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Effects of the insect growth regulator (S)-methoprene on survival and reproduction of the freshwater cladoceran Moina macrocopa
PII:SO269-7491(97)00030-4
Environmental Pollution, Vol. 96, No. 2, pp. 173-178, 1997 &J 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0269-7491/97 $17.00+0.00
ELSEVIER
EFFECTS OF THE INSECT GROWTH REGULATOR (S)-METHOPRENE ON SURVIVAL AND REPRODUCTION OF THE FRESHWATER CLADOCERAN MOINA MACROCOPA K. H. Chu, C. K. Wong and K. C. Chiu Department of Biology and The Centre for EnvironmentalStudies, The Chinese Universityof Hong Kong, Shatin, NT, Hong Kong (Received 4 June 1996; accepted 3 February
Abstract
1997)
prene can be quite toxic to non-target aquatic organisms, including insects (GelbiE et al., 1994) and crustaceans (Christiansen et al., 1977; McKenney and Matthews, 1990; Celestial and McKenney, 1994). In crustaceans, JH analogues like methoprene may affect reproduction and development, because methyl farnesoate, a JH-like compound in crustacean tissues, has been suggested to play a regulatory role in these processes (Borst et al., 1987; Laufer et al., 1987a,b). Indeed, methoprene has been shown to inhibit or retard larval development of some estuarine crustaceans (Christiansen et al., 1977; Celestial and McKenney, 1994; Chu et al., 1995). The objectives of this study are to determine the acute and chronic effects of (S)-methoprene on survival and reproduction of the freshwater cladoceran Moina macrocopa. This species occurs in ponds and rice paddies in Southeast Asia. It is sensitive to pollutants, such as heavy metals (Wong and Wong, 1990; Wong, 1993) and organophosphate insecticides (Wong et al., 1995; Wong, 1997). The short life span and high fecundity of M. macrocopa make it a good model for toxicity tests. Because cladocerans are important links in the aquatic food web, toxicants affecting their survival and reproduction would likely have indirect effects on their predators and prey, and cause changes at the community and ecosystem levels. For instance, there is ample evidence that pesticide contamination could lead to dominance of the zooplankton community by small organisms, thus reducing the efficiency of energy flow from algae to zooplankton (Havens and Hanazato, 1993) and increasing the impact of eutrophication (Hurlbert, 1975).
This study examines the acute and chronic efsects of the insecticide (S)-methoprene on the survival and reproduction of the freshwater cladoceran Moina macrocopa. In laboratory toxicity tests, the 24- and 48-h LC.50 values for (S)-methoprene were 0.51 and 0.34 mg litre-‘, respectively. Survival, longevity and fecundity were reduced at 0.05 mg litre-’ and higher concentrations. At 0.005 and 0.01 mg litre-‘, longevity and fecundity increased slightly as compared to control animals. If environmental concentrations of methoprene do not exceed 0.05 mg litre-‘, as is generally the case, application of this insecticide is unlikely to induce detrimental eflects on natural cladoceran populations. The stimulatory effect of very low levels of methoprene on reproductive performance is consistent with the hypothesis of a regulatory role of juvenile hormone-like compounds in crustacean reproduction. 0 1997 Elsevier Science Ltd Keywords: Methoprene,
insect growth regulator, juvenile hormone analogue, Moina macrocopa, toxicity.
