Genome-wide identification, characterization of RLR genes in Yesso scallop (Patinopecten yessoensis) and functional regulations in responses to ocean acidification

Highlights

• Four RLR family members were identified in Patinopecten yessoensis genome.

• Phylogenetic analysis confirmed duplication and evolutionary relationship of PyRLRs.

• Spatiotemporal expression patterns suggested the functional roles of PyRLRs.

• Constitutive and inducible divergence of PyRLRs has been arisen to the immune stress.

• The four PyRLRs showed functional differentiations in response to OA.

Abstract

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), are crucial sensors with a conserved structure in cytoplasm, inducing the production of cytokines, chemokines and host restriction factors which mediate a variety of intracellular activities to interfere with distinct PAMPs (pathogen-associated molecular patterns) for eliminating pathogens in innate immune system. Although RLR genes have been investigated in most vertebrates and some invertebrates, the systematic identification and characterization of RLR genes have not been reported in scallops. In this study, four RLR genes (PY-10413.4, PY-10413.5, PY-443.7 and PY-443.8, designated PyRLRs) were identified in Yesso scallop (Patinopecten yessoensis) through whole-genome scanning through in silico analysis, including two pairs of tandem duplicate genes located on the same scaffold (PY-10413.4 and PY-10413.5, PY-443.7 and PY-443.8, respectively). Phylogenetic and protein structural analyses were performed to determine the identities and evolutionary relationships of these genes. The expression profiles of PyRLRs were determined in all developmental stages, in healthy adult tissues, and in mantles that simulated ocean acidification (OA) exposure (pH = 6.5 and 7.5) at different time points (3, 6, 12 and 24 h). Spatiotemporal expression patterns suggested the functional roles of PyRLRs in all stages of development and growth of the scallop. Regulation expressions revealed PY-10413.4 and PY-10413.5 with one or two CARD(s) (caspase activation and recruitment domain) were up-regulated expressed at most time points, whereas PY-443.8 and PY-10413.4 without CARD were significantly down-regulated at each time points, suggesting functional differentiations in the two pairs of PyRLRs based on the structural differences in response to OA. Collectively, this study demonstrated gene duplication of RLR family genes and provide primary analysis for versatile roles in the response of the bivalve innate immune system to OA challenge.

Introduction

As the first-line of host defense that serves to resist against pathogens invasion, the innate immunity acts an evolutionary, conserved and significant defense role in immunity responses [1,2]. In invertebrates such as bivalves, the status of innate immunity is vital important due to the lack of adaptive immunity [3]. Innate immune recognition is mediated by a range of structurally distinct receptors that are predominantly expressed referred to as pattern recognition receptors (PRRs) recognizing with various pathogen-associated molecular patterns (PAMPs) [4,5]. To counteract pathogens invasion, a series of PRRs immediately get initiation, including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), nucleotide-binding oligomerization domain (NOD)-like protein receptors (NLRs), retinoic acid inducible gene I-like receptors (RLRs), etc. Among these PRRs families, the RLRs which composed of RIG-I (retinoic acid inducible gene I) [6], MDA5 (melanoma differentiation associated gene 5) [7] and LGP2 (laboratory of genetics and physiology 2) [8] that associated in TLR and NOD signaling pathway, co-ordinately triggered interferon response for eliminating the invading pathogens [9]. All members involved in RLRs family share a common functional RNA helicase superfamily c-terminal domain (HELICc) that has capacity of binding to the RNA molecules with viral origin. Two tandem caspase activation and recruitment domains (CARDs) at N-terminal of RIG-I and MDA5 proteins but not LGP2, trigger the interferon response via activation of interferon regulatory factor 3 (IRF3) and NF-κB [10]. As the major sensors to detect invading pathogens in cytoplasm, RLRs trigger immune defense program by inducing the production of cytokines, chemokines and host restriction factors that interfere with distinct PAMPs [11].

