Variations in ecdysteroid levels and Cytochrome p450 expression during moult and reproduction in male shore crabsCarcinus maenas

Abstract

Ecdysteroid levels were investigated by HPLC-MS over the moult cycle and in relation to reproduction in male shore crabs Carcinus maenas. Ecdysone (E), 20-hydroxyecdysone (20E) and Ponasterone A (PoA) were quantified in the haemolymph, hepatopancreas and testis. Also, the expression of 2 recently discovered Cytochrome p 450 genes (CYP330A1 and CYP4C39) inducible by ecdysteroids was studied in the hepatopancreas by Northern blot hybridisation analysis. In the haemolymph and hepatopancreas, all 3 ecdysteroids varied over the moult cycle with high levels in premoult and low levels in postmoult and intermoult. In the testis, 20E and E were present at high levels except in Postmoult Stage A, where low levels were observed. PoA was never observed in the testis. Ecdysteroids were quantified in the red and green colour forms of late intermoult C 4 crabs. In both phenotypes, 20E was the dominating ecdysteroid in late intermoult. In the haemolymph, 20E levels did not vary between the 2 phenotypes, but haemolymph 20E levels were negatively related to size. Also, haemolymph 20E levels varied with season in late-intermoult crabs, with higher levels during spring and autumn than during summer. Green crabs had significantly higher testicular E levels than red crabs. Ecdysteroid levels were negatively related to CYP330A1 and CYP4C39 gene expression. CYP330A1 and CYP4C39 mRNA levels were low during intermoult and premoult but high during postmoult. The results suggest that E and 20E are involved in both growth and reproduction whereas PoA is involved in moulting but not in reproduction and that the testis of male shore crab may be a possible source of ecdysone production in addition to the Y-organ. The results also support the concept that the probability of male shore crabs entering a new moult cycle decreases with increasing size.

Molt regulation in green and red color morphs of the crab,Carcinus maenas: gene expression of molt-inhibiting hormone signaling components

“…Hemolymph ecdysteroid titers in molting animals peaked between 400 and 1000 pg μl −1 prior to ecdysis ( Fig. 5B-D) (see also Styrishave et al, 2004b).…”

“…There are few published reports on the effects of ESA and MLA on adults (Skinner, 1985) and none have investigated possible differences between color morphs. As red morphs molt less frequently than green morphs (Styrishave et al, 2004b), we hypothesized that red morphs would be less responsive to molt induction than green morphs. MLA induces precocious molting (Skinner and Graham, 1972), but the effects of ESA are inconsistent.…”

“…As the green morphs molt to larger sizes, they transition to red morphs during a prolonged intermolt period . Older red morphs stop molting entirely and enter a blocked or terminal anecdysis state (Baghdassarian et al, 1996;Carlisle, 1957;Crothers, 1967;Lachaise et al, 1988;Saïdi et al, 1994;Styrishave et al, 2004a;Styrishave et al, 2004b;Webster, 1986). Large males (CW >70 mm) never show signs of molting (e.g.…”

“…limb regeneration) and ecdysteroid titers never exceed 70 pg μl −1 (Carlisle, 1957;Webster, 1986). Although green morphs tend to be smaller than red morphs, there is a large overlap in size distribution Reid et al, 1997;Styrishave et al, 2004b). When red morphs molt they molt back to the green phenotype [S.N., D.L.M.…”

“…ESA may (Carlisle, 1957) or may not (Carlisle, 1954;Saïdi et al, 1994;Skinner and Graham, 1972) stimulate molting. As older red morph adults occur in a blocked or terminal anecdysis state (Baghdassarian et al, 1996;Carlisle, 1957;Lachaise et al, 1988;Saïdi et al, 1994;Styrishave et al, 2004a;Styrishave et al, 2004b;Webster, 1986), animals (estimated ages between 1 and 2 years) that were capable of molting were used. The effects of ESA and MLA on the color morphs were determined by measuring hemolymph ecdysteroid levels and YO expression of the three GCs, Cm-NOS, and the housekeeping gene Cm-elongation factor 2 (EF2), using quantitative reverse transcription-PCR (RT-PCR or qPCR).…”