INTRODUCTION In recent years, the toxicity of insecticides to humans and wildlife has caused much public concern and led to the use of more target-specific chemicals. One commonly used group of insecticides consists of insect growth regulators, such as analogues of juvenile hormones (Staal, 1975). Juvenile hormones (JH) regulate both morphogenetic and reproductive development in insects, and the JH analogues mimic the action of JH by disrupting the developmental processes in insect pests (Sehnal, 1983). Methoprene (Altosid@) was the first insect growth regulator to be approved by the United States Environmental Protection Agency for use as a mosquito larvitide, and it is now widely used in other pest control and agricultural programmes (Sehnal, 1983). While some studies have demonstrated only minor adverse effects of methoprene on the aquatic biota (Kikuchi et al., 1992; Hershey et al., 1995), others have shown that metho-
MATERIALS
AND METHODS
M. macrocopa, in a continuous laboratory culture, were raised from a single parthenogenetic female. Animals were kept in aged tapwater (pH 6.5-7.0) from a 300-litre aquarium and fed Chlorella pyrenoidosa at about lo6 cells ml-’ from batch cultures. Newborn animals ( < 18 h) for experiments were collected by isolating eggbearing adults from the stock culture. 173
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(S)-methoprene (lot No. 26787) was acquired from Karlan Research Products Corporation, USA. This isomer is the only one used in Altosid Liquid Larvitide@ (ALL), a slow-release formulation designed for extended potency after application. A stock solution of 1000mglitreel was prepared every week by dissolving (S)-methoprene in acetone. Solutions for experiments were prepared by adding aliquots of stock solution to 20-km filtered aquarium water. All experiments were conducted in 100ml beakers. Each beaker contained 50ml of test solution and 10 test animals. C. pyrenoidoss cells were centrifuged, resuspended in filtered aquarium water and added to test containers at a density of about lo6 cells ml-’ to feed the test animals. Acute toxicity of methoprene was studied at concentrations of 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0 and 10.0 mg litre-‘. Six replicates of 10 animals were studied for each concentration. Controls using uncontaminated aquarium water or aquarium water containing 10 ml litre-’ of acetone were tested simultaneously with each replicate experiment. Acetone was added to test solutions to provide the same concentration of acetone for each concentration of (S)-methoprene. The animals were examined under a dissecting microscope after 24 h. Individuals without heart beat were considered dead. The number of dead animals in each beaker was recorded and the live animals were transferred to fresh test solutions with algal food. The number of dead animals was determined again after 48 h when the experiment was terminated. All experiments were conducted at 26 * 2°C. The chronic effects of (S)-methoprene on survival and reproduction were tested at 0.005, 0.01, 0.05, 0.1 and 0.5 mglitre-‘. Each beaker contained 80ml of test solution and 10 animals. Two replicates were used for each concentration. Animals were transferred at the same time daily to fresh test solutions with C. pyrenoidosa at
4 x 1O5 cells ml-‘. Preliminary experiments indicated that a higher food concentration did not enhance survival or reproduction. Controls with uncontaminated aquarium water or 0.5ml acetone litre-’ were conducted simultaneously with each replicate experiment. The number of dead animals was determined at the time of transfer. Newborn animals produced during the 24 h interval were counted and discarded. Each experiment was terminated in about two weeks when all individuals had died. Life table parameters were calculated according to Hedrick (1984).
RESULTS Mortality of M. macrocopa increased with methoprene concentration in acute toxicity experiments (Fig. 1). Complete mortality was observed at 5 and 10 mg litre-’ of methoprene after 48 h of exposure (not shown in Fig. 1). No significant mortality was observed at 0.1 mglitre-’ and lower concentrations. Data points at concentrations lower than 0.1 mglitre-’ were not included in the calculation of LC50 values. The 24 and 48 h LC50 values were 0.51 and 0.34 mg litre-‘, respectively. Because results on survival and reproduction from duplicate chronic toxicity experiments were within 10% of each other, data were pooled for analysis. Survivorship of M. macrocopa at different concentrations of methoprene is shown in Fig. 2. Difference in survivorship between treatments was compared with the day of death as observation. Non-parametric tests (KruskalWallis test followed by Student-Newman-Keuls test) were used because the data did not meet the requirement for normality (Zar, 1984). The results show no significant difference between control and solvent control. Significant effects (p < 0.05) of methoprene on r
24 h
7
48 h
0.001
0.01
0.1
1.0
10
Methoprene Concentration (mg I-‘) Fig. 1. Mortality of Moina macrocopa exposed to different concentrations of methoprene for 24 and 48 h. Percentage of mortality is expressed as probit and concentrations are plotted on logarithmic scale. Each point represents the mean of 6 replicates of 10 animals. The lines are based on analysis of mortality data at 0.1 mg litrec’ and higher concentrations of methoprene. C, control with uncontaminated water; S, control with acetone (10 mg litre- ‘).
