The Society for Integrative and Comparative Biology
Ecdysteroid Responses of Estuarine Crustaceans Exposed Through Complete Larval Development to Juvenile Hormone Agonist Insecticides1
1 Appalachian State University, Department of Biology, 572 Rivers Street, Boone, North Carolina 28608
2 U.S. Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, Gulf Ecology Division, 1 Sabine Island Dr., Gulf Breeze, Florida 32561-5299
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Fenoxycarb and pyriproxyfen are insecticides that gain their toxicity by specifically acting as insect juvenile hormone agonists (JHA), and so are endocrine disruptors by design and effectively prevent larvae from maturing into adults. Efforts to assess the environmental effects of JHAs on nontarget populations of invertebrates have resulted in the utilization of several established estuarine crustacean models. This work was conducted to test the hypothesis that the mortality, inhibition of development and decreased fecundity reported previously in these animals from JHA exposure coincides with abnormal circulating titers of ecdysteroids. Gravid female grass shrimp (Palaemonetes pugio) and mud crabs (Rhithropanopeus harrisii), species with different developmental plasticity and JHA tolerances, were collected and held at wet lab conditions (20 ppt salinity, 25°C) until larval release. Larvae were collected <12 hr after hatch and exposed to JHAs during a static renewal test through end of development with seawater or nominal concentrations of JHA previously shown to induce significant developmental delays and/or decreased body weights. Larvae were subsampled (10 larvae/sample, n = 2 to 8) at each developmental stage, lyophilized, and ecdysteroids extracted by homogenization in 80% methanol and elution from C18 Sep-Pak cartridges with 25%, 60% and 100% methanol to capture the polar, free, and apolar conjugates, respectively, and then quantified by ELISA. As was expected significant differences in successful completion of development (larval survival), developmental duration, and growth (dry weight) were observed. These physiological perturbations were linked with significantly altered ecdysteroid titers, supporting a newly emerging theory that juvenoids possibly act as anti-ecdysteroids through a novel molecular mechanism involving inhibition of ecdysteroid signaling.
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INTRODUCTION |
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The focus of the symposium for which this paper was written was the use of endocrinology and immunology to aid in understanding population declines of species in peril as well as elucidate new mechanisms of toxicity that may contribute to future population declines. The link between conservationists and physiologists has slowly begun to strengthen yet there are only a few examples of collaboration between these important fields. Although many toxicologists would also consider themselves physiologists, few of them would define their work as conservation biology, yet they may hope their work would be applied to such a noble cause. We hoped that by creating a new field named Conservation Physiology we could help foster the types of integrative collaborations necessary to best serve efforts to save declining populations of organisms and gain a better understanding of factors that eventuate population losses. As a growing field of toxicology, the study of endocrine disrupting xenobiotics on both vertebrates and invertebrates has contributed significantly to both the basic and applied sciences. The interdisciplinary nature of endocrine disruption research seems to lend itself well to further application as a sub-field of conservation physiology since it has as one of its goals elucidation of mechanisms of reproductive and developmental toxicities which can be major contributors to population declines.
Application of insecticides to salt marshes in efforts to control problem insect populations and contamination by land-drainage and erosion from adjacent pesticide-treated agricultural lands are not new threats to the many non-target species which use estuarine environments for nurseries and feeding grounds. For decades the chemical and pesticide industries have labored to find compounds that are nontoxic to birds, fish, mammals and non-target invertebrate species in order to diminish the adverse effects of pest control on the delicate balance that is an ecosystem.
Recent advances in insect nueroendocrinology have led to the application of insect hormones and their analogues as "third generation pesticides" for use as biochemical biological control agents (Keeley et al., 1990
). Methoprene, pyriproxyfen, and fenoxycarb are members of the juvenile hormone analogue (JHA) class of insecticides that gain their toxicity through modulation of the physiological processes in insects known to be regulated by endogenous juvenile hormones of insects (Downer and Laufer, 1983; Miyamoto et al., 1993; Dhadialla et al., 1998).
Since juvenile hormones (JH) are not present in vertebrate species, JHAs have minimal acute toxicity to many non-target populations at the maximum field concentrations (Knuth, 1989; Lee and Scott, 1989; Ross et al., 1994a, b). However, there is some evidence that methoprene and its derivitive, methoprene acid, are capable of binding to the retinoid X receptor (RXR) and therefore may be able to effect mammalian gene transcription (Harmon et al., 1995). Furthermore, metabolites of methoprene were shown to produce deformities in Xenopus laevis embryos following developmental exposures (LaClair et al., 1998).
