Ecdysteroid levels in Daphnia magna during a molt cycle: Determination by radioimmunoassay (RIA) and liquid chromatography–mass spectrometry (LC–MS)

Abstract

Despite their abundance and their enormous signiWcance for various ecological processes, endocrine systems of microcrustaceans have been poorly investigated. Here, we used a highly sensitive radioimmunoassay (RIA) to determine free and conjugated ecdysteroid levels in whole-body extracts of adult Daphnia magna during a complete molt cycle. Ecdysteroid levels were predominated by free ecdysteroids. Starting from basal levels in the postmolt stage the concentration of free ecdysteroids increased sharply in the early premolt stage, followed by a sharp decline back to basal levels just prior to ecdysis. Polar and apolar ecdysteroid conjugates were found in rather low amounts with little variation during the molt cycle. Only small amounts of ecdysteroids were found in newly deposited eggs of D. magna, which suggest a sparing investment of maternal ecdysteroids into the eggs for early embryogenesis. As in whole-body extracts, free ecdysteroids were the predominant ecdysteroids found in eggs of D. magna, together with small amounts of polar and intermediary amounts of apolar conjugates. Hence, the pathways leading to polar and apolar ecdysteroid conjugates appear to be of minor importance in D. magna. Analyses of the immunodetectable peak in free ecdysteroids by liquid chromatography-mass spectrometry (LC-MS) revealed that molting is induced most probably by an increased level of 20-hydroxyecdysone. Microcrustaceans of the genus Daphnia are key components in freshwater food webs. Examination of the functional role of ecdysteroids in controlling developmental processes might help to understand the performance of the herbivorous grazer in its environment, in particular with regard to the adverse eVects of environmental xenobiotics acting as endocrine disrupters.

Roles of ecdysteroids for progression of reproductive cycle in the fresh water crustacean Daphnia magna

“…On the other hand, 20E inhibits vitellogenesis in D. magna [36]. In support of this, endogenous ecdysteroids titre is low when moulting and ovulation are about to occur in D. magna [11]. Ovulation occurs immediately after moulting when ecdysteroids titre has decreased.…”

“…In Drosophila melanogaster, ecdysteroids are required for differentiation of germ line stem cells [7,8] and prepupal development [9,10]. A pioneering study reported that a titre of ecdysteroids increased at the premoult stage and decreased back to the basal level before moulting in Daphnia magna [11], indicating that ecdysteroids are predominant moulting hormones. However, in crustaceans, reports on ecdysteroidal regulation in both moulting and reproduction are scarce [4].…”

“…Spacio-temporal expression analyses of these genes during the reproductive cycle indicated that ecdysone is possibly synthesised in the gut, and that the decline of the 20E titre before moulting [11] might be regulated by Cyp18a1. In addition, exogenous 20E treatment during the reproductive cycle resulted in a failure of moulting and ovulation, suggesting that a low level of ecdysteroids before moulting is required for successful moulting and ovulation in D. magna.…”

Effects of temperature and dietary sterol availability on growth and cholesterol allocation of the aquatic keystone speciesDaphnia

“…Furthermore, cholesterol serves as a precursor for multiple steroid hormones, e.g. ecdysteroids in arthropods, which are involved in the moulting process (Goad, 1981;Grieneisen, 1994;Martin-Creuzburg et al, 2007). Arthropods lack the ability to synthesize cholesterol and other sterols de novo and therefore have to acquire these essential compounds from their diet (Svoboda and Thompson, 1985;Martin-Creuzburg and Von Elert, 2009).…”

Dopamine is a key regulator in the signalling pathway underlying predator-induced defences inDaphnia

“…The key to this is the temporal and spatial regulation of growth and moulting through the counterbalanced actions of juvenile hormones and ecdysteroids. Whereas juvenile hormones control growth and suppress reproduction, ecdysone derivatives induce moulting and embryogenesis [31][32][33][34]. Thus in Crustacea, a proper balance between methyl farnesoate (a juvenile hormone) and 20-hydroxyecdysone (the active form of ecdysone) is important for the normal progression of oogenesis.…”