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(Christiansen et al., 1977; Celestial and McKenney, 1994) and shrimps (McKenney and Matthews, 1990; Chu et al., 1995). However, it should be noted that such methoprene concentrations are considerably higher than typical environmental concentrations resulting from applications for control of mosquitoes and other insect pests. Ross et al. (1994) pointed out that if Altosid Liquid Larvicide@ with 5% (S)-methoprene is applied at the maximum label rate (293 ml ha-‘) to water 15 cm deep, the expected environmental concentration will be 0.01 mglitree’. This concentration has no effect on the survival of M. macrocopa and was also considered to be a safe dosage for D. magna (Templeton and Laufer, 1983). In contrast to various field studies which documented zooplankton community responses to pesticide applications (Havens and Hanazato, 1993 review), field trials of methoprene have generally produced little stress on non-target aquatic organisms (Kikuchi et al., 1992; Hershey et al. 1995). Of particular interest is that concentrations comparable to those in field application are not toxic to biological control agents such as planarians (Nelson et al., 1994), predatory copepods and Bacillus thuringiensis var. israelensis (Tietze et al., 1994), indicating that integrated pest control using methoprene and biological control agents is feasible. Overall, these studies confirm that methoprene is relatively nonhazardous to the environment when compared to conventional insecticides. Other than lethal effects, the present study showed that methoprene concentrations of 0.05 mg litree’ or higher reduced the longevity, reproductive capacity and population growth of M. macrocopa. Similar effects of methoprene on longevity and fertility of insects have been reported (Bouchard and Wilson, 1987). The detrimental effect on the reproductive performance of crustaceans was possibly related to the ovicidal properties of juvenile hormone analogues, as previously observed in insect studies (e.g. Kinawy and Hussein, 1987; Keil and Othman, 1988). Methoprene at 1.3 mglitree’ has been shown to induce gametogenesis disorders in the crab Rithropanopeus harrissi (Payen and Costlow, 1977). Yet the most intriguing finding from our study is the apparent stimulatory effect of 0.005 and 0.01 mglitre-’ methoprene on longevity, reproductive performance and population growth of M. macrocopa. This may partially be attributed to the presence of acetone in the test medium. A possible explanation for the stimulatory effect of acetone is that the solvent acted as a substrate for bacteria which served as food for the cladocerans. Beneficial effects of low level exposure to toxicants have been previously reported (Calabrese, 1994). Some of the biological effects include enhanced survival, growth, longevity and reproductive performance. A range of cellular and molecular mechanisms activated at low levels of toxicants could produce such effects (Mehendale, 1994). In the case of methoprene, the stimulatory responses observed at low concentrations may be related to the proposed role of JH-like hormone in stimulating crustacean reproduction (Laufer et al., 19873). JH in conjunction with ecdysteroids controls vitellogenesis
in insects (Engelmann, 1979; Bownes et al., 1993). Like JH, methoprene also has been shown to induce vitellogenin synthesis and release in many insects (e.g. Dhadialla et al., 1987; Adams and Filipi, 1988; Satyanarayana et al., 1994; Kim and Krafsur, 1995). Methyl farnesoate, a JH-like substance, has been postulated to play a similar role in crustacean reproduction (Laufer et al., 1987a,b). Such a role for a JH-like hormone would explain the shorter initial age of reproduction and higher fecundity in M. macrocopa exposed to 0.005 and 0.01 mg litree’ methoprene, and impairment of reproductive performance at higher concentrations. Sehnal (1983) noted that JH analogues also can suppress or stimulate insect reproduction dependent on the dose applied. While our results support a role for JH-like hormones in crustacean reproduction, studies on the inhibitory effects of methoprene on crustacean larval development (Christiansen et al., 1977; Celestial and McKenney, 1994; Chu et al., 1995) suggest that they may also control development (Borst et al., 1987). Methoprene and its derivatives can stimulate gene transcription in mammals by activating retinoid X receptors (Harmon et al., 1995). Interestingly, retinoids have juvenile hormonelike effects on insect metamorphosis, embryogenesis and reproduction (Nemec et al., 1993), suggesting their regulatory role in morphogenesis is common to all animals. Although retinoid receptors were identified in crustaceans (Durica and Hopkins, 1996), the role of retinoids in crustacean biology remains obscure. As JH analogues can act as retinoid analogues, studies of their action on morphogenesis and reproduction in a variety of animals is crucial in understanding their effects on non-target organisms.
ACKNOWLEDGEMENTS We thank K. C. Cheung and L. S. Leong for their technical assistance. We are grateful to an anonymous reviewer for constructive comments on the manuscript. The work is supported by a research grant from the Centre for Environmental Studies, The Chinese University of Hong Kong.
