly. The average standing
crops of each life stage in the two stock cultures are shown in Table 4.
T-tests showed no significant difference between the two cultures with
respect to either the standing crops or the percentage composition of
the three-life stages, and no significant differences due to changes in
medium composition during the life of the cultures. The two cultures
appeared as part of the same population throughout their existence. The

V7


EPA-660/3-75-010
MAY 1975

Ecological Research Series



SECTION IIT
BOSMINA LONGIROSTRIS

Attempts to isolate Bosmina longirostris from field populations were begun
in July 1972. Two sources of specimens were found:

1. A shallow, marshy area near the headwaters of Lewis and Clark
Lake, comprising part of the area known locally as the
"Springfield Bottoms".

2. Several of the fish rearing ponds at the Gavins Point National
Fish Hatchery near Yankton, South Dakota.

Since these two areas are separated by only about 56 kilometers, and since
both receive water (and plankton stock) directly from the Missouri River,
no attempt was made to separate organisms from the two habitats in any

of the experiments. B. longirostris were abundant in the plankton from
both localities from mid-June until mid-September.

The first method used to collect and transport field populations of B.
longirostris has been used successfully for other cladocerans and copepods.
First, the zooplankton are concentrated by slowly towing a Wisconsin
plankton net (with removable bucket) for 10 to 20 meters. Without
completely draining the water from the bucket at the end of the tow, the
bucket is removed and the contents placed in an insulated 20-liter plastic
bucket containing water from the field source. In the laboratory, aliquots
are siphoned from the bucket and plankton are reconcentrated in the
Wisconsin-type plankton cups. This concentrate is then used as the
source for stock cultures. Stock cultures are started by removing 10 to
20 active adults with a small pipette, and placing them in 2,000 ml of

um.

After several attempts, it became apparent that B. longirostris cultures
could not be obtained in this manner. Virtually all of the specimens
collected would be air-locked and floating on the surface by the time

the laboratory concentrates were prepared. Repeated attempts to insure
that the B. longirostris never came in contact with the air-water interface
did not succeed. Observations made during these later attempts indicated
that shortly after collection, numerous B. longirostris would be swimming
normally in the water. When these organisms were Teft in any kind of
container, they would eventually swim to the surface and air lock.
Therefore the majority of the air-locked animals were not a result of the
mechanics of the procedure, but resulted primarily from this swimming
behavior.

Eventually, one modification of the method did allow organisms to survive
long enough to be placed alive in containers in the laboratory. This
involved putting about 10 liters of ice into the 20-liter bucket along
with the water from the field source. The change from 25-30° C to about


SECTION II
GENERAL METHODS

All experiments, unless otherwise stated, were carried out in Percival
Model I35L environmental chambers. Light intensity was the same for al]
experiments, and consisted of two General Electric, 20 Watt, cool white
fluorescent lamps per chamber. Photoperiod was constantly set at a 12:12-
hour light:dark cycle. Temperatures within each chamber were monitored
continuously with a Weksler, Type 12M3A5A recording thermometer.

The culture medium was varied from time to time, and these specific
conditions will be described later. However, frequent reference is made
to "filtered Missouri River water". This is water taken from the Missouri
River at Gavins Point Dam, diluted with distilled water to a total hardness
of about 50 mg/liter, and filtered through a "Millipore" Type HA filter
(0.45 micron pore size) before use.

Glass containers with a high surface to volume ratio were used for all
experiments. This was to provide for maximum oxygen exchange at the
air-water interface and reduce the possibility of oxygen depletion. Except
where noted, no aeration was provided in any of the cultures. Dissolved
oxygen was not monitored continuously in the cultures but periodic "spot
checks" were taken with a Yellow Springs Model 54 oxygen meter.

One method was used consistently throughout the studies for determining
the population density of organisms in the cultures. First, the culture
was gently stirred to disperse the organisms evenly, and then a 500 ml
sample was rapidly withdrawn. All organisms in the 500 ml sample were
counted and the result used to estimate the standing crop in the culture.


foods do not adequately satisfy the nutritional requirements of all life
stages of C. bicuspidatus thomasi. Such deficiencies, would result in
slower development, decreased reproduction or incomplete development at
One or more stages in the life history. All of the tentative presumptions
regarding the use of dried food in culturing C. bicuspidatus thomasi
represent areas where further research is needed.