Crustaceans, like insects, have JH-like compounds that are involved in the control of their larval development and reproduction. Recently methyl farnesoate (MF), a JH-like compound isolated from many crustacean species, has attracted attention as a crustacean hormone due to its ability to control larval metamorphosis (Borst et al., 1987; Rotllant et al., 2000), affect release of ecdysteroid molting hormone (Tamone and Chang, 1993), stimulate onset and maintenance of vitellogenesis (Laufer et al., 1992; Reddy and Ramamurthi, 1998; Jo et al., 1999), control sex determination (Olmstead and LeBlanc, 2002), or even reproductive behavior and morphotype (Laufer et al., 1993; Laufer and Ahl, 1995; Laufer et al., 1997, 2004). Methyl farnesoate, the unepoxidised form of JH III, is not only structurally very similar to insect JH but also to JHAs and therefore the processes controlled by MF may be adversely affected by their application (deFur et al., 1999).
There have already been many reports of the adverse effects of JHAs on nontarget insects and crustaceans alike (Payen and Costlow, 1977; Bircher and Ruber, 1988; Magadum et al., 1990; Key and Scott, 1994; Mortimer and Chapman, 1995; Chu et al., 1997; Hosmer et al., 1998; Lawler et al., 1999; Wirth, 1999; Nates and McKenney, 2000; Cripe et al., 2003; McKenney et al., 2004). However, due to the close phylogenetic relationships of several other major and minor phyla to the arthropods, often grouped into a clade called the Ecdysozoa ("molting animals") (Fig. 1) there should also be cause for concern regarding their health in environments exposed regularly to JHAs and anti-ecdysteroids. Except for recent research into the control of juvenoids on reproduction and development of C. elegans in the phylum Nematoda (Fodor and Timar, 1989) and the presence of ecdysteroid-like compounds (Mercer et al., 1988), there has been little investigation of their endocrinology or the effects of JHAs on these important invertebrate phyla, even though they may be one of the most extensively studied organisms on Earth.
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The molting hormones, ecdysone and 20-hydroxyecdysone, have long been known to play critical roles in molting and development of insects and crustaceans (Subramoniam, 2000
). Only recently has information on the molecular mechanism of their control of gene transcription been elucidated. The ecdysteroid receptor (EcR) forms a heterodimer with another member of the nuclear hormone family, ultraspiracle (USP), which is the insect homologue of vertebrate RXR (Yao et al., 1992). Although this association is unstable, further binding with ecdysteroid responsive elements (EcREs) and/or ecdysteroids create a more stable functional complex capable of mediating the effect of ecdysteroids by activating and repressing ecdysone responsive genes (Yao et al., 1993). More recently, juvenoids were shown to bind specifically to Drosophila USP (Jones and Sharp, 1997). This is the first ligand described for USP and is capable of inducing conformational changes and homo-oligomerization activity.
In this study, we endeavored to describe the shrimp, Palaemonetes pugio, and the crab, Rhithropanopeus harrisii, ecdysteroid responses to JHA exposure in order to narrow the possibilities for underlying mechanisms of the well-described physiological indicators of juvenile hormone analogue (JHA) exposure in these non-target crustacean populations (McKenney and Matthews, 1990; McKenney and Celestial, 1993; McKenney, 1999; Nates and McKenney, 2000; Cripe et al., 2003). These two species were chosen due to their extensively studied physiology and past use in this laboratory for JHA exposures as well as their well-described differences in developmental plasticity (Costlow Jr., 1968; McKenney, 1999). The JHA methoprene has been shown to affect several ecdysteroid controlled processes in insects including blocking ecdysteroid induction of mitotic arrest (Cherbas et al., 1989), small heat shock proteins (Berger et al., 1992) and microRNAs involved in metamorphosis (Sempere et al., 2003). In crustaceans, JHAs have been shown to inhibit ecdysone-controlled processes such as morphogenesis and shell formation in the blue crab Callinectes sapidus (Horst and Walker, 1999) and growth, development and reproduction of several estuarine crustaceans (Payen and Costlow, 1977; McKenney and Celestial, 1995; McKenney, 2005; McKenney et al., 2004). Since many of the growth, developmental, and reproductive endpoints that suffer from JHA exposure are known to be controlled or influenced by ecdysteroids, we hypothesized that juvenoids cause changes in normal levels of ecdysteroids during larval development and oogenesis. Therefore, we extracted and quantified free and conjugated classes of ecdysteroids from whole larvae from larval stages of two estuarine crustaceans in order to ascertain whether exposure to juvenoids caused disruptions in normal ecysteroid expression.
