Low Exposure Concentration Effects of Methoprene on Endocrine-Regulated Processes in the Crustacean Daphnia magna

Allen W. Olmstead and Gerald L. LeBlanc,1

Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695–7633

Received January 22, 2001; accepted April 27, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Methoprene is a growth-regulating insecticide that manifests its toxicity to target organisms by acting as a juvenile hormone agonist. Methoprene similarly may exert toxicity to crustaceans by mimicking or interfering with methyl farnesoate, a crustacean juvenoid. We hypothesized that methoprene interferes with endocrine-regulated processes in crustaceans by several mechanisms involving agonism or antagonism of juvenoid receptor complexes. In the present study, we evaluated this hypothesis, in part, by characterizing and comparing the concentration-response curves for methoprene and several endpoints related to development and reproduction of the crustacean Daphnia magna. Our results demonstrate that methoprene has multiple mechanisms of toxicity and low-exposure concentration effects. Methoprene reduced the growth rate of daphnids with evidence of only a single concentration-response line, having a threshold of 12.6 nM. Molt frequency was reduced by methoprene in a concentration-dependent manner, with a response curve corresponding to a 2-segmented line and thresholds at 4.2 and 0.21 nM. An endpoint related to reproductive maturation, the time of first brood deposition, was also affected by methoprene, with a clear concentration-dependent response and a NOEC of 32 nM. Methoprene reduced fecundity according to a 2-segmented line, with thresholds of 24 and <=0.18 nM. These results demonstrate that methoprene elicits significant toxicity to endocrine-related processes in the 5–50 nM concentration range. Furthermore, molting and reproduction were impacted at significantly lower methoprene concentrations, with a distinct concentration response and a threshold of <=0.2 nM. The different concentration-dependent response from that of methoprene could involve agonism or antagonism of various juvenoid receptor configurations.

Key Words: methoprene; daphnid; crustacea; endocrine disruption; methyl farnesoate.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Insect growth regulators are insecticides that are designed to disrupt specific physiological processes of target insects. These insecticides typically perturb enzymatic and hormonally regulated processes that are relatively specific to insect physiology (Dhadialla et al., 1998Go; Retnakaran et al., 1985Go). These designer pesticides usually have low toxicity to non-target organisms, because these species generally lack the enzymatic or hormonally regulated processes that these insecticides disrupt. Methoprene is one of the most widely used and successful insect growth regulators, which elicits toxicity to target insects by acting as a juvenile hormone agonist (Retnakaran et al., 1985Go). One of the main uses of methoprene is in the control of mosquitoes, where it prevents the metamorphosis of mosquito larvae into adults, and in this capacity, is applied directly to the aquatic environment, often as pelleted, sustained-release, or liquid formulations (Dhadialla et al., 1998Go; Retnakaran et al., 1985Go).

Juvenile hormone (JH) is a sesquiterpenoid that bears structural similarity to the terpene component of retinoic acid. JH modulates ecdysteroid activity in insects and also influences reproduction, caste determination, behavior, diapause, and metabolism (Nijhout, 1998Go). In vertebrates, retinoids are ligands to retinoic acid receptors (RAR) and retinoid-x receptors (RXR; Mangelsdorf et al., 1990). Liganded RXR can homodimerize or can heterodimerize to RAR, some steroid receptors, and other receptors to form transcriptional activators (Harmon et al., 1995Go; Mangelsdorf and Evans, 1995Go). USP is an insect homolog of the RXR (Yao et al., 1992Go), which heterodimerizes to the ecdysone receptor (EcR) to form the ecdysone transcriptional activator (Yao et al., 1993Go). USP also can heterodimerize with partner receptors to RXR (Yao et al., 1992Go), demonstrating its functional homology to RXR. Like RXR, it is possible that USP also stimulates transcriptional activation by homodimerization. The endogenous ligand to USP has not been unequivocally established; however, USP was shown to bind juvenile hormones in vitro and the complex was shown to induce USP-dependent transcription of a reporter gene assay (Jones and Sharp, 1997Go). JH analogs also have been shown to bind and activate RXR in mammals (Harmon et al., 1995Go). Taken together, these observations indicate that (1) USP functions as an insect homolog to RXR, (2) JH is the ligand to USP, and (3) USP mediates the regulatory influence of JH, either by heterodimeric combination with other receptors such as EcR or through homodimerization.

