chemical responses to temperature changes.

For experimental work on the physiology and biochemistry, larvae of each
species were mass cultured in 12 cm diameter finger bowls under previously
determined optimal temperature and salinity combination producing the max-
imum survival. The larvae were transferred frequently to fresh sea water and
provided with a diet of freshly hatched Artemia salina nauplii as previously

described.

TABLE 2 EGG INCUBATION AND LARVAL CULTURE CONDITIONS
USED FOR SIX SPECIES OF CRUSTACEA

Larval Culture Conditions

Species Egg Incubation Temperature (°C) Salinity (0/00) Number of Combinations
Gancer irroratus 15°C, 30 o/oo 10,15, 20,25 10,15,20,25,30,35 26
Cancer borealis 15°C, 30 o/oo 10,15,20,25 10,15,20,25,35 26
Homarus americanus 20°C, 30 ofoo 10,15,20,25 15,20,25,30,35 20
Pagurus longicarpus 15°C, 30 ofoo 10,15,20,25 15,20,25,30,35 20
ahi theopanopeue 20°C, 25 of/oo 20,25,30 10,15,20,25,30,35 18

20°c, 30 o/oo 10,15,20,25,30 5,10,15,20,25,30,35,40 40

Effects of Daily Cyclic Temperatures on Larval Development

Effects of daily cyclic and comparable constant temperatures on develop-
mental and survival rates of C. irroratus larvae hatched in 30 o/oo at 15 C
and P. pugio larvae hatched in 30 o/oo salinity at 20 C were determined.

The larvae were reared in 10-20, 15-25, 12.5-17.5 and 17.5-22.5 C daily cyclic
temperatures and comparable constant 15 and 20 C under a 14:10 LD photoperiod.
The same culturing methods were used as for the temperature and salinity
studies cited above. Molting and deaths were recorded daily to determine the
patterns of survival and duration of development.

Metabolic Responses of Larvae to Temperature

The temperature effects on metabolism were determined by measuring the
oxygen consumption rates for the larval stages of C. irroratus, H. americanus
and P. pugio over a graded series of temperatures. For the metabolic rate
determinations, larvae from mass cutures of C. irroratus in 30 o/oo salinity
at 15 C and H. americanus and P. pugio in 30 o/oo salinity at 20 C were used.
The oxygen consumption rates of C. irroratus and P. pugio were measured with
all glass differential microrespirometers (Sastry and McCarthy, 1973). Met-
abolic rates were measured using 10-12 individuals of the first, second and
third stage zoeae, 4-5 individuals of 4th and 5th stage zoeae and one of
megalops for C. irroratus. For P. pugio, one to four individuals of each
larval stage, depending upon the stage of development, were introduced into
each flask for respiration measurements. The metabolic rates of H. americanus

T


Larvae of P. pugio cultured at 12.5 to 17.5 and 10-20 C daily cyclic
temperatures survived to the post-larval stage slightly better than those at
comparable constant 15 C (Fig. 13). No significant differences in the rate
of survival to the post-larval stage were observed at 17.5-22.5 and 15-25 C
daily cycles and at comparable constant 20 C. Duration of development to the
post-larval stage was slightly longer at constant 15 C compared to that at
10-20 or 12.5-17.5 C daily cycles. No significant differences were observed
in the duration of development at constant 20 C and 15-25 or 17.5-22.5 C
daily cyclic temperatures (Fig. 14).

Mean weight of the developmental stages of C. irroratus increased pro-
gressively from first zoeal stage to the crab stage. There were no sig-
nificant differences in the weight of larval stages cultured at 10-20 C
daily cycle and comparable constant temperature (p<0.01). Mean weight of the
larval stages of P. pugio also increased progressively with development.

METABOLIC-TEMPERATURE RESPONSES

Larvae Cultured at Constant Temperatures

Larvae cultured at a temperature and salinity producing highest survival
to the post-larval stage (C. irroratus,15C~30 o/oo; C. borealis, 20 C-30 0/00;
P. pugio, 20 C-30 o/oo) showed both inter and intraspecific variation in
their metabolic responses to temperature. The first stage larvae of C.
irroratus were metabolically active over a temperature range of 5-25 C (Fig.
15), with a Qo close to 2. For successive stages, the temperature range
for depression (Q9<1) of metabolic rate was about the same, but differences
in temperature ranges for sensitivity (Q,9>2) and compensation (1<Q,9<2) were
observed. The temperature range for sensitivitv in the second and third zoeal
stage was from 5-15, but this shifted to 10-15C in the fourth and fifth stages.
The temperature range for compensation has also narrowed from 10-20C in the
second and third stage to 15-20 C in the fourth and fifth stages. The meg-
alops showed no compensation over the 10-20 C range. The metabolic rate of
all the stages was depressed between 20-25 C.

The larval stage of C. borealis showed a distinctly different metabolic
response to temperature. Larvae of this species were metabolically active
over the entire temperature range of 5-25 C and there was no depression of
metabolic rate at warmer temperatures (Fig. 16 and 17). The first and sec-
ond stage zoeae showed compensation over a temperature range of 5-20 C and
5-15 C, respectively. Sensitivity was observed at warmer temperatures. The
third stage showed no compensation over the entire temperature range of 5-
25 C. Metabolic-temperature patterns of the fourth, fifth and megalops
stage are reversed from those of the first two with sensitivity to colder
temperatures and compensation to warmer temperatures.

The metabolic response patterns of P. pugio to temperature are shown as

values (Fig. 17). Larvae of this species were metabolically active over
a wider temperature range and at warmer temperatures than the larvae of the
two sublittoral species. The first and second larval stages of P. pugio
compensated between 15-20 C and 20-25 C, respectively. The temperature range

28


Effects of constant and daily cyclic temperatures

The effects of daily cyclic and comparable constant temperatures on the
duration of larval development and survival were determined for the larvae
of C. irroratus and P. pugio.

Larvae of Cc. irroratus cultured at 10 to 20 and 25 C
daily cyclic temperatures experienced greater survival
than those at comparable constant temperatures (Fig. 10 and Tables 8 and 9).
Survival of larvae was less at 12.5-17.5 C cycle than that at the 10-20 C
eycle. Larvae cultured at the 17.5-22.5 C cycle developed only to the meg-
alops stage. Duration of the zoeal stages at both 12.5-17.5 C and 10-20 C
daily cycles decreased and megalops duration increased compared to that at
constant 15 C (Fig. 11 and Table 10). The duration of the megalops stage in-

100

i Constant a+ 4 i: 4
m 10°C cycle Ik i 4
[~ 6 8'c cycte

x
%

% SURVIVAL
8
T
SSG

SEO

3

bS

<p

oS

05
SS

-
"ee

"es

33

XP
fetes

Ei

1 15 20 10 15 12,5- 17.5- 15 20 10- 15 125- 15° 20 10 15 12.5-

x

20 25 «(17.5 225 200-25 «(17.5 200 25 17.5
TEMPERATURE, °C TEMPERATURE, °C TEMPERATURE, °C

ta) (b) (e}

Figure 10. Percentage survival of C. irroratus larvae at constant and cyclic
temperatures. (a) Hatch to megalops, (b) Megalops to crab, (c) Hatch to crab.

TABLE 8. SURVIVAL OF C. IRRORATUS LARVAL STAGES AT CONSTANT AND CYCLIC
TEMPERATURES.

‘Survival at ‘Survival at
constant temperature, % cyclic temperatures, %

stage* 10°C 15°C 20°C 25°C 10-20°C 15-25°C 125-17.5°C 17.5-22.5°C

I 54 857639 88 87 82 76
u 37 78 «68 «7 86 83 72 60
mm 32 72:0«SBC 84 78 66 40
Iv 20 66 #47 «0 79 75 58 27
v 1 60 31 OF 78 n 49 20
Megalops 0 40 17 0 63 397 12 CY)

*Stages I to V are zoeal stages.

24



C. irroratus
—, .
Vi) cr et Seren | CYCLIC 23
CONSTANT [3
— 728)
ne Ec is)
o 1<Q9<2 @
=v) Gomme
5 cf peeewet2 3<!
di
gin
ec
<=
4
u
i| &
x
L 1 1 a 2 4
0 5 10 15 20 25 30

TEMPERATURE, C

Figure 18. Metabolic-temperature responses (as Q19 values) of laryal gtages
of C. irroratus cultured in 3C o/oo at constant 15°C and cyclic 10°-20°C.

Larvae experiencing the daily cyclic temperature regime exhibited an exten-
sion of the temperature range for compensation, with a shift towards the
higher temperatures. Oxygen consumption rate for larvae cultured with cyclic
temperatures was higher than those from constant temperatures at warmer test
temperatures. The temperature range for depression of metabolic rate shifted
from 20-25 C range for constant larvae to 25-30 C range for the cyclic larvae.

Larval stages of the American lobster, H. americanus, cultured at 15-25C
and 17.5-22.5 C daily cyclic temperatures and constant 20 C also showed inter
and intra~stage variation in their metabolic-temperature response patterns
(Pig. 19). Larvae cultured at constant 20 C showed no compensatory response
of metabolic rate in the first larval stage, but compensatory response was
observed between 15-25 C for the second stage, 10-15 C for the third stage
and 15-25 C for the fourth stage. Metabolic rate of the first and third
stage larvae was depressed between 20-30 C, compared to the 25-30 C range
for the second and fourth stages. Culture at the daily cyclic regimes has
altered the metabolic-temperature response patterns of the larval stages rel-
ative to those at constant temperature. These differences were reflected
in the zones of thermal sensitivity and compensation, but the pattern of
these changes were different for the larval stages.

Larval stages of the estuarine grass shrimp, P. pugio, cultured at 15-
25 C daily cyclic and 20 C constant temperatures showed both inter and intra-
stage variation in their metabolic responses to temperatures. However, inter-
stage variation between the two culture regimes had no constant or predict-
able pattern (Fig. 20). Culture at cyclic temperatures enhanced the res-
piration rate in some stages and reduced the rate in other stages. First
stage larvae at the cyclic regime showed compensation between 10-15 C

33


SECTION 1
INTRODUCTION

The possible effects of thermal alterations of the coastal and estuarine
environments from conventional and nuclear power plants has become a concern
to the public during the past several years. The constraints of thermal in-
erease in these bodies of water are determined by consideration of the effects
on the biotic communities. The adaptive capacities of organisms either to a
direct increase of water temperature, or the changes in the physico-chemical
properties of sea water due to increased temperature are important consider-
ations for determining water quality criteria. An important issue then in
thermal pollution of the environment is to assess its short and long term
impact on biotic communities which may eventually result in alterations in
the structure and functioning of these ecological systems. To develop the
capability for assessment and prediction of thermal alterations of the en-
vironment, there is a need for a comprehensive understanding of the effects
of thermal changes, singly or in combination with other factors, on all the
life stages of populations within the communities.

An extensive body of literature evaluating the effects of thermal pol-
lution on organisms has been already published (Clark, 1969; Jensen, et al.,
1969; Krenkel and Parker, 1969; Nylor, 1969; Hargis and Warinner, 1970;
Gibbons and Shiritz, 1974; Esch and McFarlane, 1976; Coutant and Talmage,
1977). It is generally recognized that temperature changes in the environ-
ment affect metabolism, growth, reproduction and development, and activity
and behavior of the organisms. The functional activities of organisms are
performed within a temperature range characteristic to each species (Prosser,
1971; Vernberg and Vernberg, 1972). Within the tolerance range, physiologi-
cal rate processes are altered relative to changes in the ambient water
temperature. However, many organisms have capabilities to regulate their
phsiological rate functions within a certain temperature range (Bullock,
1955; Hazel and Prosser, 1974; Wieser, 1973). These adaptations to changes
in the thermal environment play an important role in their ecology and dis-—
tribution. At either extreme of the tolerance range, there is a point beyond
which the organisms are unable to survive. The adaptations which favor
regulation of physiological rate processes within the tolerance range and the
limits may vary with the stage of life cycle and also with interactions of
other environmental factors (Sastry and Vargo, 1977). The synergistic inter-
action of multiple factors in the environment makes it difficult to describe
and evaluate the complex relationship between organisms and their environment-—
al alterations under natural conditions. One approach to determine the ef-
fects of temperature changes on organisms is to measure alterations in their
physiological rate processes within the tolerance range as well as measuring
the thermal limits for survival. Since a number of physical, chemical and
biological factors interact with the organisms under natural conditions to

1


SECTION 6

DISCUSSION

Larvae of epibenthic crustacea play an important role in the distribu-
tion, gene exchange, pelagic food web and recruitment of young to the adult
populations. Generally, larvae are released into the pelagic environment
when conditions are optimal for their development and growth. During their
pelagic existence larvae are exposed to continuously varying temperatures
along with other fluctuating environmental parameters. Larval development
occurs within a range of environmental conditions which is characteristic
to each species. Interaction of environmental parameters can affect larval
development as reflected in varying survival and developmental rates when
cultured under different combinations of temperature and salinity (Costlow
and Bookhout, 1964; Sastry and Vargo, 1977). A species may exhibit an opti-
mal survival with a given combination of conditions. Then as environmental
conditions deviate from this optimum, survival rate is reduced. Limits, as
well as the optimal combination for development, vary interspecifically. In
the present study, the coastal species (high salinity) completed development
over a narrower range of temperature and salinity than the predominantly es-
tuarine species. These limits varied from a single temperature and salinity
combination, as seen with C. borealis, to a wide range of suitable tempera-
ture and salinity conditions as occurred with P. longicarpus and P. pugio.
Limits for complete development may also be a function of the environmental
history of the eggs prior to their hatching. Larvae of C. irroratus hatched
from eggs incubated during the period they are normally released in nature
survived in laboratory culture better than those hatched at other seasons.
It appears then that stage of embryonic development at which the eggs are
incubated for hatching and their previous thermal history will affect the
survival rate, and the limits for complete development.

