The functional importance and significance of ecdysteroids in molt-cycle regulation of the blue crab, Callinectes sapidus

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This study aims to expand our understanding of how ecdysteroids and neuropeptide hormones (MIH/CHH) regulate molting in crustaceans using the blue crab Callinectes sapidus as a model animal. The hypothesis of this study is that ecdysteroids have a stimulatory effect on MIH/CHH production in eyestalks while generating both positive and negative feedback on ecdysteroidogenesis in Y-organs. Since ecdysteroids exert their signals through an ecdysteroid receptor complex, composed of an ecdysone receptor (EcR) and its partner, the retinoid-X receptor (RXR), the functional activity of ecdysteroids on tissues of interest is examined through EcR expression. Endogenous levels of ecdysteroids as well as expression in EcR, RXR and MIH/CHH are assayed in both embryonic and juvenile molt cycles. Similar patterns of changes in these two cycles suggest that molting control is driven by the same mechanisms in each. During embryonic molt, the correlation of ecdysteroid levels and MIH/CHH expression suggests that ecdysteroids are involved in MIH/CHH production. Using an in vitro system, stimulation of MIH expression occurs in eyestalks incubated with ponasterone A (PoA) and 20-hydroxyecdysone (20-HE) in the ratio found endogenously (3:1) and in ecdysteroid concentrations of 75 and 150 ng/ml. The in vivo response of MIH expression to depletion of ecdysteroid signals was examined by RNAi knockdown of an ecdysone receptor that was found to reduce the levels of MIH to those found by MIH knockdown, providing support for the hypothesis that ecdysteroids stimulate MIH expression through an ecdysone receptor. This study also found evidence suggesting that crustaceans have long-loop feedback for ecdysteroids in the neuroendocrine axis, which is similar to that found in insects.