Search for hepatopancreatic ecdysteroid-responsive genes during the crayfish molt cycle: from a single gene to multigenicity

Abstract

SUMMARY The expression of the vitellogenin gene of the red-claw crayfish Cherax quadricarinatus (CqVg) was previously demonstrated in male crayfish during an endocrinologically induced molt cycle. The hypothesis that this expression is under the direct control of ecdysteroids was tested in this study both in vivo and in vitro. Unlike vitellogenin of insects, CqVg was not found to be ecdysteroid-responsive. Thus, a multigenic approach was employed for the identification of other hepatopancreatic ecdysteroid-responsive genes by a cDNA microarray. For the purposes of this study, a multi-parametric molt-staging technique, based on X-ray detection of gastrolith growth, was developed. To identify ecdysteroid-responsive genes during premolt, the molt cycle was induced by two manipulations, 20-hydroxyecdysone administration and X-organ–sinus gland complex removal; both resulted in significant elevation of ecdysteroids. Two clusters of affected genes (129 and 122 genes, respectively) were revealed by the microarray. It is suggested that only genes belonging to similarly responsive (up- or downregulated) gene clusters in both manipulations (102 genes) could be considered putative ecdysteroid-responsive genes. Some of these ecdysteroid-responsive genes showed homology to genes controlling chitin metabolism, proteases and other cellular activities, while 56.8% were unknown. The majority of the genes were downregulated, presumably by an energetic shift of the hepatopancreas prior to ecdysis. The effect of 20-hydroxyecdysone on representative genes from this group was confirmed in vitro using a hepatopancreas tissue culture. This approach for ecdysteroid-responsive gene identification could also be implemented in other tissues for the elucidation of ecdysteroid-specific signaling pathways during the crustacean molt cycle.

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

“…1 summarizes the premolt progress, presenting the relationship between the molt mineralization index (MMI), the circulating levels of ecdysteroids and the presence of a new cuticle under the existing one. It agrees with a similar relationship that was established in previous astacid studies (Nakatsuji et al, 2000;Shechter et al, 2007). Eighteen males were selected for expression analysis and were divided into seven premolt stages as follows: males at stages P1 and P2 exhibited no gastroliths, and were divided according to the time from eyestalk removal (1 day versus 4 days) and by the small difference in ecdysteroid level.…”

“…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.…”

“…In astacid crayfish, transient calcium structures named gastroliths are deposited during premolt in a cavity between a specialized hypodermis and the cuticular layer of the cardiac stomach, experimentally serving as molt stage markers. At postmolt they are dissolved and used as a mineral source for the cuticle (Nakatsuji et al, 2000;Shechter et al, 2007).…”

Effect of dietary chitin on digestive enzyme activity, growth and survival of Macrobrachium tenellum juvenile prawns

“…However, in crustaceans, this did not occur with diets containing purified chitin, but this chitinase activity has occurred in the basal region of the peritrophic membrane in the gut (Ceccaldi, 1989;Alexandre et al, 2014). It is likely that the same mechanism occurs in M. tenellum because chitin is re-absorbed when chitinase dissolves the old exoskeleton into a more soluble form in the integument, which is common in decapods (Dall et al, 1990;Chang, 1995;Shechter et al, 2007;Stillman et al, 2008). …”

Growth and Moulting in Northern Krill (Meganyctiphanes norvegica Sars)

“…Previous nomenclature used for E. superba is also included for comparison (Table 6.2; Buchholz, 1991). Combining morphological moult staging ( other crustaceans, for example such as in the elucidation of chitin turnoveror cuticle-digestion processes (Seear et al, 2009;Shechter et al, 2007). In turn, the morphological stage definitions may be validated and refined further in this way (see also Fig.6.1B).…”