In addition to these variations, larvae of C. irroratus cultured under
constant and daily cyclic regimes also differed in their survival and time
required for complete development. Those cultured under a suitable ampli-
tude and rate of temperature change showed an increase in survival compared
to those at comparable constant temperatures. Beyond these limits, as with
the 15-25 C cylce for C. irroratus, larval development was delayed compared
to that at constant 20 C. The affects of daily cyclic and constant tempera-
tures on survival and development time also varied inter-specifically. The
estuarine grass shrimp, P. pugio larvae showdd no significant differences in
either the duration of development or survival at daily cyclic and comparable
constant temperatures. Clearly, the affects of fluctuating temperatures
from observations on one species should not be generalized for others.

43


number) + b.

3 1orc r 20°¢
L _/ 3k
a F z
L a A
VX
iL vo
=
x ~w
i}
E re) 1 1 L 1 L 1
° °
eab t 15°C
4
Sb
sk ir 25sec
L x1
lr a a ® ob
ak Is °
L / Ya,
q
re \ Ir
| Can , 30°c
po gL TT “Ie

'-Z 2-2 3-2 4-Z5-Z M -Z 22.3-2 4-2 5-2 M
LARVAL STAGE

Figure 24. Cancer irroratus. Effect of stage on LD., value for oxygen.

Dots: 120 min exposure (solid line); crosses: 240 min exposure (dashed line).
1 - 5Z: Zoeal stages; M: megalops.

Interspecific Comparison of Temperature Tolerance limits

The larval stages of three species exhibited different thermal tolerance
limits to extremes. The upper limits for 50% survival of C. irroratus, H.
americanus and P. pugio, each cultured in their respective optimal tempera-
ture and salinity combinations, are presented in Fig 25. The temperature
tolerance limits for P. pugio larvae were much higher than those for the
other stages, Between the two coastal species, C. irroratus larvae exhib-
ited lower temperature tolerance limits than H. americanus larvae. The tol-
evrance limits for successive larval stages remained fairly constant for each
species, with the exception of certain stages. The limits for the fourth and
seventh stages of P. pugio were 2 and 2.3 C higher, respectively, than for
other species. Second stage H. americanus larvae were less tolerant than
was any other stage. For C. irroratus larvae, the second and fourth stages

AL


Gibbons, J. W. and R. R. Sharitz. Thermal ecology. Conf. 730505, U.S.
Atomic Energy Commission, 1974. 670 pp.

Goldstein, A. 1964. Biostatistics: An introductory text. 272 pp. The
McMillan Co., New York, 1964. 272 pp.

Gunter, G. Temperature, Chapter 8. Geol. Soc. America, Memoir 67, 1:159-18h.
1957.

Hadley, P. B. Additional notes upon the development of the lobster.
Fourtieth Ann. Rep. Commissioners Inland Fish. Rhode Island, E. L.
Freeman Co., Providence, R.I., 1909.

Hargis, W. J. and J. E. Warinner. Impact of thermal alterations on estuarine
and coastal environments. Program on thermal considerations in the
production of electric power, Atomic Industrial Forum Inc., and Electric
power council on environment, Washington, D. C. 1970. 53 pp.

Hazel, J. R. and C. L. Prosser. Molecular mechanisms of temperature com—
pensation in poikilotherms. Physiol. Rev., 54: 620-677, 1974.

Herrick, F. H. The habits and development of the lobster and their bearing
upon its artificial propagation. Bull. U.S. Fish. Comm. 13: 75-86,
1894,

Hughes, J. T. and G. C. Matthiessen. Observations on the biology of the
American lobster, Homarus americanus. Limonl. Oceanogr. 7: 414-421,
1962.

Jensen, L. D., R. M. Davies, A. Brooks and C. D. Meyers. The effects of
elevated temperature on aquatic invertebrates. Edison Electric Inst.,
Res. Projept No. 49, The John Hopkins Univ., Baltimore, 1969. 232 pp.

Kinne, 0. The effect of temperature and salinity on marine and brackish
water animals. I. Temperature. Oceanogr. Mar. Biol., Ann. Rev., 1:
301-340, 1963.

Kinne, 0. The effect of temperature and salinity on marine brackish water
animals. II. Salinity and temperature-salinity combinations. Oceanogr.
Mar. Biol. Ann. Rev., 2: 181-339, 1964.

Kinne, 0. Temperature, animals, invertebrates. In: Marine Ecology, 0.
Kinne, ed. Wiley-Interscience, London, 1970. pp. 407-514.

Krenkel, P. A. and F. J. Parker. Biological aspects of thermal pollution
Vanderbuilt Univ. Press., Nashville, Tenn. 1969.

Meleikovsky, S. A. ‘the influence of pollution on pelagic larvae of ‘bottom

invertebrates in marine nearshore and estuarine waters. Mar. Biol.,
6: 350-356, 1970.

47


Sastry. A. N. Physiological adaptation of Cancer irroratus larvae to cyclic
temperatures. In Physiology and Behavior of Marine Organisms, D. S.
Mclusky and A. J. Berry, eds. Pergamon Press, Oxford, 1978, pp. 57-65.

Sastry, A. N. and W. R. Ellington. Lactate dehydrogenase during the larval
development of Cancer irroratus: Effect of constant and cyclic thermal
regimes. Experentia, 34: 308-309, 1978.

Sastry, A. N. Metabolic adaptation of Cancer irroratus developmental stages
to cyclic temperatures. Mar. Biol. 51: 243-250, 1979.

Sastry, A. N. Effects of temperature on the larval development of crustacea.
In: Reproduction and growth in Marine Invertebrates (Ed. V. L. Kasyanov).
XIV Pacific Science Congress. In Russian (in press).

PAPERS PRESENTED AT SCIENTIFIC MEETINGS

Sastry, A. N. Effects of constant and cyclic temperature regimes on the
larval development of a brachyuran crab. Second Thermal Ecol. Symposium,
Augusta Georgia, April 2-5, 1975.

Sastry, A. N. Metabolic adaptation of pelagic larvae of a crustacean to
constant and cyclic temperatures. Ann. Meeting, AIBS, Corvallis,
Oregon, August 17-23, 1975.

Sastry, A. N. and S. L. Vargo. Tolerance of Cancer irroratus and Palaemonetes
pugio larval stages to acute temperature and low dissolved oxygen. 38th
Ann. Meeting, Am Soc. Limnol. Oceanogr., Halifax, Canada, June 22-27,
1975.

Sastry, A. N. and S. L. Vargo. Variation in physiological responses of
crustacean larvae to temperature. Pollution and physiology of marine
organisms, Symposium, Milford, Conn. November 4-6, 1975.

Sastry, A. N. and John Laczak. Metabolic adaptation of estuarine grass
shrimp, Palaemonetes pugio larvae cultured at constant and cyclic temp-
eratures. Am. Soc. Physiol., Ann. Meeting, Philadelphia, Pa. August,
1976.

Sastry, A. N. Temperature variation and physiological responses of crusta—
cean larvae. Joint Oceanogr. Assembly, Edinburgh, U.K., September 12-
2h, 1976.

Sastry, A. N. Physiological adaptation of Cancer irroratus larvae to cyclic
temperatures. 12 European Symp. Mar. Biol. Stirling, Scotland, U.K.,
Sept. 4-13, 1977.

Sastry, A. N. Physiological adaptations in reproduction and larval develop-
ment of coastal and estuarine organisms, U.S. - USSR meeting on
Physiology and Biochemistry of marine organisms. Georgetown, S.C., Oct.
6-10, 1977.

51


ACUTE TEMPERATURE AND LOW DISSOLVED OXYGEN TOLERANCES

Tolerance limits to acute temperature, alone and in combination with low
dissolved oxygen stresses were determined for five zoeal stages and the meg-
alops of Cancer irroratus. Temperature tolerance at saturated dissolved
oxygen levels varied with stage, with this variation dependent on exposure
time (Fig. 22). Little interstage variation was observed for the 120 minute
time interval, with all stages having an LDe for temperature of about 29 C
(Table 13). For a 240 minute exposure, there was an overall decline in
tolerance and more interstage variation. The second and fourth zoeae were
slightly more tolerant (28.2-28.5 C), while the others were less tolerant
(2%.2-27.5) (Table 13). No significant correlation was found between stage
and temperature tolerance to indicate that there was a continuous relationshir
between morphologically distinct stages and temperature tolerance.

2 a

LDgg TEMP °C

1 L 1 1 1
25 Wz 2-2 3-2 HZ SZ

LARVAL STAGE |

Figure 22. Cancer irroratus. Effect of larval stage on LD_. values for
temperature. Open circles: 120 min exposure; filled circleS: 240 min ex-
posure. 1-52; Zoeal stages; M: megalops.

TABLE 13. CANCER LRRORATUS. LDe9 VALUES FOR TEMPERATURE FOR 120 MIN AND
240 MIN EXPOSURE TIMES, BY STAGES NO STATISTICALLY SIGNIFICANT CORRELATION
WAS FOUND BETWEEN LD,,. AND STAGE. OXYGEN CONCENTRATIONS WERE KEPT AT SAT-

URATED LEVELS IN ALL fEsTS

Stage LDsq temperature (°C)
120 min 240 min

1 29.0 + 0.2 27.5 40.1
2 29.0 + 0.02 28.2 t0.1
3 29.0 $0.1 27.5 to.
4 28.9 $0.1 28.5 $0.2
5 29.0 + 0.3 27.5 + 0.2
Megalops 29.0 + O.08 27.2 + 0.2

39


ll

M. megalops.

irroratus I to V, zoeal stages;

Life cycle stages of C.

Figure 1.


62

Figure 13.

(oo

all ll

0-10 25-15 225-175 175-125
CONSTANT ne ULTURE CYCLING
TEMPERATURE

SURVIVAL
°

%

io]

Percentage survival of Palaemonetes pugio at constant and daily cyclic temperatures.


FOREWORD

The Environmental Research Laboratory of the U.S. Environmental
Protection Agency is located on the shore of Narragansett Bay, Rhode Island.
In order to assure the protection of marine resources, the laboratory is
charged with providing a scientifically sound basis for Agency decisions on
the environmental safety of various uses of marine systems. To a general
extent, this requires research on the tolerance of marine organisms and
their life stages as well as the tolerance of ecosystems to many forms of
pollution stress.

This report describes a three-year study undertaken to determine the
environmental requirements for development of pelagic life stages of some
epibenthic crustaceans from the coastal and primarily estuarine environ-
ments. The effects of temperature changes on the development, metabolism
and survival of the larval stages of the crustaceans are presented.

Tudor T. Davies
Director

ili


TABLE 6. VARIATION IN THE SURVIVAL AND DURATION FOR COMPLETE DEVELOPMENT
OF GEOGRAPHICALLY SEPARATED POPULATIONS OF THREE SPECIES OF CRUSTACEANS.