Crustaceans apparently do not utilize juvenile hormones as do insects (Chang, 1993Go). However, the related juvenoid, methyl farnesoate, the unepoxidated form of juvenile hormone III, may be an important reproductive hormone in crustaceans (Homola and Chang, 1997Go; Laufer et al., 1993Go; Lu et al., 2000Go). Methyl farnesoate has been shown to stimulate ovarian maturation in crustaceans (Laufer et al., 1998Go; Reddy and Ramamurthi, 1998Go), induce larval metamorphosis in the barnacle (Yamamoto et al., 1997Go), and increase molt duration in the larvae of lobster (Borst et al., 1987Go) and shrimp (Abdu et al., 1998Go).

In light of the structural and functional homology between insect juvenile hormones and crustacean methyl farnesoate, we hypothesized that the juvenile hormone analog methoprene may specifically target processes in crustaceans that are regulated by methyl farnesoate. Methoprene has been reported to reduce fecundity of mysid shrimp (McKenney and Celestial, 1996Go) and cladocerans (Chu et al., 1997Go; Templeton and Laufer, 1983Go) and interfere with normal juvenile development of grass shrimp (McKenney and Matthews, 1990Go), mud crab (Celestial and McKenney, 1994Go), and daphnids (Templeton and Laufer, 1983Go). These effects have commonly been reported to occur in the 30 to 300-nM-exposure range. In contrast, EC50 values for emergence of mosquitoes have been reported in the aqueous concentration range of 0.03 to 3 nM (Schaefer and Wilder, 1973Go; Ritchie et al., 1997Go). These observations would suggest that methoprene is a significantly less potent analog of methyl farnesoate when compared to juvenile hormone, and crustaceans are less susceptible to the endocrine-disrupting toxicity of methoprene as compared to insects.

We have hypothesized that the toxicity of methoprene to crustaceans is mediated by its interaction with one or more RXR/USP family members, resulting in agonism or antagonism of receptor dimers that utilize RXR/USP. Some of these interactions may result in toxicity at exposure concentrations rivaling those that affect juvenile hormone-regulated processes in target insects. Modeling of concentration responses of the effects of methoprene on several endocrine-regulated processes in the crustacean, Daphnia magna, was performed, using a 2-segmented linear approach in an effort to discern multiple mechanisms of methoprene toxicity and establish threshold concentrations for the observed effects.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Daphnid cultures.
Daphnids were cultured in medium as described previously (Baldwin and LeBlanc, 1994Go). Daphnids were fed green algae (Selenastrum capricornutum) supplemented with Tetrafin fish food (Pet International, Chester Hill, New South Wales, Australia). Algae were cultured in Bold's basal medium (Nichols, 1973Go). The fish food supplement was prepared by homogenizing 10 g of fish food in a blender with 1 liter of water for 10 min. Solids were settled out overnight, and aliquots of the resulting supernatant were dried at 100°C to determine the suspended-solids content. Daphnid cultures (1 L medium containing 40 daphnids) were fed 1.4 x 108 cells of algae twice daily and 4 mg (dry weight) of fish food suspension twice daily. Daphnids were transferred to new medium and offspring removed 3 times weekly. Cultures and all experiments were maintained at 20°C at a 16-h photoperiod.

Influence of Methoprene on Growth, Maturation, and Reproduction
All experiments were conducted at methoprene exposure levels of <300 nM. Prior studies conducted in our laboratory have indicated that daphnids would tolerate these exposure levels without signs of overt toxicity (Olmstead and LeBlanc, 2000Go). Definitive concentration-response analyses of the effects of methoprene on growth, molting, attainment of reproductive maturity, and fecundity were evaluated using one of two experimental designs.

