Metabolic effects of acute exposure to methoprene in the American lobster, Homarus americanus.

ABSTRACT 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.

KEY WORDS: methoprene, lobster, endocrine disruption, juvenile hormone, methyl farnesoate, Homarus americanus

INTRODUCTION

Methoprene is a pesticide that acts as a juvenile hormone analog in insects (Restifo & Wilson 1998). It also mimics the action of methyl farnesoate (MF), the crustacean analog of juvenile hormone (Laufer et al. 1987). Although developed initially against insects, it has since been shown in a number of investigations to have toxic effects on larval and adult forms of various crustaceans (Christensen et al. 1977, Payen & Costlow 1977, Templeton & Laufer 1983, Ahl & Brown 1990, 1991, Horst & Walker 1999).

During the warm weather months of 1999, concerns about the spread of the mosquito-borne West Nile Virus led to increased application of pesticide compounds in the New York City and Connecticut area. In mid September of that year, the region experienced extremely heavy rainfall due to Hurricane Floyd. One month later, lobstermen in Western Long Island Sound (WLIS) began to report sightings of gravid female lobsters (Homarus americanus H. Milne Edwards, 1837) dying in the throes of abortive molts. The combination of circumstances and observations led us to question whether methoprene, a known constituent of the pesticide cocktail applied to the WLIS watershed, had disrupted the normal hormonal balance in ovigerous female lobsters and triggered molting at inappropriate times. The subsequent decline since 1999 in the seasonal lobster catches would likewise indicate that one or more harmful events had increased morbidity and mortality among adult lobsters and their offspring. In addition to the reduction in lobster populations, once abundant adult mysids are now reportedly absent from the WLIS. (Dr. Lance Stewart, pers. com.).

We undertook to study the acute effects of methoprene exposure on the survival of larval lobsters and to determine whether such an exposure would exert changes in the tissues of adult animals or lead to bioaccumulation of the pesticide in specific tissues. We also exposed adult mysids to methoprene to determine whether methoprene was a cause of morbidity and/or mortality in this crustacean.

MATERIALS AND METHODS

Seawater

Filtered seawater was obtained at the Skidaway Institute of Oceanography, Savannah, Georgia and returned to Macon in 120-L plastic barrels. Seawater was stored at 23[degrees]C in a 400-L storage tank attached to a purification system consisting of a magnetic pump, a 1-[micro]m bag filter and an ultraviolet sterilizing unit. After addition to the storage tank, the seawater was routinely recirculated in this system for 3 days. Before use, seawater was filtered through wound fiber and activated charcoal filters and adjusted to 31 ppt salinity by addition of Instant Ocean salts.

Animals

Gravid lobsters were obtained from commercial sources near Vineyard Haven, Massachusetts and shipped in moist seaweed at 4[degrees]C to Macon. Juvenile lobsters were obtained from Long Island Sound and from Round Pond, Maine and shipped as indicated earlier. Upon arrival, animals were equilibrated to 16[degrees]C and held in a recirculating seawater system maintained at 31 ppt salinity at 16[degrees]C. Individual juvenile lobsters were housed in slotted plastic crates fitted with a lid made from a perforated PVC sheet. Crates were placed into insulated tanks equipped with seawater and air delivery hoses as well as an outlet drain. Gravid lobsters were housed in glass aquaria; the outlet from the tank was covered with a Nytex screen (#124) to prevent loss of larvae into the recirculating system. Hatched larvae (Stage I) were collected daily and transferred to a Hughes Pot (plankton kreisel) attached to a recirculating filter/pump (Fluval, model 204); the entire system was housed in a refrigerated cold case equipped with a thermostatic regulator and maintained at 16.5[degrees]C. Larvae were fed live, adult brine shrimp twice daily and were removed to individual containers when they reached postlarval stage (Stage IV). Postlarvae were routinely maintained in refrigerated wine coolers at 16[degrees]C. The racks were removed and replaced with plastic development trays (Pfaff, B & H Photo, # PFT810Q) containing 1.5 L of fresh seawater. Individual postlarvae were housed in 18-compartment plastic storage boxes; the bottom of each 3.4 x 3.4 cm compartment was perforated with 1-mm holes. When placed in the development trays, each compartment maintained 1.5-cm depth of seawater. Postlarvae were fed live, adult brine shrimp ad libitum and the seawater was changed weekly. Molts were recorded daily and postmolt postlarvae were used for metabolic studies within 3 days of ecdysis.

