vvEPA
United States
Environmental Protection
Agency
Environmental
Research Laboratory
Gulf Breeze, FL 32561
Research and Development EPA/600/M-87/004 June 1987
ENVIRONMENTAL
RESEARCH BRIEF
Optimization of Environmental Factors During
the Life Cycle of Mysidopsis Bahia
Charles L. McKenney, Jr.
Introduction
The estuarine mysid, Mysidopsis bahia, has been
established through numerous toxicity tests during the last
decade as members of the estuarine community most
sensitive to low-level toxicant exposure (for recent reviews
refer to Nimmo and Hamaker, 1982; McKenney, 1986a).
Because of the relatively short life cycle, this crustacean is
an excellent test species for life-cycle toxicity tests
permitting not only a comparative evaluation of differential
toxicity among the various life stages, but also an
assessment of the impact of chronic exposures on
ecologically important biological responses, such as brood
size and time to sexual maturity. Indeed, for the majority of
toxicants examined in life-cycle toxicity tests using this
species, a sublethal reduction in reproductive success has
proven to be the most sensitive criterion for chronic
biological effect.
As an estuarine organism, however, M. bahia must be able
to functionally adapt to a host of dynamically changing
environmental variables, characteristic of the complex and
harsh estuarine environment (Vernberg and Vernberg, 1972,
1981; Lockwood, 1976). A vast number of continually
changing environmental factors (physical and chemical,
abiotic and biotic) modify the physiological performance of
estuarine organisms, such that, when viewed as a highly
integrated system of multiple functional components, the
organism is differentially influenced by these environmental
variables acting in concert. Tolerance limits for these
environmental parameters are controlled genetically and,
within these limits, conditions exist for optimal physiological
performance. The physiological capacity of these estuarine
organisms, in turn, dictates the ecological performance of
the population in the natural environment. Therefore, the
responses of an estuarine organism to a toxicant are
dictated by the simultaneous influences of a number of
exogenous and endogenous variables (Figure 1) (Vernberg
et a/., 1974; Vernberg, 1975, 1985; Lockwood, 1979).
Unfortunately, little is presently known about the basic
biological requirements and physiological capacity of M.
bahia (Mauchline, 1980), due in part to its rather recent
identification as a species (Molenock, 1969). With its
continued use as a test organism in marine toxicity tests, an
increased understanding of this species' optimal values for
the dominant environmental variables should further its
successful culture while also ensuing adequate assessment
of the ecological hazards of the various potential
contaminants tested.
Temperature and salinity represent the dominant ecological
master factors within the estuarine environment; i.e., these
two factors may act either singly or in combination to
modify both the physiological and ecological properties of
estuarine species (Kinne, 1970, 1971; Alderdice, 1972).
Furthermore, temperature and salinity stress have been
shown to modify the expression of toxic responses of
estuarine crustaceans and to decrease their resistance to
toxicant exposure (Vernberg et a/., 1973, 1977; McKenney
and Neff, 1979, 1981; McKenney and Costlow, 1982).
Empirical determination of the optimal salinity-temperature
conditions for M. bahia should improve laboratory culture of
this organism and benefit the application of this species in
toxicity assessment by eliminating extraneous
environmental stress.
The availability of food has been shown to influence a
number of vital life processes of marine and estuarine
crustaceans, including functional rates of ingestion,
assimilation, growth, and reproduction (Frost, 1972;
Grahame, 1983). In addition, several recent studies have
demonstrated the interrelationships between alterations in
the energy metabolism of assimilated food energy and
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Figure 1. Responses of an estuarine organism to a
toxicant are dictated by the simultaneous
influences of a variety of endogenous and
exogenous variables acting in concert.
Exogenous Factors
(i.e., Temperature, Food Levels, Dissolved Oxygen, etc.)
Sublethal
Toxicant
Stress
Changing
Salinity
Endogenous Factors
(Size, Sexual Maturity, Stage in Life Cycle, etc.)
reductions in growth and reproduction of M. bahia during
sublethal exposure to pesticides (McKenney, 1982, 1985).
Identification of the optimal food concentration for the
growth and reproduction of M. bahia will ensure maximum
expression of these physiological processes during their
use in the hazard assessment of potential toxicants with this
species.