Experimental design 1.
Daphnids (<5 h old) were exposed to 70 concentrations of methoprene (Chem Service, West Chester, PA). One daphnid was exposed to each concentration of methoprene and each methoprene concentration was 90% of the next greater concentration. Ten daphnids (controls) were also individually exposed to the same amount of carrier solvent (0.005% absolute ethanol) as was present in all methoprene treatments. One daphnid was exposed to each treatment level in a 50-ml beaker containing 40 ml of test solution. Algae (3.5 x 106 cells) and fish food supplement (100 µg dry weight) was provided to each beaker twice daily during the first week of each experiment. Subsequently, 7.0 x 106 cells of algae and 200 µg fish food supplement (dry weight) were provided to each beaker twice daily. Daphnids were transferred to fresh media 3 times weekly. Offspring were counted and removed from the beakers daily.

The impact of methoprene exposure on offspring production was assessed for each brood through the first 5 broods. Solutions were examined hourly for the presence of molted exoskeletons. Growth rates of the daphnids were established by measuring molted exoskeletons from the base of the shell spine to the top of the carapace. Since growth rates of daphnids are linear over the first week of life (Olmstead and LeBlanc, 2000Go); these rates were determined as the slope of the linear fit of the graph of molt size versus molt number for the first 5 molts (~6 days). This design provided for definitive characterization of the methoprene concentration-response lines.

Experimental design 2.
Daphnids (<5 h old) were exposed to 6 different concentrations of methoprene or carrier solvent (0.025% absolute ethanol) with 10 daphnids individually exposed to each treatment. The experimental design was otherwise the same as described for Experimental Design #1. Time to reproductive maturation was assessed using this design. Time to reproductive maturation was denoted as the time at which the first clutch of eggs was transferred from the ovaries to the brood chamber. Transparency of the daphnids allowed for visual determination of this endpoint without having to manipulate the organisms in any way, and represents the end of the reproductive-maturational period, which is when oocyte development begins in the ovaries. This design was necessary in order to assess the effects of methoprene on the time required for daphnids to attain reproductive maturity, since high methoprene concentrations were found to delay the transfer of eggs from the ovaries to the brood chamber by one molt period (6th versus 5th instar). As a result, concentration-response lines generated using experimental design #1 did not represent a continuous concentration response, but rather, resulted in a best fit between daphnids that attained reproductive maturity during the 5th and the 6th instars.

Data Analysis
Concentration-response curves were fitted with a 2-segmented linear model using the PROC NLIN, Marquardt method using SAS 7.0 software (SAS Institute, Cary, NC). The following model was fit to the data:


where y is the measured variable, x is the logarithm of the methoprene concentration, t is the logarithm of the methoprene concentration at which the 2 linear segments intersect, and m1 and m2 are the slopes of the lower- and higher-concentration segments, respectively. Each segment of the model fits were then analyzed by linear regression in order to test whether the slopes were significantly different from zero, using an F-test (Zar, 1996Go). Threshold concentrations were defined as the level at which the concentration-response line intersected the line representing the appropriate control.

Differences in reproductive maturation time were deemed statistically significant using an analysis of variance and Dunnett's t-test (Zar, 1996Go) at an alpha level of 0.05 with JMP software (SAS Institute, Cary, NC). Growth rates were calculated as the slope of the linear-regression fit of molt length vs. molt number for individual daphnids.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Juvenile Molt and Growth Rates
Molting and growth are closely coordinated in arthropods, since molting of the exoskeleton must occur to allow for the expansion of body mass associated with growth (Pennak, 1953Go). Molting is stimulated by 20-hydroxyecdysone in conjunction with the ecdysone transcriptional activator (Chang, 1993Go). Growth also is likely to be regulated by this or a similar hormone-receptor complex. We therefore hypothesized that methoprene would coordinately interfere with molting and growth.