Acute Exposure Studies

All solutions for larval exposure studies were prepared using filtered seawater. S-Methoprene (Welmark Corp.) was diluted in acetone prior to adding it to seawater; seawater used to maintain control animals contained an equivalent amount of acetone. The highest concentration of acetone used in the present study was 0.79 g/L, which is five times lower than the maximum allowable carrier acetone concentration suggested for crustacean studies (Rayburn & Fisher 1997). All methoprene solutions were prepared fresh just before use. Plastic development trays were half filled with seawater containing various concentrations of methoprene (see Results) and aerated with a small airstone. Trays were maintained in the dark at 18[degrees]C in a walk-in cold room. Plastic 18-compartment storage boxes were set within the development trays for exposure studies. Each storage box had multiple 1-mm holes drilled in the bottom; each compartment (4 x 5 cm, with a depth of 3 cm) housed one postlarval lobster (see Results). Eighteen postlarvae (Stage IV) were tested at 25 ppb and 18 were tested at 50 ppb methoprene. Animals were fed live, adult brine shrimp ad libitum and were scored daily for molting and survival. Dead animals were removed from the trays immediately.

Bioaccumulation Studies

Adult intermolt lobsters were equilibrated in the laboratory for at least 7 days. Individuals were placed in plastic buckets containing 8 L of filtered seawater. Dilutions of methoprene (50 ppb final concentration) were made from a stock solution (3000 ppm) prepared in acetone. Control animals were treated with an equal volume of acetone in seawater. Exposures were conducted at 18[degrees]C for 4 h. Thereafter, animals were anesthetized by packing in ice, sacrificed, and tissues were dissected, snap frozen in liquid nitrogen and stored at -80[degrees]C. Tissue samples were prepared from a total of six lobsters (average weight/animal: 567 g) and transported to the Pesticide Analysis Laboratory at the University of Georgia, Athens, Georgia, where they were extracted and analyzed for methoprene content by gas chromatography-mass spectrometry (GC/ MS). Sample preparation for GC-MS was essentially as described by Reed et al. (1977). Briefly, tissues (5 g) were homogenized in [Na.sub.2]S[O.sub.4] (50 g) and ethyl acetate (300 mL) for 1 min in a motorized blender. After passage through glass fiber filter paper, the filtrate was concentrated using a rotary vacuum evaporator. The residue was redissolved in ethyl acetate: toluene (3:1), vortexed and clarified. The supernatant was defatted by gel permeation chromatography on BioBeads SX-3 (100-200 mesh; Bio-Rad); the eluting solvent was ethyl acetate: toluene (3:1). The sample was further purified by Fluorisil chromatography (serial elution with 6%, 15% and 50% ether in hexane); material in the 15% fraction was turbo-evaporated and redissolved in 1 mL methylene chloride. Internal calibration standards were added and the extract was analyzed by gas chromatography-mass spectrometry as described by Noakes et al. (1999); the MS instrument was operated in selected ion monitoring (SIM) mode. Gas chromatographic conditions: column: RTX-5Mcol (Restec, Inc.) 30 m by 0.25 mm ID megabore capillary column; oven temperature program: initial temperature = 70[degrees]C, initial hold 2 min; temperature programmed to increase at 20[degrees]C per minute to 210[degrees]C with a final hold of 10 min. Under these conditions, methoprene had a retention time of 29 min, phenanthrene d-10 retention time = 25.5 min; chrysene d-12 retention time = 33.9 min. Data were expressed as parts per million. The methoprene minimal detection limit varied dependent on sample size but was approximately 0.05 ppm (wet weight).

Tissue Homogenization, Differential Centrifugation and Extraction

Day 1 Stage I larvae were exposed for 18 h to varying concentrations of methoprene (6.1, 12.5, 25, 50 ppb) and then homogenized. Juvenile animals were exposed to 50 ppb for 4 h and then tissues were removed and homogenized. Samples of the resultant subcellular fractions were used for heat shock, phosphoprotein and chitin synthase studies. Each tissue was homogenized with a Potter-Elvejhem homogenizer fitted with a Teflon plunger. Samples were homogenized 10 strokes at 50% maximal rpm setting in homogenization buffer: 20 mM Tris, pH 7.8, containing 0.4 M NaCl, 10 mM Mg[Cl.sub.2], 0.2 mM phenylmethanesulfonylfloride (PMSF) and protease inhibitor cocktail (Calbiochem). Shell samples were pre-extracted with 0.5 M EDTA, pH 7, containing PMSF and protease inhibitor cocktail for 12 h at 18[degrees]C. Homogenates were centrifuged (500xg for 15 min) to remove cell debris and nuclei. The supernatant was centrifuged at 5500xg (15 min) to remove mitochondria and finally at 30,000xg (45 min) to obtain a crude microsomal fraction (16Kp) and cytosol (16Ks). The latter fraction was dialyzed against distilled water using 12 kDa cutoff dialysis membranes.