Rhode Island North Carolina®
Duration Duration
Species T (¢), § (o/oo) % survival Days T (Cc), 8 (ofoo) % Survival Daye
20 52 30-38 20 ¢ 0 -
32 ofoo 31.1 0/00
Panopeus herbatii
25¢ 48 20-32 25 3 33-35
32 ofoo 31.1 ofoo
23 20.7 20-30 25¢ 83 15-23
25 ofoo 25 ofoo
256 2.0 22 256 23 16-21
35 0/oo 35 ofoo
30¢ 1.89 23-24 30.¢ 14 15-22
15 0/oo 15 o/oo
30 ¢ 4.08 23-30 30 ¢ 60 11-15
25 ofoo 25 ofoo
Pala cee pugio 2 oh 91.8 18-31 25-27 ¢ 65 17-21
* Data from Costlow, Bookhout and Monroe (4966); Broad (1957)

22


larvae were measured for 4-5 individuals of first, second and third stages,
and one or two individuals of the fourth stage larvae introduced into each
flask. A number of replicate runs (3-12) were made for larval stages of each
species over a graded series of temperatures between 5 and 30 C. Larvae were
then weighed to the nearest microgram on a Cahn electrobalance. The mean
oxygen consumption rate and the standard deviation were computed. The regres-—
sion analysis of oxygen consumption as a function of weight of each life
cycle stage was performed and tested for significance by F test. The mean
weight-specific oxygen consumption rates, or those values determined from
the regressions when those were significant, were plotted against temperature
to represent the metabolic temperature response patterns. These values were
used to compute the Q19 for 5 C test temperature intervals to reveal changes
in metabolic response to temperature.

Fatty Acid Methyl Esters

A known weight of each larval stage of C. irroratus and H. americanus
were placed in 25 ml centrifuge tubes with teflon lined screw caps. Ten ml
of 0.5 N KOH in methanol, and 5.0 ml of benzene were added to each tube, the
tubes were flushed with nitrogen, sealed and heated at 100 C for 30 minutes
with shaking for 10 min. The saponified samples were cooled, 5.0 ml of 0.3
NHCL were added, and the tubes were shaken and centrifuged. The benzene lay—
er was drawn off and transferred to 50 ml pearshaped flasks and the aqueous
methanol phases were twice re-extracted with 5 ml portions of petroleum ether.
The petroleum-benzene extracts were evaporated to dryness under reduced pres—
sure.

The extract residues were transferred to screw cap centrifuge tubes with
teflon lined caps, using 1 ml methanol and 1 ml of benzene. One ml of BF3
methanol was added to each tube, the tubes were flushed with nitrogen, sealed
and heated in boiling water for 5 minutes. Methyl esters were isolated by
adding 4 ml of Ho70 and 4 ml of petroleum ether. Solvent was evaporated under
reduced pressure.

Fatty acid methyl esters were separated by thin layer chromatography
using Me-palmitate (16:0) and Me-decosahexaenate (22:6) as spotting standards.
The silica~gel plates were developed for 30 minutes in 95:5:1 petroleum ether,
ethyl ether and NaOH. After drying the plates, they were sprayed with
bromophenol blue solution and the bands corresponding to the saturated and
unsaturated fatty acids were scraped. The scrapings were added to a micro-
filter apparatus attached to an aspirator and eluted with 30 ml of CHC13.

The elutant was evaporated to dryness in a 50 ml pearshaped flask. The sam-—
ple material was resuspended in a small amount of CHC13.

Fatty acid methyl esters of the sample of each larval stage were an-
alyzed by Hewlet-Packard Model No. 5 1 A Gas Chromatograph, equipped with
flame ionization detectors. Fatty acid methyl esters were quantitatively
identified by comparison of their relative retention time with those of
standard methyl esters. The fatty acids in samples were determined by in-
corporating a fatty acid internal standard into the extraction with the ,
sample prior to saponification. The peak area (height X width at half height)
of the internal standard methyl esters was then compared with the peak areas

8


©. irroratus ZA

H. americanus (| P. pugio KN
€. borealis © PB longicarpus Rharrisii FT 4
40
e NX
= 30 )
x ob.
ra . i
F 29 1
2 ‘
4
aq 10 —
n
obras Lirias Litiiis

° 10 20 fo 10 20 ie) 10 20 «6030
TEMPERATURE, C

Fig. 3. Temperature and salinity limits for complete larval development of
six species of crustaceans from Narragansett Bay and vicinity.

4
: F longicorpus

30 C.irroratus

Pig. 4, Percentage survival of larvae to the post-larval stages for five

species of crustaceans reared in different combinations of salinity and
temperature.

1h


SECTION 5

EXPERIMENTAL RESUL!'S

LARVAL DEVELOPMEN'!

The methods for culture and descriptions of larval stages during complete
development of C. irroratus and C. borealis were not previously available.
Therefore, the larvae of both these species have been cultured and morpho-
logical features of the larval stages have been described (Sastry, 1970; 1977
a, b). Larval development of both species includes five zoeal stages and a
megalops stage before metamorphosis to the crab stage (Figs. 1 and 2).

Larval stages of H. americanus have been previously described (Herrick,
1896; Hadley, 1909). ‘The development of this species includes four plankton-
ic larval stages. Connolly (1925) described the four zoeal stages and a meg-
alops stage in the development of R. harrisii. Larvae of P. pugio were de-
scribed by Broad (1957). In the present study, the number of larval stages
in the development of this species varied from 6 to 14 in various combinations
of temperature and salinity (Table 3). Larval stages of P. longicarpus cul-
tured in the laboratory have been described by Roberts (1970). The larvae
pass through four zoeal stages and a megalops stage before metamorphosis to
the post-larval stage.

TABLE 3. VARIABILITY IN THE NUMBER OF LARVAL STAGES DURING DEVELOPMENT
OF PALAEMONETES PUGIO CULTURED AT DIFFERENT COMBINATIONS OF
TEMPERATURE AND SALINITY

Temperature Salinity 0/00
c 10 45 20 25 30 35
15 8-14 7-12 8-12 8-12 9-12 8-14
(10) (10) (10) (10) (10) (10)
20 6-9 6-9 6-9 6-10 7-11 6-11
(8) (8) (8) (8) (8) (8)
25 7-11 6-11 6-11 6-10 6-9 7-12

(8) (9) (8) (8) (7) (10)

Numbers in parenthesis indicate the model frequency

10


TABLE 7. DURATION (DAYS) FOR DEVELOPMENT TO THIRD AND FOURTH LARVAL STAGE
OF H. AMERICANUS FROM DIFFERENT GEOGRAPHICAL REGIONS (THE FOOD FOR LARVAE
AND CULTURE CONDITIONS ARE SOMEWHAT DIFFERENT).

Geographical Culture Days to Days to Reference
region temperature 4th stage Sth stage
c
St. Andrew's 6-7 120
Canada 10 54.5 100
4 26.5 50 Tempiem an (1936)
19 13.5 28
24 10.5
Nova Scotia & 15 25 Sherman and Lewis
Maine 19-20 15 1967; Scaratt, 1968
Martha's 19-20 13.6 Hughes and Mattheissen,
vineyard, Mass. 1962
Rhode Island 10 7 109
15 29 76 This study
20 23 46

23


SECTION 4

MATERIALS AND METHODS

CULTURE OF LARVAE

Larvae of six species of crustaceans, Cancer irroratus, Cancer borealis,
and Homarus americanus from the coastal zone (high salinity), and Pagurus
longicarpus, Palaemonetes pugio and Rhithropanopeus harrisii from predomin-
antly estuarine waters in Narragansett Bay have been cultured in the labora-
tory. Details of their habitat, temperature and salinity conditions are sum-
marized in Table 1. The general methods for incubation and larval cuture are
the same as described in detail by Sastry (1970) and Sastry and Vargo (1977).
Ovigerous adults were brought to the laboratory, eggs removed and incubated
until hatching in 30 o/oo salinity at a temperature suitable for each species
(Table 2). In the cases of H. americanus and P. pugio, eggs were not removed
and the ovigerous animals were held at the incubation temperatures until
hatching.

Within 24 hours from hatching of eggs in the laboratory, the larvae were
transferred to different temperature and salinity combinations for rearing.
Larvae were reared individually in compartmented plastic boxes containing
filtered sea water or in the case of H. americanus in glass crystallizing
dishes containing sea water. Within 24 hours after hatching of eggs, the
larvae were maintained in Sherer controlled environmental chambers and were
exposed to a 14:10 LD cycle. The sea water was changed on alternate days
and the larvae were fed daily on a diet of freshly hatched Artemia salina
nauplii. Molting and deaths were recorded daily to quantify the patterns of
survival to complete development for each species at the various conditions.
Temperature and salinity effects were determined using a factorial design
to produce graded series of responses to constant 10, 15, 20 and 25 C and
salinities 10, 15, 20, 25, 30 and 35 o/oo. The factorial design allowed
estimation of the range of overall effects and the interaction between dif-
ferent experimental factors (Alderdice, 1972).

The morphological descriptions of the larval stages of each species ex~
cept C. irroratus and C. borealis have been reported in the literature.
Drawings and descriptions of larval stages of C. irroratus and C. borealis
were made from larvae of known stages cultured in the laboratory under optimal
temperature and salinity conditions and placed in Permount on a slide.

Whole mounts were made of fresh material, and appendages also dissected from
each stage and similarly mounted. Scale drawings were made of whole mounts
and larval appendages with the aid of camera lucida (Sastry, 1977a, b). The
descriptions of these larvae and those of other species available from liter-
ture were used for recognizing different stages in the development of a
species in conducting the experimental work on their physiological and bio-

5



Thorson, G. Reproduction and larval ecology of marine bottom invertebrates
Biol. Rev., 25: 1-45, 1950.

Vargo, S. L. and A. N. Sastry. Acute temperature and low dissolved oxygen
tolerances of brachyuran crab (Cancer irroratus) larvae. Mar. Biol.,
4o: 165-171, 1977.

Vernberg, F. J. and W. B. Vernberg. The animal and the environment. Holt,
Reinhart and Winston, New York, 1970. 398 pp.

Vernberg, W. B. and F. J. Vernberg. Environmental physiology of marine
animals. Springer-Verlag, New York, 346 p. 1972.

Wieser, W. Effects of temperature on ectothermic organisms, ecological
implications and mechanisms of compensation. Springer Verlag, New York,
1973. 298 pp.

Williams, A. B. Marine decapod crustaceans of the Carolinas. Fishery
Bulletin, Vol. 65 (1): 1-298, 1965.

49


FIGURES (cont'd)

Number Page

14

15

16

17

18

19

20

22

23

ah
25

Duration of larval development of Palaemonetes pugio at
constant and daily cyclic temperatures. ..cccccccecceseceeccesccees 30

Metabolic-temperature response of different larval stages of
Cancer irroratus...csceceeeccec cece cere ccerececcceeseescccereecees Sh

Hetabolic-temperature response of different larval stages
of Cancer DOrealis....cseececcccccsceevcccrccccccneseesceseeeesees Sb

Metabolic temperature responses (as QQ values) of larval stages
of Cancer borealis and Palaemonetes pugio cultured at constant
20 C and 30 0/00 Salinity... cece cece eee eeeceeeeeceeeeeeteeeneeee 32

Metabolic-temperature responses (as Q)Q values) of larval stages
of Cancer irroratus cultured in 30 o/oo at constant 15 C and
CYCLIC 10-20 Creve ccceseceeccccccesecsecccccceetesscesseseseseees 33

Metabolic-temperature responses (as Qu values of larval stages
of Homarus americanus cultured in 30 oyoo at constant 20 C
and daily cyclic 15-25 and 17.5-22.5 C temperature.....seeeceeeeee 3h

Metabolic-temperature responses (as Qj9 values) of larval stages
of Palaemonetes pugio cultured in 30 o/oo salinity at constant
20 C and daily cyclic 15-25 C temperatures......sseeeeeeeeseesceee 39

The effects of daily cyclic and constant temperatures on the
activities of lactate dehydrogenase, malate dehydrogenase

and glucose 6-phosphate dehydrogenase in Cancer irroratus

larval stages cultured at constant 20 C and 10-20 C daily

Cyclic temperatures. ..ceccecceccvecencccecccccccsesccsccccescacees 38

Cancer inroratus. Effect of larval stage on LDs59 values for
temperature. Open circles: 120 min exposure; filled circles:
2h0 minute exposure. 1-5Z: zoeal stages; M, megalops.........2+-- 39

Cancer irroratus. Effect of temperature on LD50 of oxygen
for different larval stages.........ececeseeesccsceeeeesceerensses HO

Cancer irroratus. Effect of larval stage LD59 value for oxygen... Wa
Acute temperature tolerance limits for three species of crustaceans
cultured under constant conditions optimal for their survival. (c.
irroratus, 15 C-30 o/oo; H. americanus and P. pugio, 20 C-30 o/oo. 42

ix


TABLE 5. EQUATIONS DESCRIBING THE DEVELOPMENT TIME
FOR LARVAL STAGES OF CANCER IRRORATUS A‘! DIFFERENT
CONSTANT TEMPERATURES (FROM SASTRY, 1976).