The objective of the research summarized herein was to
determine for M. bahia optimal values of several dominant
environmental variables. Optimal salinity-temperature
conditions will be determined for survival of the various life
stages of M. bahia in a complete life cycle through
reproductive maturation, as indicated by appearance of the
female marsupial pouch. Feeding densities of Artemia
nauplii will be determined which will result in maximum
growth of M. bahia through its life cycle and which,
ultimately, will be responsible for maximum reproductive
effort. Optimization of these environmental variables during
life-cycle toxicity tests with this species should enhance
the ecological validity of using M. bahia for assessing the
ecotoxicological hazards of compounds to the estuarine
community.
Approach
Salinity and Temperature Study
The experimental design for the temperature-salinity study
was a 5x3 factorial with salinities of 3, 10, 17, 24, and 31 %o
and temperatures of 18, 25, and 32°C. Three replicates of
15 mysids each were reared at each of the 15 salinity-
temperature combinations for at least 28 days or until
appearance of the marsupial brood pouch in female mysids
occurred.
The study was initiated by obtaining newly released juvenile
M.bahia (<24 h old) from ovigerous females maintained in
a flowing seawater culture at 24 ± 2%o S and 25 ± 1 °C.
To avoid osmotic shock, juvenile mysids to be reared in
either lower or higher salinities were transferred at hourly
intervals in graded steps of 3-4%0 S from the original
salinity of 24%o S to the appropriate experimental salinity.
Mysids to be reared in 24%0 S were transferred directly
into an aquarium for the appropriate temperature condition.
Water temperatures for all aquaria, originally at 25°C, were
allowed to equilibrate to the test temperatures after
placement in constant temperature water baths maintaining
either 18, 25, and 32 ± 1°C.
Mysids were reared in groups of five in chambers
constructed of a 10-cm glass petri dish to which a 15-
cm-high cylinder of nylon mesh screen was attached by
silicone cement. Throughout the study, all groups of mysids
were fed an abundance of freshly hatched Memia nauplii
daily. Every third day, the 20 L of seawater in each
aquarium was renewed with freshly prepared seawater of
the apropriate salinity. Daily observations were made of
mysid mortality and day of appearance of the female
marsupium.
Feeding Study
Juvenile mysids (<24 h old) were reared in a flowing
seawater system at 20 ± 2%o S and 25 ± 1 °C through an
entire life cycle (29 days) under various feeding regimes
using procedures described by McKenney (1986b). For the
reproductive part of the study, three replicates of 15
juveniles each were reared in retention chambers receiving
four different feeding regimes until maturation of the female
(appearance of the marsupium), at which time the mature
female was paired with a male in a smaller brood cup for
observations on young production. For growth observations,
other groups of mysids were reared in the same manner
and subsampled at seven-day intervals for subsequent dry
weight measurements.
Since past experience has shown that the feeding rates of
mysids increase as they increase in size through their life
cycle, feeding levels in each feeding regime progressively
increased through the various life stages. Identical
procedures were used for daily incubation of hydrated
Artemia cysts to produce nauplii densities of approximately
600 per ml (mean ± standard error = 598 ± 33). For the
four feeding regimes, different volumes of these nauplii
were added to each replicate retention chamber in the
manner described in Table 1 . For the paired adults, 0.5, 1 .0,
1 .5, and 2.0 mis were added daily to each replicated brood
cup for feeding regimes 1-4, respectively. Since the water
level within the seawater system varied in the isolated
replicate aquaria, water volumes in the juvenile retention
chambers varied from approximately 500 to 1700 mis and
in the brood cups from 100 to 300 mis.
Statistical Treatment of the Data
Differences in biological responses under the various
salinity, temperature, and feeding regimes were analyzed
by analysis of variance, using Duncan's procedure for
multiple comparisons of treatment means (Zar, 1974). Data
from the salinity-temperature study was regressed on the
quadratic function of salinity and temperature:
Y = b0x0
b2TEMP
b12(SALxTEMP)
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Table 1. Feeding regimes (in mis) employed in the
growth and reproduction study with Mysidopsis
bahia. Each ml contained about 600 Artemia
nauplii.
