vvEPA
                             United States
                             Environmental Protection
                             Agency
                         Environmental Research
                         Laboratory
                         Gulf Breeze, FL 32561
Research and Development    EPA/600/M-88/003    Feb.  1988
ENVIRONMENTAL
RESEARCH   BRIEF
      Influence of an Insect Growth Regulator on  Larval Development of a
                                     Marine  Crustacean
                         Charles L. McKenney, Jr. and Edward Matthews*
 Introduction
 Increased understanding of the role of hormones in  the
 regulation of a number of life processes of insects gained
 through intensive pioneer research during the first half of
 this century has introduced  these hormones and their
 analogues as "third  generation  pesticides"  used  as
 biochemical biological control  agents for  insect  pests
 (Williams, 1967, Slama et al., 1974). Of particular interest in
 strategies for insect control are certain hormones  known as
 insect growth regulators, which are involved  in  the
 regulation of developmental and growth processes  of
 insects (Bowers, 1982; Mian and Mulla, 1982; Staal, 1982;
 Jennings, 1983).

 It has now been established that several hormones  are
 involved  in regulating the  larval development of insects
 through the critical and complex process of metamorphosis
 (Hoar, 1975;  Downer  and  Laufer,  1983)  In addition to
 natural insect juvenile hormones, several  major types  of
 insect growth regulators have been studied to date (Mian
 and Mulla, 1982; Downer  and Laufer, 1983). The most
 extensive group are the juvenile hormone analogues, which
 are  substances of natural or synthetic origin that act as
 endogenous juvenile  hormone and  disrupt  insect  larval
 development.

 When these insect growth regulators are used  in pest
 control, residues of these compounds may enter the marine
 environment either by direct application toward  aquatic-
 borne pests (such as mosquitoes) or indirectly  through
 land-drainage or  erosion from  the  adjacent pesticide-
 treated agricultural lands. With these usage patterns, it
 "The authors are with the US  Environmental Protection Agency's
 Environmental Research Laboratory, Gulf Breeze, FL 32561
                      seems of particular interest to determine whether  these
                      compounds may affect marine or estuarine populations of
                      closely related nontarget organisms. Substances that act as
                      growth hormones for insects are found not only in insects,
                      but also in other invertebrates (Tombes, 1970,  Barnngton,
                      1979). Of particular relevance are the crustaceans, the
                      dominant marine member of the same phylogenetic group
                      containing insects, Arthropoda. In  fact, compounds with
                      juvenile hormone  activity in insects have  been extracted
                      from  crustaceans  (Schneiderman and  Gilbert, 1958),
                      suggesting  that  juvenile  hormone  may  function  in
                      crustacean larval development and metamorphosis.
                      Since the mode of action  of juvenile hormone in insects
                      entails regulation of the complex process of metamorphosis
                      during larval development,  it is not surprising  that  larval
                      stages of insect pests  are routinely  used  as  bioassay
                      organisms in testing the  activity of juvenile  hormone
                      analogues as potential insecticides (Downer and Laufer,
                      1983;  Mian  and  Mulla,  1982)  To  best  predict the
                      environmental  impact of  this type  of insecticide on
                      estuarine biota, an analogous examination of their influence
                      on the larval  development of a ubiquitous and ecologically
                      important estuarine crustacean  appears appropriate  For
                      this purpose  a series  of studies were  initiated to examine
                      the effects of methoprene,  a juvenile  hormone analogue
                      used in mosquito control, on developing larvae of the grass
                      shrimp Palaemonetes pugio

                      Approach
                      To obtain larvae for  the study,  ovigerous grass shrimp
                      (Palaemonetes pugio) were collected  from shallow grass
                      beds of Santa Rosa Sound, FL, and held in the laboratory in
                      flowing, filtered (20 urn) seawater at 25 ± 1 °  C and 20 ±
                      1o/oo S,  optimal temperature  and salinity conditions for
                      larval development of P. pugio (McKenney and Neff, 1979).

