Summary Expression and release patterns of neuropeptides during embryonic development and hatching of the green shore crab , Carcinus maenas

Embryogenesis in most marine decapod crustaceans, except Euphausiacea and Dendrobranchiata is epimeric in that the naupliar stage is rapidly superseded by a period of extensive embryonic morphogenesis, widely known as a ‘metanauplius’ stage, irrespective of the developmental phenotype exhibited at hatching (Goudeau and Lachaise, 1983). With regard to embryonic moulting, for crabs ( Carcinus maenas ) progression from the nauplius to metanauplius is recognised by the addition of a fourth egg envelope, in addition to two distinctive fertilisation envelopes. Subsequent metanaupliar development is associated with development of a loosely fitting cuticle, which is rapidly shed just after hatching (the prezoea), and a cuticle belonging to the first free-swimming planktonic stage (the zoea) (Goudeau and Becker, 1982; Goudeau and Lachaise, 1980a; Goudeau and Lachaise, 1980b). Thus, during development, the embryo essentially completes three moult cycles, one concerned with early events, which occur without exuviation, and a second, during which considerable morphoand neurogenesis occur, as illustrated by eye development which has long been used to stage ontogenic processes during this period (Perkins et al., 1972). The third moult, and the only true ecdysis, occurs just after hatching (the prezoeal moult). Subsequent important refinements have been used to completely temporally define the metanaupliar moult cycle in developing lobster embryos (Helluy and Beltz, 1991). An important issue concerns the hormonal control of embryonic moulting. The fertilised egg of crustaceans contains large quantities of maternally derived ecdysteroids and corresponding polar metabolites, which decline during naupliar development (Goudeau and Lachaise, 1983; Lachaise et al., 1981; Okazaki and Chang, 1991; Wilder et al., 1990). These are presumed to be important in naupliar development. However, during metanaupliar development, dramatic Crustacean ecdysis is controlled by at least three neuropeptides: moult-inhibiting hormone (MIH), which represses ecdysteroid synthesis; crustacean hyperglycaemic hormone (CHH), which not only influences ecdysteroid synthesis but also water uptake during moulting; and crustacean cardioactive peptide (CCAP), which is involved in stereotyped ecdysis behaviour. During embryonic development, moulting takes place in the egg, but there is little information regarding developmental expression of these neuropeptides during this period or during hatching – an event that is analogous to eclosion in insects. To address this problem, we determined expression profiles of MIH and CHH mRNA by quantitative RTPCR, together with developmental peptide expression studies [confocal immunocytochemistry (ICC) and radioimmunoassay (RIA)]. Likely homologous events relating to neuropeptide surges of both CHH and CCAP were seen during larval hatching, when compared to the adult moult, and cell-specific copy concentration of both MIH and CHH mRNAs was identical to that of the adult during late embryonic development. We measured parallel mRNA and peptide expression of two neuropeptides (red pigment-concentrating hormone RPCH) and pigmentdispersing hormone (PDH) during development, as these have roles as neuromodulators and as classical neurohormonal roles. For MIH and CHH, gene expression was in accordance with peptide expression, but novel sites of CHH expression were found (abdominal peripheral neurones), the expression and release patterns of which may be related to larval eclosion and water uptake necessary for eggshell rupture and hatching. For RPCH and PDH, gene transcription and peptide expression were not in accordance. A significant contribution of maternally derived (non-translated) PDH mRNA to the embryo was seen, and for RPCH, high-level mRNA and peptide expression during late embryogenesis is related to a long ignored, but potentially important release site – the enigmatic post-commissural organs – which are the most prominent structures expressing RPCH during late embryogenesis.