Methoprene Exposure Research Articles

Limb regeneration and molting processes under chronic methoprene exposure in the mud fiddler crab, Uca pugnax

Insect growth regulator application for wetland mosquito control remains controversial due to the potential for disruption of normal development and growth processes in non-target crustaceans and beneficial arthropods, e.g. Apis mellifera. Concerns include slow-release methoprene formulations and its environmental breakdown products which mimic an endogenous crustacean hormone and retinoids, respectively. Our primary objective was to evaluate the effect that a chronic methoprene exposure would have on male and female Uca pugnax limb regeneration and molting. After single limb autonomy, limb growth and molt stage were monitored every two days while eyestalk ablation was used to induce proecdysis. Dorsal carapace was collected 6 days post-molt to determine protein and chitin content. In post-molt crabs, methoprene-exposed individuals displayed lower percent gain in body weight. Male crabs lost more weight per body volume than females, took significantly longer to proceed through proecdysis than females exposed to 0.1 µg/L methoprene and exhibited significantly elevated frequency for abnormal limb formation at 1.0 µg/L while females displayed no such trend. Methoprene did not significantly alter extractable exoskeleton protein or chitin content. However, variable water-soluble protein expression increased with exposure at 1.0 µg/L (1 ppb) which contributed to overall variability in total protein content. Our findings suggest that adult male U. pugnax possess greater sensitivity to chronic methoprene exposure during limb regeneration and molting, potentially affecting their post-molt fitness. Furthermore, methoprene has the potential to impact post-molt biomass and exocuticle quality.

METABOLIC EFFECTS OF ACUTE EXPOSURE TO METHOPRENE IN THE AMERICAN LOBSTER, HOMARUS AMERICANUS

Methoprene was a constituent of the pesticide cocktail applied to the Western Long Island Sound (WLIS) watershed area during the summer of 1999. Subsequently, the seasonal lobster catches from the WLIS have decreased dramatically. We have been engaged in ongoing studies of the effects of methoprene on larval, juvenile and adult lobsters. Most recently, we found that Stage IV larvae exposed to 50 ppb methoprene experience >90% mortality rate after 3 days. Bioaccumulation studies on adult lobsters showed that methoprene concentrated against the gradient of the surrounding seawater (50 ppb) in hepatopancreas (1.55 ppm), gonad (5.18 ppm), epithelial tissue (6.17 ppm) and, most significantly, the eyestalks (28.83 ppm). Exposure to methoprene altered the expression of the stress proteins and the pattern of ubiquitinylation of cytosolic proteins by Day 1 Stage I larvae and by epithelial tissue of postmolt juvenile lobsters. Postmolt juvenile animals also demonstrated an altered pattern of protein phosphorylation in their epithelial tissues following methoprene exposure, indicating that it may interfere with cell signaling pathways. Increasing concentrations of methoprene were associated with increasing chitoproteins in the microsomal fractions of Day 1 Stage I larvae, suggesting that methoprene may compromise the exocytosis of shell matrix precursors from the epithelial cells. Methoprene did not, however, alter the activity of chitin synthase in these larvae. Although it is likely that a combination of harmful events and exposures led to the reduced lobster population in WLIS, methoprene may have contributed to the decline both by direct toxic effects and by disrupting homeostatic processes.

Expression of nuclear receptor-transcription factor genes during Aedes aegypti midgut metamorphosis and the effect of methoprene on expression

Exposure of mosquito 4th instars to the juvenile hormone analogue methoprene prevents the emergence of adults by interfering with metamorphosis. One metamorphic processes that is disrupted is midgut remodeling. To investigate the molecular mechanisms by which this occurs, the pattern of transcription factor gene expression during the Aedes aegypti (L.) 4th instar was investigated by the method of real time PCR. The results indicate that in untreated larvae, expression of transcription factors genes AHR3 and AaE75B increases within 24 h after the last larval–larval molt, transcription of AaEcR-B, AaUSP-a and AaßFTZ -F1 increases approximately 24 h later, and transcription of AaE75A increases just before the larval–pupal molt. There is uniform expression of AaUSP-b throughout the 4th instar. The effect of methoprene exposure on transcription factor gene expression during midgut remodeling was investigated. The results indicate that, in a dose and stage dependent manner, methoprene affects increases in expression that normally occur during midgut remodeling. The coincident effects of methoprene on metamorphic midgut remodeling and on transcription factor gene expression suggests that the two processes are related.