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Upon release, larval grass shrimp were separated from  tne
adult shrimp, using a flow-through hatching apparatus.
Newly released P.  pugio larvae were randomly distributed
among replicate exposure  conditions either individually in
compartmented plastic boxes as previously described by
McKenney  and Neff (1981)  or in  groups  of  10 in  glass
beakers as described  by  McKenney and Hamaker (1984).
The exposure media was renewed daily for each exposure
condition in each  of  the  three  replicates (6  larvae  per
replicate) in the plastic boxes.  Larvae in the glass beakers
were placed in  a flow-through  exposure system similar to
that described  by McKenney (1986) utilizing a proportional
diluter described by Schoor and McKenney (1983).
Larvae exposed  in  boxes were  individually reared through
completion of  metamorphosis  in  a  range  of  nominal
concentrations  of the  juvenile  hormone analogue
methoprene  Both technical  grade  (R.S)-methoprene,
isopropyl  [2E,4E]-1 1 -methoxy-3,7,1 1 -tnmethyl-2,4-
dodecadienoate, and  the  single isomer (S)-methoprene,
isopropyl  [2E,4E,7S]-11-methoxy-3,7,11-tnmethyl-
2,4-dodecadienoate as contained in the formulation Altosid
Liquid Larvicide (ALL)  were used in these static exposures
For the flow-through studies larvae  were exposed only to
(S)-methoprene  in  ALL  All methoprene  concentrations
referred  to   throughout  this  paper  are nominal
concentrations  In  static  exposures, daily  records  were
maintained on  survival and molting frequency  Molting  rate
was determined by the presence of the cast exuvia for each
larva  Observations for individual larva were  ended  upon
completion of larval development through metamorphosis to
the postlarval  form (Broad,  1957). Similarly,  survival  and
developmental  duration to  completion  of  metamorphosis
were recorded for  all larvae in  the flow-through exposures.
On days corresponding with the mtermolt of  various  larval
stages,  8  larvae for  each exposure  concentration  were
individually  sealed  in  all-glass  syringes  with  fully
oxygenated media  from the appropriate exposure condition;
for each larva,  oxygen consumption and ammonia excretion
rates were determined  using  methods  described by
McKenney (1982) and Shirley and McKenney  (1987)  Each
larva was then briefly rinsed in  distilled water and placed in
an oven to dry at 60° C for  48 hours  The weight of each
larva was subsequently determined to the nearest 01  ng on
an electronic  microbalance O:N ratios were calculated for
each  larva as  the  ratio of atoms of oxygen  consumed to
atoms of nitrogen excreted
Growth rates   of  larvae  were  determined,  using  their
respective dry weights  and  formulae  described by
McKenney (1986).  In  addition,  bioenergetic terms for daily
production (P)  and maintenance (R)  costs  were derived
from the caloric equivalents of  daily growth and respiration
rates of the  larvae as described by  McKenney (1982)
These values  were used  to derive net growth efficiency
values (K.%) (Winberg, 1971)  or larvae under the various
methoprene exposure conditions by the formula


                         ~
 Values  for the  various biological responses are presented
 as means  ±  standard  errors.  Significant  differences
 between control and  exposed larvae were determined by
 analysis of variance with arcsine transformation to stabilize
 variable survival percentage data (Zar, 1984) and William's
 procedure for  multiple comparisons  between  means
 (Williams, 1972).

 Results and Discussion
 During exposure to technical grade  concentrations  of the
 isomeric  mixture  (R.S)-methroprene  and  equivalent
 concentrations  of the  single  isomer (S)-methoprene in  a
 liquid  formulation  (Altosid  Liquid  Larvicide), differential
 survival values  were  found  through the complete larval
 development of Palaemonetes pugio (Figure 1). No larvae
 survived completion of metamorphosis  while  exposed to
 10000 ng  methoprene/l in  a static-renewal  system,
 regardless of the isomeric form  Even though larval survival
 was significantly reduced by  exposure to  100.0  pg/l of
 (R,S)-methoprene and not  by  this concentration  of  (S)-
 methoprene,  an analysis of variance of the entire  data set
 showed no significant difference in  larval survival  between
 the two isomers for exposure  concentrations  from  0.1 to
 1000 0 ug/l
 An examination of the survival  of discrete larval stages of
 grass  shrimp  during  exposure   to  (R,S)-methoprene
.showed that  larval toxicity to 100 ng/K was  stage specific
 with the  second and  final larval  stages  responsible for
 significant mortality (Table 1). This apparent stage-specific
 sensitivity to juvenile hormones  and their analogues is seen
 during insect larval development (Downer and Laufer, 1983)
 and accounts  for  the  predominant  use  of the final,
 premetamorphic  larval  instar of insects  during  efficacy
 testing of these compounds as  insecticides (Mian  and
 Mulla, 1982, Downer and Laufer, 1983).