In every phase of the work with C. bicuspidatus thomasi one problem that
constantly reoccurred was the predation by adults (and late-stage
copepodites) on nauplii. This was a variable that could not be controlled
or measured in any of the mass culture experiments. Considerable time
was spent in trying to devise a method for separating the adults from

the nauplii. A chamber was designed to keep the brood stock (adults)

in one section and the nauplii in another. Segregation was achieved by
size selection. The chamber consisted of an upright 3 liter cylinder
with a 175 micron mesh screen on the bottom. Below the screen was
attached a 500 ml cylinder and an outlet that was closed off by a piece
of rubber tubing and a pinch clamp. The adults were placed in the upper
section. A culture volume of about 2 liters was maintained and about
2,000 Paramecium sp. per liter per week were added to the upper section
for food. The culture was drawn down at various intervals (ranging from
daily to weekly) from the outlet at the bottom. In this manner, the

brood stock were retained in the upper section by the screen, but the
nauplii were drawn through the screen. Various volumes were drawn out
ranging from 500 to 1,500 ml.

These experiments were carried out over about a 6-month period, and at
best, the results were erratic. A few nauplii were harvested but the
number obtained was not comparable to the number produced. The amount
of Paramecium sp. added seemed to be in excess to the amount required by
the C. bicuspidatus thomasi since numerous Paramecium $2 would always
appear in the harvested portion of the culture. Nauplii were being
produced in the upper chambers, because frequent examination of the
brood stock showed a good population of adults (approximately 75-150/
liter) including ovigerous females. Probably the nauplii were consumed
shortly after hatching and most did not survive long enough to be drawn
through the screen and away from the adults. A continuous movement of
water through the screen may result in better survival of nauplii but
this technique was not achieved.

In addition to the mass culture experiments, the life history of C.
bicuspidatus thomasi was studied by observing individual specimens in
cultures of 10-100 ml volume. The medium in all of the small culture
experiments was filtered Missouri River water. Copepodites and adults

were fed Paramecium sp., and nauplii were given small portions of the
bacterized CerophyT] (from the Paramecium sp. cultures). In all experiments
food was considered to be in excess.

24


Table 8. Average standing crop and average percentage composition for
each life stage of Cyclops bicuspidatus thomasi in 2-liter cultures
fed different size particles of LIV, brewer's yeast, and Cerophyll. Culture

temperature was 15° C, duration was 191 days, and 9 mg of food were
added per week.

Less than
37_microns 37-90 microns 90-150 microns
Number Percent Number Percent Number = Percent
. per of per of per of
Life stage liter total liter total liter total
Adults . 2 10.5 20 10.5 28 16.1
Copepodi tes 2 10.5 78 41.1 34 19.5
Nauplii 15 79.0 92 48.4 2 64.4
Total 19 100.0 190 100.0 174 100.0

A t-test showed significantly lower (P>.05) standing crops for all three
life stages in the culture receiving food less than 37 microns in size
(Table 8). No significant difference was found between the standing
crops in the other two treatments. Therefore, food particle size was
shown to be an important factor in culture maintenance.

Although, C. bicuspidatus thomasi can be cultured with non-living foods,
standing crops in the cultures receiving dried foods have not reached
the levels obtained in the Paramecium sp.-fed cultures. Several factors
could be responsible. First, it appears that the animals must obtain
the non-living foods from the bottom of the cultures, since the larger
particles they prefer sink rapidly. This restricts the availability of
food since animals usually search for food in the open water portions and
along the side walls of the cultures. Secondly, the larger particles
break down rapidly. This problem cannot be solved simply by adding more
particles since too much organic matter will soon "foul" the cultures.
These problems, associated with the stability of prepared foods, have
recently been investigated by Balazs, Ross and Brooks (1973), who
experienced similar difficulties in mass culturing shrimp. Recently,
these investigators have made some progress in the preparation of water-
stable foodstuffs.

Another factor resulting in lower standing crops might be an increase in
predation by adults on nauplii in cultures without live food. This is
probable since, in the absence of Paramecium sp., nauplii would be left

as the only moving "natural" food source jn the cultures. The proportion
of encounters between adults and nauplii would be increased in the .
cultures without Paramecium sp. There is also a possibility that artificial

23


ACKNOWLEDGMENTS

The assistance of Merlin H. Bittner throughout this study is gratefully
acknowledged.

vi


manner as that described for the stock culture (i.e., 9 mg/week); and
(c) 1B and 2B received one-half the amount of the same foods. One hundred
mature individuals were placed in each 2-liter culture at 15° C. Cultures

were sampled every week and fresh medium was added to maintain a 2-liter
volume. The results of this experiment are shown in Table 2.