Projecting Population-Level Responses of Mysids Exposed to an Endocrine Disrupting Chemical

To fully understand the implications of a chemical's effect on the conservation of a species, effects observed at the physiological or individual level must be expressed in terms of the population. Since long-term field experiments are typically not feasible, vital rates such as survival and reproduction of individual organisms are measured in life table response experiments (LTRE) and employed to extrapolate the effects of a pollutant on the population. The population-level response of the mysid, Americamysis bahia, to varying concentrations of methoprene (0, 4, 8, 16, 31, 62 μg/L) was determined using age-structured population models. Models were parameterized from the results of an LTRE conducted throughout the entire mysid life cycle. A density-independent matrix model with time invariant demographic parameters was developed to measure the change in population growth rate, λ, with change in methoprene concentration. The values of λ were greater than one for all methoprene concentrations, indicating that populations exposed to the concentrations reported here would not become extinct. However, a general decrease in λ occurred with increasing methoprene concentration and would result in reduced population sizes. Sensitivity and decomposition analyses were conducted to determined the relative roles of the vital rates on altered population growth rates and determined that impaired reproduction was the primary influence on the observed decrease in λ. The model constructed was a useful tool for linking the individual-level effects to the population-level consequences of methoprene exposure on mysids, as well as defining the mechanism (reduced reproduction) responsible for the observed effects on population.

Testosterone and energy metabolism in the estuarine mysid Neomysis integer (Crustacea: Mysidacea) following exposure to endocrine disruptors.

A diverse set of reference compounds suspected of having an endocrine-disrupting mode of action (i.e., testosterone, flutamide, ethinylestradiol, precocene, nonylphenol, fenoxycarb, and methoprene) were tested for acute toxicity to the estuarine mysid Neomysis integer (Crustacea: Mysidacea). Neomysis integer was very sensitive to all tested compounds, with 96-h median lethal concentrations in a narrow range between 0.32 and 1.95 mg/L. The pesticides methoprene and fenoxycarb, both synthetic insect juvenile hormone analogs, were most toxic to N. integer. In addition, the short-term sublethal effects of methoprene and nonylphenol (an estrogen agonist) on the energy and steroid metabolism of N. integer were evaluated. Both compounds significantly affected energy and testosterone metabolism of N. integer at concentrations below acute toxicity levels. Energy consumption in methoprene- and nonylphenol-exposed mysids was significantly induced at 100 microg/L, resulting in a lower cellular energy allocation in these animals. Testosterone phase I metabolism was affected at 10 microg/L, whereas glycosylation was the most important phase II pathway affected in mysids exposed to 100 microg/L of both compounds. Methoprene exposure resulted in a concentration-dependent increase in the metabolic androgenization ratio. Mysids exposed to nonylphenol at 10 microg/L had a significantly higher metabolic androgenization ratio. The present study indicates that energy and testosterone metabolism of mysids, as endpoints, are able to detect endocrine-disruptive activity of chemicals after short-term exposure to environmentally realistic levels of endocrine disruptors.

Methoprene interferes with mosquito midgut remodeling during metamorphosis.

Many juvenile hormone analogs interfere with insect metamorphosis, a property that makes them useful in insect control and as investigatorial tools with which to study metamorphic mechanisms. We report that one such analog, methoprene, interferes with mosquito metamorphic midgut remodeling. Methoprene treated Aedes aegypti (L.), Aedes albopictus (Skuse), and Culex quinquefasciatus (Say) fourth instars pupate, but the pupal midguts are morphologically similar to larval midguts. The degree of midgut remodeling is affected by the dose of methoprene applied and the extent of fourth instar development when methoprene exposure is initiated. DNA staining indicates that high methoprene concentrations interfere with diploid cell division and programmed death of polytene cells. Lower methoprene concentrations do not interfere with diploid cell division but the removal of polytene cells is incomplete. The effect of methoprene dose on the extent of midgut remodeling correlates well with its effect on pupal and adult survival. Metamorphic midgut remodeling offers a mosquito system with which to study the effect of ecdysone, juvenile hormone and juvenile hormone analogs on metamorphosis. An understanding of the molecular mechanisms by which methoprene affects mosquito metamorphosis might lead to the development of more effective mosquito control chemicals having fewer deleterious environmental effects and to genetic strategies by which mosquito populations might be controlled.