Larval stage* Equation
I D=500/(T + 1.53)'-*4
u D = 498/(T + 1.72)!-33
sing D=500/(T + 1.63)'-*>
Iv D=500/(T + 6.6)'-*°
v D = 999/(T + 2,06)-7°
Megalops D = 500/(T — 3.19)" 79

*Stages I to V are zoeal stages.

nae STAGE
y% le
- “ mx 4
\ IIo
Ss \ Wa
3 MEGALOPS. Yo
~20- 4
uw
=
-
e Lb 4
2
é
=
oy
grok 4
>
Ww
oa
Lo) 1 i L L
° 15 25

TEMPERATURE, C

Figure 8. Effect of temperature on development time for larval stages
of C. irroratus. Dashed lines on the curves represent extrapolated valu

20


Duration of larval development

Temperature significantly affected the duration of larval development
of C. irroratus, H. americanus and P. pugio (Fig 7). In all these species,
the duration for complete larval development decreased with the increase of
temperature. In comparison, the developmental rate of P. longicarpus was
less affected by temperature. The larvae of R. harrisii showed still anoth-
er pattern of temperature effect on the rate of development, developing at a
slower rate at 30 C compared to those at 25 C. The duration of larval de-
velopment of all the species is affected by the temperature, but the rates
vary interspecifically (Fig. 7 and Table 4).

The effect of temperature on the rate of development of each larval
stage of C. irroratus was determined to examine whether successive larval
stages in the development of a species are differentially affected by the
temperature. ‘he first zoeal stage of C. irroratus developed at a slower
rate than the succeeding zoeal stages (Fig. 8 and Table 5). The rate of de-
velopment of the zoeal stages decreased significantly at temperatures below
10 C; developmental rate remained about the same at temperatures above 25 C.
The duration of megalops stage is much longer than any of the preceeding
zoeal stages. The duration of the megalops stage also became significantly
increased below 15 C and decreased slightly above 20 C (Pig. 8).

In contrast, salinity had much less effect on the rate of development
than temperature (Fig. 9 and Table 4). The duration of development of each
species was different at comparable salinities. Contrasting salinity con-
ditions within the range of tolerances did not significantly affect the rate
of development of P. longicarpus and P. pugio. Larvae of R. harrisii de-
veloped at about the same rate in salinities between 30 and 35 o/oo and at
slightly slower rate in 25, 30 or 35 o/oo salinity. In contrast, the larvae
of H. americanus developed at a slower rate in salinities above and below
25 o/oo.

Variation! in geographically separated populations

All the species selected for this study are distributed over a wide
geographical range (Table 1). Information on the duration of complete de-
velopment and survival of larvae to the post-larval stage of geographically
separated populations are not available for most species. Data are avail-
able for R. harrisii and P. pugio populations from North Carolina (Costlow,
et al., 1966; Broad, 1957) and are compared with Rhode Island populations
cultured under somewhat similar conditions (Table 6). Rhode Island larvae
of R. harrisii took longer to complete development and their survival was
much lower compared to those from North Carolina. In comparison, survival
of P. pugio larvae from Rhode Island was higher (91.8%) compared to those
from North Carolina (65%). Larval development of the Rhode Island population
was completed between 18-31 days compared to 17-21 days required for the
North Carolina population (Table 6). ‘lhere is an indication that the dura—
tion of H. americanus larval development also varies for geographically
separated populations (Table 7).

17


stresses were combined, the thermal tolerance limits of C. irroratus larval
stages were altered.

Environmental thermal alterations can affect survival, developmental
rates and metabolism of developing larvae of crustaceans. The specific ef-
fects of thermal alteration will vary during the course of larval develop-
ment in a manner characteristic to each species. The nature of these effects
will be influenced by the organisms recent thermal history, as illustrated
by the higher survival rate of larvae reared under cyclic temperature con-
ditions than at a single constant temperature. Further, these effects are
modified as other environmental parameters, such. as dissolved oxygen and
salinity interact with temperature. Generally, estuarine species have a-
daptive capacities for development and growth over a wider range of environ-—
mental conditions than coastal species.

This report was submitted in fulfillment of Grant number R 800981 under
the sponsorship of Environmental Protection Agency. Work was completed as
of September 1976.


ABSTRACT

Studies have been conducted to determine the effects of temperature
singly and in combination with other environmental factors on the develop-
mental and survival rates, metabolic adaptation and tolerance limits of
larvae of selected epibenthic crustaceans from the New Hngland region. Six
species, Cancer irroratus, Cancer borealis and Homarus americanus from the
coastal area’ (high salinity), and Palaemonetes pugio, Pagurus longicarpus
and Rhithropanopeus harrisii, from the primarily estuarine region (variable
salinity were used for this study. Larvae of each species were cultured at
various combinations of temperature and salinity to establish the combina-
tion contributing to highest survival rates and to determine the limits for
complete development of each species. ‘lemperature and salinity limits and
the optimal combination for development varied interspecifically. Generally,
coastal species had a more restrictive temperature range for complete devel-
opment than estuarine species.

The effects of daily cyclic temperatures vs. a comparable constant re-
gime on the development and survival rates of larvae were variable. Survival
of larvae of C. irroratus cultured under certain daily cyclic regimes was
better. In contrast, larvae of P. pugio cultured under daily cyclic re-
gimes showed no significant differences in either survival or developmental
rate when compared to those at constant temperatures.

Metabolic responses of C. irroratus, H. americanus and P. pugio larvae
cultured at temperature and salinity combinations optimal for their max-
imum survival rate were determined over a graded series of test temperatures.
Larval stages of the three species exhibited thermal sensitivity (Q, >2),
insensitivity or compensation (1<Q)9<2) and depression (Q,,<1) of mét—
abolic rates over the range of tested temperatures. The patterns of met-
abolic response to temperature varied inter-and intraspecifically. Gen-
erally, the larvae of coastal species C. irroratus and H. americanus
were metabolically active over a narrower temperature range compared to the
primarily estuarine species, P. pugio. Metabolic-temperature response pat-—
terns of the larval stages of each species cultured at daily cyclic temper-
atures were altered relative to those cultured at comparable constant temp-
eratures. The differential effects of daily cyclic and constant temperatures
were also exhibited in the fatty acid methyl esters in larval stages of H.
americanus and in the enzyme systems examined in the larval stages of C.
irroratus. :

Acute temperature and low dissolved oxygen tolerances were determined
for larval stages of C. irroratus, H. americanus and P. pugio. The thermal
tolerance limits for the larval stages of the primarily estuarine grass
shrimp, P. pugio were higher compared to those for larvae of coastal spegies,
C. irroratus and H. americanus. When temperature and low dissolved oxygen

iv


Table 1 Distribution, ecology and breeding period of seven species of crustaceans used in the study

eer

Narrnyansett Bey and Vicinity,
Habitat
‘Temperature °c

Species Geographical Distribution Habitat Collection — Rreeding Period and Salinity (o/s0)
(Wi 1Ldams 1965) Tawa ity during breeding period
Pagurus longicarpus Nova Scotia to Northern Common on harbor beaches, Boat basin Late April to 7.5-17.0 €
Florida and from Sanibel and in shallow littoral mid-June 3 ofoo
' Island, Florida to Texas on a variety of bottoms.

Palaewonetes pugio Massachusetts to Texas Estuarine waters espe- Bissell's Mid-June to 20-25 ¢
cially in submerged Cove early September 10-30 o/oo
vegetation

Rhithropanopeus Canada to Mexico, north- —- Ewtuarine, found in Narrow River Hid-June to Late 22-27 ¢

erat aa east Brazil, introduced places always providing August 13-26 0/00

to West Coast of U.S. shelter
and in parts of Europe

Cancer irroratus Labrador to South Carolina Low water mark to 600m, ‘Narragansett April to early 6-19 C
shallow bay in north and Bay July 31 of/oo
deep waters in south

Gancer borealis Nova Scotia to Tortugas, Between tides in rocks Narragansett July 18-23 ¢

Florida and Bermuda to 870 m; shallow baya Bay 31 of/oo
in north and deep waters
in south
Nova Scotia to New Jersey © Sub-Littoral rocky bottom Narragansett July 18-23 ¢
Bay and 31 ofoo
vicinity
Panopeus herbstii Massachusetts to Brazil; Estuarine, bottom com- Pawcatuck July 18-23 ¢
Bermuda posed of soft mud and River 31 ofoo

oyster shells


Number Page
1 Larval stages of Cancer irroratus. I-V, zoeal stages; M, megalops..11
2 Larval stages of Cancer borealis. I-V, zoeal stages; M, megalops...12
3 Temperature and salinity limits for complete larval development
of six species of crustaceans from Narragansett Bay and vicinity....14
4 Percentage survival of larvae to post-larval stages for five species
of crustaceans reared in different combinations of salinity and
PEMPCTALULE. . ce eesece cree ceecccccceecccnseneccsecenccccccesccccsucce LH
5 Survival of Cancer irroratus larvae to the post-larval stage in
different combinations of salinity and temperature. The larvae
resulted from eggs hatched in 30 o/oo salinity at 15 C in dif-
ferent SCASONS...cceeececeeeeccc ence ececceeesstncsceenccserssseeeee le
6 Isopleths of mortality of larvae to the post-larval stage for
primarily estuarine and coastal crustaceans in relation to
temperature and salinity conditions.. 16
7 ‘Effect of temperature on the duration of larval development of
primarily estuarine and coastal CYUSEACEANS. cece eee e eee eecececnce ee LB
8 Effect of constant temperature on development time for larval
stages of Cancer irroratus.cersccccscseccccceececccccccccscsecsesess 20
9 Effect of salinity on the duration of larval development of
primarily estuarine and coastal crustaceans. .cccececesccccccccceces ob
10 Percentage survival of Cancer irroratus larvae at constant and
Cyclic temperatures. .ccccecccccecccceccccceeccceescceeecssesssceeas ot
11 Duration of larval development of C, irroratus at constant and
cyclic temperatures....ccececcececcccncceccccuseeceseesesssseseesess lO
12 Survival of Cancer irroratus larvae to the post-larval stage
under constant and comparable cyclic temperature regimes (A)
The duration of larval development under constant and comparable
cyclic temperature regimes (B)....c.c cece eee e cece cece cececeesceeeeclT
~
13 Percentage survival of Palaemonetes pugio larvae at constant

FIGURES

and daily cyclic temperatures. ...cececccccecccccenccccccceccesseres od

viii


Figure 2.

Larval stages of Cancer borealis I to V, zoeal stages; M. megalops.


United States Environmental Research EPA 600 3-80.064
Environmental Protection Labor July 1980

ratory
Agency Narragansett Ri 02882

Research and Development

GEPA Effects of Thermal
Pollution on Pelagic
Larvae of Crustacea

>



and depression between 25-35 C ranges. Metabolic rate of the second stage
was depressed over both the 10-15 and 30-35 C ranges, with compensation be-
tween 25-30 C. The third stage compensated between 10-15 C, with depression
between 30-35 C. For the fourth stage metabolic rate was compensated be-
tween 10-15 and 25-30C and depressed between 30-35 C. The fifth stage com-
pensated over a broader range than the earlier stages, i.e. between 20-35 C.
The sixth stage compensated between 15-25 C and has depressed between 30-35C.
In the seventh stage metabolic rate compensation was observed between 15-20
and 25-30 C and depresssion between 30-35 C. The eighth stage larvae com-
pensated between 10-20 and depression occurred between 25 and 35 C. Larval
stages cultured at constant 20 C showed equally variable metabolic tempera-
ture responses as those at the daily cyclic regime.

8 [ESS WY crc.
i ll. SSS con

7 C_ RES ce
a BSS con

, ——_ NSSS cre
— | —] con

; —___ BE xc
> TT con

———= RSS crc
CON

3 S crc
co —n a CON

2 SX MESS crc
? RSS CT CON

\ [ESSSYS| AS CC

KN con
io = 20 25 30 35
TEMPERATURE °C

%>2, ME N< Qi9< 2, Gc!

STAGE

Figure 20. Metabolic-temperature responses (as Q)y values) of larval stages
of Palaemonetes pugio cultured in 30 o/oo salinity at constant 20 C and
daily cyclic 15-25 and 17.5-22.5 C temperatures.