The synthetic medium appeared to be at least as good as Missouri River
water for culturing C. sphaericus. Population densities at both food
levels in the defined median were higher than those at either of the food
levels in the Missouri River water. This situation may have resulted from
an uncontrolled variable that appeared near the end of the experiment.
Apparently, the synthetic medium was more favorable for the growth of
algae. The contribution of the algae to the food supply of the C.
sphaericus is unknown, but it could account for the increased standing
crops observed in the synthetic medium. Despite the algae problem, it

was concluded that the artificial medium was suitable for C. sphaericus
and that it should be used in place of natural waters for future experiments.

The quantity of food added also had a measurable effect on standing

crops within each medium treatment (Table 2). The lower level of feeding
appeared less than that needed for maximum production. It still cannot
be determined if the upper level was sub-optimal or excess for this
temperature.

Table 2. A comparison of Missouri River water (MRW) and synthetic
medium (SM) for culturing Chydorus sphaericus at two levels of feeding.

Average Average
harvested standing

Total (Number crop
Culture Culture Food Duration number per liter (Number
number medium (mg/week) (Days) harvested per day) per liter)
1A MRW 9.0 63 7,030 224 1,568
1B MRW 4.5 63 3,696 118 826
2A SM 9.0 70 13,250 378 2,646
2B SM 4.5 70 8,334 238 1,666

The metabolism of poikilothermic animals is assumed to be temperature
dependent. Normally production increases with temperature up to an
optimum, above which increased temperature results in decreased production
and eventually death. The effects of 10° and 20° C on reproduction and
population growth were carried out with C. sphaericus. The experiments
were started by placing 25 individuals in duplicate 2-liter cultures.
Synthetic medium was used, and feeding was carried out as previously
described for the stock cultures. Cultures were sampled each week, and
all eggs within the brood pouches of adults were also counted.

14


I,

APPENDIX

Recommended Procedures for Batch Culturing Chydorus sphaericus and
Cyclops bicuspidatus thomasi.

1

Containers - Glass containers with a minimum volume of two
Titers and a high surface to volume ratio should be used.
Larger containers with proportionate amounts of food could
be maintained using the same methods and growing conditions.

Medium - Static cultures are reared in the synthetic medium
described by Sheer and Armitage (1973):

Compound Stock Solution Add Per Liter
MgS04:7 H20 36.97 g/1 1.4 cce/liter
NaHC03 12.6 g/1 6 cc/liter
KHCO3 6.4 g/1 1 ce/liter
CaClo:2 Hod 2.5 g/1 8.8 cc/liter
KoHPOg 8.7 9/1 0.1 cc/liter
NagSi03:9 H20 18.1 9/1 1 cce/liter

Photoperiod - The photoperiod should be a standard 12 hour
Tight-12 hour dark cycle. It may be desirable, when using
the cultures to feed fish, to adjust the light-dark cycle to
correspond to fish photoperiod. We found that two G.E., 20
watt, cool white fluorescent lamps produced satisfactory
illumination.

Oxygen requirements - No aeration is necessary as long as the
air-water interface is sufficiently large to provide maximum
oxygen exchange. Usually this can be accomplished by using

a container with a high surface to volume ratio; A periodic
"spot check" of oxygen content should be taken.

Maintenance - At least one-fourth of the culture medium
Should be removed each week along with organisms and replaced
with fresh synthetic medium. Our samples were removed with

a vacuum device after thoroughly mixing the contents of

the culture. Cultures were maintained in environmental
chambers in which we were able to control photoperiods and
temperatures.

31


Obtaining brood stock - A Wisconsin plankton net (with removable
bucket) can be towed through the water to collect and

concentrate the plankton. Taking care not to drain the bucket,
it can be removed and the contents emptied into an insulated
container containing water from the field source. This plankton
can then be transported to the laboratory. After reconcentrating
the plankton into a smaller container, desired species may

be removed using a small pipette.

II. Methods for Chydorus sphaericus.

1

Brood stock - The brood stock can be obtained from field
Populations of C. sphaericus. Usually a dense population of
this species can be found in shallow, temperate ponds during
the warmer summer months. Cultures should initially be stocked
with approximately 50 adults per liter.