Developmental toxicity of methoprene and several degradation products in Xenopus laevis

Methoprene is an insect juvenile growth hormone mimic, which inhibits pupation and is used for the control of emergent insect pests such as mosquitoes. Researchers have hypothesized that methoprene use in US may be a contributing factor to the recent increase in malformed amphibians. However, little is known concerning the developmental toxicity of methoprene and its degradation products in amphibians. In these studies, the aqueous stability and developmental toxicity of methoprene and several degradation products (methoprene acid, methoprene epoxide, 7-methoxycitronellal, and 7-methoxycitronellic acid) were examined. Xenopus laevis embryos (stage 8) were exposed to the test chemicals for 96 h. Assays were conducted under static renewal (24 h) conditions and chemical concentrations in water were measured at the beginning and end of the renewal periods. Methoprene exposure did not result in developmental toxicity at concentrations up to 2 mg/l, which is slightly higher than its water solubility. Methoprene acid, a relatively minor degradation product, produced developmental toxicity when concentrations exceeded 1.25 mg/l. Methoprene epoxide and 7-methoxycitronellal caused developmental toxicity at concentrations of 2.5 mg/l and higher. 7-Methoxycitronellic acid was not developmentally toxic at a test concentration as high as 30 mg/l. The five test chemicals had differential stability in aqueous solution that was in some instances affected by the presence of test organisms. These data indicate that methoprene and its degradation products are not potent development toxicants in X. laevis. This, in combination with the fact that field applications of sustained-release formulations of methoprene result in methoprene concentrations that do not typically exceed 0.01 mg/l, suggests that concerns for methoprene-mediated developmental toxicity to amphibians may be unwarranted.

Determination of acute mortality in adults and sublethal embryo responses of Palaemonetes pugio to endosulfan and methoprene exposure

Adult grass shrimp ( Palaemonetes pugio) were exposed to endosulfan or methoprene for 96 h and LC 50 values were calculated. Male and female P. pugio cohorts were also exposed to endosulfan for 96 h in an attempt to determine potential differences in sensitivity between the sexes. Results from the methoprene exposure indicated that this pesticide was not acutely toxic to adult grass shrimp at 1 mg l −1. Due to the lack of sensitivity, sex specific tests with methoprene were not performed. The calculated LC 50 for a population of grass shrimp, including both males and females exposed to endosulfan, was 0.62 μg l −1. The LC 50 determinations for the sex specific tests were 0.92 μg l −1 for males and 1.99 μg l −1 for females. Following these acute exposures, reproductively active grass shrimp were chronically exposed to 200 ng l −1 endosulfan or 1 mg l −1 methoprene and were allowed to produce embryos. The resulting embryos were assessed for potential sublethal toxicity. There were no observed differences in the percent successfully hatching or larval mortality 3-days post hatch among the treatments. However, endosulfan treated embryos had a significantly increased hatching time (9.76 days compared to 8.72 days in controls). Methoprene treated embryos also took longer to hatch (9.55 days), but this delay was not significantly different from controls. These findings suggest that endosulfan may preferentially affect male grass shrimp and exposed female grass shrimp may produce embryos with delayed hatching times.

Effects of the Pesticide Methoprene on Morphogenesis and Shell Formation in the Blue Crab Callinectes sapidus

The juvenile hormone analog methoprene causes both cytologic and biochemical alterations in larval and adult stages of the blue crab Callinectes sapidus. This insect growth regulator, used for mosquito control, caused (at a concentration of 10 gM) profound ultrastructural changes in the cuticular epithelial cells of postmolt adult blue crabs studied in vitro; these changes included loss of secretory organelles as well as distention and blebbing of the outer membrane of the nuclear envelope. Biochemically, 10 gM methoprene caused decreased deposition of extracellular cuticular chitin and protein, as well as a remarkable intracellular accumulation of chitoprotein precursors. These findings suggest that methoprene alters exocytosis and deposition of cuticular components. In vivo studies indicated that 5-10 gM methoprene is able to penetrate the embryonic investment coat to localize in lipovitellin. Exposure to methoprene at environmental concentrations (2-10 giM) produced morbidity and mortality in the form of an overall reduction in the number of successful hatching and lethargic behavior exhibited by the surviving zoeae. Methoprene exposure (3.3 gM) was also toxic to megalopae, delaying the molt to the first crab form and resulting in death of 80% of larvae after 10 days. The blue crab Callinectes sapidus Rathbun is a crustacean of both ecologic and economic importance to the coastal zones of the eastern and southern United States. Its life cycle and feeding habits bring it into estuarine environments subjected to pesticide treatments for mosquito control. It is logical to assume that the blue crab might be impacted by compounds active in other arthropods. The pesticide methoprene, a compound frequently applied to wetlands and salt marshes, belongs to the group of pesticides known as insect growth regulators (IGRs), which, in general, exert their toxic effects by disrupting insect development and/or reproduction. We offer in this report evidence that methoprene can penetrate the embryonic cuticle and can produce morbidity and mortality in larval forms of the blue crab. We also present in vitro findings indicating that methoprene may be toxic to the adult blue crab as well by altering the exocytosis of cuticular material.