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MATERIALS AND METHODS |
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Animals and exposure protocol
Gravid female Palaemonetes pugio and Rhithropanopeus harrisii were collected from estuaries near Pensacola, Florida in June 2003 and transported to the US EPA Gulf Ecology Division laboratory in Gulf Breeze, Florida and treated as described previously (McKenney and Celestial, 1993
; Nates and McKenney, 2000). Females were gradually acclimated to filtered seawater (20
salinity, 25°C) and fed <24 hr Artemia nauplii. Two culture scenarios were used for each species: the first was mass culture of larvae in 20 cm glass culture bowls containing 100 ml of test media to produce the extensive numbers required for multiple subsamples (N = 3 to 5, 10 larvae/sample) at each larval stage. The other cultures (108 individuals/treatment) were raised in compartmentalized plastic boxes (18 individuals/box) containing 10 ml of exposure media to allow assessment of developmental period durations and percent survival.
The primary stocks were prepared by dissolving fenoxycarb and pyriproxyfen (U.S. EPA, Research Triangle Park, NC) in acetone to a final concentration of 1,000 µg/ml. Nominal exposure concentrations for P. pugio were 10 µg pyriproxyfen/L and 50 µg fenoxycarb/L, while for R. harrisii they were 10 µg pyriproxyfen/L and 100 µg fenoxycarb/L. These concentrations were specifically chosen due to their report as the minimal concentrations required to elicit significant effects on growth and development in static renewal exposures conducted in this laboratory (Cripe et al., 2003; McKenney et al., 2004) (McKenney, unpublished data). P. pugio treatments of fenoxycarb were one half the concentration of those of R. harrisii due to the more sensitive responses of P.pugio to this JHA (McKenney et al., 2004). Exposure solutions were prepared daily by serial dilution of primary stocks with 20 salinity filtered seawater. An acetone control was prepared with seawater with 0.02% acetone. Water changes were performed daily by carefully pipetting the larvae into new bowls containing fresh media. After water changes, the larvae were fed <24 hr Artemia nauplii.
With the aid of a dissecting microscope, larvae were monitored daily for mortality and developmental stage by noting the presence/absence of the molted exoskeleton (exuvia). Cultures were maintained until all larvae had completed metamorphosis to the terminal larval stage (R.harrisii- 1st crab; P. pugio-postlarvae) or died. Three to five samples of ten larvae each were collected for as many stages as the cultured populations allowed and then rinsed in deionized water and lyophilized (12 hr –58°C) in pre-weighed and labeled cryovials. Dry weights were recorded to the nearest 0.01 mg.
Ecdysteroid extraction
Following a modified ecdysteroid extraction protocol (Lafont et al., 1982; Espig et al., 1989; Young et al., 1991), homogenized samples were twice extracted in 80% methanol followed by clarification in a biphasic solvent solution with equal parts hexane and 80% methanol (Fig. 2). The methanolic fraction was then concentrated to dryness in a Centri-Vap (Labconco, USA) and resuspended in water to be loaded on a C18 SepPak Vac RC cartridge (Waters, USA). The ecdysteroid fractions were eluted from the cartridge with 25%, 60% and 100% methanol in order to capture the polar, free, and apolar conjugates, respectively, and then concentrated again by Centri-Vap. Polar and apolar conjugates underwent enzymatic hydrolysis with Helix pomatia arylsulphatase (1.5 mg/ml in MES buffer, pH 6.5; 24 hr, 37°C) or porcine liver esterase (3 units/ml in 0.2 M Borate buffer, pH 8.4; 48 hr, 37°C), respectively. Finally the free ecdysteroids liberated from the enzymatic hydrolysis step were eluted using Sep-Pak separation as described above and dried by Centri-Vap. Validation of the extraction method recovery efficiency was performed by the addition of 3H-ecdysone (New England Nuclear Life Science Products, Inc.) spikes to shrimp homogenates that were then extracted and the amount of 3H-ecdysone determined by scintillation counter for each elution fraction. The free ecdysone fraction contained 97.2% of the radiolabeled hormone while the polar and apolar fractions contained 0.3% and 2.1%, respectively.