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Ejects
of (S)-methoprene
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Environmental Pollution, Vol. 96, No. 2, pp. 173-178, 1997 &J 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0269-7491/97 $17.00+0.00
ELSEVIER
EFFECTS OF THE INSECT GROWTH REGULATOR (S)-METHOPRENE ON SURVIVAL AND REPRODUCTION OF THE FRESHWATER CLADOCERAN MOINA MACROCOPA K. H. Chu, C. K. Wong and K. C. Chiu Department of Biology and The Centre for EnvironmentalStudies, The Chinese Universityof Hong Kong, Shatin, NT, Hong Kong (Received 4 June 1996; accepted 3 February
Abstract
1997)
prene can be quite toxic to non-target aquatic organisms, including insects (GelbiE et al., 1994) and crustaceans (Christiansen et al., 1977; McKenney and Matthews, 1990; Celestial and McKenney, 1994). In crustaceans, JH analogues like methoprene may affect reproduction and development, because methyl farnesoate, a JH-like compound in crustacean tissues, has been suggested to play a regulatory role in these processes (Borst et al., 1987; Laufer et al., 1987a,b). Indeed, methoprene has been shown to inhibit or retard larval development of some estuarine crustaceans (Christiansen et al., 1977; Celestial and McKenney, 1994; Chu et al., 1995). The objectives of this study are to determine the acute and chronic effects of (S)-methoprene on survival and reproduction of the freshwater cladoceran Moina macrocopa. This species occurs in ponds and rice paddies in Southeast Asia. It is sensitive to pollutants, such as heavy metals (Wong and Wong, 1990; Wong, 1993) and organophosphate insecticides (Wong et al., 1995; Wong, 1997). The short life span and high fecundity of M. macrocopa make it a good model for toxicity tests. Because cladocerans are important links in the aquatic food web, toxicants affecting their survival and reproduction would likely have indirect effects on their predators and prey, and cause changes at the community and ecosystem levels. For instance, there is ample evidence that pesticide contamination could lead to dominance of the zooplankton community by small organisms, thus reducing the efficiency of energy flow from algae to zooplankton (Havens and Hanazato, 1993) and increasing the impact of eutrophication (Hurlbert, 1975).
This study examines the acute and chronic efsects of the insecticide (S)-methoprene on the survival and reproduction of the freshwater cladoceran Moina macrocopa. In laboratory toxicity tests, the 24- and 48-h LC.50 values for (S)-methoprene were 0.51 and 0.34 mg litre-‘, respectively. Survival, longevity and fecundity were reduced at 0.05 mg litre-’ and higher concentrations. At 0.005 and 0.01 mg litre-‘, longevity and fecundity increased slightly as compared to control animals. If environmental concentrations of methoprene do not exceed 0.05 mg litre-‘, as is generally the case, application of this insecticide is unlikely to induce detrimental eflects on natural cladoceran populations. The stimulatory effect of very low levels of methoprene on reproductive performance is consistent with the hypothesis of a regulatory role of juvenile hormone-like compounds in crustacean reproduction. 0 1997 Elsevier Science Ltd Keywords: Methoprene,
insect growth regulator, juvenile hormone analogue, Moina macrocopa, toxicity.
