A molting-inhibiting hormone-like protein from Pacific white shrimp Litopenaeus vannamei is involved in immune responses

doi:10.1016/j.fsi.2017.11.031

Fish & Shellfish Immunology

Volume 72, January 2018, Pages 544-551

https://doi.org/10.1016/j.fsi.2017.11.031 Get rights and content

Highlights

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Identification of a molting-inhibiting hormone-like gene (MIHL) from Litopenaeus vannamei.
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The expression of MIHL is regulated by Relish but not Dorsal.
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MIHL is positively involved in antiviral and antibacterial immune responses.

Abstract

The molting-inhibiting hormones (MIHs) from the crustacean hyperglycemic hormone (CHH) family are a group of neuropeptides that are implicated in regulation of molting and reproduction in crustaceans. In this study, a novel protein containing a typical crustacean neuropeptide domain was identified from Litopenaeus vannamei. The protein showed high homology with other shrimp MIHs and was then designated as a MIH-like protein (MIHL). Among the detected tissues, the heart expressed the highest level of MIHL. The expression of MIHL could be significantly up-regulated after infection with white spot syndrome virus (WSSV), gram-negative bacterium Vibro parahaemolyticus and gram-positive bacterium Staphylococcus aureus, indicating that MIHL could be involved in immune responses. The promoter of MIHL was predicted to contain two NF-κB binding sites and could be regulated by the NF-κB family protein Relish but not Dorsal, suggesting that MIHL could be an effector gene of the IMD/Relish pathway. Silencing of MIHL in vivo by RNAi strategy significantly down-regulated the expression of many immune effector genes and increased the mortalities of shrimp infected by V. parahaemolyticus and WSSV and their copy numbers in tissues. These confirmed that MIHL could play a role in antiviral and antibacterial immune responses in shrimp.

Section snippets

Instruction

Neuropeptides are a group of bioactive peptides generally synthesized and secreted by neurons or neuroendocrine cells that serve as intercellular signaling molecules to regulate the functions of the brain and the body through activation of specific receptors [1], [2]. Since the first invertebrate neuropeptide, red pigment concentrating hormone (RPCH) [3], was characterized in shrimp almost half a century ago, crustaceans have been commonly used as a research model for neuropeptide research [4],

Shrimp and pathogens

L. vannamei (∼5 g) were obtained from a shrimp farm in Zhanjiang city, Guangdong Province. Before experiments, shrimp were acclimated in a recirculating water tank system filled with air-pumped seawater (2.0% salinity) at ∼27 °C for at least 7 days. The stocks of white spot syndrome virus (WSSV) and Vibro parahaemolyticus were prepared as previously described [20].

Cloning of MIHL gene

An expressed sequence tag (EST) containing a putative neuropeptide domain homologous sequence was retrieved from a L. vannamei

Cloning and bioinformatics analysis of MIHL

The full length of MIHL mRNA is 1114 bp with a 245 bp 5′ untranslated region (UTR) and a 402 bp ORF encoding a protein of 133 amino acids with a calculated molecular weight of 14.94 kDa and a theoretical isoelectric point of 9.00 (GenBank Accession No. MF358695.1) (Fig. 1A). MIHL was predicted to possess a crustacean neuropeptide domain covering the 59–126 residue region. Prediction by SignalP 4.1 software suggested that MIHL may have no signal peptide, which needs further confirmation.

Discussion

The CHH family shares similar amino acid sequences with six conserved Cys residues in a similar arrangement [28]. Members of the MIH subfamily lack the CHH precursor-related peptide (CPRP) region of 30 + amino acids, which is a marker of the CHH subfamily [13], [29]. The novel identified protein MIHL in this study contained a typical crustacean neuropeptide domain but lacks the CPRP, and was clustered with other shrimp MIHs, thus could be classified as a novel member of the MIH subfamily.

Acknowledgements

This work was supported by National Natural Science Foundation of China under grant No. 31572649; China Postdoctoral Science Foundation 2015M580746; Natural Science Foundation of Guangdong Province, China 2014A030313125; China Agriculture Research System CARS47; The Key Fundamental Research Funds for the Central Universities of China 17lgzd27.