The 5500xg and 16Kp pellets were extracted with 8 M urea containing 0.2% dithiothreitol and PMSF. After centrifugation (10,000xg for 15 min) the urea-soluble supernatant was removed and dialyzed. After washing with 10 mM Tris, pH 7.4, the crude microsomes were collected by centrifugation (30,000xg for 45 min). The microsomal pellet was then extracted with boiling 2% SDS in 10 mM Tris buffer, pH 7.4 for 5 min. The samples were filtered through Nytex screen and the residue was washed sequentially with water, ethanol and acetone.

SDS-PAGE Procedures

Samples of control and pesticide treated fractions were prepared for SDS-PAGE by boiling in 10 mM Tris, pH 7.0 containing 2% SDS, 15% glycerol, 0.001% bromophenol blue and 0.2% dithiothreitol for 3 min. Samples (15 [micro]L) were applied to either precast 4% to 20% gradient gels (Cambrex) or 10% acrylamide gels and separated according to Laemmli (1970). At the completion of the experiment, gels were either fixed and stained for total protein with Sypro Ruby (Molecular Probes) or Coomassie Blue, or for phosphorylated proteins with ProQ Diamond (Molecular Probes) or electroblotted to PVDF membranes using a semidry technique. The membranes were then blocked with 5% Blotto (Bio-Rad) in Tris-buffered saline containing 0.05% Tween 20 (TBS-Tw) at 4[degrees]C overnight.

Western Blot Probing

After blocking, Western blots were probed either with biotinylated lectin for specific carbohydrate groups or with primary antibodies against stress proteins or cellular marker proteins. Blots were probed (1 h/room temperature) with biotinylated Tomato lectin (Vector Laboratories, Burlingame, CA) diluted 2 [micro]L to 20 mL TBS-Tw. After washing, bound lectin was detected with Streptavidin-HRP conjugate (Dako Corp.).

The following primary antibodies were obtained from StressGen (Vancouver, British Columbia): heme oxygenase (OSA-155), ubiquitin (SPA-200), Hsp 60 (Spa 828). A primary antibody against HSP 70 (MA3-006) was obtained from Affinity Bioreagents (Golden, CO). Western blots were probed with primary antibodies (1 h/room temperature) diluted 1:4000 in TBS-Tw containing 1% Blotto. After washing, blots were probed with secondary antibody--HRP conjugate (Sigma) diluted 1:20,000 in TBS-Tw. After final washing, the blots were treated for 5 min with ECL Plus reagent (Amersham) and positive bands detected using Probe Plus X-ray film (Pierce). After exposure for 1-10 min, films were developed using an automated (Xomat) processor.

Chitin Synthase Assay

The assay used is based on a previous method developed for assay of chitin synthase activity in larval brine shrimp (Horst 1983). Crude microsomes were prepared as described earlier from Day 1, Stage I larvae after exposure for 4 h to methoprene (0, 50 ppb). Microsomal fractions were resuspended in 25 mM Hepes buffer, pH 7.4, containing 0.4 M NaCl, 30 mM Mg[Cl.sub.2] and 100 [micro]M allosamidin, a chitinase inhibitor. After addition of the substrate, UDP-6-[sup.3]H N-acetyl-D-glucosamine (Perkin Elmer; Sp. Act. = 39.7 Ci/mmol; 1 [micro]Ci/sample), duplicate samples were incubated for 4 h at 18[degrees]C. The incubations were placed on ice and precipitated with an equal volume of ice cold 100% methanol. After standing on ice for 15 min, samples were centrifuged (3,000 rpm/ 30 min/4[degrees]C) and the supernatant was discarded. The pellets were washed with cold 50% methanol and centrifuged as before. The methanol insoluble residues were boiled for 3 min in 10 mM borate buffer, pH 8.0 containing 2% SDS. After centrifugation (3000 rpm/15 min/24[degrees]C), the SDS supernatants were removed and the pellets, containing the insoluble chitin residue, were resuspended in water and re-centrifuged. The washed pellets were transferred to glass vials and 10 mL scintillation fluid (Ecolume; RPI) was added to each vial. Radioactivity was determined in a liquid scintillation spectrometer. Total protein in soluble and microsomal samples was measured by a modified fluroescamine assay following solubilization of proteins in boiling 2% SDS (Horst 1981).

Exposure of Adult Mysids to Methoprene

Adult mysid shrimp, Mysidopsis bahia (Sachs Systems Aquaculture, St. Augustine, FL), were exposed to various concentrations of methoprene (1-25 ppb) in dilute seawater (22 ppt) for 3 days and observed for mortality as compared with the control population.