Methoprene significantly reduced growth rate among juvenile daphnids in a concentration-dependent manner with a highly significant slope (p < 0.0001) and a clear threshold evident at 12.6 nM (Fig. 1Go). Methoprene also significantly reduced juvenile molt frequency, as measured by the duration of the intermolt period (instar) between the first and second molt (Fig. 2Go). This response conformed to a 2-segmented line, each having a significant non-zero slope. The threshold concentration for this response at higher concentrations was 4.2 nM, with a highly significant slope (p < 0.0001). The second concentration-response line was evident at lower methoprene concentrations, with a significant slope (p = 0.012). This response exhibited a threshold at 0.21 nM.



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FIG. 1. Growth rate of daphnids exposed to concentrations of methoprene. Each point represents a single daphnid. Growth rate was calculated as the slope of the linear regression line of a plot of molt length versus molt number for each individual daphnid. The dashed and dotted lines represent mean and standard deviation, respectively, of the growth rate associated with the control daphnids. Solid lines represent the 2-segmented line-model fit of the data.

 


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FIG. 2. Molt frequency of daphnids exposed to concentrations of methoprene as measured by the duration of the 2nd intermolt period (instar). Each point represents a single daphnid. The dashed and dotted lines represent mean and standard deviation, respectively, of the duration of the 2nd instar associated with the control daphnids. The solid lines represent the 2-segmented line-model fit of the data.

 
Reproductive Maturation
Reproductive maturation of daphnids involves multiple coordinated events that are likely to be influenced by juvenoid hormones. These include growth—the organism must be of adequate size to accommodate the brood of eggs deposited into the brood chamber from the ovaries—and the timing of molting—egg maturation in the ovaries, deposition of eggs into the brood chamber, and release as fully developed offspring must all be timed with the occurrence of a molt. The effect of methoprene on the time of first deposition of eggs into the brood chamber was evaluated. Methoprene significantly increased the time to first brood deposition according to a single concentration-response line exhibiting an NOEC of 32 nM (Fig. 3Go).



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FIG. 3. Age at which daphnids exposed to concentrations of methoprene attained reproductive maturation as measured by the initial transfer of eggs to the brood chamber. Data are presented as the mean and standard deviation (n = 10). An asterisk indicates a significant (<=0.05) difference from the control (ANOVA, Dunnett's t-test).

 
Fecundity
Modeling of the concentration responses for the impact of methoprene on brood sizes yielded a segmented line with 2 non-zero slopes. At high exposure concentrations (>24 nM), methoprene reduced fecundity of daphnids in a manner characterized by a steep concentration-response line (Fig. 4Go) that progressively increased in magnitude with increasing cumulative brood number (Fig. 5Go). The threshold concentration for this effect was 24 nM after 5 cumulative broods, with a highly significant slope (p < 0.0001; Fig 4Go).



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FIG. 4. Concentration-response lines for cumulative offspring production by daphnids exposed to concentrations of methoprene. Each point represents a single daphnid. The dashed and dotted lines represent mean and standard deviation, respectively, of offspring production associated with the control daphnids. Solid lines represent the 2-segmented line model fit of the data.

 


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FIG. 5. Two-segmented line model slopes of the concentration response curves for cumulative offspring production of daphnids exposed to concentrations of methoprene. The slopes of the models in Figure 1Go were plotted against the cumulative brood number to which the 2-segmented line models were fit. Squares represent the slopes of the lines generated with methoprene concentrations <24 nM and circles represent the slopes of the lines generated with methoprene concentrations >24 nM. Brackets represent the 95% confidence interval associated with each slope.

 
Methoprene adversely affected fecundity of daphnids at lower exposure concentrations (<24 nM) with a concentration-response line appreciably more shallow than that observed at higher concentrations (Fig. 4Go). The slopes of the concentration-response lines at the low methoprene exposure concentrations did not appreciably change with progressing cumulative broods (Fig. 5Go). The slope for this effect after 5 cumulative broods was significant (p = 0.03) with a threshold of <0.18 nM, the lowest concentration evaluated.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study was based upon the premise that methoprene can elicit toxicity to crustaceans through various receptor-mediated interactions. Concentration-response lines were generated for several endocrine-regulated processes using a 2-segmented linear model, in an attempt to discern differences in the concentration responses that would be indicative of different mechanisms of toxicity and would perhaps reveal effects of methoprene at low exposure levels. Results provided evidence for both multiple mechanisms of methoprene toxicity to crustaceans and threshold concentrations for some of these effects, which were significantly lower than previously reported for crustaceans.