 Further examination of this  phenomenon as it exists in the
 response  of  crustacean larvae  to insect growth regulators
 resulted in a study  demonstrating differential survival rates
 of early and final larval stages of P. pugio when exposed to
 (S)-methoprene in  a flow-through system  (Figure  2).
 Larval viability during the first two  larval  stages  was
 reduced by four days exposure  to concentrations of 31 and
 62 jig methoprene/l, but not  for exposure during  the  final
 larval stage. An analysis of variance of the 96-hour survival
 patterns of these  larval stages  separately  exposed to
 concentrations of (S)-methoprene from  31  to 250  ug/l
 revealed  no  significant differences in  sensitivity  between
 these  larval stages.
 The type  of exposure  system  used to evaluate the  effects
 of  insect growth regulators  on  crustacean larvae  can
 influence  the  results of these studies Survival of  P. pugio
 through  total  larval  development  was several orders of
 magnitude more sensitive to methoprene in  a flow-through
 exposure  system  than when using  a  static-renewal
 exposure system. A concentration  of  100  jag/l(S)-
 methoprene  produced  no significant larval  mortality  in  a
 static-renewal  system  (Figure  1), while  a  concentration of
 8  ug/l reduced  the  number  of larvae  completing
 metamorphosis from  82 to 28 percent in a  flow-through
 system (Table 2). It is also interesting to note in  the latter
 study that the developmental duration of P. pugio larvae
 was  prolonged by methoprene exposure,  suggesting  a
 similar  hormonal function  of  juvenile-hormone-like
 compounds in crustaceans as seen in insects.
 Modifications in the  energy  metabolism  of  grass  shrimp
 larvae  occurred  with  exposure  to  methoprene

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  Figure 1.    Comparative survival percentages of Palaemonetes pugio from hatch through completion of metamorphosis while
              exposed in a static-renewal system to a range of concentrations of (R,S)- and (S)-methoprene. Asterisk denotes
              significant differences from the control (0) survival (P<0.05).
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                      1000
                                   10000
Table 1.  Survival Percentage of Discrete Larval Stages of
Palaemonetes pugio Reared Through Larval Development to
Completion  of  Metamorphosis While Exposed  in  a Static-
Removal System  to  a  Range  of  Concentrations  of  (R,S)-
Methoprene.
Concentration
(UQ/D
0.0
0.1
1 0
10.0
100.0
1000.0
Larval Stage
I
100
100
100
100
100
173
II
100
100
100
100
923
443
III
98
100
98
100
100
100
IV
98
100
91
94
94
100
V
98
91
92
96
100
100
VI
95
97
90
94
90
100
VII
96
91
93
92
79a
100
3Significantly less than the control (0.0) survival at that larval stage
  (p<0.05).
concentrations  which prevented  successful development
through  completion  of  metamorphosis  (Table  3 a,b).
Respiration rates of larvae were significantly  elevated  as
early as 24 hours after exposure to these concentrations of
methoprene For unexposed larvae, O.N ratios suggested a
shift from predominant usage of lipid as major metabolic
substrate to a significant increase in protein as the energy
source during premetamorphic larval stages  Similar energy
substrate patterns  toward greater  reliance  on  protein
catabohsm just prior to metamorphosis have  been observed
during the larval development of other marine crustaceans
(Capuzzo  and  Lancaster,  1979;  Anger,  1986) and  may
represent a physiological prerequisite necessary for
successful  completion of  metamorphosis.  However,  lipid
catabohsm remained  dominant for  premetamorphic larvae
of P. pugio  exposed  to methoprene,  as  indicated  by  their
significantly  higher O.N  ratios (Table 3b).  Modification of
this premetamorphic energy  utilization  pattern by
methoprene  exposure  could  indicate  an  important
physiological mechanism of toxicity of these substances to
developing crustacean larvae.

