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
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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
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Frost, B.W. 1972. Feeding processes at lower trophic levels
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Pacific.  C.B. Miller (ed.). Oregon  State  University  Press,
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                                               Kinne,  O.  1970. Temperature: animals-invertebrates.  In:
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                                               Kinne,  O. 1971. Salinity:  animals-invertebrates. In: Marine
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                                               Lockwood, A.P.M.  1976. Physiological adaptations to life 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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McKenney,  C.L.,  Jr.  1986a.  Critical  Responses  of
Populations  of  Crustacea to  Toxicants.  Environmental
Research  Brief,  EPA/600/M-86/004,  U.S.  EPA,  Cincinnati,
OH, 8 pp.
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:
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                                               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

                         Feeding Regime
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 resistance  adaptation  and metabolic  compensation  to
 salinity  and temperature by  the  caridean shrimp,
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