35


TABLES (cont'd)

Number Page

13 Cancer irroratus: IDsg values for temperature for 120 min and
2h0 min exposure times, DY Stages. ccccsccccccccccccceccccensessseees 39


Number

1

10

12

TABLES

Distribution, ecology and breeding period of seven species
of crustaceans used in the Study...ccecccccvcccccccccecveccccscece 6

Egg incubation and larval culture conditions for six species
Of CLUSTACEA. . cece cece c ccc c eee c erence cence stneeesteeeessesessceee T

Variability in the number of larval stages during development
of Palaemonetes pugio cultured at different combinations of

temperature and salinity........ weee 10

Mean (+ SD) days for complete development to post-larval stage
in various combinations of temperature and salinity for five
species of crustaceana...scccsccccccccccncccsecccecsccceeseseeee nde

Equations describing the development time for larval stages
of C. irroratus at different constant temperatures.......ee+000+420

Variation in the survival and duration for complete development
of geographically separated populations of three species of
crustaceans. nnn 1

Duration for development to the third and fourth larval stage
of H. americanus from different geographical regions.........0++.33

Survival of C. irroratus larval stages at constant and cyclic
Gemperaturesscsccccccccccceccccccccecececececececeseccececscssess fl

Statistical analysis testing the significance of differences
between survival of larval: stages of Cancer irroratus cultured
at constant and cyclic temperatures.....cceccecccccccccecccsseses lo

Mean days and the proportion of total development time (hatch
to crabs) for each larval stage of C rroratus at constant
and cyclic temperatures...

for)

Fatty acid methyl esters identified in developing larval stages of

H, americanus cultured at constant 20 and 15-25 C cyclic temperatures. 37

Activities of lactate dehydrogenase determined from crude cell-
free homogenates of Cancer irroratus larvae cultured under cyclic
and constant temperatureS.ceccccccccccaccccccccccccccscceesseseeed

Pag: e


Nylor, E. Effects of heated effluents upon marine and estuarine organisms
Adv. Mar. Biol., 3: 63-103, 1965.

Prosser, C. L. ‘llemperature. In: Comparative Animal Physiology, C. L.
Prosser, ed. Saunders, Philadelphia, Pa., 1971. pp.. 362-428.

Roberts, M. H. Larval development of Pagurus longicarpus Say reared in
the laboratory, I. Description of larval instars. Biol. Bull., 139:
188-202, 1970.

Sastry, A. N. Culture of brachyuran crab larvae using a recirculating sea
water system in the laboratory. Helgolander wiss. Meeresunters, 20:
406-416, 1970.

Sastry, A. N. Effects of constant and cyclic temperature regimes on the
larval development of a brachyuran crab. In: Second. Termal Ecol.
Symp. G. W. Esch and R. W. McFarlane, eds. ERDA Symp. Ser., (Conf.
750425), 40: 81-87, 1976.

Sastry, A. N. Larval development of the rock crab, Cancer irroratus Say
1817, under laboratory conditions (Decapoda, Brachyuran). Crustaceana,
32: 155-168, 1977a.

Sastry, A. N. Physiological adaptation of Cancer irroratus larvae to cyclic
temperatures. In: Physiology and Behavior of Marine Organisms.
D. S. McLusky and A. J. Berry, eds. Pergamon Press, Oxford, 1978.
pp. 57-65.

Sastry, A. N. and W. R. Ellington. Lactate dehydrogenase during the larval
development of Cancer irroratus: Effect of constant and cyclic thermal
regimes. Experentia, 34: 308-309, 1978.

Sastry, A. N. and J. F. McCarthy. Diversity of metabolic adaptation of
pelagic larval stages of two sympatric species of brachyuran crabs.
Neth. J. Sea Res., 7: 434-Lh6, 1973.

Sastry, A. N. and S. L. Vargo. Variation in physiological responses of
crustacean larvae to temperature. In: Physiological responses of
marine biota to pollutants. F. J. Vernberg, A. Calabrese, Thurberg
and W. B. Vernberg, eds. Academic Press, New York, 1977. pp. 401-23.

Scarratt, D. J. Distribution of lobster larvae (Homarus. americanus) off
Pictous, Nova Scotia. J. Fish. Res. Bd. Canada, da, 25: 427-430 pp. 1968.

Sherman, K. and R. D. Lewis. Seasonal occurrence of larval lobsters in
coastal waters off central Maine. Proc. Nat. Shellfish, Assoc.
57: 27-30, 1967.

Templeman, W. ‘lhe influence of temperature, salinity, light and food con-
ditions on the survival and growth of the larvae of the lobster
(Homarus americanus). J. Biol. Bd. Canada, 2: 485-97, 1937.

48


j

{ uw

j

q

nO, /hs mg DRY WEIGHT

zr zz

7m tz MEGALOPS:
18
or:
as 4

o 8 2 30 5 0 8 O30 5 OB BH 2

TEMPERATURE “C

Figure 15. Metabolic-temperature response of different larval stages of
Cancer irroratus; ZI to ZV zoeal stages.

oo, 22 3 <4
42.
3”
g q
% 07; 3
r4 q
te
s f
z
e zm 2z MEGALOPS:
a «0
20.
Io:
ar:
as.
o s Ly Bs 202 wo s 0 os * es pcnaTURe ms

Figure 16. Metabolic-temperature response of different larval stages of
Cancer borealis; ZI to ZV zoeal stages.


TABLE 4.

MEAN (+ SD) DAYS FOR COMPLETE DEVELOPMENT TO POST-
LARVAL STAGE IN VARIOUS COMBINATIONS OF TEMPERATURE
AND SALINITY FOR FIVE SPECIES OF CRUSTACEANS

Species Temp _C Salinity
10 15 20 25 30 35 40
Palaemonetes TS TOT + 96.5 84.46 86.34 BT.374 94.72
3.0 11.68 6.87 6.37 18.34 15.03
20 34.14 34.6+ 31.664 32.09% 32.064 33.274 = -
3.2 3.3 2.28 2.21 2.21 2.87
25 26.324 30.07+ 22.64+ 23.914 23.56+ 26.194
. 3.66 2.52 3.07 3.04 4.70 -
Paugurs 10 46.5 44.0 35.5 41.0 38.5
ongucarpus 15 39.0% 31.64 32.0+ 2 ?
3.46 3.81 4.12
20 - 31.6 29.8 = 26.3% 25.34
5.06 4.02 2.8T 2.73
Rhithropanopeus 25 - - 36 24 22 22
Aarristt 30 - - 24 26 27 -
\i
H. americanus 15 - - 79.674 97.0% 2? 76.0#
7.02 4.24 2.77
20 - 55 53.7+ 42.274 46.364 -
8.27 7.89 7.28
C. irroratus 15 - —48.33+ 56.39% 55.734 60.26+
3.51 §.13 4.24 7.42
20 - - 40.02+ 36.03+ 38.194
9.94 3.83 6.69

19


FATTY ACID METHYL ESTERS

Fatty acid methyl esters were determined for the developmental stages
of C. irroratus beginning with eggs to post-larvae cultured at constant 15 C
and H. americanus cultured at constant 20 C and 15-25 daily cyclic tempera-
tures. In general, the fatty acid methyl esters in the developmental stages
of both species showed minor qualitative differences, except for the presence
of detectable amounts of 14:0 and 22:5 chain length fatty acids in C.
irroratus (Table 11).

Fatty acids 16:0, 16:1, 18:1, 20:5 and 22:6 were present in higher
concentrations than 14:0, 18:0, 20:1 and 20:4 and 22:5 in the eggs of C.
irroratus. In the succeeding larval stages, the fatty acids 16:0 and 20:5
remained at a fairly constant concentration, while 22:6 decreased from a
high concentration in the eggs and first zoeae to low levels in the later
megalops and crab stages. Fatty acids 18:2 and 18:3 were observed in only
trace amounts in eggs and first zoeae, but they increased to detectable
amounts in the megalops and crab stages. The 22:5 chain length fatty acid
present in detectable amount in eggs and first stage zoeae decreased to
trace amounts in the megalops and crab stage.

In H. americanus cultured at constant 20 C, fatty acids 16:0 and 18:1
were present in high concentrations and 16:1, 18:0 and 20:4 in low concen-
trations through all the developmental stages from eggs to post-larvae.

20:5 and 22:6 chain length fatty acids were initially present in high con-
centrations in eggs and first larval stage then decreased progressively with
development to the post-larval stage. Fatty acid chain lengths 18:2 and 18:3
in trace amounts in eggs and first larval stage increased to detectable a-
mounts in the later stages of development, while 20:1 in high concentrations
in the eggs and first stage decreased beginning with the second stage.

In H. americanus cultured with 15-25 C daily cyclic temperatures, the
16:1, 18:1 and 20:5 chain length fatty acids were present in fairly high
concentrations through all stages beginning with eggs to the post-larvae.
Fatty acids 16:1, 18:0 and 20:4 were in low concentrations through all the
stages of development. Initially high concentrations of 22:6 fatty acid in
eggs steadily decreased with the progress of development to the post-larval
stage, whereas 18:2 and 18:3 were present in only trace amounts in the eggs,
then increased to detectable amounts beginning with the first larval stage.
Fatty acid 20:1 was present in detectable amounts in the eggs, then de~
creased to trace amounts beginning with the first larval stage.

A comparison of fatty acid methyl esters in H. americanus larvae cul-
tured at constant and daily cyclic temperatures showed that certain fatty
acids had a tendancy for greater unsaturation under fluctuating thermal re-
gime in some stages of development (i.e. 16:0). The other difference ob-
served was an increase of certain fatty acids present in only trace amounts
at constant temperature to detectable amounts in those cultured under cyclic
temperatures (i.e. 18:2 and 18:3). Fatty acid (i.e. 20:1), present in only
detectable amounts in H. americanus cultured at constant 20 C, were only pre-

sent in trace amounts in those under cyclic regime. 7

36


80r
70} oa
6O0F
SOP
7)
> ar
<
a
30
20F
0° H.americanus
© C. irroratus
AP. pugio
lok 8 A Aarrisii
&P. longicarpus
fo) L L 1 1 1.
5 10 1s 20 25 30

TEMPERATURE C

Fig. 7. Effect of temperature on the duration of larval development of
primarily estuarine and coastal crustaceans.

18


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Abstract.
Figures.......
Tables...eeeeeee

CONTENTS

Acknowledgements. .ccccccccccccsccccccccccccccccvccsessvcessecceees XiL

Au rwne

Introduction. ccesecececcccccccccccccneccecsccnccscssceseseedl
Recommendations.
Conclusions........
Materials and Methods sec e eee eee ee D

Experimental Results. «-10
Discussion... cececscccccccccceceecccccceccseececsseeceseee HS

REPETENCES. cece eee een cence ccc eee e tee tee tense eeenseeseesecees ee HG

vil


of the sample methyl esters on the gas chromatogram. The fatty acid methyl
esters were expressed as weight percent. All solvents used were distilled
and blank solvents showed negligible quantities of fatty acids.

Enzyme Assays

Specific activities of lactate dehydrogenase, malate dehydrogenase and
glucose 6-phosphate dehydrogenase were assayed for pooled groups of larval
stages of C. irroratus cultured at 10-20 C daily cycle and 15 C constant
temperature. Larvae were homogenized in 1:5 (weight/volume) of 5 mM Tris-
HCl (pH 7.5) and the resulting homogenate was centrifuged at 25,000 g ina
refrigerated centrifuge for 20 minutes. The resulting supernatant was used
as the source of enzymes. The assays were performed using the following re-
action mixtures: LDH, 0.01 mM NADH, 5 mM sodium pyruvate and 50 mM Tris-HCl
(pH 7.5) in a total volume of 3.0 ml;

MDH, 0.01 mM HADH, 0.2 mM oxaloacetate and 50 mM Tris-HCl (pH 7.5) in a total
volume of 3.0 ml;

GOPDH, 0.15 mM NADP, 1.66 mM glucose 6 phosphate, 5 mM MgClo and 50 mM Tris-
HCl (pH 7.5) in a total volume of 3.0 ml. Enzyme assay mixtures were equiil-
ibrated to 15 C prior to assay in a Zeiss PMQ II Spectrophotometer. Enzyme
activities were expressed as y moles per mg Lowry protein.

Acute Temperature and Dissolved Oxygen Tolerances

Acute temperature and low dissolved oxygen tolerances for the larvae
of C. irroratus, H. americanus and P. pugio were determined for each stage
of the three species. ‘Ihe larval stages were placed in flat walled 25 ml T-
type glass flasks designed for tissue culture, which allow direct micro-
scopic examination of the larvae for testing. Lethal temperature limits were
determined under saturated dissolved oxygen conditions by bubbling with com-
pressed air. ‘lhe tests were conducted at 2 C intervals between 27 and 33 C,
using 10 larvae at each temperature. After the flasks had been equilibrated
to the test temperature, the larvae were introduced and the number surviving
was determined at 30, 60, 120 and 240 minute intervals. Cessation of heart
beat was the criterion of death. After testing, the larvae from each flask
were returned to their culture temperature and the number of survivors was
determined after 24 hours. The tolerance of larval stages to low dissolved
oxygen was determined by reducing the dissolved oxygen from saturation (4.2
to 6.2ml 05/1) to 0.2 ml 09/1 at 5 test temperatures (10, 15, 20, 25 and 30
C) were used. After the test, two 60 ml oxygen samples were drawn from each
flask and analyzed by modified winkler method (Carritt and Carpenter, 1966).
The time intervals and criterion of death were the same. ‘the LD5g values for
temperature and dissolved oxygen were determined by the graphical method
(Goldstein, 1964). Because the tolerance to low dissolved oxygen has been
measured, thé greater the LDcq value the lower the tolerance; therefore, the
LDso value is plotted as 1/1Be9 . With this transformation, the higher values
in the plot represent greater Porerances (Vargo and Sastry, 1977). In the
temperature and low dissolved oxygen tests, tolerances of individual larval
stages were highly variable for the 30 and 60 min. time sampling intervals;
hence only the data for the 120 and 240 min. time intervals are given. This
variability is probably due to differing activity levels resulting from
handling when the larvae were. transferred to the test flasks.