EFFECTS OF THE PESTICIDE METHOPRENE ON MORPHOGENESIS AND SHELL FORMATION IN THE BLUE CRAB CALLINECTES SAPIDUS

The juvenile hormone analog methoprene causes both cytologic and biochemical alterations in larval and adult stages of the blue crab Callinectes sapidus. This insect growth regulator, used for mosquito control, caused (at a concentration of 10 µM) profound ultrastructural changes in the cuticular epithelial cells of postmolt adult blue crabs studied in vitro; these changes included loss of secretory organelles as well as distention and blebbing of the outer membrane of the nuclear envelope. Biochemically, 10 µM methoprene caused decreased deposition of extracellular cuticular chitin and protein, as well as a remarkable intracellular accumulation of chitoprotein precursors. These findings suggest that methoprene alters exocytosis and deposition of cuticular components. In vivo studies indicated that 5-10 µM methoprene is able to penetrate the embryonic investment coat to localize in lipovitellin. Exposure to methoprene at environmental concentrations (2-10 µM) produced morbidity and mortality in the form of an overall reduction in the number of successful hatching and lethargic behavior exhibited by the surviving zoeae. Methoprene exposure (3.3 µM) was also toxic to megalopae, delaying the molt to the first crab form and resulting in death of 80% of larvae after 10 days.

Modified survival, growth and reproduction in an estuarine mysid ( Mysidopsis bahia) exposed to a juvenile hormone analogue through a complete life cycle

The influence of methoprene, a juvenile hormone analogue used in mosquito control, on survival, growth, and reproduction of the estuarine mysid, Mysidopsis bahia, was examined during exposure through a complete life cycle in the laboratory. Total lethality occurred among all juvenile mysids exposed to 125 μg methoprene/l for 4 days. Mysids reared at a sublethal concentration (62 μg/l) weighed significantly less than unexposed mysids as they matured after 15 days of exposure. Release of the first brood was significantly delayed by as much as 3 days in mysids exposed to low μg/l concentrations of methoprene. The total number of young produced by groups of mysids during their first brood was significantly reduced when mysids were reared in methoprene concentrations ≥8 μg/l. The most sensitive response of mysids to methoprene exposure was a significant reduction in number of young produced per female in concentrations ≥2 μg/l. Ecological concern exists because the responses of mysids in this study to very low concentrations of a synthetic pesticide resemble responses of insects to juvenile hormone and juvenile hormone analogues. Similarity in these responses suggest that methoprene may be interfering with an endogenous endocrine system in this crustacean which utilizes juvenile hormone-like compounds.

Variations in larval growth and metabolism of an estuarine shrimp Palaemonetes pugio during toxicosis by an insect growth regulator

1. Exposure of the estuarine shrimp, Palaemonetes pugio, to a juvenile hormone analogue (⩾8 μg methoprene 1 −1) throughout larval development inhibited successful completion of metamorphosis. 2. Methoprene exposure retarded growth in early larval stages and postlarvae, but enhanced growth in premetamorphic larvae. 3. Respiration rates of early larvae were elevated by methoprene exposure, but not so older larvae or post larvae. 4. Lower net growth efficiency ( K 2 values) in methoprene-exposed early larvae suggests that increased metabolic demands reduced assimilated energy available for growth. 5. Modifications in O:N ratios of premetamorphic larvae and postlarvae suggest that methoprene altered substrate utilization patterns during metamorphosis.

Influence of an insect growth regulator on the larval development of an estuarine shrimp

The influence of methoprene, an insect growth regulator used in mosquito control, on larval development of the estuarine grass shrimp ( Palaemonetes pugio) was examined in the laboratory. No grass shrimp larvae successfully completed metamorphosis when continuously exposed to 1000 μg methoprene litre −1. Completion of larval metamorphosis was significantly reduced by exposure to 100 μg litre −1 of the isomeric mixture (R,S)-methoprene but not the single isomer formulation (S)-methoprene. No statistically significant difference was revealed, however, in ability to inhibit metamorphosis between these two isomeric types across the broad range of exposure concentrations from 0·1 to 1000·0 μg litre −1. The first two larval stages and the final premetamorphic larval stage were more sensitive to methoprene toxicity than intermediate larval stages. Methoprene exposure did not alter either the duration of total larval development or the total number of larval stages prior to metamorphosis.