Functional and evolutionary implications from the molecular characterization of five spermatophore CHH/MIH/GIH genes in the shrimp Fenneropenaeus merguiensis

Abstract

The recent use of RNA-Seq to study the transcriptomes of different species has helped identify a large number of new genes from different non-model organisms. In this study, five distinctive transcripts encoding for neuropeptide members of the CHH/MIH/GIH family have been identified from the spermatophore transcriptome of the shrimp Fenneropenaeus merguiensis. The size of these transcripts ranged from 531 bp to 1771 bp. Four transcripts encoded different CHH-family subtype I members, and one transcript encoded a subtype II member. RT-PCR and RACE approaches have confirmed the expression of these genes in males. The low degree of amino acid sequence identity among these neuropeptides suggests that they may have different specific function(s). Results from a phylogenetic tree analysis indicated that these neuropeptides were likely derived from a common ancestor gene resulting from mutation and gene duplication. These CHH-family members could be grouped into distinct clusters, indicating a strong structural/functional relationship among these neuropeptides. Eyestalk removal caused a significant increase in the expression of transcript 32710 but decreases in expression for transcript 28020. These findings suggest the possible regulation of these genes by eyestalk factor(s). In summary, the results of this study would justify a re-evaluation of the more generalized and pleiotropic functions of these neuropeptides. This study also represents the first report on the cloning/identification of five CHH family neuropeptides in a non-neuronal tissue from a single crustacean species.

Fig 1. Alignment of the spermatophore CHHs with homologous sequence from different shrimps.

The same horizontal colors indicate homologues that share a much higher degrees of amino acid identity in the mature peptide region. The predicted signal peptide of each member is in bold letters. The potential cleavage site is shown (in black bold letters) is the location to produce the mature peptide (i.e. KR and ER). No signal peptide was predicted for translate product of transcript 14056 and MrGIH and the glycine at position 11 is highlight in red. The identity (%) on the left of the table indicated the overall amino acid identity of the mature peptides between members of the same color horizontal block. Un-identical amino acid of the aligned sequence within the same horizontal color blocks are indicated by the bolded white or red letters.

Fig 2. Phylogenetic analysis of spermatophore CHH subtype I transcripts of F. merguiensis.

The spermatophore sequences and selected homologous sequences from other shrimps were analyzed by constructing a neighbor-joining phylogenetic tree using the program Mega 6.0. Transcript ID was highlighted and all the sequences can be divided into six major groups according to their reported functions.

Fig 3. Phylogenetic analysis of spermatophore CHH subtype II transcripts.

Transcript 14056 and selected type II neuropeptides of other shrimps were analyzed using the Mega 6.0. Transcript 14056 and a previously reported MIH gene (FmMIH) of F. merguiensis were highlighted.

Fig 4. Tissue expression profile of the five CHH-family gene transcripts.

Transcript ID was shown on the left and tissue include epidermis (Ep), eyestalk (Es), gill (Gi), mid-gut gland (Hp), heart (Ht), hemolymph (Hl), nerve cord (Nc), brain (Br), thoracic ganglion (Tg), ovary (Ov), testis (Te), muscle (Mus), spermatophore (Sph), no template control.

Fig 5. Expression of the five spermatophore CHH genes.

Expression level of the gene in intact (left bar) and unilateral eyestalk ablated shrimp (right bar). Sample size is N = 6 for each group.Transcript ID was shown on the X-axis are for Unigenes 4459, 8101, 28020, 32710 and 14056. The relative expression level for each gene was shown on the Y-axis. Different letters above the standard error bar indicated significant difference (P<0.05) in expression level of each gene.