INTRODUCTION In recent years, the toxicity of insecticides to humans and wildlife has caused much public concern and led to the use of more target-specific chemicals. One commonly used group of insecticides consists of insect growth regulators, such as analogues of juvenile hormones (Staal, 1975). Juvenile hormones (JH) regulate both morphogenetic and reproductive development in insects, and the JH analogues mimic the action of JH by disrupting the developmental processes in insect pests (Sehnal, 1983). Methoprene (Altosid@) was the first insect growth regulator to be approved by the United States Environmental Protection Agency for use as a mosquito larvitide, and it is now widely used in other pest control and agricultural programmes (Sehnal, 1983). While some studies have demonstrated only minor adverse effects of methoprene on the aquatic biota (Kikuchi et al., 1992; Hershey et al., 1995), others have shown that metho-
MATERIALS
AND METHODS
M. macrocopa, in a continuous laboratory culture, were raised from a single parthenogenetic female. Animals were kept in aged tapwater (pH 6.5-7.0) from a 300-litre aquarium and fed Chlorella pyrenoidosa at about lo6 cells ml-’ from batch cultures. Newborn animals ( < 18 h) for experiments were collected by isolating eggbearing adults from the stock culture. 173
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(S)-methoprene (lot No. 26787) was acquired from Karlan Research Products Corporation, USA. This isomer is the only one used in Altosid Liquid Larvitide@ (ALL), a slow-release formulation designed for extended potency after application. A stock solution of 1000mglitreel was prepared every week by dissolving (S)-methoprene in acetone. Solutions for experiments were prepared by adding aliquots of stock solution to 20-km filtered aquarium water. All experiments were conducted in 100ml beakers. Each beaker contained 50ml of test solution and 10 test animals. C. pyrenoidoss cells were centrifuged, resuspended in filtered aquarium water and added to test containers at a density of about lo6 cells ml-’ to feed the test animals. Acute toxicity of methoprene was studied at concentrations of 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0 and 10.0 mg litre-‘. Six replicates of 10 animals were studied for each concentration. Controls using uncontaminated aquarium water or aquarium water containing 10 ml litre-’ of acetone were tested simultaneously with each replicate experiment. Acetone was added to test solutions to provide the same concentration of acetone for each concentration of (S)-methoprene. The animals were examined under a dissecting microscope after 24 h. Individuals without heart beat were considered dead. The number of dead animals in each beaker was recorded and the live animals were transferred to fresh test solutions with algal food. The number of dead animals was determined again after 48 h when the experiment was terminated. All experiments were conducted at 26 * 2°C. The chronic effects of (S)-methoprene on survival and reproduction were tested at 0.005, 0.01, 0.05, 0.1 and 0.5 mglitre-‘. Each beaker contained 80ml of test solution and 10 animals. Two replicates were used for each concentration. Animals were transferred at the same time daily to fresh test solutions with C. pyrenoidosa at
4 x 1O5 cells ml-‘. Preliminary experiments indicated that a higher food concentration did not enhance survival or reproduction. Controls with uncontaminated aquarium water or 0.5ml acetone litre-’ were conducted simultaneously with each replicate experiment. The number of dead animals was determined at the time of transfer. Newborn animals produced during the 24 h interval were counted and discarded. Each experiment was terminated in about two weeks when all individuals had died. Life table parameters were calculated according to Hedrick (1984).
RESULTS Mortality of M. macrocopa increased with methoprene concentration in acute toxicity experiments (Fig. 1). Complete mortality was observed at 5 and 10 mg litre-’ of methoprene after 48 h of exposure (not shown in Fig. 1). No significant mortality was observed at 0.1 mglitre-’ and lower concentrations. Data points at concentrations lower than 0.1 mglitre-’ were not included in the calculation of LC50 values. The 24 and 48 h LC50 values were 0.51 and 0.34 mg litre-‘, respectively. Because results on survival and reproduction from duplicate chronic toxicity experiments were within 10% of each other, data were pooled for analysis. Survivorship of M. macrocopa at different concentrations of methoprene is shown in Fig. 2. Difference in survivorship between treatments was compared with the day of death as observation. Non-parametric tests (KruskalWallis test followed by Student-Newman-Keuls test) were used because the data did not meet the requirement for normality (Zar, 1984). The results show no significant difference between control and solvent control. Significant effects (p < 0.05) of methoprene on r
24 h
7
48 h
0.001
0.01
0.1
1.0
10
Methoprene Concentration (mg I-‘) Fig. 1. Mortality of Moina macrocopa exposed to different concentrations of methoprene for 24 and 48 h. Percentage of mortality is expressed as probit and concentrations are plotted on logarithmic scale. Each point represents the mean of 6 replicates of 10 animals. The lines are based on analysis of mortality data at 0.1 mg litrec’ and higher concentrations of methoprene. C, control with uncontaminated water; S, control with acetone (10 mg litre- ‘).