RLRs belong to RNA helicases family and have been found in almost all organisms. RIG-I was first identified as a gene induced by retinoic acid in promyelocytic leukemia cells in 1997 [12]. Subsequent studies on RLR family members have been investigated in vertebrates and invertebrates. Nowadays, three distinct RLR members including RIG-I, MDA5, LGP2 have been demonstrated and systematically analyzed through human and mouse genomes [13], two RLRs with the absence of RIG-I were found in Gallus gallus genome [14,15], and three RLRs were genome-wide identified from Danio rerio [[16], [17], [18]]. However, the number of RLR genes in most invertebrates is varied, with three homologs (DRH-1, DRH-2, DRH-3) in Caenorhabditis elegans genome [19], only one DjRIG-I in Dugesia japonica [20] and single LGP2 in Ciona intestinalis [21]. Similarly, in marine invertebrates Crassostrea gigas, one homolog named CgRIG-I was cloned, and then thirteen putative RLR genes were predicted in genome level [22,23]. In addition, a single RIG-I was found in Mytilus galloprovincialis genome [24]. Furthermore, twelve genes referred as potential homologous of RLRs were found by database searches of purple sea urchin Strongylocentrotus purpuratus genome [25]. This dramatic difference in gene copy number makes RLRs(-like) genes a potential ideal model for exploring innate immune divergence and gene duplication in vertebrates and invertebrates.

Indeed, numerous researches have been conducted about the biological function of RLRs on pathogens response in the innate immune system [[26], [27], [28]]. In vertebrates, RIG-I was found to be acted as an important role in vascular pathology after human endothelial cells treated with lipopolysaccharide (LPS) [27]. RIG‐I knock out mice (Mus musculus) showed an increase in effector T cells and down-regulation of G protein αi2 subunit (Gαi2) in various tissues which are potential factors associated with colitis [29]. In addition, RLRs are also reported as required for the innate immune response to viral hemorrhagic septicemia and ictalurid herpesvirus 1 in channel catfish, Ictalurus punctatus [30]. Compared to the role in vertebrates, RLRs were broadly reported as one of the pivotal components in RIG-like receptor signaling pathway in invertebrates, such as Dugesia japonica, and the transcription level of DjRIG-I were all up-regulated after in vivo challenges with gram-negative (Pseudomonas aeruginosa) and gram-positive (Staphylococcus aureus) bacteria [20]. In bivalves, CgRIG-I expression showed an abrupt increase after stimulation by LPS, peptidoglycan (PGN), heat-killed Listeria monocytogenes (HKLM) and heat-killed Vibrio alginolyticus (HKVA), especially noticeable by poly (I:C) stimulation [22]. RIG-I from mussel (M. coruscus) was also significantly induced in hemocytes after Vibrio alginnolyficus challenge [31].

Although the identification and importance of RLRs in innate immune regulation have been reported in both vertebrates and invertebrates, systematic analysis of the RLRs has not been carried out in scallops, particularly in Yesso scallop (Patinopecten yessoensis), one of the most economic aquaculture species cultured in northern China and consumed worldwide [32]. In the last decade, elevated level of atmospheric CO₂ penetrated into the oceans resulted the ocean acidification (OA), and has always been a major global problem that caused great economic losses in scallop aquaculture industry [33,34]. Substantial evidence has proved that elevated partial pressure of carbon dioxide (pCO₂)-levels negatively affected fertilization [35], metabolism [36], behavior [37], immune function [38,39] and even survival [40] of economic marine mollusks. For instance, the fertilization rate and sperm velocity of Tegillarca granosa fell by a third and two-fifths respectively when pH decreased from 8.1 to 7.4 [35]. Continuous exposure to acidified seawater led to an obvious decline of byssal threads number in mussel (M. coruscus), making the mollusk decrease in attachment strength [36]. In another simulated acidification experiment, razor clam (Sinonovacula constricta) presented significant reduction in digging depth if sea water pCO₂ elevated [37]. Specifically, elevated pCO₂ significantly suppressed almost all candidate genes related to immune systems of the Pacific oyster (C. gigas), which make it vulnerable to diseases [38]. In addition, when blood clam (Tegillarca granosa) were exposed to pCO₂ acidified seawater, the inducer and key response genes of NF-κB signaling and toll-like receptor pathways generally performed a down-regulated expression pattern in which process depressed regulation of RIG-I displayed [41]. All investigations above demonstrated that OA served as a potential threat to immune response of bivalves and made them more vulnerable to pathogens’ attack. However, the knowledge about how OA impacts the immunity of scallops remains limited. The aim of this paper was to provide rudiment understanding of RLRs in the complex innate immune regulation system of Yesso scallop and help in facilitating the long-term sustainable development of scallop industry.