The earliest and most sensitive  response of grass shrimp
larvae to methoprene exposure  was a retardation  of  their
growth rates  (Table 4 a,b). Reduced net growth efficiency
values suggest that  retarded  larval  growth  rates  resulted
from less assimilated energy being  available for tissue
production. As indicated by elevated respiration  rates of
methoprene-exposed  larvae, proportionally  more  of the
physiologically available energy was channeled into energy
required for metabolic  maintenance.  In  that  juvenile
hormones are thought to also  play a functional role in the
regulation of insect energy metabolism (Downer and Laufer,
1983),  these metabolic  and  bioenergetic  responses  of

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 Figure 2.    Comparative 96-hour survival percentages of Palaemonetes pugio larvae after exposure to (S)-methoprene during
            first larval stages and final premetamorphic larval stage. Asterisk denotes significant differences from the control
            (0) survival (P<0.05).
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                                          31
        62

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                                                                              125
                                                                                                250
Table 2. Survival Percentage and Developmental  Duration of
Pa/aemonefes pugio Reared  Through Larval Development to
Completion of Metamorphosis While  Exposed  in  a  Flow-
Through  System to a Range of  Concentrations  of (S)-
methoprene.  Each Value Represents the  Mean  ±  Standard
Error.
Concentration
(Ml/0
0
8
16
32
62
Survival
Percentage
82 ± 4
28a ± 6
8a ± 3
7a ± 4
2a ± 2
Developmental
Duration in Days
18.8 ± 0.7
18.5 ± 0.4
17.5 ± 0.7
20 5 ± 0.5
29.0a ± -
aSignificantly different from the control (0 0) value (p<0.05).
crustacean  larvae to  an insect growth  regulator  with
juvenile-hormone activity  may suggest  similar  endocrine
      control of these functions in  this closely related  group of
      organisms.

      Conclusions
      Larval  survival,  growth,  and  energy  metabolism of  an
      estuarme  shrimp (Palaemonetes pugio) were  altered  by
      exposure  to  low ug/l concentrations  of  an insect  growth
      regulator  (the juvenile hormone  analogue, methoprene)
      Larvae were several orders of magnitude more sensitive to
      methoprene in  a flow-through exposure  system than in a
      static-renewal  exposure  system.
      The  first two larval stages and the final premetamorphic
      larval stage were more sensitive to methoprene toxicity than
      the intermediate larval stages  As indicated by reduced net
      growth efficiency values,  elevated  metabolic maintenance
      demands  of exposed  larvae were related to retarded larval
      growth rates. A premetamorphic shift in substrate  utilization
      patterns,  thought to  be  a physiological  prerequisite  for
      successful metamorphosis  in marine  crustaceans,  was
      altered  by exposure  to  methoprene concentrations  that
      prevented completion  of larval development  through
      metamorphosis.
      These findings  support an analogous functional approach in
      the  selection  of  an appropriate  testing  procedure to