Feeding
Regime
1
2
3
4
Days in Life Cycle
1-3
1.00
2.00
3.00
4.00
4-6
1.25
2.50
3.75
5.00
7-9
1.50
3.00
4.50
6.00
10-12
1.75
3.50
5.25
7.00
>12
2.00
4.00
6.00
8.00
where Y = the estimated biological response (either
percentage survival or days to sexual maturity in this case);
XQ = the intercept; SAL and TEMP = the linear effect of
salinity and temperature; SAL2 and TEMP2 = the quadratic
effect of salinity and temperature; SAL x TEMP = the linear
by linear interaction between salinity and temperature; and
bo, b-|, etc. = the regression coefficients (Alderdice, 1972).
Response surface curves were generated at a range of
levels of these two factors by the General Linear Model
(GLM) and PLOT procedures available in the SAS statistical
package (Barr et at., 1976).
Results and Discussion
Salinity-Temperature Study
The combined effects of salinity and temperature
differentially modified survival of the estuarine mysid, M.
bahia, dependent on stage in the life cycle. As depicted by
the isopleths of percentage survival on the various response
surfaces (Figure 2), generated from empirical observations
at discrete salinity-temperature conditions, survival of M.
bahia was influenced by a salinity-temperature-life stage
interaction. Juvenile mysids during their first week of life
were least resistant to lower salinities and temperatures,
being particularly sensitive to salinities below 10%o S and
temperatures below 20°C (Figure 2A). Optimal salinity-
temperature conditions for survival of these young mysids
was at 20%0 S and 28°C (center of the 95% isopleth), while
95 % survival occurred at the approximate range of 15-
28%o S and 23-32°C. During their second week of
existence, juvenile mysids were more resistant to the
combined effects of a broad range of salinity and
temperature conditions (Figure 2B) and optimal levels for
both of these factors was lowered to 17%o S and 23°C. As
shown by the survival isopleths, salinity was the dominant
factor affecting survival of juveniles during their second
week. After mysids matured during the end of this second
week, adults were more resistant to salinity and survival
was principally affected by temperature (Figure 2C). These
dramatic changes in the resistance patterns of M. bahia to
salinity during the third week of its life cycle suggest the
development of osmoregulatory mechanisms in this
estuarine organism concurrent with its maturation period.
Older adult mysids, during the fourth week of their life
cycle, appeared to develop a susceptibility to higher
salinities at temperatures above 20 °C (Figure 2D).
The cumulative effects of salinity and temperature on
survival of M. bahia through a complete life cycle are shown
on Figure 3. Through a 28-day period, optimal salinity-
temperature conditions for survival of this estuarine species
occurred at a salinity of 20%o S and a temperature of 23°C.
The finding that the optimal salinity-temperature values for
survival of this estuarine mysid are located at intermediate
values within the broad range of these environmental
factors seen in a temperate estuary, are consistent with
those for other estuarine crustaceans (McKenney and Neff,
1979; McKenney and Costlow, 1982; and references
therein).
The time required for reproductive maturation of M. bahia
was influenced by the salinity-temperature conditions in
which this estuarine species was reared (Figure 4).
Marsupial brood pouches developed in females in 10.5
days at 18%o S and 29 °C. Both lower and higher salinities
delayed the development of these pouches in females by
as much as 2 to 3 days. Lower temperatures more than
doubled the time necessary for the appearance of the
marsupium in female mysids.
Feeding Study
Food availability altered growth patterns of M. bahia through
its life cycle (Table 2). The four progressively higher
feeding regimes produced a linear increase in the size of
juvenile mysids after one week. These weights, however,
were not significantly (P<0.05) different between the
various feeding regimes. Two weeks of exposure to the
different feeding levels produced significantly smaller
mysids at the next to lowest level. At the end of three
weeks, again, a direct linear relationship existed between
feeding level and dry weights of the mysids. Only the
weights of mysids in the lowest feeding level, averaging
approximately 0.5-1.0 Artemia nauplii per ml of seawater,
were significantly lower than the three higher feeding
regimes after three weeks of growth.
The availability of food during the complete life cycle of M.
bahia influenced its reproductive capacity. The lowest
feeding regime delayed the onset of reproduction in this
mysid species (Figure 5). Mysids being provided with food
densities averaging greater than 1 nauplii per ml (Feeding
Regimes 2-4) released their first brood a minimum of five
days prior to those fed lower average food densities. Young
production, both of individual females and of the total mysid
population (Figures 6A and 6B), was modified by feeding
level. The two highest feeding regimes, receiving average
food densities greater than 2 Artemia per ml of seawater,
resulted in more young being produced. As has been
observed in functional responses of other aquatic
crustaceans to food concentration (Frost, 1972; Grahame,
1983), there appeared to exist a "threshold" concentration
of food, above which the functional response remains
maximally stable. For maximum reproductive success in M.
bahia, this "threshold" concentration appears to be a
feeding regime averaging approximately 2-3 Artemia
nauplii per ml of seawater.