RESULTS

Acute Toxicity Studies on Postlarval (Stage IV) Forms

Three days of exposure to 25 ppb methoprene were not associated with increased numbers of deaths versus the control population. In contrast, 3 days exposure to 50 ppb methoprene resulted in >90% mortality compared with the control population.

Bioaccumulation Results

The tissues where methoprene was concentrated against the gradient of the surrounding seawater (50 ppb) were hepatopancreas (1.55ppm), gonad (5.18 ppm), epithelial cells (6.17 ppm) and, most significantly, the eyestalks (28.83 ppm), See Table 1.

Heat Shock Proteins

Expression of several HSPs was studied: HSP 60, HSP 70, heme oxygenase and ubiquitin, comparing fractions from control animals and methoprene-exposed. Larvae were exposed to 25 ppb for 18 h, postmolt juveniles to 50 ppb methoprene for 4 h.

HSP 60

Day 1 Stage I larvae; preliminary data showed no difference in the expression of HSP 60 between control and methoprene-exposed Day 1 Stage I larvae or between control and methoprene-exposed postmolt juvenile animals.

HSP 70

An HSP 70 immunoreactive band was present at the appropriate position in microsomal fractions from both control and methoprene-exposed postmolt juveniles. Cytosolic fractions from control animals also contained an HSP 70 immunoreactive band at the same molecular weight; this band was markedly decreased in intensity in the cytosolic fractions of methoprene-exposed postmolt juveniles (Fig. 1).

[FIGURE 1 OMITTED]

Heme Oxygenase

Preliminary data indicated that heme oxygenase expression was increased in Day 1 Stage I larvae and postmolt juvenile lobsters following exposure to methoprene (data not shown).

Ubiquitin

Day 1 Stage I larvae exposed to methoprene had more ubiquitinylated proteins in the cytosolic fraction compared with control animals. Both populations had detectable free ubiqutin as well. Postmolt juveniles exposed to methoprene showed increased ubiquitinylation of proteins from epithelium, especially in the cytosolic fraction, when compared with control animals. The cytosolic fraction from methoprene-exposed juveniles also contained detectable free ubiquitin whereas the comparable fraction from the control animals did not (Fig. 2).

[FIGURE 2 OMITTED]

Synthesis of Phosphoproteins

Soluble (cytosolic) and membrane bound proteins were separated by SDS-PAGE and the gel was stained with ProQ Diamond to detect phosphoproteins in each sample. As shown in Figure 3, there were differing patterns of protein phosphorylation in the comparable fractions from the control animals and the postmolt juvenile lobsters exposed to 50 ppb methoprene.

[FIGURE 3 OMITTED]

Chitin Synthase Assay

Day 1 Stage I larvae were exposed to 50 ppb methoprene for 4 h and crude microsomes were prepared and assayed for chitin synthase as described in Methods.

We observed a linear increase in control activity with added microsomal enzyme as evidenced by accumulation of radiolabeled product. When the chitin synthase activity in the specimens from exposed animals was studied, there was no significant difference in the specific activity of the enzyme from exposed animals versus controls. (Control = 1364 cpm/mg; 50 mM methoprene = 1378 cpm/mg).

Mysids

We exposed adult mysids to various concentrations of methoprene (1-25 ppb) for 3 days, but did not observe increased mortality rates over those of the control population.

DISCUSSION

Juvenile hormone (JH) is a regulator of insect development. It modifies the response to the molting hormone, 20-hydroxyecdysone, at the molecular, cellular and organismal level. In larval insects, JH-JH receptor interaction insures that the outcome of a molt is to another larval stage, while absence of JH-JH receptor binding results ultimately in a pupal or adult molt. (Riddiford 1993, 1996, Wyatt & Davey 1996). Methyl farnesoate (MF), synthesized in the mandibular organ of crustaceans, is the unepoxidated precusor of insect Juvenile Hormone III (JH III). Methoprene is a pesticide that acts as a juvenile hormone analog in insects (Restifo & Wilson 1998) and also mimics the action of MF in crustaceans (Laufer et al. 1987). Although developed initially against insects, it has since been shown in a number of investigations to have toxic effects on larval and adult forms of various crustaceans (Christensen et al. 1977, Payen & Costlow 1977. Templeton & Laufer 1983, Ahl & Brown 1990, 1991, Horst & Walker 1999). 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 questioned whether methoprene, through its effects on larvae, adults or both, could have contributed to this decline. In our initial studies, we found that low levels of methoprene had adverse effects on lobster larvae. Stage II larvae experienced increased mortality rates at methoprene concentrations as low as 2 ppb. Stage IV larvae were more resistant, but did exhibit significant increases in molt frequency beginning at exposures of 5 ppb. Juvenile lobsters exhibited variations in tissue susceptibility to methoprene. The hepatopancreas appeared to be the most vulnerable, with environmental concentrations of methoprene inhibiting almost all protein synthesis in this organ (Walker et al. 2005).