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Ecdysteroid quantification
Rabbit anti-ecdysteroid and peroxidase labeled 20-hydroxyecdysone were purchased from Dr. Tim Kingan (UC-Riverside, Riverside, CA, USA) and the enzyme immuno assay (EIA) protocol accompanying the antibody was adopted (Kingan and Adams, 2000
). The secondary antibody (Goat anti-rabbit IgG, Jackson Immunoresearch Labs) was diluted in PBS (10 mM sodium phosphate/0.15 M NaCl, pH 7.5) to 0.5 µg/90 µl and 90 µl/well were pipetted onto 96 well ELISA plates and allowed to stand overnight at RT. The secondary antibody solution was discarded and the plates blocked with 300 µl/well blocking solution (assay buffer: 25 mM sodium phosphate,0.15 M NaCl,1 mM Na2 EDTA, pH 7.5 containing 0.1% BSA, 0.002% sodium azide) for 1 hr at RT. Dried ecdysteroid extracts gained from crustacean larvae were resuspended and vortexed in 50 µl 0.1% BSA in assay buffer. A 20-hydroxyecdysone stock solution was made in ethanol (1 mg/ml) and standards were prepared by dilution of the stock in 0.1% BSA assay buffer. Concentrations of standards included most of our unknown concentrations (1, 5, 10, 50, 100, 200, 400, 800, 1600 femtomoles, each in 50 µl buffer) although further dilution of extracts was occasionally necessary. The blocking solution was discarded and the plate washed three times with PBS-T(300 µl/well, PBS containing 0.05% Tween-20; 3–5 min/wash). 50 µl of samples and standards (duplicates) were pipeted to wells. To all wells, 50 µl of 1:1,100 diluted anti-ecdysone antibody was added, followed by the addition of 50 µl 1:1,100 diluted ecdysone-peroxidase. Plates were placed on an orbital shaker for 5 min., followed by incubation at 4°C overnight. The plates were again washed 3 times with PBS-T. Next, 100 µl of the TMB:H2O2 (Kirkegaard and Perry Labs) enzyme substrate was added to all wells. The reaction was stopped by adding 100 µl 1 M H3PO4. Plates were immediately read at 450 nm, and a 4-parameter logistic fit of the standard absorbance was utilized resulting in typical correlation coefficients of 0.99–1.00.
Statistical analyses
All statistical analyses were performed using SAS V8 (SAS for Windows, 2001, Cary, NC). Ecdysone data were log transformed prior to analyses to homogenize variance. Mean separations after one-way ANOVA were determined by Tukey's adhoc multiple comparisons test. Statistical significance was defined as P < 0.05.
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RESULTS |
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R. harrisii and P. pugio larvae were reared through complete larval development during static renewal exposures to fenoxycarb or pyriproxyfen. Significant differences occurred by way of reduced growth (dry weight), reduced successful completion of development, and increased developmental duration of both crabs and shrimp following static renewal exposure to fenoxycarb and pyriproxyfen treatments. Furthermore these significant differences in growth and development are reported as early as days 6–10 of exposures, a period concomitant with the third and/or fourth instar larvae of both species' larval development, indicating possible stage specific toxicity. At the same stages, or the stage just prior to those reported with significant reduction of dry weight and retarded development, there are also significant changes in ecdysteroid titers.
Growth and survival
R. harrisii larvae exposed to 100 µg/L fenoxycarb, but not 10 µg/L pyriproxyfen had significantly reduced dry weights by the fourth zoeal stage, a condition which continued onto the end of larval development at the 1st crab stage (Fig. 3). P. pugio larvae showed significantly smaller 3rd instars when exposed to pyriproxyfen, but interestingly only 4th instars were effected by fenoxycarb exposures and they were significantly larger (Fig. 4). There were no further effects of JHA exposure in the 5th instar larvae through metamorphosis to postlarvae.
