Elsevier

Gene

Volume 769, 15 February 2021, 145244
Gene

Research paper
Identification, characterization, and expression profiles of insulin-like peptides suggest their critical roles in growth regulation of the Pacific oyster, Crassostrea gigas

https://doi.org/10.1016/j.gene.2020.145244Get rights and content

Highlights

  • Four insulin-like peptide genes (ILPs) were characterized in the Crassostrea gigas.

  • The ILPs were expressed at higher levels in the fast-growing oysters.

  • Expressions of ILPs were greatly affected by food abundance and temperature.

Abstract

The insulin/insulin-like growth factor signaling (IIS) pathway is well-known in regulation of cell growth and proliferation in vertebrates, while its role in invertebrates such as mollusks remains largely unknown. In this study, we performed an extensive multi-omics data mining and identified four insulin-like peptide genes, including ILP, MIRP3, MIRP3-like and ILP7, in the Pacific oyster, Crassostrea gigas. Their potential roles in growth regulation were further investigated using the selectively bred fast-growing C. gigas variety “Haida No.1”. Expression profiling and in situ hybridization of these insulin-like peptides suggested their distinct tissue-specific expression pattern, with dominant expression in the neural enrichment tissues such as labial palp, visceral ganglia, adductor muscle, and digestive gland. The expressions of insulin-like peptides were significantly altered by food abundance in a gene-specific fashion. The expression of ILP was reduced during fasting and increased after re-feeding, the expressions of MIRP3 and ILP7 were generally induced during fasting and down-regulated after re-feeding, while the expression of MIRP3-like was firstly up-regulated and then down-regulated during the fasting and re-feeding process. Furthermore, the expressions of all four insulin-like peptide genes were significantly suppressed at low temperature, in accordance with the growth inhibition. These results indicated that all four insulin-like peptides would play critical but different roles in regulation of growth in the oysters. This work provides valuable information for further investigation on growth regulation mechanism in mollusks and molecular assisted breeding of growth with other production traits in the Pacific oyster.

Introduction

Growth is under control of both genetic and environmental factors in all organisms. At cellular level, the growth rate of an organism is determined by cell proliferation and growth. Therefore, several genes and pathways involved in cell proliferation and growth have been revealed to play indispensable roles in regulation of growth in many organisms, such as homeobox genes (Crickmore and Mann, 2008), and components of the Insulin signaling (Hyun, 2013), Wnt signaling (Wells et al., 2007), and TGF-β signaling (Dineen and Gaudet, 2014) pathways. Environmental factors, such as ambient temperature and nutrient level, are also critical to affect growth of organisms (Vijendravarma et al., 2012, Mortzfeld and Taubenheim, 2019).

Growth regulation in vertebrates has been well-studied, which is mainly under control of the growth hormone (GH) and insulin-like growth factors (IGFs) axis. The IGFs are regulated by the GH which is secreted by neuroendocrine cells of the anterior pituitary gland (Bianchi et al., 2017, Blum et al., 1993, Duan et al., 2010, Radcliff et al., 2005, Moore et al., 2019, Soliman et al., 2017). In addition, the insulin secretion from pancreatic β cells are acutely altered in response to levels of nutrients such as glucose, specific amino acids, free fatty acids and other hormones (Fu et al., 2013, Plum et al., 2006). Increased levels of circulating insulin finally activate the IIS pathway in responsive target tissues, leading to the promotion of nutrient uptake and storage (Fu et al., 2013, Nässel et al., 2015). However, in invertebrates such as mollusks, no GH have been identified, but the insulin-like peptides are reported to function in multiple biological process including the metabolisms of glucose, lipid, and amino acids in several mollusk species (Geraerts, 1976, Gomot et al., 1992, Satake et al., 1997, Duret et al., 1998, Gricourt et al., 2003, Gricourt et al., 2006, Hamano et al., 2005, Zhang and He, 2020).