All of the physiological endpoints measured were negatively impacted by methoprene at concentrations greater than 30 nM. These effects are consistent with those typically reported for methoprene and crustaceans (Celestial and McKenney, 1995; Chu et al., 1997Go; McKenney and Celestial, 1996Go; McKenney and Matthews, 1990Go; Templeton and Laufer, 1983Go). The generalized adverse response of all of the measured physiological endpoints to these exposure concentrations suggests that methoprene impacted processes that were critical to the general fitness of the organisms. For example, the RXR family of receptors is involved in regulating several aspects of lipid metabolism and utilization in vertebrates (Repa, 2000). Methoprene has been shown to activate gene transcription from an RXR response element (Harmon et al., 1995Go). At the higher exposure concentrations used in this study, perhaps methoprene interacted with an RXR/USP family member in a manner that resulted in aberrant lipid metabolism. Limitations in fuel (lipids) for energy may have resulted in uniform adverse effects on the measured growth and reproductive endpoints.

The concentration responses generated with molting and fecundity conformed to a 2-segmented line with each segment having a non-zero slope. The common concentration response of these endpoints at the low methoprene concentrations suggests that these two endpoints share a common target of methoprene toxicity that is different from that responsible for the high-concentration effects. The molting process in arthropods is initiated by a drop in ecdysteroid levels, and the receptor heterodimer EcR-USP mediates this response (Yao et al., 1993Go). EcR-USP also is known to regulate oogenesis in insects (Carney and Bender, 2000Go). Thus, a high affinity interaction of methoprene with USP/RXR (Harmon et al., 1995Go) may have modulated EcR-USP activity, resulting in the observed low-concentration effects of this compound on molting and reproduction.

Reproductive maturation denotes the end of the juvenile phase and the beginning of the adult phase in daphnids. Energy resources previously allocated to growth during the juvenile stage are largely directed towards reproduction during the adult stage. Among daphnids, reproductive maturation commences with the maturation of the ovaries and the development of diploid eggs that develop via parthenogenesis (Pennak, 1953Go). These eggs are deposited into the brood chamber following the first adult molt. Reproductive maturation therefore begins one instar before that at which eggs are deposited into the brood chamber. Methyl farnesoate has been shown to stimulate ovarian growth and maturation in crustaceans (Laufer et al., 1992Go, 1998Go). Exposure to the higher concentrations of methoprene in the present study delayed reproductive maturation. This endpoint was not amenable to definitive concentration-response analyses, as were the other endpoints. However, results suggest that methoprene may have affected this endpoint through the competitive inhibition of methyl farnesoate or in a manner similar to the high-concentration effects observed with the other endpoints (i.e., altered lipid metabolism).

The definitive concentration-response analyses performed in the present study identified responses of daphnids that were unique to molting and fecundity, exhibiting thresholds <0.2 nM. These observations demonstrate that crustaceans do respond to methoprene at concentrations known to affect aquatic insects (Ritchie et al., 1997Go; Schaefer and Wilder, 1973Go). The use of methoprene at recommended application rates would be expected to result in environmental concentrations of ~30 nM (Ingersoll et al., 1999Go). Methoprene concentrations in natural and experimentally enclosed surface waters, following application at recommended rates, have typically ranged from 3.0–30 nM (Knuth, 1989Go; Ross et al., 1994Go). Results from the present study indicate that methoprene adversely impacts some endocrine regulated processes in daphnids at concentrations significantly below environmental concentrations at application. Establishing whether the effects elicited at these environmentally relevant concentrations are sufficient to adversely impact crustacean populations awaits additional study.


    ACKNOWLEDGMENTS
 
This work was supported by U.S. EPA grant #R826129 to G.A.L.


    NOTES
 
1 To whom correspondences should be addressed. Fax: (919)515-7169. E-mail: ga_leblanc{at}ncsu.edu. Back


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