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Crab survival was not effected by JHA exposure (Fig. 5) but shrimp exposed to 50 µg/L fenoxycarb but not 10 µg/L pyriproxyfen had significantly lower survivorship by day 6 (4th instar) and continued through day 11 (Fig. 6).
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Developmental duration
Significantly longer developmental durations were observed in both crabs and shrimp exposed to the JHAs (Figs. 7, 8). In control crabs the period between hatch and metamorphosis to megalopae lasted an average of 9.5 days compared to 10.4 and 10.9 days, respectively, for the pyriproxyfen and fenoxycarb exposed larvae (Fig. 7A). There was also a significant increase in the developmental duration from megalopae to 1st crabs for crab larvae exposed to fenoxycarb (5.6 days), but not pyriproxyfen (3.8 days), when compared with the controls (4 days) (Fig. 7B). Significant increases in the total duration of crab development from hatch to 1st crab was also observed (Fig. 7C). In comparison to the mean duration of development for control crabs (13.6 days), those exposed to pyriproxyfen or fenoxycarb required 14.2 and 16.5 days, respectively. The mean duration for complete shrimp development from hatch through 9 instars to postlarvae was 19.6 days in controls (Fig. 8). Pyriproxyfen and fenoxycarb exposed shrimp had significantly increased total developmental durations (20.8 and 21.8 days, respectively) when compared with controls.
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Ecdysteroid concentrations
Figure 9 depicts the titers of the apolar conjugated, free, polar conjugated, and total ecdysteroids during subsequent stages of control R. harrisii larval development. In general, the apolar conjugated ecdysteroids (primarily storage forms) decreased slightly in each subsequent stage of development (103 to 13 ng/g dry wt) until the 1st crab stage was reached at which time there was a slight increase (29 ng/g dry wt), while the polar conjugated ecdysteroids (programmed for elimination) remained at very low concentrations throughout development (0–50 ng/g dry wt) (Fig. 9). Free ecdysteroids (active forms) were the most common and increased in titer during the first 3 crab larval stages (to 213 ng/g dry wt) followed by a precipitous decline in titer in Z4 and megalopae (to 20 ng/g dry wt) and a large increase in 1st crabs (194 ng/g dry wt), a titer similar again to Z1–3 larvae (Fig. 9). Total ecdysteroid profiles mirror those of the free ecdysteroids, primarily since the free ecdysteroids comprised between 51 and 80 percent of the total ecdysteroids.
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Figure 10 depicts the titers of the apolar conjugated, free, polar conjugated, and total ecdysteroids during subsequent stages of control P. pugio larval development. The titers of apolar conjugated ecdysteroids of shrimp were remarkably similar to those of crabs although, unlike in crabs, they were often the most common class of ecdysteroid. As was the case in crabs, apolar ecdysteroids diminished in each subsequent stage of development (except instar 4) from hatch through postlarvae (103 to 16 ng/g dry wt) (Fig. 10). The polar conjugated ecdysteroids remained at very low concentrations throughout development (10–22 ng/g dry wt) (Fig. 10). Free ecdysteroid titers were roughly half the concentration of apolar conjugates at instar 1 (53 ng/g dry wt) and there was a further decrease in titer to 21 ng/g dry wt at the 2nd instar. The largest peak in shrimp free ecdysteroid occurred at instar 3 (84 ng/g dry wt) followed again by diminished titers in instars 4 and 5 (to 27 ng/g dry wt) and finally an increased titer during the postlarval stage (49 ng/g dry wt) (Fig. 10). Again the total ecdysteroid profiles mirror those of the free ecdysteroids even though they only comprised between 21 and 64 percent of the total ecdysteroids. When comparing the shrimp ecdysteroid profiles with those of the crabs, crabs have greater concentrations of total ecdysteroid at the onset of development (45% more than shrimp at the 1st instar), but the lowest observed total ecdysteroid levels of shrimp (69 ng/g dry wt; instar 5) are 2 times that of the crabs at the megalopal stage (35 ng/g dry wt). Furthermore, crabs had 3 times the free ecdysteroids at the onset of development than did shrimp (164 and 53 ng/g dry wt, respectively). These relative differences in ecdysteroid profiles may suggest a hormonal basis for the difference in developmental plasticity demonstrated by these two species.
