Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 20 (1), 74

Characterization of G-protein Coupled Receptors From the Blackback Land Crab Gecarcinus Lateralis Y Organ Transcriptome Over the Molt Cycle

Affiliations

Characterization of G-protein Coupled Receptors From the Blackback Land Crab Gecarcinus Lateralis Y Organ Transcriptome Over the Molt Cycle

Nhut M Tran et al. BMC Genomics.

Abstract

Background: G-protein coupled receptors (GPCRs) are ancient, ubiquitous, constitute the largest family of transducing cell surface proteins, and are integral to cell communication via an array of ligands/neuropeptides. Molt inhibiting hormone (MIH) is a key neuropeptide that controls growth and reproduction in crustaceans by regulating the molt cycle. It inhibits ecdysone biosynthesis by a pair of endocrine glands (Y-organs; YOs) through binding a yet uncharacterized GPCR, which triggers a signalling cascade, leading to inhibition of the ecdysis sequence. When MIH release stops, ecdysone is synthesized and released to the hemolymph. A peak in ecdysone titer is followed by a molting event. A transcriptome of the blackback land crab Gecarcinus lateralis YOs across molt was utilized in this study to curate the list of GPCRs and their expression in order to better assess which GPCRs are involved in the molt process.

Results: Ninety-nine G. lateralis putative GPCRs were obtained by screening the YO transcriptome against the Pfam database. Phylogenetic analysis classified 49 as class A (Rhodopsin-like receptor), 35 as class B (Secretin receptor), and 9 as class C (metabotropic glutamate). Further phylogenetic analysis of class A GPCRs identified neuropeptide GPCRs, including those for Allatostatin A, Allatostatin B, Bursicon, CCHamide, FMRFamide, Proctolin, Corazonin, Relaxin, and the biogenic amine Serotonin. Three GPCRs clustered with recently identified putative CHH receptors (CHHRs), and differential expression over the molt cycle suggests that they are associated with ecdysteroidogenesis regulation. Two putative Corazonin receptors showed much higher expression in the YOs compared with all other GPCRs, suggesting an important role in molt regulation.

Conclusions: Molting requires an orchestrated regulation of YO ecdysteroid synthesis by multiple neuropeptides. In this study, we curated a comprehensive list of GPCRs expressed in the YO and followed their expression across the molt cycle. Three putative CHH receptors were identified and could include an MIH receptor whose activation negatively regulates molting. Orthologs of receptors that were found to be involved in molt regulation in insects were also identified, including LGR3 and Corazonin receptor, the latter of which was expressed at much higher level than all other receptors, suggesting a key role in YO regulation.

Keywords: Crustacean Hyperglycemic Hormone family of neuropeptides; Decapod crustaceans; Ecdysis regulation; Ecdysteroids; G protein-coupled receptor; Molting; Neuroendocrine signalling pathways; Rhodopsin-like receptors; Secretin-like receptors.

