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
-------
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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T
l
t
-j-
j i;
(R S) (S)
6 6
0 1
10 100
fjg Methoprene/l (LOdo)
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
-------
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).
CD
Q.
-C
to
10U -
on
60 -
40 -
20 -
T
:
•
*
T
;
:
»
0
i
:
-
*
T
S&8S&;
IBS
First Final
Larval Larval
Stages Stage
r.L
*
0
31
62
/jg (S)-Methoprene/!
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
-------
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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