In this study, we elected to examine further the potential toxicity of higher concentrations of methoprene to the more resistant Stage IV larvae. Stage IV larvae did not exhibit increased mortality over a period of 3 days while exposed to 25 ppb methoprene. This population, however, did experience significantly increased numbers of deaths when exposed to 50 ppb methoprene, with more than 90% of the animals dying during the 3 days of exposure. Thus, although the Stage IV larvae are more resistant to the effects of methoprene, they may still experience increased mortality at environmental concentrations of the pesticide.

The increased frequency of molts in the stage IV larvae and the historical observation of berried females dying while attempting to molt raise the possibility that methoprene could be responsible for endocrine disruption in larval and adult lobsters. Other investigators have shown interplay between MF and ecdysone in other crustaceans (Laufer et al. 1987, Chang 1993). Demeusy (1975) suggested that large premolt increases in the hemolymph ecdysteroid titer were due to an ecdysiotrophic action of methyl farnesoate. Tamone and Chang (1993) demonstrated that, during the premolt, the mandibular organ in Cancer magister undergoes ultrastructural changes indicative of increased synthetic activity. In addition, they showed that ecdysteroid synthesis by Y organs is increased both by incubation with mandibular organ-conditioned media or with methyl farnesoate. If methoprene is acting as an MF analog, it is reasonable to suggest that methoprene could affect the synthesis of ecdysone, and accordingly influence the timing and frequency of molts.

The synthesis of ecdysone in the Y-organ is also under inhibitory control by MIH (molt-inhibiting hormone) released by the sinus glands located in the eyestalks. Eyestalk ablation generally results in increased synthesis of ecdysteroids and increased hemolymph levels of ecdysteroids in crustaceans (Webster 1998). Not all animals, however, respond in that fashion. Eyestalk ablation does not accelerate molting in female crustaceans undergoing seasonal vitellogenesis (Lachaise et al. 1992). This last observation would suggest that endocrine changes associated with reproduction can ordinarily override the interplay of MIH and ecdysteroids that would otherwise bring on a molt. The impact that endocrine disruptors might have at such a time is currently in need of additional study.

Our first bioaccumulation studies revealed that methoprene concentrates in the hepatopancreas, nervous tissue and epidermal cells of the adult lobster. As we have continued our bioaccumulation studies, we have found that high concentrations of methoprene accumulate preferentially in several tissues, including the eyestalks. This finding has led us to question whether methoprene could alter or influence the balance of MIH, ecdysteroids, methylfarnesoate and other hormones, and bring on a molt at an inappropriate time. Molting while berried would obviously result in complete loss of the female animal's brood.

In addition to the significant concentrations of methoprene in the eyestalks of adult lobsters, we also found high levels of the pesticide concentrated in epidermal tissue and hepatopancreas. Our findings are very similar to those of De Guise et al. (Dr. Sylvain De Guise, pers. com.). Those investigators have observed methoprene accumulation in the hepatopancreas of adult lobsters following acute exposure. Other investigators have observed accumulation of various toxicants in the hepatopancreas of lobsters, including methyl mercury (Guarino & Anderson 1976), benzo[a]pyrene (James et al. 1995), polychlorinated biphenyls (King et al. 1996) and heavy metals (Chavez-Crooker et al. 2003). The last authors showed that methyl mercury, which is very lipophilic, persisted in the eggs of ovigerous females for longer than 1 mo; levels in the hepatopancreas were initially high after an acute exposure, but decreased with time possibly due to the rapid turnover of hepatopancreatic cells.

"Heat shock" protein is a somewhat generic term applied to a diverse group of proteins that are characteristically expressed or expressed in increased amounts by cells exposed to stress of various types. These proteins are, in general, conserved across phylogeny and have been identified in insects (Berger et al. 1992) and more recently in lobsters. (Spees et al. 2002a, 2002b) Methoprene has been shown to influence the expression of a heat shock protein in Drosophila (Berger et al. 1992). We have begun to study the effects of methoprene exposure on the expression of various heat shock proteins by larval and/or by juvenile lobsters. We detected HSP 60 expression but our preliminary data showed no difference in its expression by either population following methoprene exposure. HSP 60, however, is a constitutively expressed chaperone and changes in its intracellular concentration might be difficult to assess by the semiquantitative technique of Western blotting.

HSP 70 was present in microsomal fractions of epithelial tissues from both control and methoprene-exposed postmolt juveniles. There was, however, a decrease in the cytosolic concentration of HSP 70 in the methoprene-exposed population, suggesting sequestration of this protein, which functions as a chaperone, into other subcellular compartments. We also observed an apparent increase in the expression of heme oxygenase in methoprene-exposed Day 1 Stage I larvae and postmolt juveniles.