Transfer and stocking cultures - Unlike some cladocerans, C.
sphaericus can be easily transferred from one vessel to another
with no adverse affects. This may be accomplished using a
dissecting microscope and glass pipettes.

Temperature - Optimum temperatures for culturing C. sphaericus
were not specifically determined, but cultures maintained at
15° © (+ 1° C) were more productive than those at 10° or 20° C.
Food - Food consisting of equal proportions of LIV, brewer's
yeast, and Cerophyll (less than 37 microns) should be added

to the culture at least 3 times a week. The concentration
added to the culture should be equivalent to 4-5 mg (dry weight)
per liter per week. Specific sized foods can be obtained from
stock supplies by first using a grinder to reduce the material
to powder and then separating the powder into size categories
with a micro-sifter. In our studies we used a micro-mill
(Chemical Rubber Co.) to grind the foods to powder, and an
Allen-Bradley "Sonic-Sifter" to separate the powder into various
size categories.

Stock solutions of the three foods were prepared by
adding known quantities to distilled water. Each week fresh
feeding solutions should be made from the stock solutions.
Feeding solutions can be maintained for one week in the
refrigerator without spoiling. However, stock solutions should
be kept frozen prior to use.

32


After two trials with this experiment, it was concluded that the adult
C. bicuspidatus thomasi would consume all the Paramecium sp. they
encountered within the first 24 hr; and this behavior was not related to
the temperature used nor to the original number of prey introduced.
Apparently, these C. bicuspidatus thomasi were feeding in excess of their
requirements for normal maintenance, and this made it impossible to
determine the quantity of food required.

Another experiment was set up to determine the effects of temperature on
standing crops of C. bicuspidatus thomasi in culture. Three 2-liter
cultures containing filtered Missouri River water, and fed 1,500 Paramecium
Sp. per liter per week, were kept at 10°, 15°, and 20° C. Each culture
was started with 100 adults and sampled weekly. Fresh medium was added
each week to maintain a volume of 2 liters.

No significant differences (t-test) in the average standing crops of any
life stage were found between the cultures at 15° and 20° C (Table 6).

A significantly lower average number of adults and copepodites was found
in the culture at 10° C (P>.05). However, there was no difference in the
standing crop of nauplii among any of the cultures. These results show
that there was a measurable shift in the population age structure at

10° C, with a higher proportion of juvenile organisms. This would be
expected, since development of the various instars would be slower at
this temperature (see later discussion on life history). Also, fewer
adults would result in less predation on the nauplii, and the ratio of
Paramecium sp. to adults would also be greatly increased. It appears
that Tower temperatures are more favorable for the production of nauplii,
and higher temperatures are probably best for overall production. Also,
large variations in standing crop and percentage composition of the
various life stages discussed for previous experiments were observed in
the 15° and 20° C treatments, but there seemed to be less variation in
the standing crops at 10° C.

Table 6. Average standing crop and average percentage composition

for each life stage of Cyclops bicuspidatus thomasi in 2-liter cultures
at three experimental temperatures. Feeding rate was 1,500 Paramecium
sp. per liter per week and duration was 210 days.

TO" C TS? C
Number Percent Number Percent Number Percent
per of per of per of
Life stage liter total liter total liter total
Adults 12 3.1 46 8.7 39 7.3
Copepodites 65 16.6 149 28.2 77 33.1
Nauplii 314 80.3 334 63.1 319 59.6
Total 391 100.0 $29 100.0 535 100.0

20


ABSTRACT

Studies to determine laboratory methods for culturing unispecific
populations of Bosmina longirostris, Chydorus sphaericus and Cyclops
bicuspidatus thomasi were carried out. These cultures are to provide a
source of animals to be used as live food for fish and as bioassay test
organisms. B. longirostris was not successfully cultured, High mortalities,
apparently associated with the phenomenon of "air-locking", always
occurred during handling in the laboratory. C. sphaericus was successfully
maintained in relatively dense cultures (approximately 1,000 per liter)
using a mixture of dried foods, less’ than 37 microns in size. One-fourth
of the standing crop was harvested each week without apparently reducing
the production in the culture. C,. bicuspidatus thomasi could be grown
using both dried food and live Paramecium multimicronucleatum as an

energy source. However, the latter resulted in higher standing crops.
Total standing crop as well as the proportion of each life stage in the
population fluctuated greatly in the C. bicuspidatus thomasi cultures.