9


SECTION 7
REFERENCES
Alderice, D. F. Factor combination. In: Marine Ecology, Vol. 1, Pt. 3.
Wiley-Intersceince, New York, 1972. pp. 1659-1722.
Bliss, C. I. The statistics of bioassay. Academic Press, N.Y., 1952. 628 pp.

Borad, A. C. Larval development of Palaemonetes pugio. Holthius. Biol.
Bull., 112: 144-161, 1957.

Bullock, T. H. Compensation for temperature in the metabolism and activity
of poikilotherms. Biol. Rev., 30: 311-342, 1955.

Carritt, D. E. and J. H. Carpenter. Comparison and evaluation of currently
employed modification of the Winkler method for determining dissolved
oxygen in sea water, a NASCO report, J. Mar. Res., 2h: 286-318, 1966.

Clark, J. R. Thermal pollution and aquatic life. Sci. American, 220: 19-
2T, 1969.

Connoly, C. J. The larval stages and megalops of Rhithropanopeus harrisii.
Fish. Res. Bd. Canada, Contributions to Canadian Biology and Fisheries,
N 2: 329-337, 1925.

Costlow, J. D. and C. G. Bookhout. An approach to the ecology of marine
invertebrate larvae. Symp. Expt. Ecol., Grad. Sch. Oceanography,
Occassional Publ., No. 2, 1964. pp. 69-75.

Costlow, J. D. and C. G. Bookhout. The effects of cyclic temperatures on
development in the crab, Rhithropanopeus harrisii. In: 4th European
Symp. Mar. Biol., D. J. Crisp, ed. Cambridge Univ. Press, Cambridge,
Mass., 1971. pp. 211-220.

Costlow, J. D. and C. G. Bookhout and R. Monroe. Studies on the larval
development of the crab, Rhithropanopeus harrisii (Gould). I. The
effect of temperature and salinity on larval development. Physiol.
Zool., 39: 81-100, 1966.

Coutant, C. C. and S. S. Talmage. ‘lhermal effects. J. Water. Poll. Cont.
Fed., 49: 1369-1425, 1977.

Esch, G. W. and R. W. McFarlane. Thermal ecology II. Conf. 750225. ERDA
Symposium Series, No. 40, Tech. Infor. Center, ERDA, 1976. oh pp. ~

46


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

1, REPORT NO. 2. 3. RECIPIENT'S ACCESSIONNO.
EPA-600/3-80-064
4. TITLE AND SUBTITLE 5. REPORT DATE
JULY 1980 ISSUING DATE.
Effects of Thermal Pollution on Pelagic Lar
gic ‘vae of

Crustacea 6. PERFORMING ORGANIZATION CODE

17. AUTHOR(S) IB. PERFORMING ORGANIZATION REPORT NO.

A. N. Sastry

9. PERFORMING ORGANIZATION NAME AND ADDRESS 10, PROGRAM ELEMENT NO,
Graduate School of Oceanography
University of Rhode Island 11. CONTRACT/GRANT NO.
Kingston, Rhode Island 02881
R-800981
12, SPONSORING AGENCY NAME AND ADDRESS 13. TYPE OF REPORT AND PERIOD COVERED
Env ironmental Research Laboratory ~ Narragansett Final
Office of Research and Development 14, SPONSORING AGENCY CODE
U.S. Environmental Protection Agency EPA/600/05

Narragansett, RI 02882
15. SUPPLEMENTARY NOTES

16. ABST RA - * . 5 =
8 “T Larvae of six species, Cancer irroratus, C. borealis and Homarus americanus

of coastal waters (high salinity), and Palaemonetes pugio, Pagurus longicarpus and
Rhithropanopeus harrisii, from the estuarine region (variable salinity) were studied.
Larvae were cultured at various combinations of temperature and salinity and highest
survival rates and limits for complete development determined. Coastal species have
& more restrictive temperature range. Thermal tolerance limits for larvae of the
primarily estuarine P. pugio were higher compared to larvae of coastal species, ,.

C. irroratus and H. americanus. When temperature and low dissolved oxygen stresses
were combined, thermal tolerance limits of C. irroratus larvae were altered. Sur-
vival was better for C. irroratus larvae cultured under certain daily cyclic

regimes vs. a constant temperature. In contrast, larvae of P. pugio showed no
significant differences in either survival or developmental rate when under cyclic
vs. constant temperatures.

Metabolic responses of C. irroratus, H. americanus and P. pugio were determined
for a series of temperatures. Larvae of coastal C. irroratus and H. americanus were
metabolically active over a narrow temperature range. The estuarine species P.
pugio and response patterns of larvae cultured at cyclic temperatures differed from
those at constant temperatures. Differential effects of daily cyclic vs. constant

temperatures also occurred in fatty acid methyl esters in H. americanus and in e~zyme
ystems of C. irroratus larvae

ka KEY WORDS AND DOCUMENT ANALYSIS
la. DESCRIPTORS b.IDENTIFIERS/OPEN ENDED TERMS jc. COSATI Field/Group
Crustacea Larval develupment
Larvae Limiting factors O6F
Physiological Effects Cyclic temperatures
Temperature Estuarine
Salinity Coastal
Oxygen
78. DISTRIBUTION STATEMENT 19. SECURITY CLASS (This Report) 21. NO, OF PAGES
UNCLASSIFIED 65
‘ RELEASEsTO PUBLIC 20. SECURITY CLASS (This page) 22. PRICE
UNCLASSIFIED

EPA Form 2220-1 (9-73)
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creased considerably at’ the 15-25 C cycle compared to constant 20 C. The
megalops was the most thermally sensitive stage in the larval development of
Cc. irroratus.

Larval survival from eggs of C. irroratus hatched under daily eyclic
temperatures and reared under the same temperature cycle showed an increase
in the survival to the crab stage, compared to larvae hatched at constant
temperature and reared under daily cyclic regime (Fig. 12.) Survival in-
creased by 9% for the 10-20 C cycle, 7% for the 15-25 C cycle and 1.5% for
the 12.5-17.5 C cycle. Larval development time whether eggs hatched under
cyclic or constant temperature, was not significantly different for the 10-20
and 12.5-17.5 cycles. However, for the 15-25 C cycle, the development time
of larvae hatched under cyclic temperatures was less than that for larvae
hatched under constant conditions (Fig. 12).

60
(a) A

40-- |
20+ 4

=

30

ui f(b)

=

-

20h 4

a

=

§

go

Fa

a 60 Ex) Constant 7

ZI 10°C cycle
BS 8°c cycle

40 4
: y 4 : |
C)

20 10 15- 125- 17.5-
20 25 175 225
TEMPERATURE, °C.

Figure.11. Duration of larval development of C. irroratus at constant and
cyclic temperatures. (a) Hatch to crab. (b) Megalops. (c) Hatch to meg-
alops.

26


Lz

80 80

A Bo. |
Z Eggs & Larvae t
Cycled — 6o-
7 60/- oO Larvae Cycled { | i
g | | 4
= o |
& 40- x 40ob i
2 a |
x |
20h 20- |
A Eggs & Larvae
¥ | Cycled
° 4 0 i} © Larvae Cycled
15 20 10-20 15-25 125-175 15 20 10-20 15-25 12.5-175
CONSTANT CYCLIC CONSTANT CYCLIC

TEMPERATURE C

Figure 12. Survival of C. irroratus larvae to the post-larval stage under constant and comparable
cyclic temperature regimes (A). The duration of larval development under constant and comparable
cyclic temperature regimes (B).


tolerated slightly higher temperatures and the megalops lower temperatures
than the other stages. It would appear that although certain stages in the
larval development of a species may be slightly more resistant or sensitive
to temperature, the general temperature tolerance limits for overall devel-
opment remains the same for a species and is related to the temperature
range of their habitat.

P pugio

oe
+
T

H. americanus

LIMIT, LDgo in C
T

sol
26 Lo 1 1 1 1 a
1 2 3 4 5 6 7

LARVAL STAGE

Figure 25. Acute temperature tolerance limits for three species of crus-
taceans cultured under constant conditions optimal for their survival. C.
irroratus, 15°C - 30 0/oo; H. americanus and P. pugio, 20°C - 30 o/oo.

ke


WINTER

80.

To
SURVIVAL 40.
°

Fig. 5. Survival of C. irroratus larvae to the post-larval stage in different
combinations of salinity and temperature. The larvae resulted from eggs
hatched in 30 o/oo salinity at 15 C in different seasons.

15


(Sastry and Ellington, 1978).

Acute temperature tolerance limits varied intra- and inter-specifically
with estuarine species tolerating higher temperatures than the coastal
species under saturated oxygen conditions (Sastry and Vargo, 1977). These
limits remained fairly constant for all C. irroratus larval stages; some
H. americanus and P. pugio stages showed differential sensitivity. However,
when low dissolved oxygen and temperature stress were combined more inter~
stage variation was evident with C. irroratus.

The metabolism, development and growth and survival of larvae may vary
with stage, season of hatching, culture conditions, and geographic origin of
the population. These intra-specific variables have to be taken into con-
sideration in the application of laboratory bioassay results for the eval-
uation of the potential effects of thermal and other pollutants on a natural
population or community.

45


DISCLAIMER

This report has been reviewed by the Environmental Research Laboratory,
Narragansett, U.S. Environmental Protection Agency, and approved for publi-
eation. Approval does not signify that the contents necessarily reflect the
views and policies of the U.S. Environmental Protection Agency, nor does the
mention of trade names or commercial products constitute endorsement or re-
commendation for use.

di


ITTTA 20°C
J 15-25°C

La, Ene
WZZZ7777A
Es I

L3 GES ETET
wi CLOT: J
oO
2 7A
e
on
2
TITTINILTITTIZA (J Aig >2
ZQi9<!
LIC I WTI FA <Qq <2
{ 5 VLLL2 J
ra
10 15 20 25 30

TEMPERATURE °C

Figure 19. Metabolic-temperature responses (as Q,, values)of larval stages of Homarus americanus
cultured in 30 o/oo temperature-salinity at constant 20 C and cyclic 15-25 and 17.5-22.5 C.


for metabolic compensation was much wider for the third through seventh
stage larvae. In the fourth stage compensation extended to warmer tempera-
tures than in the earlier stages. The fourth stage showed no compensation
from 10-25 C and compensated from 25-35 C. The fifth stage was the reverse.
In the eighth stage, compensation occurred between 15-20 C and 25-30 C. At

the extreme warm temperatures of 30-35 C, the metabolic rate of the third,
sixth, and eighth stages was depressed.

Larvae Cultured at Daily Cyclic Temperatures

Oxygen consumption rates of the developmental stages of C, irroratus
cultured at the constant 15 C and 10-20 C daily cyclic temperatures were
different at some of the test temperatures. These differences in rate were
significant (p<0.01) for only first and second zoeal stages and the first
crab stage at 15 C. At the 20 and 25 C test temperatures, almost all stages
from the cyclic regime respired faster than those from constant culture temp-
erature. Wo obvious correlation of the variation of oxygen consumption rates
with the stage of development was evident.

Vu P pugio
C. borealis
vil Q9>2 oO
Qig<! 2
1<Qi9<2 a
y 0
- v
oO
2 Iv
z=
<i
a
i
1

Oo 5 10 15 20 25 30 35
TEMPERATURE, C

Figure 17. Metabolic-temperature responses (as Q10 values) of larval stages
of C. borealis and P. pugio cultured at constant 20°C and 30 o/oo salinity.

Larvae of C. irroratus cultured at 10-20 C daily cyclic temperatures had
different metabolic-temperature response patterns from those larvae from con-
stant 15 C as reflected in the temperature range of sensitivity (Q19?2). com-
pensation (1<Q,9<2), and depression (Q9<1) of metabolic rate (Fig. 18). TM

32


affect their distribution, abundance and survival, laboratory studies of
temperature effects must: be extrapolated with caution to a field situation,
keeping in view the experimental limitations. None the less, laboratory
studies provide an understanding of the effects of environmental alterations
on the adaptive capacities of organisms and give some insight into the pro-
bable consequences to organisms under natural conditions.