Our most remarkable finding from the stress protein studies was the effect of methoprene on the ubiquitinylation of cytosolic proteins. Methoprene exposure was associated with an increase in ubiquitinylation of epithelial proteins from Dayl Stage I larvae and from post molt juveniles. Ubiquitin is small (8-10 kDa) protein that serves as the intracellular label to target abnormal or senescent proteins for degradation by the 26S proteasome complex. Stressful conditions associated with protein damage or abnormal protein synthesis result in increased ubiquitinylation of cellular constituents. Thus, increased ubiquitinylation following methoprene exposure is another indication that methoprene is a stressor for immature lobsters.

We found that methoprene exposure produced qualitative and quantitative differences in the phosphorylation of proteins extracted from epithelial tissue of postmolt juvenile lobsters when compared with tissues of control animals. Although preliminary, these results imply that methoprene could affect intracellular signaling pathways. Alterations in protein phosphorylation have been reported in nervous tissue of lobsters exposed to deltamethrin (Miyazawa & Matsumura 1990).

In our earlier report (Walker et al. 2005) and in this study, we observed that methoprene exposure was associated with changes in the chitoprotein production. Previously we had found that methoprene altered the synthesis and incorporation of chitoproteins into adult postmolt shells. SDS PAGE analyses of adult post-molt shell extracts revealed changes in chitoprotein expression in the methoprene-treated specimens, suggesting that methoprene blocks the normal pathway of lobster cuticle synthesis and affects the quality of the adult postmolt shell. In this study we examined microsomal fractions from Day 1 Stage I larvae and compared fractions from control animals with fractions from animals exposed to differing concentrations of methoprene. We found that increasing concentrations of methoprene were associated with increasing chitoproteins in the microsomal fractions, as assessed by binding of Tomato Lectin on SDS PAGE/Western blot analysis. This finding suggests that methoprene may compromise the exocytosis of shell matrix precursors from the epithelial cells.

We also compared the activity of the enzyme chitin synthase in control and methoprene-exposed Day 1 Stage I larvae. There was no difference in the activities of chitin synthase in the microsomal fraction from animals exposed to 50 ppb methoprene for 4 h compared with the enzyme in the microsomal fraction of the control population. This exposure time may not have been sufficient to affect existing levels of the enzyme in microsomes, but should have been sufficient to elicit any direct impact in enzymatic activity.

At a recent meeting on the lobster population of WLIS, another investigator made mention of the absence of once abundant adult mysids from the WLIS after the events of 1999 (Dr. Lance Stewart, pers. com.). We exposed adult mysids to various concentrations of methoprene (1-25 ppb) for 3 days, but did not observe increased mortality rates. McKenney and Celestial (1996) examined the toxicity of methoprene to adult mysids and observed effects on fecundity at 2 ppb. They did not report acute mortality of adult mysids, but since the lifespan is only 7-14 days, the overall population could be affected by impaired reproductive success.

At this point in our investigations, we do not believe that there was one single factor responsible for the lobster die-off of 1999 in WLIS. A unifying concept that does emerge from our work and from the findings of many other WLIS investigators is that the lobsters were stressed by short- and long-term harmful stimuli, including a variety of environmental and man-made conditions. Of the pesticides that likely washed into the WLIS, methoprene had actually been applied in the lowest amounts to the surrounding watershed during the summer of 1999 (Miller et al. 2005). Our data, however, and those of Deguise et al. (pers. com.) indicate that methoprene bioaccumulates as much as 250-fold in some tissues. Thus, in combination with other known and unknown factors such as increased temperature, decreased salinity, hypoxia and combinations of other pesticides, methoprene may well have contributed to the overall decline of the lobster population. The net effect of these insults may be greater than the simple sum of the individual parts.

Studies are currently underway to elucidate the subcellular localization of and ultrastructural changes that result from treatment with sublethal and lethal concentrations of methoprene. Future aims in the investigation of the effect of methoprene on lobster populations include defining the portal(s) of entry for the pesticide into the animal and the pathologic consequences of its preferential accumulation in selected tissues.

ACKNOWLEDGMENTS

The authors acknowledge the technical assistance of Mr. Kenneth Holloway, Ms. Tammy Mifflin, and Mr. John Knight. Portions of this research were conducted at the Darling Marine Center, Walpole, ME; The authors thank the Director, Dr. Kevin J. Eckelbarger, for providing laboratory space and ship time. This research was supported by the Connecticut Sea Grant College Program, Grants No. LR/LR-6 and LR/LR-1, through the US Department of Commerce, National Oceanic and Atmospheric Administration (NOAA), Award NA16RG1364.