Both C. bicuspidatus thomasi and C. sphaericus were grown at 15° C, ata
light:dark cycle of 12:12 hours, and in a synthetic medium of known
chemical composition. C. sphaericus was recommended as being best suited
for live fish food and as a bioassay test animal.

This report was submitted in fulfillment of Grant/Contract EPA~IAG-0152(D)
by the U.S. Fish and Wildlife Service, Yankton, S.D. under the (partial)
sponsorship of the Environmental Protection Agency. Work was completed
as of June, 1974.

ii


The history of this culture is shown in Figure 1. Initially, the culture
contained: C. bicuspidatus thomasi, 170 per liter; Simocephalus Sp., 107
per liter; Diaptomus ‘Sp., 2 per liter; Ceriodaphnia ulchella, 72 per liter;
Pleuroxus denticulatus, 7 per liter; and B. Ton Trostris, 5 per liter.

The original medium was unfiltered pond water (rom the source of the
organisms), and no food was added during the first 14 weeks. From week-

14 to week-30, a mixture of C. reinhardtii and C. ellipsoidea was added
once a week. During the remaining 30 weeks, additions of algae were
discontinued, and a mixture of LIV, Cerophyll and brewer's yeast (less

than 37 microns) was added three times a week.

Only three of the original species persisted throughout the life of the
culture. P. denticulatus increased steadily during the first several
weeks, reaching a maximum of 1,137/liter at week-22; and fluctuated
thereafter between about 200 and 800 per liter. C. bicuspidatus thomasi
was at a minimum of 36/liter at week-12 and reached a maximum of 847/liter
at week-30. 8B. longirostris fluctuated between 5 and 75 per liter during
the first two feeding phases (i.e., no food and algae); while during the
last phase, an increased standing crop (between 42 and 265/liter) was
observed.

Diaptomus sp. reached a maximum of 4/liter at week-4 and was not found
again. C. pulchella increased initially to a high of 401/liter at week-2,
then declined rapidly and was not found again after week-10. Simocephalus
sp: also reached a maximum at week-2 (491/liter) and then declined.

This last species persisted in the culture for 36 weeks, but was not
found after that time.

The significance of Figure 1, as it relates to the original objectives of
the study, is negligible. It has been included for three reasons. First,
it illustrates the present status of this investigator's ability to
culture B. longirostris, after one year of rather intensive effort.
Second, after looking at hundreds (maybe thousands) of dead or dying B.
longirostris floating on the surface of uncounted cultures, the impulse
to taclade something in this report about living B. longirostris could
not be resisted. Third, Figure 1 suggests a principle Frown as enrich-
ment culturing that may well be worth pursuing in other studies of
zooplankton ecology and/or culture. This technique was first used by
Winogradsky and Beijerinck in the early 1900's to isolate micro-organisms
important in geochemical transformations, and the method has since been
applied extensively in bacterial and algal studies. Essentially, it is
an application, on a microscale, of the Darwinian principle of natural
selection. The investigator devises a culture with a particular, defined
set of conditions, inoculates it with a mixed population, and then
ascertains which species come to predominate as time continues. Since
their predominance is caused precisely by their ability to flourish in
the "enrichment medium", the same species can usually be isolated and


maintained in a medium of this same composition. An application of this
principle will be shown in the next section.

The reasons for the repeated failure of all efforts to establish
unispecific cultures of B. longirostris seem to all relate to the phenom-
enon of air-locking. The carapace of many cladoceran species is
hydrophobic, and thus, when the animals are exposed to an air-water
interface they become trapped by the surface tension and are unable to
submerge themselves again. In field collections of plankton, air-locked
Daphnia sp. are often seen floating on the surface. These animals become
air-locked during the collection process. It is not really known how
frequent air-locking occurs in natural populations under field conditions.

The tendency for B. longirostris to become air-locked during laboratory
manipulation has been observed by other investigators attempting to
culture this organism. Richard Applegate, South Dakota State University,
(personal communication), has cultured a number of zooplankton species
during the past several years using pond water and mixed cultures of
natural foods. He has repeatedly tried to establish cultures of B.
longirostris only to find that all of the organisms would appear air-locked
at the surface of the cultures, usually during the first few hours
following isolation. He has not been successful in establishing cultures
of B. longirostris under any conditions. James S. Murphy, Rockefeller
University, has successfully cultured about 14 species of the family
Daphnidae, under rigidly defined, monoxenic conditions (Murphy 1970).

