Isolation and Tissue Distribution of an Insulin-Like Androgenic Gland Hormone (IAG) of the Male Red Deep-Sea Crab, Chaceon quinquedens

Abstract

The insulin-like androgenic gland hormone (IAG) found in decapod crustaceans is known to regulate sexual development in males. IAG is produced in the male-specific endocrine tissue, the androgenic gland (AG); however, IAG expression has been also observed in other tissues of decapod crustacean species including Callinectes sapidus and Scylla paramamosain. This study aimed to isolate the full-length cDNA sequence of IAG from the AG of male red deep-sea crabs, Chaceon quinquedens (ChqIAG), and to examine its tissue distribution. To this end, we employed polymerase chain reaction cloning with degenerate primers and 5' and 3' rapid amplification of cDNA ends (RACE). The full-length ChqIAG cDNA sequence (1555 nt) includes a 366 nt 5' untranslated region a 453 nt open reading frame encoding 151 amino acids, and a relatively long 3' UTR of 733 nt. The ORF consists of a 19 aa signal peptide, 32 aa B chain, 56 aa C chain, and 44 aa A chain. The putative ChqIAG amino acid sequence is most similar to those found in other crab species, including C. sapidus and S. paramamosain, which are clustered together phylogenetically.

Keywords: Chaceon quinquedens; androgenic gland; cold water species; insulin-like androgenic gland hormone (IAG); red deep-sea crab.

Figure 1

The full-length cDNA and deduced amino acid sequence of ChqIAG isolated from the androgenic gland (AG) of male C. quinquedens (GenBank Accession No. KY497474). Predicted signal peptide is italicized. The predicted start codon (ATG) is bolded and underlined, and the stop codon (TAG) is bolded, double underlined and marked with “*”. The nucleotide number is on the right and the amino acid number is on the left hand side of the figure. Two predicted cleavage sites (RHKR/RFRR) are boxed and a polyadenylation signal (AATAAA) is underlined in the 3′ untranslated region (UTR). The six conserved cysteine residues are circled. The B chain is bold and underlined, the C chain is bold and italicized and the A chain is bold and double underlined. The predicted N-glycosylation site is noted with a triangle.

Figure 2

Maximum likelihood tree (using phylogeny.fr) of the open reading frame (ORF) sequence of crustacean insulin-like androgenic gland hormones (IAGs) and androgenic gland hormones (AGHs) obtained from the following species: C. quinquedens, KY497474; M. rosenbergii, ACJ38227.1; P. pelagicus, HM459854; E. sinensis, KU724192; S. paramamosain, JQ681748.1; Jasus edwardsii, KF908794; Macrobrachium lar, AB579012.1; Callinectes sapidus, KF792074.1; M. nipponense, KC460325; Sagmariasus verreauxi, KF220491; Cherax destructor, ACD91988; Palaemon pacificus, AB588014.1; Procambarus clarkii, KT343750. Armadillidium vulgare, BAA86893.1. Cherax quadricarinatus, DQ851163.1; F. chinensis, JQ388277.1; P. monodon, GU208677.1; P. paucidens, AB588013.1; M. japonicus, AB598415.1. The scale bar indicates the number of amino acid sequence substitutions per site. Distance represents neighbor-adjoining bootstrap values.

Figure 3

Spatial expression analysis of ChqIAG in adult male (a) and female (b) tissues 1: eyestalk ganglia; 2: thoracic ganglia complex; 3: brain; 4: hypodermis; 5: muscle; 6: gill; 7: midgut; 8: hindgut; 9: heart; 10: hemocytes; 11: hepatopancreas; 12: testis/ovary; 13: androgenic gland/spermatheca; 14: no template control. Arginine kinase (ChqAK) was used as a reference gene for amplification.