LITERATURE CITED

Ahl, J. S. B. & J. J. Brown. 1990. Salt-dependent effects of juvenile hormone and related compounds in larvae of the brine shrimp. Artemia. Comp. Biochem. Physiol. 95A:491-496.

Ahl, J. S. B. & J. J. Brown. 1991. The effect of juvenile hormone III, methyl farnesoate, and methoprene on Na/K-ATPase activity in larvae of the brine shrimp, Artemia. Comp. Biochem. Physiol. 100A:155-158.

Berger, E. M., K. Goudie, L. Klieger, M. Berger & R. DeCato. 1992. The juvenile hormone analogue, methoprene, inhibits ecdysterone induction of small heat shock protein gene expression. Dev. Biol. 151:410-418.

Chang, E. S. 1993. Comparative endocrinology of molting and reproduction: Insects and crustaceans. Annu. Rev. Entomol. 38:161-180.

Chavez-Crooker, P., P. Pozo, H. Castro, M. S. Dice, I. Boutet, A. Tanguy, D. Moraga & G. A. Ahearn. 2003. Cellular localization of calcium, heavy metals, and metallothionein in lobster (Homarus americanus) hepatopancreas. Comp. Biochem. Physiol. C. 136:213-224.

Christensen, M. E., J. D. Costlow & R. J. Monroe. 1977. Effects of the juvenile hormone mimic ZR-515 (Altosid) on larval development of the mud crab Rhithropanopeus harrisii in various salinities and cyclic temperatures. Mar. Biol. 39:269-279.

Demeusy, N. 1975. Observations sur le functionnement des glandes mandibulaires du Decapode brachoure Carcinus maenas L.: animaux temoins et animaux sans pedoncles oculaires. Contes Rendus de l'Academie des Sciences Paris. Serie D 281:1887-1889.

Guarino, A. M. & J. B. Anderson. 1976. Tissue distribution of [sup.14]C methyl mercury in the lobster, Homarus americanus. J. Toxicol. Environ. Health 2:13-24.

Horst, M. N. 1983. The biosynthesis of crustacean chitin. Isolation and characterization of polyprenol-linked intermediates from brine shrimp microsomes. Arch. Biochem. Biophys. 223:254-263.

Horst, M. N. & A. N. Walker. 1999. Effects of the pesticide methoprene on morphogenesis and shell formation in the blue crab, Callinectes sapidus. J. Crust. Biol. 19:699-707.

James, M. O., A. H. Altman, C. J. Li & J. D. Schell, Jr. 1995. Isotransformation, hepatopancreas DNA binding and pharmacokinetics of benzo[a]pyrene after oral and parenteral administration to the American lobster, Homarus americanus. Chem. Biol. Interact. 95:141-160.

King, T. L., B. K. Haines & J. F. Ulthe. 1996. Non-, mono-, and di-ochlorobiphenyl concentrations and their toxic equivalents to 2,3,7,8-tetrachlorodibenzo[p]dioxin in Aroclors and digestive glands from American lobster (Homarus americanus) captured in Atlantic Canada. Bull. Environ. Contam. Toxicol. 57:465-472.

Lachaise, F., M. Goudeau, G. Carpenter, B. Saidi & H. Goudeau. 1992. Eyestalk ablation in female crabs: effects on egg characteristics. J. Exp. Zool. 261:86-96.

Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(259):680-685.

Laufer, H., D. Borst, F. C. Baker, C. Carrasco, M. Sinkus, C. C. Reuter, L. W. Tsai & D. A. Schooley. 1987. Identification of a juvenile hormone-like compound in a crustacean. Science 234:202-205.

McKenney, C. L., Jr. & D. M. Celestial. 1996. Modified survival, growth and reproduction in an estuarine mysid (Mysidopsis bahia) exposed to a juvenile hormone analogue through a complete life cycle. Aquatic Toxicol. 35:11-20.

Miller, R. L., J. R. Wands, K. N. Chytalo & R. A. D'Amico. 2005. Application of water quality modeling technology to investigate the mortality of lobsters (Homarus americanus) in western Long Island Sound during the summer of 1999. J. Shellfish Res. 24(3):859-864.

Miyazawa, M. & F. Matsumura. 1990. Effects of deltamethrin on protein phophorylation and dephosphorylation process in the nerve fibers of the American lobster, Homarus americanus L. Pesticide Biochem. Physiol. 36:147-155.

Noakes, J. E., S. E. Noakes, D. K. Dvoracek, R. A. Culp & P. B. Bush. 1999. Rapid coastal survey of anthropogenic radionuclides, metals and organic compounds in superficial marine sediments. The Science of the Total Environment 237/238:449-458.