B. longirostris is not a member of the family Daphnidae, but Bosminids

and Daphnids are closely related (i.e., they belong to the tribe Anomopoda).
In Murphy's medium large quantities of protein (Bovine albumin, fraction

v, at 200 mg/liter) are added to decrease the hydrophobic condition at

the surface of the carapace, and hence reduce the tendency for the

animals to air-lock. Murphy reported (personal communication) however,
that he had repeatedly tried to establish cultures of B. longirostris,
using the technique that proved successful with the other cladocerans,

and that he was not able to do so. In Murphy's technique, cultures are
not started by isolating live individuals. Instead, eggs are removed

from the parent's brood pouch, sterilized, and subsequently hatched in

a bacteria-free medium to produce the first generation. Murphy's

experience suggests that even if B. longirostris can be kept alive
through the first few critical hours, problems in maintaining cultures

may still exist.

The possibility of unsuitable culture conditions such as light, tempera-
ture, container size, medium composition, and food supply cannot be —
completely disregarded as reasons for failure to establish unispecific
cultures. However, the prolonged maintenance of mixed cultures containing
B. longirostris (such as the one described in Figure 1), suggest that

the physical and chemical environment was within the limits of tolerance


EPA~-660/3-75-010
May 1975

STUDIES TO DETERMINE METHODS FOR

CULTURING THREE FRESHWATER ZOOPLANKTON SPECIES

By

Dan B. Martin
Jerry F. Novotny
North Central Reservoir Investigations
U.S. Fish and Wildlife Service
P.O. Box 139
Yankton, South Dakota 57078

EPA-IAG-0152(D)
Program Element 1BA021
ROAP/Task No. 16AAI/10

Project Officer
Richard E. Siefert
National Water Quality Laboratory
National Environmental Research Center
6201 Congdon Boulevard
Duluth, Minnesota 55804

NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330

For Sale by the National Technical Information Service
U.S. Department of Commerce, Springfield, VA 22151


Semenova, L.M. 1968. Some data on the biology of Bosmina coregoni
Baird in Rybinsk Reservoir. Akademiya Nauk SSSR, Institut
Biologii Vnutrennikh Vod. 17:21-26.

Sheer, C.A. and K.B. Armitage. 1973. Preliminary studies of the effects
of dichromate ion on survival and oxygen consumption of Daphnia
pulex (L.). Crustaceana 25:51-69,

Siefert, R.E. 1972. First food of larval yellow perch, white sucker,
bluegill, emerald shiner, and rainbow smelt. Transactions of
the American Fisheries Society 101:219-225,

Starr, R.C. 1964. The culture collection of algae at Indiana University.
Amer, J. Botany 51:1013-1044.

Taub, F.B. and A.M. Dollar. 1964. A Chlorella-Daphnia food chain study:
The design of a compatible chemically defined culture medium.
Limnol. Oceanog. 9:61-74.

Whitehouse, J.W. and 8.G. Lewis. 1973. The effect of diet and density
on development, size, and egg production in Cyclops abyssorum
Sars, 1863 (Copepoda, Cyclopoida). Crustaceana 25:225-236.

Zhdanova, G.A. 1969. Comparative characteristics of the life cycle

and productivity of Bosmina longirostris 0.F. Muller and B.
coregoni Baird in the Kiev Reservoir. Hydrobiol. J. 5:8-15,

30


standing crop of adults ranged from 2 to 222/liter, and the percentage of
adults in the total population varied between 0.7 and 52.1. Copepodites
ranged from 4 to 510/liter and comprised 1.8 to 79.2% of the total
population. Nauplii standing crop varied from undetectable numbers (on
two occasions) to 1,678/liter, and they comprised from 0.0 to 92.6% of
the total C. bicuspidatus thomasi present on various sampling dates. The
total combined life stages varied from 128 to 1,812 per liter.

Table 4. Average standing crop and average percentage composition for each
life stage of Cyclops bicuspidatus thomasi in 10-liter stock cultures.

Cultures were maintained at 15° C and were fed 1,500 Paramecium Sp. per
liter per week.