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Comparison of the JHA treatments with control titers of the apolar conjugated, free, polar conjugated, and total ecdysteroids during subsequent stages of R. harrisii larval development are included in Figure 11. Significant (P < 0.05) differences in the Zoeal 2 stage free and total ecdysteroids were observed in both the fenoxycarb and pyriproxyfen treatments when compared to the controls. Free ecdysteroids increased by an average of 270% over controls following JHA exposure. Although not significant (P = 0.23), there were increased titers of 1st crab free and total ecdysteroids as well. The Zoeal 3 stage polar conjugated ecdysteroids were significantly increased only for the fenoxycarb exposed crabs, likely from elimination of the significantly increased free ecdysteroids from the Zoeal 2 stage. There were no significant differences in apolar conjugated ecdysteroids in the crab treatments.
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The apolar ecdysteroids were significantly reduced in fourth instar P.pugio larvae exposed to both fenoxycarb and pyriproxyfen. Although not significant (P = 0.065), there was an increase in the mean apolar ecdysteroids in the pyriproxyfen treated 3rd instar larvae. Free ecdysteroids were significantly increased in 2nd instars of pyriproxyfen treated shrimp, while they were significantly reduced in 4th instars treated with fenoxycarb, but not pyriproxyfen. Postlarvae exposed to pyriproxyfen had reduced mean free ecdysteroid titers as well, but not significantly so (P = 0.08). Polar ecdysteroids of 4th instar shrimp were also significantly impacted when exposed to pyriproxyfen. The total ecdysteroid profiles were significantly reduced in 4th instars exposed to either JHA, while only postlarvae exposed to pyriproxyfen had significantly reduced total ecdysteroids.
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DISCUSSION |
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SYNOPSISINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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The results of the static renewal JHA exposures on crustacean larval growth, survivorship and developmental duration are entirely consistant with observations reported earlier from this lab (McKenney and Celestial, 1996
; McKenney et al., 1998; McKenney, 1999; Cripe et al., 2003; McKenney, 2005; McKenney et al., 2004). However, concomitant observations of changes in decapod crustacean ecdysteroid titers in response to JHA exposure are unique to this work and consequently add another important line of evidence necessary for determining the molecular mechanism of action of juvenoids during crustacean development.
Significantly altered free and conjugated ecdysteroids were found in both crabs and shrimp exposed to the JHAs fenoxycarb and pyriproxyfen. These data support our hypothesis that juvenoids can cross communicate with ecdysteroids by an undescribed manner. Specific mechanisms of cross-talk between these two endocrine systems were proposed by Berger et al. (1992) who hypothesized that JH may activate ecdysteroid catalytic activity by way of a kinase or phosphatase or through activation of a separate target gene that encodes for an enzyme capable of inhibition of ecdysteroid-mediated gene expression. A review of the morphogenic action and modulation of ecdysteroid action by JH was later published (Riddiford, 1994) and included the premise that JH has no specific action of its own, but rather it alters the molecular responses to ecdysteroid signals. Just recently, Mu and Leblanc (2004) have proposed a mechanism involving the interference of molecular action of ecdysteroids by juvenoids (i.e., anti-ecdysteroid activity). They have evidence to support the premise that there is a subsequent reduction in ecdysteroid receptor gene expression following juvenoid exposure, possibly by competitive binding of the receptor partner protein USP. This postulate may be useful in linking the observed changes in ecdysteroid titers to JHA exposure. Blocked ligand-receptor binding or elimination of either polar conjugation of free ecdysteroids or a negative feed-back mechanism regulating ecdysteroid levels could explain our observations. Furthermore, the promiscuous nature of USP in forming heterodimers with other receptors (e.g., RXR, EcR, DHR38, BHR38, XR78E) (Thomas et al., 1993; Antoniewski et al., 1994; Sutherland et al., 1995; Zelhof et al., 1995b) and its reported ability to bind to two juvenoid ligands that each cause unique final USP conformations (Jones and Sharp, 1997) creates a great number of possible mechanisms for transcriptional activation and inhibition during arthropod development. These variations in USP form and function may explain the differential sensitivities of these two crustacean models to pyriproxyfen and fenoxycarb. Futhermore, the inhibitory modulation of USP by transcription factors (Svp and COUP-TF) demonstrates that ecdysone-dependent transactivation by the ecdysone receptor complex can be inhibited by both DNA binding competition and protein-protein interactions (Zelhof et al., 1995a). Similar USP binding mechanisms or upregulation of USP inhibiting factors could explain the anti-ecdysteroid effects of juvenoids on normal growth, reproduction and development.