The Pacific oyster (Crassostrea gigas) is one of the most widely farmed mollusks, which has been introduced from Asia to many other countries around the world (Zhu et al., 2016). Due to its economic importance, many genetic breeding programs have been established toward genetic improvement of growth rate. Genetic studies have also been performed and identified several QTL associated with growth traits (Li and Guo, 2004, Hedgecock et al., 2007, Wang and Li, 2017, Zhang et al., 2019). However, the identified growth-related QTL could only explain a limited portion of the phenotypic variation in the Pacific oyster. The growth of oyster in both soft body parts and the shell showed obvious seasonal variations with higher growth rate of soft tissue during spring and increased growth rate of shell in summer and autumn (Gricourt et al., 2003), suggesting a complex mechanism underlying growth. Previous studies have been performed toward identification of the genes participated in growth regulation of the Pacific oyster and reported a number of growth-related genes (Li et al., 2018), while the genes or pathways playing major roles remain unexplored. The association of insulin-like peptides with growth of the Pacific oysters has been suggested in previous studies (Gricourt et al., 2003, Hamano et al., 2005, Choi et al., 2018, Cherif-Feildel et al., 2019, Zhang and He, 2020), while genome-wide identification and functional characterization of these insulin-like peptide genes have not been conducted.

In this study, we performed an extensive multi-omics data mining and identified four insulin-like peptide genes, including ILP, MIRP3, MIRP3-like and ILP7, in the Pacific oyster genome. We further utilized the selectively bred fast-growing C. gigas variety “Haida No.1” as research model to investigate their potential involvement in growth regulation. Additionally, expression profiles of these insulin-like peptide genes were determined under conditions of food deprivation and low temperature, which further supported their critical roles in regulation of growth. This work provides valuable information for further investigation on growth regulation mechanism in mollusks and molecular assisted breeding of growth with other production traits in the Pacific oyster.

Section snippets

Sequence analysis

The amino acid sequences of insulin/insulin-like peptides from Homo sapiens, Gallus gallus, Xenopus laevis, Anolis carolinensis, Danio rerio, Limulus polyphemus, Musca domestica, Ctenocephalides felis, Mizuhopecten yessoensis, Octopus bimaculoid, Pomacea canaliculata, Crassostrea virginica and Crassostrea gigas were retrieved from the NCBI database followed by further manual curation and phylogenetic analysis. The detailed information of these sequences was provided in Supplementary Table 1.

Identification of insulin-like peptides in C. gigas

Four insulin-like peptide genes were identified in C. gigas, including insulin-like peptide (ILP), molluscan insulin-related peptide 3 (MIRP3), molluscan insulin-related peptide 3 like (MIRP3-like) and insulin-like peptide 7 (ILP7). Their gene names, sequence characteristics and accessions were provided in Table 1. Phylogenetic analysis suggested that the C. gigas ILP was clustered into one clade with ILP3 of C. virginica, which had a closer homology to the clade of insulin/insulin-like growth

Discussion

The insulin/insulin-like peptides are essential in regulation of cell growth and proliferation in vertebrates, while their roles in invertebrates such as mollusks remains largely unknown. In the present study, we conducted an extensive analysis of multi-omics data in the public database and identified four insulin-like peptide genes in the C. gigas. Phylogenetic analysis confirmed their identities and reconstructed their evolution relationships with insulin/insulin-like peptides from other

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the grants from National Natural Science Foundation of China (Nos. 31802293, 31741122 and 41976098), the Young Talent Program of Ocean University of China (No. 201812013), Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology (No. 2017-2A04), and China Postdoctoral Science Foundation (No. 2017M622283).

Author contributions

SL conceived and designed the study. YL, HF, FZ, LR, and JT collected the samples and executed the experiments. YL, HF and SL analyzed the data. YL drafted the manuscript, and SL revised the manuscript. QL provided reagents and materials and supervised the study. All authors have read and approved the final version of the manuscript.

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