Payen, G. G. & J. D. Costlow. 1977. Effects of a juvenile hormone mimic on male and female gametogenesis of the mud crab, Rhithropanopeus harrisii (Gould) (Brachyuran: Xanthidae). Biol. Bull. 152:199-208.

Rayburn, J. R. & W. S. Fisher. 1997. Developmental toxicity of three carrier solvents using embryos of the grass shrimp, Palaemonetes pugio. Arch. Environ. Contam. Toxicol. 33:217-221.

Reed, D. L., P. B. Bush, N. H. Booth, J. T. Kiker, D. D. Goetsch & R. L. Farrell. 1977. Tissue residues from feeding pentachloronitrobenzene to broiler chickens. Tox. Appl. Pharm. 42:433-441.

Restifo, L. L. & T. G. Wilson. 1998. A juvenile hormone agonist reveals the distinct developmental pathways mediated by ecdysone-inducible broad complex transcription factors. Dev. Genet. 22:141-159.

Riddiford, L. M. 1993. Hormone receptors and the regulation of insect metamorphosis. Receptor 3:203-209.

Riddiford, L. M. 1996. Juvenile hormone: the status of its "status quo" action. Arch. Insect Biochem. Physiol. 32:271-286.

Spees, J. L., S. A. Chang, M. J. Snyder & E. S. Chang. 2002a. Thermal acclimation and stress in the American lobster, Homarus americanus: equivalent temperature shifts elicit unique gene expression patterns for molecular chaperones and polyubiquitination. Cell Stress Chaperones 7:97-106.

Spees, J. L., S. A. Chang, M. J. Snyder & E. S. Chang. 2002b. Osmotic induction of stress-responsive gene expression in the lobster Homarus americanus. Biol. Bull. 203:331-337.

Tamone, S. L. & E. S. Chang. 1993. Methyl farnesoate stimulates ecdysteroid secretion from crab Y-organs in vitro. Gen. Comp. Endocrin. 89:425-432.

Templeton, N. S. & H. Laufer. 1983. The effects of a juvenile hormone analog (Altosid ZR-515) on the reproduction and development of Daphnia magna (Crustacea: Cladocera). Internat. J. Invert. Reproduct. 6:99-110.

Walker, A. N., P. Bush, J. Puritz, T. Wilson, E. S. Chang, T. Miller & M. N. Horst. 2005. Bioaccumulation and metabolic effects of the endocrine disruptor methoprene in the lobster, Homarus americanus. Int. Comp. Biol. 45:118-126.

Webster, S. G. 1998. Neuropeptides inhibiting growth and reproduction in crustaceans. In: G. M. Coast & S. G. Webster, editors. Recent Advances in Arthropod Endocrinology. Cambridge, UK: Cambridge University Press. pp. 33-52.

Wyatt, G. R. & K. G. Davey. 1996. Cellular and molecular actions of juvenile hormone. II. Roles of juvenile hormone in adult insects. In: P. D. Evans, editor. Advances in Insect Physiology, vol. 26., San Diego: Academic Press. pp. 1-155.

ANNA N. WALKER, (1) PARSHALL BUSH, (2) THOMAS WILSON, (3) ERNEST S. CHANG, (4) TIM MILLER (5) AND MICHAEL N. HORST (6) *

(1) Department of Pathology, School of Medicine, Mercer University, Macon, Georgia 31207; (2) Agricultural and Environmental Services Labs, University of Georgia, 2300 College Station Road, Athens, Georgia 30602; (3) Department of Entomology, Ohio State University, Columbus, Ohio; (4) Bodega Marine Laboratory, University of California, Davis, Bodega Bay, California 94923; (5) Darling Marine Center, University of Maine, Walpole, Maine 04573; (6) Division of Basic Medical Sciences, School of Medicine, Mercer University, 1550 College Street, Macon, Georgia 31207

* Corresponding author. E-mail: [e-mail]

TABLE 1.
The bolded results are the latest additions to our compilation of
bioaccumulation data. Methoprene was concentrated against the
gradient of the surrounding seawater (50 ppb) in several tissues.
The concentration of methoprene in the eyestalks was approximately
a thousand-fold greater than the concentration in the seawater.
Lesser, but still significant concentrations were present in the
hepatopancreas, epithelial tissue and gonads.

Sample                   Weight (g)    Methoprene Conc. (ppm)

Calibration standard       30                   3.97
Hepatopancreas             10.39                1.55
Gills                       7.52                0.14
Epithelial cells            0.81                6.17
Muscle, abdominal           6.3                 0.16
Gonad                       1.08                5.18
Stomach                     7.17                n.d.
Connective tissue           1.40                n.d.
Eyes                        0.35               28.83
Heart                       0.70                n.d.