Conclusions
When considering both survival capacity of M. bahia
through a complete life cycle and time required for juvenile
mysids to become reproductively mature, salinity-
temperature conditions of 20%o S and 25°C appear optimal
for this estuarine crustacean. Optimization of growth and
reproduction in this species requires a feeding density of
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Figure 2.
Estimated percentage survival of Mys/dops/s bahia at weekly intervals through its life cycle based on fitted
response surface to observed survival under 15 combinations of salinity and temperature. Curved areas of the
isopleths cover those salinity- temperature ranges within which specified survival percentages are predicted.
A 1st Wk
B. 2nd Wk
I
5
I
20-
15
35
30-
25-
20-
35 ~
30-
25-
20-
75-I
35
/5H
C. 3rd Wk
20%
,35%
—I 1
5 10
D. 4th Wk
35 H
30-
25-
20-
-35% .
15
80%
20
—T—
25
30
35
5 10 15 20 25 30 35 05 10 '5 20 25 30 35
Salinity (0/00)
2-3 Artemia nauplii per ml of seawater. For M. bahia this
food density results in maximum growth, shortest duration
prior to initiation of reproduction, and maximum young
production.
References
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marine poikilotherms to environmental factors acting in
concert. In: Marine Ecology. Volume 1. Environmental
Factors, Part 3. 0. Kinne (eds.). Wiley Interscience,
London, pp. 1659-1722.
Barr. A.J., J.H. Goodnight, J.P. Sail, and J.T. Helwig. 1976.
A User's Guide to SAS 76. Statistical Analysis Systems
Institute, Inc., Raleigh, NC, 329 pp.
Frost, B.W. 1972. Feeding processes at lower trophic levels
in pelagic communities. In: The Biology of the Ocean
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Grahame, J. 1983. Adaptive aspects of feeding
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Kinne, O. 1970. Temperature: animals-invertebrates. In:
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Figure 3.
Estimated percentage survival of Mysidopsis
bahia through a complete life cycle in 28 days
based on fitted response surface to observed
survival under 15 combinations of salinity and
temperature.
35 H
30-
o
Q)
75H
Figure 4.
5 10 15 20 25 30 35
Salinity (0/00)
Estimated number of days required for
appearance of marsupium in female Mysidopsis
bahia based on fitted response surface to
observed values under 15 combinations of
salinity and temperature.
35-
5 10 15 20 25 30 35
Salinity (0/00)
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Table 2. Dry weights (ug) of different aged Mysidopsis
bahia as influenced by different feeding
regimes through an entire life cycle. Each value
represents the mean ± standard error.
Feeding -
Regime
1
2
3
4
7
148 ± 7
155 ± 9
166 ± 22
183 ± 9
uays in Lilt; v_-yuie
14
424 ± 16
359a ± 20
479 ± 25
470 ± 29
21
508a + 35
663 ± 41
690 ± 24
723 ± 33
a Significantly lower than other dry weights at that day in the life
cycle (P < 0.05).
Figure 5. Influence of four different feeding regimes on
onset of reproduction in Mysidopsis bahia.
Columns not sharing same letter (A or B) are
significantly different (P < 0.05).
Days to Release
of First Brood
1234
Feeding Regime
McKenney, C.L., Jr. 1986b. Influence of the
organophosphate insecticide fenthion on Mysidopsis bahia
exposed during a complete life cycle. I. Survival,
reproduction, and age-specific growth. Dis. aquat. Org. 1:
131-139.
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harrisii (Gould). I. Interactions of temperature, salinity, and
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development of the grass shrimp Palaemonetes pugio. I.
Survival and developmental duration through
metamorphosis. Mar. Biol. 52:177-188.
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Figure 6. Influence of four different feeding regimes on
the reproductive success of Mys/dops/s bahia.
Columns not sharing same letter (A or B) are
significantly different (P<0.05).
Young Per Female
in 29 Days
1234
Feeding Regime
Total No. of Young
Released in 29 Days
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7234
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