Conflict of interest statement

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Gene expression (RPKM) heat map of GPCRs in different molt stages. Clustered by gene expression profile in a transcriptome dataset based on 5 different stages. Scores are coloured on a log2 scale with the red maximum and white minimum. Putative GPCR receptors are predicted based on a phylogenetic study and domain analysis
Fig. 2
Fig. 2
Statistical analysis of transcript RPKM expression. Molt-related GPCRs in G. lateralis YO were differentially expressed in five different stages of molt cycle (P < 0.05 and FDR < 0.05, highlighted in green in Additional file 2). Abbreviations: IM, intermolt stage; EP, early premolt stage; MP, mid premolt stage; LP, late premolt stage; and PM, postmolt stage. Significance level is marked as: * = P < 5E-02; ** = P < 5E-04; *** = P < 5E-06
Fig. 3
Fig. 3
Pruned tree of Ast receptors and their amino acid sequence arrangement. a G. lateralis Ast A receptors in comparison with P.clarkii Ast A receptor and b G. lateralis Ast B receptors in comparison with P. clarkii Ast B receptor
Fig. 4
Fig. 4
Phylogenetic tree of class A GPCRs presented as circular cladogram with different identified protein groups. The tree was constructed by the neighbor joining method with bootstap 1000 following multiple sequence alignment of 7TM regions in CLC workbench. Abbreviations: Aa=Aedes aegypti, Ad = Anopheles darlingi, Ag = Anopheles gambiae, Am = Apis mellifera, Bd = Bactrocera dorsalis, Bm= Bombyx mori, Bt = Bombus terrestris, Cq = Culex quinquefasciatus, Cs= Callinectes sapidus, Dm= Drosophila melanogaster, Dp= Daphnia pulex, Es = Eriochier sinensis, Gl= Gecarcinus lateralis, Ha = Homarus americanus, Haa = Hasarius adansoni, Lp = Limulus polyphemus, Lv = Litopenaeus vannamei, Mr = Macrobrachium rosenbergii, Nl = Nilaparvata lugens, Nv = Nephrops norvegicus, Ob = Ooceraea biroi, Pa = Periplaneta americana, Pc= Procambarus clarkia, Pm = Penaeus monodon, Px = Plutella xylostella, Sm = Strigamia maritima, Sp = Scylla paramosain, Sv= Samariasus verreauxi, Tc= Tribolium castaneum, Tu= Tetranychus urticae
Fig. 5
Fig. 5
Pruned tree of Crz receptors and their amino acid sequence arrangement on their membrane. a G. lateralis Crz receptors and their sequence arrangement. b Statistical analysis of gene expression in term of RPKM for GPCRA6&7 in G. lateralis which were expressed in five different stages of molting cycle (P < 0.05 and FDR < 0.05, highlighted in red in Additional file 2)
Fig. 6
Fig. 6
Putative CCHamide, FMRamide, GRL 101 like and LGR3 receptors. a & b Pruned tree of CHHamide and FMRFamide receptors and amino acid sequence arrangement of their putative receptors on their membrane. B) RPKM expression of FMRF receptor in five different molt stages. c RPKM expression of both putative FMRFamide receptor through molt stages. d LGR receptors with the number of LDLa motif, and LRR motif in the ectodomain. Abbreviations: IM, intermolt stage; EP, early premolt stage; MP, mid premolt stage; LP, late premolt stage; and PM, postmolt stage
Fig. 7
Fig. 7
Putative CHH receptors and their tissue distribution. a Pruned tree of CHHRs and amino acid sequence arrangement of putative CHHRs. Transmembrane domains of both Gl_GPCRA9 and Gl_GPCRA12 were predicted using TMHMM online tool. b RT-PCR was carried out using cDNA from ten different organs of G. lateralis. Primers were designed to amplify two putative CHHR receptors (Gl_GPCRA9 and Gl_GPCRA12) (Table 1). Tissue expression pattern obtained from RT-PCR gel image visualized under UV light
Fig. 8
Fig. 8
Phylogenetic tree of class B GPCRs presented as circular cladogram. Five protein groups were identified, including latrophilin, lipoprotein, methuselah, PDF, and DH44 receptor. The phylogenetic trees were constructed by neighbor joining method with bootstrap 1000 following multiple sequence alignment of 7-TM regions in CLC workbench. Abbreviations: Cs= Callinectes sapidus, Dm= Drosophila melanogaster, Dp= Daphnia pulex, Gl= Gecarcinus lateralis, Pc= Procambarus clarkii, Tc= Tribolium castaneum, Tu= Tetranychus urticae
Fig. 9
Fig. 9
Phylogenetic tree of class C GPCRs presented as circular cladogram. Two protein groups were identified, including mGlu receptor and boss receptor. The phylogenetic trees were constructed by neighbor joining method with bootstrap 1000 following multiple sequence alignment of 7-TM regions in CLC workbench. Abbreviations: Cs= Callinectes sapidus, Dm= Drosophila melanogaster, Dp= Daphnia pulex, Gl= Gecarcinus lateralis, Pc= Procambarus clarkii, Tc= Tribolium castaneum, Tu= Tetranychus urticae

Similar articles

See all similar articles

Cited by 1 article

References

    1. Von Reumont BM, Jenner RA, Wills MA, Dell'ampio E, Pass G, Ebersberger I, Meyer B, Koenemann S, Iliffe TM, Stamatakis A, et al. Pancrustacean phylogeny in the light of new phylogenomic data: support for Remipedia as the possible sister group of Hexapoda. Mol Biol Evol. 2012;29(3):1031–1045. doi: 10.1093/molbev/msr270. - DOI - PubMed
    1. Zhou X. FAO Fisheries and Aquaculture. 2015. An overview of recently published global aquaculture statistics.
    1. Holdich DM, Pöckl M. Invasive crustaceans in European inland waters. In: Gherardi F. (eds) Biological invaders in inland waters: Profiles, distribution, and threats. Invading Nature - Springer Series In Invasion Ecology. Dordrecht: Springer; 2007;2.
    1. Gherardi F. (2007) Understanding the impact of invasive crayfish. In: Gherardi F. (eds) Biological invaders in inland waters: Profiles, distribution, and threats. Invading Nature - Springer Series In Invasion Ecology. Dordrecht: Springer; 2007;2.
    1. Gherardi F. Measuring the impact of freshwater NIS: what are we missing?. In: Gherardi F. (eds) Biological invaders in inland waters: Profiles, distribution, and threats. Invading Nature - Springer Series In Invasion Ecology. Dordrecht: Springer; 2007;2.

MeSH terms

Substances

LinkOut - more resources

Feedback