Hemolymph ecdysteroids during the last three molt cycles of the blue crab, Callinectes sapidus: quantitative and qualitative analyses and regulation

Abstract

The profiles of circulating ecdysteroids during the three molt cycles prior to adulthood were monitored from the juvenile blue crab, Callinectes sapidus. Ecdysteroid patterns are remarkably similar in terms of peak concentrations ranging between 210-330 ng/ml hemolymph. Analysis of hemolymph at late premolt stage revealed six different types of ecdysteroids with ponasterone A (PoA) and 20-OH ecdysone (20-OH E) as the major forms. This ecdysteroid profile was consistent in all three molt cycles. Bilateral eyestalk ablation (EA) is a procedure that removes inhibitory neurohormones including crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) and often results in precocious molting in crustaceans. However, the inhibitory roles of these neuropeptides in vivo have not yet been tested in C. sapidus. We determined the regulatory roles of CHH and MIH in the circulating ecdysteroid from ablated animals through daily injection. A daily administration of purified native CHH and MIH at physiological concentration maintained intermolt levels of ecdysteroids in the EA animals. This suggests that Y organs (YO) require a brief exposure to CHH and MIH in order to maintain the low level of ecdysteroids. Compared to intact animals, the EA crabs did not exhibit the level of peak ecdysteroids, and the major ecdysteroid turned out to be 20-OH E, not PoA. These results further underscore the important actions of MIH and CHH in ecdysteroidogenesis, as they not only inhibit, but also control the composition of output of the YO activity.

Expression of cytoskeletal and molt-related genes is temporally scheduled in the hypodermis of the crayfish Procambarus clarkii during premolt

“…Hence, it is still unresolved how one hormone initiates this complex sequence of events. A crucial time point of the premolt is P7, in which the ecdysone level drops and in which other putative hormones are involved, as already shown in insects and crustaceans (Chung, 2010;Mesce and Fahrbach, 2002;Phlippen et al, 2000). The genes of which the expression peaked at P7 may, for future studies, serve as indicators of the hormonal effects toward the end of premolt and across postmolt.…”

“…The latter two thresholds were used with the aim of avoiding misinterpretation of natural variability as differential expression. The selected genes were responsible for 65±5.1% of the total normalized reads, and the percentage of total induction or attenuation of reads, as well as the Shechter et al (Shechter et al, 2007) and ecdysteroid levels according to Chung (Chung, 2010). The widely accepted Drach index was assigned to P1-P7 by comparison with the findings of Durliat et al (Durliat et al, 1988).…”

“…The supernatants were retrieved, dried by centrifugation under vacuum and stored at -20°C until further analysis. The hemolymph samples were assayed to evaluate the circulating levels of ecdysteroids according to Chung (Chung, 2010).…”

“…It has to be noted that the removal of the eyestalk eliminates a variety of peptide hormones and, although inducing premolt, the moltExpression of cytoskeletal and molt-related genes is temporally scheduled in the hypodermis of the crayfish Procambarus clarkii during premolt independent physiology of the individual may be affected as well. Just before ecdysis, the level of circulating ecdysteroids is sharply decreased (Chung, 2010;Nakatsuji et al, 2000;Shechter et al, 2007). The level of two additional peptide hormones is increased from this period across ecdysis and postmolt.…”

Ecdysteroid metabolism in crustaceans

“…A precipitous drop in hemolymph ecdysteroids triggers the shedding of the exoskeleton [90]. Eyestalk ablation, which removes the primary source of inhibitory neuropeptides (e.g., MIH), activates the YO and increases hemolymph ecdysteroid levels [1,2,21,23,70,79,86,91,92]. In addition to the peak in hemolymph titers at the end of the premolt stage, there are smaller peaks during postmolt and early premolt stages, which may have physiological functions [30,48,[93][94][95].…”