“Culture No. | Culture No.2
Number Percent Number Percent
; per liter of total per liter of total
Life stage Ave. Std.Dev. Ave. Std.Dev. Ave. Std.Dev. Ave. Std.Dev.
Adults . 46 36 13.9 12.6 73 67 18.6 14.8
Copepodites 125 105 34.2 24.7 130 142 29.6 23.8
Nauplii 325 442 51.9 32.9 166 86 51.8 31.5
Total 496 435 100.0 - 369 134 100.0 -

Large variations in standing crop as well as shifts in the population

age structure occurred in these cultures. At several points the population
was dominated by nauplii, while at other times, adults and copepodites
were the most abundant forms. These changes in the population may result
from the fact that adult and late-stage copepodites are heavily predaceous
on the nauplii. This predation was repeatedly observed in our laboratory,
and apparently is a common occurrence in natural populations (McQueen,
1969). The familiar cycle of numbers often reported for predator-prey
Populations involving two species may be operative in these cultures

where both predator and prey are of the same species.

Two other characteristics of the stock culture populations did appear to
be comparable to those observed in nature. The average clutch size of
ovigerous females in the cultures was 29 (range 15-42), and the average
ratio of adult females/males was 1.7. Martin and Novotny (unpublished
manuscript) found the average clutch size for females in Lewis and Clark
Lake was 26 (range 1-78), and the average adult female/male ratio was 1.2.

The efforts to determine optimum culture conditions for C. bicuspidatus
thomasi began with an experiment to measure the effects of different
Tevels of food supply on standing crop at 15° C. Three 2-liter cultures
were started in filtered Missouri River water by adding 100 adults to each.
Each culture was then given a different number of Paramecium sp, three
times a week. The three rates of feeding used were (a) 1,000; (b) 1,500;

18


RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development,

U.S. 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 STUDIES
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
include formation, 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.

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
policies of the Environmental Protection Agency, nor does mention
of trade names or commerical products constitute endorsement or
recommendation for use.


Considerable difficulty was encountered in rearing the individuals in the
small containers, especially at the colder temperatures. The eggs would
hatch readily regardless of temperature and there did not ‘seem to be

any significant reduction in the percentage of eggs hatched at colder
temperatures. However, there was a noticeable increase in nauplii
mortality associated with the lower temperatures. Food seemed to be
plentiful but there was no way of determining if the nauplii were feeding.
Eventually, the individuals would cease to become active and die. The
reasons for this lack of development could be several. Provasoli and

his group have shown that nutritional deficiencies often manifest
themselves in the failure of an organism to develop beyond a certain

life stage. These workers have also shown that in the case of certain
deficiencies (e.g., vitamins) normal development may occur for several
generations in laboratory populations, then finally the trace amounts
required are finally exhausted and development stops. Ewers (1936) listed
the following factors as important in laboratory culture of Cyclops sp.:
(1) size of living space; (2) temperature; (3) food; (4) bacteria; and
(5) Og and CO content. Coker (1934) listed temperature, food, and
genetics as the three most important factors on rate of development in
Cyclops sp. More recently, Lewis, et al. (1971) and Whitehouse and
Lewis (1973) have shown the importance of temperature, food, and container
size on culturing Cyclops abyssorum.

Perhaps the most conspicuous property of the C. bicuspidatus thomasi cultures
in this study, both large and small, was their striking variability.

Part of this variation was due to lack of control over important
environmental factors during the culture experiments. However, one
should not overlook the wide range of adaptability demonstrated by this
species in nature, and the probable genetic basis for some of this
observed variation. C. bicuspidatus thomasi is extremely widespread
geographically, and its habitat 1s known to range from the open water

of large lakes to the littoral areas of small ponds. In many areas, it
is known as a perennial species, and in some localities all life stages
are present the year around. Field studies of this species show that it
is capable of existence under a wide variety of environmental conditions.
These broad limits of tolerance and adaptability are undoubtedly brought
into play under culture conditions. Such inherent properties within the
species would make it more difficult to demonstrate the effects of some
environmental variable (e.g., temperature), and to ultimately determine
“optimum" culture conditions. This same variation also seriously weakens
attempts to describe the effects of environmental variables from
observations on a few individuals (e.g., the small culture experiments).

