Characterizing the blackback land crab molt gland in search for the molt inhibiting hormone receptor

Minh Nhut Tran

doi:10.25907/00017

Molting is the cyclic process through which arthropods grow and develop by shedding their exoskeleton, then hardening a new one which was previously formed underneath. Molting (also known as ecdysis) is orchestrated by ecdysteroids – a class of arthropod steroid hormones playing a key role in molting, development and, to a lesser extent, reproduction. Ecdysteroids are synthesized primarily in the Y-organs (YOs) in decapod crustaceans, where their production and secretion into the hemolymph increases at the early premolt and drops to the baseline level at mid-late premolt. This spike mediates several physiological and biological processes that set the molt in motion. The molt inhibiting hormone (MIH) inhibits YO ecdysteroidogenesis in crustaceans via a yet to be identified G protein-coupled receptor (GPCR) present on the YO membrane. In crustaceans, MIH is produced predominantly by the X organs (XO) and stored in the sinus glands (SG). The XOSG neuroendocrine complex is situated bilaterally in the eyestalk ganglia. MIH is a member of the crustacean hyperglycemic hormone (CHH) neuropeptide family which also includes CHH itself, the vitellogenesis-inhibiting hormone (VIH), mandibular organ-inhibiting hormone (MOIH), and insect ion transport protein (ITP), all of which are also predominantely produced in the XO-SG. Receptors for these important neuropeptides have not been identified to date, although molt and reproduction can be manipulated by eyestalk removal. In recent years, identification of ITP and ITP-L receptors in Bombyx mori created a foundation for the discovery of CHH receptors (CHHR) in decapod species using insect characterized ITP receptors. In the quest to identify these elusive CHHRs, in my PhD I identified 99 putative GPCRs in the YO of the blackback land crab Gecarcinus lateralis transcriptome over the molt cycle. Phylogenetic analysis classified 49 as class A (Rhodopsin-like receptor), 35 as class B (Secretin receptor), and 9 as class C (metabotropic glutamate). Three GPCRs clustered with recently identified putative CHHRs and showed differential expression over the molt cycle, suggesting that they are associated with ecdysteroidogenesis regulation. Two putative Corazonin (Crz) receptors showed much higher expression in the YOs compared with all other GPCRs, suggesting an important role in molt regulation (Chapter II). The peak of ecdysteroids at premolt mediate changes in several neuropeptides including Crz and ecdysis triggering hormone (ETH) which participate in the ecdysis sequence and enable the completion of the molt cycle. Following the finding of high Crz receptors expression in the YOs compared to all other GPCRs (Chapter II), a bioassay was conducted with Crz and ETH in the redclaw crayfish Cherax quadricarinatus which revealed a function of Crz and ETH in mediating ecdysis behavior (Chapter III). A single injection of Crz and ETH resulted in a clear and immediate eye twitching response. The Crz injection induced eye twitching slowly and asynchronously, while ETH injection caused a relatively fast and synchronous eye twitching. A single injection of ETH to intermolt crayfish resulted in a remarkably prolonged molt cycle, with twice the molt interval of the control group, suggesting that ETH plays a key role in controlling the molt cycle in decapod crustaceans. Given the key significance of ETH in molt regulation and its plausible application in pest control, we characterized ETH across the pancrustacean orders. The bioinformatic analysis shows the mature ETH sequence is identical in all studied decapod species, suggesting the conserved function of this neuropeptide across pancrustacea (Chapter III). With the attempt to deorphanize the putative CHHRs identified in Chapter II, I produced recombinant G. lateralis CHH and MIH in yeast (Chapter IV). An ex vivo assay was carried out in which YOs were incubated with recombinant neuropeptides (rCHH, rMIH), showing down-regulation of ecdysone when incubated with high level of rMIH, but no change with rCHH, supporting the biological function of rMIH. Following the ex vivo assay, a receptor assay was carried out using two of our predicted CHHRs (CHHR1 and CHHR3) with rCHH and rMIH. The results of multiple attempts were inconclusive and warrant further study. Although not conclusive, both CHHR1 and CHHR3 showed elevated reporter levels at low dose of rCHH but not rMIH. In summary, this study identified a comprehensive list of computationally annotated putative GPCRs in the YO, serving as a reference for future crustacean GPCR databases. Two predicted CHHRs were further assessed by receptor assay. Receptors for MIH and GIH/VIH are yet to be identified and could use the rMIH we produced and validated ex vivo. A behavioral study confirmed the function of ETH in ecdysis behavior for the first time in crustaceans. Further study is required to clarify the function of Crz and ETH in ecdysis behavior regulation which could contribute to our ability to regulate molting in pancrustacea.

Characterizing the blackback land crab molt gland in search for the molt inhibiting hormone receptor

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