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(Christiansen et al., 1977; Celestial and McKenney, 1994) and shrimps (McKenney and Matthews, 1990; Chu et al., 1995). However, it should be noted that such methoprene concentrations are considerably higher than typical environmental concentrations resulting from applications for control of mosquitoes and other insect pests. Ross et al. (1994) pointed out that if Altosid Liquid Larvicide@ with 5% (S)-methoprene is applied at the maximum label rate (293 ml ha-‘) to water 15 cm deep, the expected environmental concentration will be 0.01 mglitree’. This concentration has no effect on the survival of M. macrocopa and was also considered to be a safe dosage for D. magna (Templeton and Laufer, 1983). In contrast to various field studies which documented zooplankton community responses to pesticide applications (Havens and Hanazato, 1993 review), field trials of methoprene have generally produced little stress on non-target aquatic organisms (Kikuchi et al., 1992; Hershey et al. 1995). Of particular interest is that concentrations comparable to those in field application are not toxic to biological control agents such as planarians (Nelson et al., 1994), predatory copepods and Bacillus thuringiensis var. israelensis (Tietze et al., 1994), indicating that integrated pest control using methoprene and biological control agents is feasible. Overall, these studies confirm that methoprene is relatively nonhazardous to the environment when compared to conventional insecticides. Other than lethal effects, the present study showed that methoprene concentrations of 0.05 mg litree’ or higher reduced the longevity, reproductive capacity and population growth of M. macrocopa. Similar effects of methoprene on longevity and fertility of insects have been reported (Bouchard and Wilson, 1987). The detrimental effect on the reproductive performance of crustaceans was possibly related to the ovicidal properties of juvenile hormone analogues, as previously observed in insect studies (e.g. Kinawy and Hussein, 1987; Keil and Othman, 1988). Methoprene at 1.3 mglitree’ has been shown to induce gametogenesis disorders in the crab Rithropanopeus harrissi (Payen and Costlow, 1977). Yet the most intriguing finding from our study is the apparent stimulatory effect of 0.005 and 0.01 mglitre-’ methoprene on longevity, reproductive performance and population growth of M. macrocopa. This may partially be attributed to the presence of acetone in the test medium. A possible explanation for the stimulatory effect of acetone is that the solvent acted as a substrate for bacteria which served as food for the cladocerans. Beneficial effects of low level exposure to toxicants have been previously reported (Calabrese, 1994). Some of the biological effects include enhanced survival, growth, longevity and reproductive performance. A range of cellular and molecular mechanisms activated at low levels of toxicants could produce such effects (Mehendale, 1994). In the case of methoprene, the stimulatory responses observed at low concentrations may be related to the proposed role of JH-like hormone in stimulating crustacean reproduction (Laufer et al., 19873). JH in conjunction with ecdysteroids controls vitellogenesis
in insects (Engelmann, 1979; Bownes et al., 1993). Like JH, methoprene also has been shown to induce vitellogenin synthesis and release in many insects (e.g. Dhadialla et al., 1987; Adams and Filipi, 1988; Satyanarayana et al., 1994; Kim and Krafsur, 1995). Methyl farnesoate, a JH-like substance, has been postulated to play a similar role in crustacean reproduction (Laufer et al., 1987a,b). Such a role for a JH-like hormone would explain the shorter initial age of reproduction and higher fecundity in M. macrocopa exposed to 0.005 and 0.01 mg litree’ methoprene, and impairment of reproductive performance at higher concentrations. Sehnal (1983) noted that JH analogues also can suppress or stimulate insect reproduction dependent on the dose applied. While our results support a role for JH-like hormones in crustacean reproduction, studies on the inhibitory effects of methoprene on crustacean larval development (Christiansen et al., 1977; Celestial and McKenney, 1994; Chu et al., 1995) suggest that they may also control development (Borst et al., 1987). Methoprene and its derivatives can stimulate gene transcription in mammals by activating retinoid X receptors (Harmon et al., 1995). Interestingly, retinoids have juvenile hormonelike effects on insect metamorphosis, embryogenesis and reproduction (Nemec et al., 1993), suggesting their regulatory role in morphogenesis is common to all animals. Although retinoid receptors were identified in crustaceans (Durica and Hopkins, 1996), the role of retinoids in crustacean biology remains obscure. As JH analogues can act as retinoid analogues, studies of their action on morphogenesis and reproduction in a variety of animals is crucial in understanding their effects on non-target organisms.
ACKNOWLEDGEMENTS We thank K. C. Cheung and L. S. Leong for their technical assistance. We are grateful to an anonymous reviewer for constructive comments on the manuscript. The work is supported by a research grant from the Centre for Environmental Studies, The Chinese University of Hong Kong.
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