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Table 3a. The Influence of Continual Exposure to a Range of
Concentrations of (R,S)-Methoprene on the Energy
Metabolism of Various Aged Palaemonetes pugio Larvae. VO
= Weight-Specific Oxygen Consumption Rate (ng 02 mg-1
Dry Wt h'1); VN = Weight-Specific Ammonia Excretion Rate
(ug NH3-N mg~1 Dry Wt h'1); and O:N = Ratio of Atoms of
Oxygen Consumed to Atoms of Nitrogen Excreted.
Early Larvae - 1 -day old
Concentration
(ng/i)
VO VN O:N
0 107 ± 0.5 0.086 ±0.015 136 ± 21
8 12.8 ± 0.8 0.104 ± 0.012 116 ± 15
16 12.4 + 1.1 0.089 ±0.012 137 ± 15
32 14.43 + 1.4 0.125 ± 0.028 122 ± 16
62 14.0a ±20 0.189a ± 0.090 120 ± 33
125 154a + 1.0 0.2783 ± 0.049 57a ± 7
250 23.4a ± 3.6 0.3083 ± 0.047 64a + 16
Intermediate Larvae - 9-days old
Concentration
(ra/l)
VO VN O.N
0 6.0 ± 1.0 0.070 ± 0.036 120 + 30
8 5.7 ± 0.3 0.035 ± 0.008 152 + 26
16 6.0 ± 0.7 0.097 ± 0.058 159 + 64
32 8.73 ±09 0.071 ± 0.011 124 + 20
62 9.4a ±1.4 0100 ± 0028 89 ± 16
aSignificantly different from the control (0) (p<0.05)
Table 3b. The Influence of Continual Exposure to a Range of
Concentrations of (R.S)-Methoprene on the Energy
Metabolism of Various Aged Palaemonetes pugio Larvae.
Symbols are as described in Table 3a.
Premetamorphic Larvae - 1 6-days old

Concentration
(ng/l) VO VN O-N
0 5.7 ± 0.5 0.235 ±0042 25 + 4
8 5.7 ± 0.3 0.130 ± 0.051 723 ± 21
16 4.03 ± 0.2 0.0993 ± 0042 73a + 22
Table 3b (continued)
Postlarvae - 1 9-days old
Concentration
(HQ/I) VO VN O:N
0 44 ± 0.5 0252 ± 0028 16 + 2
8 4.7 ±0.2 0112 ± 0.040 66a ± 14
16 45 + 0.4 0154 + 0031 32a + TQ

aSignificantly different from the control (0) (p<0 05)
Table 4a. The Influence of Continual Exposure to a Range of
Concentrations of (R.S)-Methoprene on Growth of Various
Aged Palamonetes pugio Larvae. DW = Dry Weight (\\g); WG
= Daily Weight Gain (yg Day1); and K2 = Net Growth
Efficiency. Each Value Represents the Mean ± Standard
Error.
Early Larvae - 1 -day old
Concentration
(pg/l) DW WG K2
0 33 ±2 8 + 0 5 68 ±1
8 25a±1 23 ± 0.1 37a + 1
16 253 ±2 3a ± 0.2 42a + 2
32 223 + 2 13 + 01 14a ± 1
62 223 ± 2 13 + 0.1 19a ± 2
Intermediate Larvae - 9-days old
Concentration
(ng/i) DW WG K2
0 236 ±36 77 ± 12 83+2
8 135a + 14 37a ±4 81+1
16 1503 + 28 433 ±8 81+2
32 88a+11 193+2 69a + 2
62 563 ± 14 7a + 2 573 ± 4
aSignificantly less than the control (0) (p<0.05).
evaluate potential environmental hazards of a new type of
pesticide The results of these studies suggest that the use
of similar crustacean larval testing procedures would be
appropriate in such assessments of insect nrowth reaulators
in the marine environment

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Table 4b. The Influence of Continual Exposure to a Range of
Concentrations  of  (R.S)-Methoprene on  Growth  of Various
Aged Palaemonetes pugio Larvae.  Symbols are as described
in Table 4a.

                  Premetamorphic Larvae - 16 -days old
Concentration
(ug/l)
0
8
16

Concentration
(ng/i)
0
8
16

DW
334 ± 36
345 ± 51
415 ± 72

WG
17 ± 2
49a ± 7
65a ± 11

K2
44 ± 2
69a ±1
773 ± 1
Postlarvae - 19-days old
DW
474 ± 30
409 ± 53
511 ±65
WG
58+4
243 + 3
37a + 5
K2
71 ± 2
52a ±1
58a ± 2
aSignificantly different from the control (0) (p<0 05).
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