Bioaccumulation and Metabolic Effects of the Endocrine Disruptor Methoprene in the Lobster, Homarus americanus1

Incorporation of Tran^sup 35^S-Label. The shells from methoprene exposed and control expiants were sequentially extracted and total non-dialyzable radioactivity in each fraction was determined. The fractions extracted with EI)TA were approximately equal. The buffer extracted fraction of the experimental, however, contained 50% less radioactivity than the control, while the urea extracted fractions were approximately equal. The SDS extracted experimental fraction contained 30% less radioactivity than the control.

SDS PAGE analysis

Analysis of solubilized shell fractions from expiant cultures by SDS-PAGE indicated subtle alterations in the total protein profile as revealed by Sypro Ruby staining. As shown in Figure 1a, methoprene treatment caused decreased levels of several protein components with molecular weights of 40, 60 and 75 kDa. On the other hand, when Western blots of duplicate gels were probed with Tomato Lectin (TL, which binds chitin oligosaccharides), differences were observed (Fig. Ib), most notably in the buffer soluble fractions. After methoprene treatment, TL positive components were either absent or reduced at 6, 25, 40, 80, 160 and 240 kDa, suggesting a major effect on an early stage in the synthesis pathway (Horst et al., 1993). Increased levels of specific bands (40, 80 and 160 kDa) were observed in the urea soluble fraction. The SDS soluble fraction appears to have minor quantitative changes as a result of methoprene treatment.

Analysis of the cellular layer of the integument (which is predominantly epithelial cells) by 10% SDS PAGE showed marked differences in the total protein profiles of all extracted fractions (Fig. 2a; changes indicated by stars). Major protein alterations were observed at 45, 100 and 150 kDa. When Western blots of duplicate gels were probed with TL, we again observed alterations in TL positive proteins in the specimens (Fig. 2b). In the cytosolic fraction (lanes 1,2), methoprene treatment caused a shift of TL positive bands from high molecular weight (150-250 kDa) to low molecular weight components (20-40 kDa). SDS extracts of the 16,000 rpm pellet (lanes 5,6) showed decreased levels of 70 kDa material and increased 45 kDa material as a result of methoprene treatment. Analysis of the fractions of the 5,500 × g pellet extracted with urea and then with SDS showed remarkable alterations, but of opposite patterns. The urea soluble fraction showed marked increases in TL positive bands at 120 and 130 kDa as well as clusters of less intense bands at 90, 60, 45 and 36 kDa. By contrast, the SDS soluble material contained fewer bands following methoprene treatment in the higher molecular weight range (60-100 kDa).

DISCUSSION

The effects of JH and JH analogs in insects have been studied extensively (Wyatt and Davey, 1996; Riddiford, 1996; Hammock and Quistad, 1981; Berger et al., 1992; Jones, 1995). In a 1996 review, Riddiford noted that JH allows molting in response to ecdysteroids but alters the switch of gene expression necessary for insect metamorphosis. Methoprene, acting as a JH agonist, has been shown to disrupt the metamorphic reorganization of the insect central nervous system, the salivary glands and the musculature (Restifo and Wilson, 1998). Other investigations indicated toxicity of methoprene to crustacean larvae. Templeton and Laufer (1983) found that methropene at concentrations known to interrupt insect development also interfered with the larval development of Daphnia magna. Other investigators found that environmental concentrations of methoprene affected molting, fecundity and the production of male offspring in this species (Olmstead and LeBlanc, 2001a, b, 2003; Peterson et al., 2001). Similar results have been observed in all life stages of salt marsh copepods (Bircher and Ruber, 1988).