Effects of thermal alterations in the environment on adaptations of
adult marine organisms have been extensively studied (Kinne, 1962, 1963,
1970; Vernberg and Vernberg, 1971; Prosser, 1971; Wieser, 1973). However,
the adaptations for functional activities and tolerance limits of the plank-
tonic early life stages of benthic organisms relative to changes in the
thermal environment are not well known. Pelagic larvae are a vulnerable life
eycle link and their survival through complete development is important for
successful recruitment to the adult populations (Thorson, 1950; Meleikovsky,
1970). During pelagic existence, the larvae are exposed to varying combin-
ations of temperature, salinity, light, food and other factors. Of these
factors, temperature, acting either singly or in combination with others,
often is of major importance, to the development and growth, survival and
distribution (Costlow and Bookhout, 1964; Sastry and Vargo, 1977). In the
present project, studies have been conducted to determine the effects of
temperature, singly and in combination with other factors, on development
and survival, tolerance limits and metabolic adaptation of larvae of sev—
eral epibenthic crustaceans from the coastal (high salinity) and predom-
inantly estuarine waters of the New England region.


Larvae of geographically separated populations may also exhibit dif-
ferences in their survival and time required for complete development. For
example, larvae of R. harrisii from the New England region developed more
slowly than those from. southern geographical regions at somewhat similar
temperature and salinity combinations. In contrast, the larvae of geograph-
ically separated populations of H. americanus and P. pugio showed no pro-
nounced differences in their developmental time at somewhat similar compara-
ble culture conditions (Table 6 and 7). A comparative assessment of lati-
tudinal population differences in the effects of temperature and other en-
vironmental factors on larval development and survival would be necessary in
utilizing the present results for water quality criteria purposes.

Within the tolerance range of a species, metabolic rate may vary rela—
tive to temperature, but each species usually has a characteristic overall
response. For example, C. irroratus larvae showed a narrowing of the temp-
erature range for metabolic compensation (1<Q 9<2) with development, but the
temperature range for depression of metabolic rate remained the same. In con-
trast, C. borealis larvae showed a shift in the temperature range of compen-
sation from colder to warmer temperatures in the later stages. These larvae
also had a broader temperature rate for metabolic range compensation than
the congeneric species C. irroratus. Larvae of H. americanus were metabolical-
ly active over a narrower range and the rate was depressed above 25 C. In con-
trast, larvae of P. pugio, an estuarine species were metabolically active over
a@ much wider temperature range than those of the sublittoral species and also
showed metabolic-rate compensation over wider ranges.

Metabolic response patterns of the larvae also varied when they were
cultured under daily cyclic temperatures. A daily cyclic culture regime ex-
tended their temperature range for metabolic rate compensation for some of
the C. irroratus larval stages compared to those under constant conditions.
The cyclic regime also increased larval survival of this species. In con-
trast, the altered metabolic responses seen in the larvae of H. americanus
and P. pugio cultured under daily cyclic regimes were neither consistent nor
predictable. Therefore, we see that it is also not possible to generalize
the effects of fluctuating thermal regimes on the metabolism, growth and de-

velopment on larvae of one crustacean species by comparing with the responses
by others.

Culture of crustacean larvae under daily cyclic and comparable constant
temperatures can also alter biochemical responses as reflected in the qual-
itative and quantitative changes in free fatty acid methyl esters in larvae
of H. americanus. ‘here was a greater tendency for unsaturation of certain
chain length (i.e., 16:0) fatty acids in larvae cultured under cyclic re-
gime compared to those cultured under constant temperature. Certain chain
length fatty acids present in only trace amounts in larvae cultured at con-
stant temperature were observed in detectable amounts in those cultured under
daily cyclic regime.

The activities of enzymes are also affected differently by the thermal
culture regime in larvae of C. irroratus. However, the differences in activ-
ity of the enzymes in larvae from the two contrasting culture regimes did not
follow the same trend, suggesting that enzymes systems in different metabolic
sequences are differently affected in each stage by the cyclic temperatures*

hy


SECTION 8

PUBLICATIONS

Listed are publications and papers presented from the work completed
with partial or full support of Grant R-800981.

Sastry, A. N. Metabolic adaptation of brachyuran crab larvae cultured under
constant and cyclic temperature regimes. Am. Zool., 15: 749, 1975.

Sastry, A. N. ‘temperature variation and physiological responses of crusta-
cean larvae. Joint Oceangr. Assembly, Edinburgh, U.K. Abstract, 1976,
p 152.

Sastry, A. N. Effects of constant and cyclic temperature regimes on the
larval development of a brachyuran crab. In: Second Thermal Ecol. Symp.
G. W. Esch and R. W. McFarlane, eds., ERDA Symp. Series (Conf. 750425).
40: 81-97, 1976.

Sastry, A. N. and John Laczak, 1976. Metabolic adaptation of Palaemonetes
pugio larval stages cultured at constant and cyclic temperature regimes.
Am. Physiol. Soc., Ann. Meeting, Philadelphia (Abstract).

Sastry, A. N. Larval development of the rock crab, Cancer irroratus Say
1817, under laboratory conditions (Decapoda, Brachyura). Crustaceana,
32: 155-168, LOTT.

Sastry, A. N. Larval development of the jonah crab, Cancer borealis Stimpson,
1859, under laboratory conditions (Decapoda, Brachyura). Crustaceana,
32: 290-303, 19TT.

Sastry, A. N. and S. L. Vargo. Variation in the physiological responses of
crustacean larvae to temperature. In: Physiological responses of mar-
ine biota to pollutants, F. J. Vernberg, A. Calabrese, B. Thurberg and
W. B. Vernberg, Academic Press, New York, 1977, pp. 401-423.

Vargo, S. L. and A. N. Sastry. Acute temperature and low dissolved oxygen
tolerances of brachyuran crab (Cancer irroratus) larvae. Mar. Biol.,
40: 165-171, 1977.

Sastry, A. N. and Jan Pechenik. A review of the ecology, physiology and be-
havior of lobster larvae (Homarus americanus and H. gammarus). Div.
Fish. Oceanogr. Circ. 7, Commonwealth Scientific and Industrial Research
Org. Australia, pp. 159-173.

50


SECTION 3
RECOMMENDATIONS

Culture studies of larvae of several predominantly estuarine and
coastal (high salinity) epibenthic crustaceans show that complete develop-
ment occurs within a temperature and salinity range which is characteristic
of each species. Limits, as well as the optimal combination for survival,
vary interspecifically. Limits, as well as optimal conditions for maximum
survival also varied with the prior environmental history of eggs (i.e.
for temperature and salinity), stage of larval development and geographic
origin of the populations. Developmental and survival rates are also al-
tered under daily cyclic culture temperatures relative to those at constant
temperatures. The effects of fluctuating temperatures on development and
survival were more evident for coastal species than estuarine species.

Larvae of the crustaceans examined in this study showed abilities for
metabolic regulation over a temperature range characteristic to each species.
The metabolic rate of larvae showed sensitivity, insensitivity or compen-
sation and depression of metabolic rate over the gradient of temperatures
tested. Generally, the larvae of estuarine species were metabolically active
over a broader temperature range than coastal species. The metabolic—
temperature response patterns of larvae also varied with the stage of de-
velopment of each species. These patterns were altered for larvae cultured
under daily cyclic temperatures relative to those at constant temperatures.
The alterations in metabolic responses of larvae cultured under daily cyclic
regimes were underlined by qualitative and quantitative changes in fatty
acid methyl esters and also in the specific activities of enzyme systems
examined.

The thermal tolerance limits of larvae were found to be generally
higher for the estuarine species than coastal species. The tolerances of
larvae are modified when temperature and dissolved oxygen stress were com—
bined.

‘Biochemical and physiological responses of developing larvae to temp-
erature and other interacting factors need to be examined when evaluating
the effects of thermal alterations on the development and growth. In ad-
dition, the effects of varying environmental temperatures and their inter-
action with other physioco-chemical factors on metabolism, development and
growth of larvae can also provide some valuable information on their abil-
ities for adaptation to altered thermal environment. Studies on larvae of
species occuring in different habitats and as well those breeding in dif-
ferent seasons would provide valuable information regarding the probable con-

sequences of environmental thermal alterations on the recruitment of young
to the adult populations in a geographic region.

4


EPA~600/ 3-80-06)
July 1980

EFFECTS OF THERMAL POLLUTION ON PELAGIC LARVAE
OF CRUSTACEA

by

A. N. Sastry
Graduate School of Oceanography
University of Rhode Island
Kingston, Rhode Island 02881

Grant R-800981

Project Officer

Don C. Miller
Environmental Research Laboratory
Narragansett, Rhode Island 02882

ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
NARRAGANSETT, RHODE ISLAND 02882


Se

TABLE 9. STATISTICAL ANALYSIS TESTING THE SIGNIFICANCE OF DIFFERENCES BETWEEN SURVIVAL OF LARVAL
STAGES OF C. IRRORATUS CULTURED AT CONSTANT AND CYCLIC TEMPERATURES.

Larval stagest
Temperature,°C OOO Megas

15 vs. 10-20 7 - - - + +
20 vs, 15-25 - + + + + +
15 vs. 12.5—17.5 - - - - 7 +
20 vs, 17.5—22.5 - - + + - +
10-20 vs. 15-25 - - - - - +
12.5-17.5 vs.

17.5-22.5 - - - + + +
10—20 vs. 12.5—

175 - - + + + +
15—25 vs. 17.5—

22.5 - + + + + +

*Minus (—) means not significant; plus (+) means p < 0.05
(Pearson and Hartley, 1954).
+Stages I to V are zocal stages.

TABLE 10. MEAN DAYS AND PROPORTION OF TOTAL DEVELOPMENT TIME (HATCH TO CRABS) FOR EACH LARVAL STAGE OF
C. IRRORATUS AT CONSTANT AND CYCLIC TEMPERATURES.

Constant temperatures Cyclic temperatures

Larval 10°C 15°C 20°C 25°C 10-20°C 15-25°C 12,5-17.5°C 17,5-22.5°C

staget Days Days % Days % Days Days b Days % Days ® Days
1 15.4468 6.5214 11.6 5.2213 144 4,222.4 5.121.0 +91 3621.2 62 5642.7 10.8 6.94 4.7
YW 26.046.6 12.942.3 114 9.1420 108 8820.41 11.4435 11.3 7.9220 7.4 11.6236 11.7 13,3251
mm 38.7 + 10.3 19.42 2.7 11.6 13.242.5 11.3 13.0 17.9#3.5 11,6 13,6428 9.8 18.423.4 13.2 18.5% 6.4
Iv 41.4412.6 26.0+2.8 11.8 17.841.7 12.7 23.5435 10.0 19.1235 95 23.923.0 10.7 24.84 3,3
v 61.3244 35.04 3.2 161 24.242.0 17,7 30.3425 11.8 24.343.9 8.9 28.9224 9.7 30.44 3,7
Megalops 55.724.2 37.5 36.0238 32.8 56.045.5 46.3 57.92 11.3 58.0 51.5424 43.9

*Weighted mean,
{Stages I to V are zocal stages.


EFFECTS OF TEMPERATURE AND SALINITY ON DEVELOPMENT AND SURVIVAL

Optima and limits

The temperature and salinity requirements for complete development of
each species to the first crab or juvenile stage were determined by culturing
larvae in different combinations of temperature and salinity (Table 2).
Larvae of C. borealis completed the development only at 20 C in 30 o/oo (Fig.
3). Survival at these conditions was low (4.9%). In contrast, the larvae
of C. irroratus completed development in salinities from 25-35 o/oo at 10
and 20 C, and 20-35 o/oo salinity at 15 C (Fig. 3 and 4). The highest sur—
vival to the first crab stage was observed in 30 o/oo salinity at 15 Cc.
Ovigerous C. irroratus are found in Narragansett Bay from November to early
July (Hilman, 1964; Jones, 1973; Sastry and McCarthy, 1973). Eggs of this
species removed from ovigerous animals collected between November and July
can be hatched when incubated in 30 o/oo salinity at 15 C. Temperature and
salinity limits for complete development as well as survival under compar-
able conditions, varied for larvae hatched at different seasons (Fig. 5).
Larvae resulting from winter hatches completed development in 30 o/oo at 10C
and in 25-35 o/oo at 15 and 20 C. Larvae from spring hatches completed devel-
opment in salinities from 25-30 o/oo and 10 C and 20 C, and between 20-35
o/oo at 15 C. The larvae of summer hatches failed to complete development in
any salinity at 10 C, although at 20 C larvae completed development in 20-35
o/oo at 15 C and 25-35 o/oo salinity. ‘he previous thermal history and stage
of embryonic development prior to incubation affected the survival rate and
limits for complete larval development.