Changes in crab ecdysteroid conjugates following exposure to JHAs were associated with increased values for both polar and apolar fractions following molt to the 2nd and 3rd zoea, although fenoxycarb exposed 3rd zoea polar conjugates were the only significantly increased values when compared to the control. Shrimp exposed to JHAs initially had nonsignificantly increased conjugated ecdysteroids at instar 2 and 3, but then were followed by significantly decreased values in the 4th instar larvae. It seems probable that we are observing the elimination of the significantly increased free ecdysteroids from the 2nd stages that are initially made into apolar intermediates before conversion to the polar final elimination products. At least in crabs, it seems that the synthesis and/or elimination of apolar ecdysteroids is upregulated at the time of the significant increased free ecdysteroid titre.
There was evidence of instar specific toxicity of JHAs on decapod larval development in both crabs and shrimp. Figure 11 shows that the 2nd zoea of R. harrisii exposed to fenoxycarb and pyriproxyfen was the only stage with significantly altered ecdysteroid profiles when compared to control animals. The ecdysteroid concentrations of the 4th instar of P. pugio, a stage roughly midway through this species larval development under these culture conditions, were the most significantly impacted, although there was marked (yet not significant) changes in the 3rd instars as well (Fig. 12). Interestingly, the trend for shrimp and crab ecdysteroid titer changes were not similar, rather crab titers of free and conjugated ecdysteroids increased while there was a significant decrease in shrimp titers. We can not rule out the possibility that animals surviving the most severely impacted larval stages are genetically predisposed to counter the effects of increased JHA concentrations in vivo, possibly by increased hormone elimination pathways or decreased sensitivity to juvenoids (e.g., decreased hormone receptor expression). Therefore, subsequent stages of development following the initial impacted stages and subsequent loss of animals to mortality may be a factor in unimpacted ecdysteroid profiles later in development.
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The relative differences in R. harrisii and P. pugio ecdysteroid profiles reported here may suggest a hormonal basis for the difference in developmental plasticity demonstrated by these two species. Larvae from the Natantia (inclusive of most shrimp) have a more labile developmental pattern with a variable number of larval stages prior to metamorphosis, while the Reptantia (inclusive of crabs) are considered to have a less flexible, more stable developmental pattern with a fixed number of larval stages (Costlow Jr., 1968
; Gore, 1985; McKenney, 1999). Since these developmental variations suggest differential endocrine regulation of the metamorphic process between these decapod crustaceans, the more rigid developmental pattern of crabs could be indicative of greater hormonal control than the more variable developmental pattern of shrimp (McKenney, 1999). The greater baseline concentrations of ecdysteroids in the crabs may well support a hypothesis that crabs have greater hormonal control of their development. In conclusion, this study demonstrated that sublethal exposure to the JHAs fenoxycarb and pyriproxyfen affect free and conjugated ecdysteroid concentrations in vivo. Although we were not able to define a specific mechanism for altered ecdysteroid titers from JHA exposure, we hypothesize that there is an anti-ecdysteroid cross-talk molecular mechanism at play. The importance of JH in normal development and metamorphosis of arthropods in undeniable. Yet, the molecular mechanisms underlying its control of these processes are still very poorly understood and require further examination. Furthermore the use of JHAs for population control of arthropod vectors of mammalian diseases warrants a more in depth understanding of the mode of toxicity of these compounds on possible non-target organisms in order to best conserve natural resources.
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ACKNOWLEDGMENTS |
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Thanks to Gerri Cripe (USEPA-GED) for aid in raising and exposure of crustacean cultures. We also wish to acknowledge Drs.Vicki Martin and Robert Creed (ASU) for assistance in securing instrumentation and consultation on statistical analyses, respectively. Finally, thanks to the anonymous reviewers for their helpful comments. This work was funded in part by the Appalachian State University Graduate Studies Program and the University Research Council summer grants program.
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FOOTNOTES |
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1 From the Symposium on EcoPhysiology and Conservation: The Contribution of Endocrinology and Immunology presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 5–9 January 2004, at New Orleans, Louisiana.
2 E-mail: tubertysr{at}appstate.edu
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References |
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SYNOPSISINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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