26


When it became evident that C. bicuspidatus thomasi could be cultured using
live Paramecium sp. as food, attention was given to finding a suitable,
non-living food. Although, Paramecium Sp. satisfied the original criteria
for culture methods, there are certain disadvantages to the use of live
food. First, it is difficult to determine precisely the quantity of food
added at each feeding. The number of Paramecium sp. to be added could be
estimated by the method previously described, but Since the population
density of the protozoans varied considerably in the batch cultures,
different amounts of the bacterized Cerophyl] would accompany the
Paramecium sp. throughout the experiment. For example, if the population
density of Paramecium sp. was 80/ml, it was necessary to add about 40

ml of the food culture to a 2-liter C. bicuspidatus thomasi culture to

obtain 1,500 Paramecium sp./liter, but if the density of Paramecium sp.
was 300/m] only 10 ml of the food culture was necessary. Since it was

assumed that the bacterized Cerophyl] was important as food for the
nauplii, these variations could have had an effect on C. bicuspidatus
thomasi populations over time. Unless there is complete control over
the quantity of food added throughout an experiment it is difficult to
isolate and evaluate the effects of any other experimental variable.

The use of live food also has disadvantages in bioassay-type experiments.
If the toxicant being tested is detrimental to the food source, this
interaction can obscure the more direct effects of the toxicant on the
test organism.

An experiment was set up to determine the suitability of certain dried
foods for mass culture of C. bicuspidatus thomasi. Three 2-liter cultures
were started with 100 individuals in each, in filtered Missouri River
water at 15° C. A different combination of dried food was added to

each culture (Table 7) at the rate of 9 mg/week. The particle size

range of each food was 37 to 90 microns. (A description of these foods
and the methods of preparation can be found in the B. longirostris
section). The cultures were sampled once a week. Fresh medium was

added to maintain a volume of 2 liters.

21


0° C apparently reduced the activity of the organisms sufficiently to
Prevent them from swimming to the surface and air-locking.

No laboratory populations of B. longirostris could be Maintained, either
in unispecific or mixed cultures, during July and August. Environmental
chambers were not received until late August, and all attempts to keep
populations at room temperature (above 24° C) failed. After each attempt,
B. longirostris would be found floating on the surface of the water within
a few hours. After the environmental chambers arrived, it was noted that
a few B. longirostris were surviving in some of the mixed cultures, at
lower temperatures. These cultures were obtained by the ice-water method
but instead of individuals being isolated into pure cultures, the mixed
Populations were simply placed, (with a minimum of handling), in 4-liter
containers of pond water. Repeated attempts were made to establish
unispecific cultures of B. jongirostris from these mixed species
populations. A partial Tist of the conditions that were tried follows:

1. Temperature - 10° C; 15° C; 20° C.

2. Water Movement - In several experiments, the water in the
cultures was slowly agitated with a laboratory stirrer. In
other experiments, circulation was. achieved with an air stone.
Continuous as well as intermittent water movement was used.

3. Food - a) Enterobacter aerogenes (#15-5030 Carolina Biological
Supply Co.). Liquid cultures (100 ml) were grown aseptically
in Nutrient Broth (Difco Laboratories, 1953) at room temp-
erature for 7 days. Cells were harvested by centrifuging.
Cells were resuspended in distilled water, rinsed, centrifuged
again, and stored frozen in distilled water.

b) Enterobacter cloacae (#15-5032 Carolina Biological
Co.). Cultures were grown and harvested in the same manner

as that described for E. aerogenes.
c) Chlamydomonas reinhardtii (#90 Indiana University

Culture Collection). This alga was grown in 2,000 ml,
bacteria-free, batch cultures using media and methods des-
cribed by Starr, (1964). Sufficiently dense cultures were
obtained so that concentration of the cells was not necessary.
Media and cells were added directly to zooplankton cultures.

d) Chlorella ellipsoidea (#20 Indiana University
Culture Collection). This organism was cultured aseptically
in 2,000 ml batch cultures using methods described by Starr,
(1964) and was added directly to zooplankton cultures.

e) Cerophy1] (Cerophy11 Laboratories, Inc.)

f) Brewer's Yeast (Schiff BioFood Products, Inc.)

g) TetraMin tropical fish food (Tetra Sales, Inc.)

Cerophyl], brewer's yeast, and Tetra were ground into
powder using a “micro-mil1" (Chemical Rubber Co), The powder
was then separated into the following size categories using an



LITER

PER

NUMBER

Lisi

@———* Bosmina longirostris O ----O Simocephalus sp.
o——© Pleuroxus denticulatus *——'— Ceriodaphnia pulchella

@----Cyclops bicuspidatus thomasi

+ +—+—+—4 + + +} + +++ +} 4+ +--+ +++ + + +4
10 20 30 40 50 60
WEEKS

Figure 1. Changes in population density (No./liter) in a mixed
zooplankton culture containing Bosmina longirostris.