Combined data analyzed for all seasons and represented as response
surfaces (Alderdice, 1972) showed that maximum survival of C. irroratus larv-
ae to the crab stage occur between 28-35 o/oo at temperatures between 13-17
C (Fig. 6). One hundred percent mortality of the larvae was predicted to
occur below 18 and above 40 o/oo salinity, with 100% mortality beyond these
limits (Fig. 6).

Larvae of H. americanus completed development to the post-larval stage
in salinities between 20-35 o/oo at 15 C and 15-30 o/oo at 20 C (Fig. 3).
Survival of larvae to the post-larval stage was higher in 35 o/oo at 15 C.
Combined data for larvae resulting from different hatches showed that H.
americanus larvae survive at a maximum rate of 80% between 20-28 o/oo sal-
inity and 15-18 C. Development of larvae to the post~larval stage was lim-
ited by 9 and 2h C and 5 and 40% salinity with 100% mortality (Fig. 6).

Larvae of P. longicarpus completed development over a wider temperature
and salinity range than larvae of any other species. The post-larval stage
was reached in salinities between 15-35 o/oo at 10 and 15 C, 20-30 o/oo at
20 C and 25-30 o/oo at 25 C (Fig. 3). Survival of larvae of this species
was uniformly high in all salinities from 15-35 o/oo at 20 and 25 C (Fig. 4).
No single optimal combination of temperature and salinity for survival could
be identified for this widely tolerant species (Fig. 6). Complete develop-
ment of R. har: i larvae was observed in 20-35 o/oo at 25 C and 15-30 o/oo
at 30 C (Fig. . Survival of larvae to the first crab stage was highest in
25 o/oo at 25 C. Larvae of R. harrisii tolerated the highest temperatures to
complete development of any of the species studied (Fig. 4).

13


RESEARCH REPORTING SERIES

hand Development, US Environmental
ed into nine senes. These nine broad cate-
urther development and application of en-

reports of th
n Agency, have t
blished to fa

vironmental technology Eliminat of tradstional grouping was consciously
planned to foster technology transter and a maximum interface in related fields
The nine

1 nial Health Effects Research

2 vironmental Protection Technology

3. Ecological Research

4 Environmental Momtoring

5 Socioeconomic Environmental Studies

6 Screntific and Technical Assessment Reports (STAR)

7 Interagency Energy-Enviranment Research and Development

8 Special” Reports

9 Miscellaneous Reports

This report has been assigned to the ECOLOGICAL RESEARCH series. This series
descnbes research on the effects of pollution on humans, plant and animal spe-
cies. and materials Problems are assessed for their long- and short-term influ-
ences Investigations include formation, transport. and pathway studies to deter-
mine 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 document is available to the public through the National Technical informa-
tion Service, Springfield, Virginia 22161


TABLE 11. FATTY ACID METHYL ESTERS IDENTIFIED IN DEVELOPING LARVAL STAGES
OF HOMARUS AMERICANUS CULTURED AT CONSTANT 20 C AND 15 - 25 C CYCLIC
TEMPERATURES

Chain length

Stage 16:0 1631 18:0 18:1 18:2 18:3 20;1 20:4 20:5 22:6
20 +H +H + + T T A+ 4 H+ cea

eee 15-25 ttt 0 +e +h 4th oT T + 4 Hh HE
1 20C HE tHe + +H T tT 4 +t H+ +H
15-25 H+ + + +H + +r t + att H+

oe 20 H+ ++ + H+ +t 4 T HW 4+ +
15-25 +H 1 + tH + + T + HE +

20 HH + ca HH Hot t +H aaa ++

um 15-25 Ht + at +H + + T ++ +H +
20 +H + +h HE Hoo T + 4 +

" 15-25 tt +H 4 HH +H +H T He +4 at

+++ indicates above 10%, ++ less than 10% and I trace amounts per gram dry weight

ENZYME ACTIVITIES

The potential influence of constant vs. daily cyclic temperatures on lar-—
vae was examined by assaying the specific activities of lactate dehydrogenase,
malate dehydrogenase and glucose-6-phosphate dehydrogenase in larvae of Cc. ir-
roratus cultured at the 10-20C cycle and constant 15C Activity of the three
enzymes was Tigher in the first zoeae and the later larval stages than in the
intermediate stages for both culture regimes (Fig. 21). However, activity
of the three enzyme systems was affected differently by the cyclic tempera-—
tures for the larval stages. Lactate dehydrogenase activity was enhanced
in the third and fifth zoeal stages and megalops stage at 10-20 C cycle com-
pared to that in larvae at 15 C. The activity of lactate dehydrogenase was
significantly increased in the megalops stage compared to that of the earlier
zoeal stages, regardless of temperature regime (Table 12). The changes in
lactage dehydrogenase activity in the present study probably reflect changes
in overall glycolytic activity during larval development. LDH activity in
larvae cultured at cyclic regime was substantially higher at the last two
stages (Table 12). The malate dehydrogenase activity decreased in all except
the fifth zoeal stage at 10-20 C cycle compared to that in larvae at 15 C.
The glucose-6-phosphate dehydrogenase activity decreased in the second and
third stage zoeae, but increased in the fourth and fifth zoeal and megalops
stages at the cyclic regime.

37


60

SOF

“ “_

no

~ 3

< °F

=)

20

© 4. americanus
eC. irroratus
SP. pugio

iob | 8 harrisi’
aP. longicarpus

° 1 L L L 1
5 10 1S 20 23 30 35

SALINITY Yoo

Figure 9. Effect of salinity on the duration of larval development of
primarily estuarine and coastal crustaceans.

al


oc

DAYS

Figure 1).
eratures.

100,

80)

40

2

°o

lu

(5 20 25 30 20-10 «25-15 225-175 175-125
CONSTANT ULTURE CYCLING
P

Duration of larval development of Palaemonetes pugio at constant and daily cyclic temp-


eho
8 a
SST

24

SALINIT Y

28b

36-

40

- 100%

\20% sor%|

—C. irroratus

\=2 americanus
'--P. pugio

\

|
|
|
|

16 20 24 28
TEMPERATURE C

Fig. 6. Isopleths of mortality of larvae to the post-larval stage for
primarily estuarine and coastal crustaceans in relation to temperature-—

salinity conditions.

16


ACKNOWLEDGEMENTS

The financial support of the Environmental Protection Agency under
Grant No. R-800981 is gratefully acknowledged. Dr. Don Miller, Project
Officer, has been particularly helpful in carrying out this project.

The project was carried out at the Graduate School of Oceanography,
University of Rhode Island, Kingston, Rhode Island under the direction of
A. N. Sastry with the assistance of Dr. Sandra L. Vargo. Technical
assistance was provided by Ms. Shirley Barton, Ms. Sharon Pavignano,

Ms. Joyce Blaisdell, Mr. John Laczak, Mr. Ross Ellington and Mr. John
McCarthy. Ms. Sharon Pavignano provided invaluable assistance in the
preparation of the final report.

xii


Sastry, A. N. Metabolic adaptation of developing larvae of crustacea to the
varying thermal environment. XIV Pacific Science Congress, Khabarovsk,
USSR, August 20- September 1, 1979.

52


a

j MOLES NADP’ REDUCED
per min. per mg. protein at I5°C

=

Figure 21.

GLUCOSE
TMP DEHYDROGENASE

#2 MOLES NADH OXIDIZED

2

TMmTM-T

per min. per mg. protein

LACTATE
DEHYDROGENASE

“h

08

Pr ce ee a

CONSTANT 15°Co
CYCLIC 10-20°C®

MALATE
DEHYDROGENASE

Pies es Se

gE
yb

zz Zit

zm zm ZN ZvOOM

LARVAL STAGE

Zz om oat Ziv 2voOM

The effects of daily cyclic and constant temperatures on the
activities of lactate dehydrogenase, malate dehydrogenase and Glucose-6-
Phosphate dehydrogenase in Cancer irroratus larval stages cultured at con-

stant 20 C and 10-20 C daily cyclic temperatures.

TABLE 12. ACTIVITIES OF LACTATE DEHYDROGENASE DETERMINED FROM CRUDE
CELL-FREE HOMOGENATES OF CANCER IRRORATUS LARVAE CULTURED
UNDER CYCLIC AND CONSTANT TEMPERATURES. ACTIVITY IS EX-
PRESSED IN yw MOLES NADH OXIDIZED/MIN PER MG LOWRY PROTEIN.
ASSAY TEMPERATURE WAS 15C.
State Constant 15C Cyclic 15-20C Cyclic/constant
Zoea-I 71.2 58.6 0.82
Zoea~II 46.1 2h.5 0.53
Zoea-III 38.8 56.4 1.45
Zoea-IV 50.2 35.8 0.71
Zoea-V 51.6 103.7 2.01
Megalops 95.1 119.7 1.26
=

38


Larvae exhibited interstage variation in their pattern of response to
low dissolved oxygen tolerance over the experimental temperature range (Fig.
23). The first, second and fourth zoeae showed a different pattern of re-
sponse, with an increase in tolerance from 10 to 15 C and a decrease from
15 to 30 C (Fig. 23). Tolerance limits were maximum when the test tempera—
ture was the same as culture temperature (Fig. 24). This pattern was con-
sistent for both the 120 and 240 min time intervals, with a tendency for
lower tolerance limits for the longer time intervals.

$0

ow

1/ LD go mia: /L

x

dt 1 of
10 20 30 10 20 30 10 20 30

TEMPERATURE, °C

Figure 23. Cancer irroratus. Effect of temperature on LD, value of oxygen
for different larval stages. Curves computed from regression equations ex-
cept for megalops (M); Dots: 120 min exposure (solid line); crosses: 240 min
exposure (dashed line). 1-5Z: Zoeal stages.

The megalops stage showed the least temperature-dependent low dissolved
oxygen tolerance (Fig. 23). Tolerance decreased from 10-30 C; however, the
slope was slight compared to that of other stages. There was no significant
difference in tolerance as exposure time was increased from 120 to 240 min-
utes. Regression analysis did not yield any statistically significant re-
lationship with temperature.

A comparison between larval stages showed the megalops was least toler-
ant at most test temperatures (Figs. 23 & 24). There was interstage varia-
tion in tolerance from 10 to 20 C, however. At the extremes of 25 and 30 C,
little difference was observed between stages. At 10 C, the first, second
and fourth zoeal stages showed higher tolerance than the other stages. At 15
C, the third stage was most tolerant, while at 20 C, the fourth was the most
tolerant. No statistically significant correlation of tollerance with stage.
was found using either I/LD, 4 = (stage number) + B or log 1/LDsy = a (stage

ko


SECTION 2
CONCLUSIONS

Thermal alterations affect the development and growth, metabolism and
survival of crustacean larvae. The limits as well as the optimal conditions
for larval development are modified by the thermal history of eggs prior to
hatching, the stage of larval development and geographic origin of the
population. Different combinations of temperature and salinity interact to
affect larval development and survival. A species may exhibit an optimal
combination for survival and as environmental conditions deviate from this
optimum, survival is reduced. Limits, as well as optimal combinations for
development, vary interspecifically. Generally, complete development for
coastal (high salinity) species, Cancer irroratus, Cancer borealis and
Homarus americanus was limited to a narrower range of temperature and sal-
inity than predominantly estuarine species, Palaemonetes pugio, Pagurus
longicarpus and Rhithropanopeus harrisii.

Time for complete development and percent survival can also be influ-
enced by use of a daily cyclic temperature regime. Larvae of C. irroratus
cultured under suitable daily cyclic regimes showed increased survival com-
pared to those at comparable constant temperatures. The effects of daily
cyclic temperatures can vary interspecifically, however. For P. pugio,
development and survival rates were not significantly different under daily
cyclic and comparable constant temperatures.

Within the thermal tolerance range of a species, the metabolic responses
to temperature may vary, but each species will have a characteristic overall
response. Larval stages of each species exhibited capacities for metabolic
rate compensation (1<Q1 9<2) over a portion of the temperature range. Gen-
erally, the larvae of coastal species are metabolically active over a
relatively narrower temperature range than the estuarine species. The met-
abolic-temperature response patterns of the larvae are also influenced by
the culture conditions: cyclic or a comparable constant temperature. The
alterations in metabolic adaptation of larvae cultured under daily cyclic
temperatures were underlined by qualitative and quantitative changes in
fatty acid methyl esters in H. americanus larvae and changes in specific
activities of the enzyme systems examined in C. irroratus larvae.

Acute temperature tolerance limits of laboratory cultured larvae varied
intra- and interspecifically, with estuarine species tolerating higher
temperatures than coastal species. These thermal tolerance limits remained
fairly constant for all C. irroratus larval stages, while some of H.
americanus and P. Pugio stages showed differential sensitivity when tested
under saturated dissolved oxygen conditions.