Elsevier

Fish & Shellfish Immunology

Volume 74, March 2018, Pages 268-280
Fish & Shellfish Immunology

Full length article
The immune response of Mytilus edulis hemocytes exposed to Vibrio splendidus LGP32 strain: A transcriptomic attempt at identifying molecular actors

https://doi.org/10.1016/j.fsi.2017.12.038Get rights and content

Highlights

  • Identification of immune system transcripts such as Toll-Like receptors (TLRs), cytokines, and protease inhibitors.

  • Vibrio splendidus LGP32 regulates expression of gene encoding for proteins involved in M. edulis immune system.

  • Tumor protein D54, serine/threonine-proteine kinase SIK2 were up-regulated in hemocytes exposed to V. splendidus.

Abstract

The marine mussel Mytilus edulis, tolerant to a wide range of environmental changes, combines a key role as a sentinel species for environmental monitoring programs and a significant economic importance. Mortality events caused by infective agents and parasites have not been described in mussels, which suggests an efficient immune system. This study aims at identifying the molecular mechanisms involved in the early immune responses M. edulis' hemocytes challenged with Vibrio splendidus LGP32 strain during 2, 4 and 6 h.

A total of 149,296 assembled sequences has been annotated and compared to KEGG reference pathways. Several immune related sequences were identified such as Toll-Like receptors (TLRs), transcription factors, cytokines, protease inhibitors, stress proteins and sequences encoding for proteins involved in cell adhesion, phagocytosis, oxidative stress, apoptosis and autophagy.

Differential gene expression clustered 10 different groups of transcripts according to kinetics of transcript occurrence. Sequences were assigned to biological process gene ontology categories. Sequences encoding for galectins, fibrinogen-related proteins, TLRs, MyD88, some antimicrobial peptides, lysosomal hydrolases, heat shock proteins and protease inhibitors, as well as proteins of oxidative stress and apoptosis were identified as differently regulated during the exposure to V. splendidus LGP32.

The levels of candidate transcripts were quantified in M. edulis' hemocytes exposed to V. splendidus LGP32 and 7SHRW by using branched DNA technology. Transcripts encoding for inhibitor kappa B, inhibitor of apoptosis proteins, tumor protein D54, serine/threonine-proteine kinase SIK2 were identified as up-regulated in hemocytes exposed to both strains.

Introduction

Bivalves, in particular mussels on the genus Mytilus, tolerant to a wide range of environmental changes, combine a key role as sentinel species for environmental monitoring programs and a significant economic importance because of the aquaculture production in many areas of the world [1,2]. Reports of mortality events and diseases caused by bivalve's pathogens, in particular members of the Gram-negative bacteria genus Vibrio, have increased during the last decades [[3], [4], [5], [6], [7], [8], [9], [10], [11], [12]]. For example, Vibrio splendidus strain LGP32 has been associated to massive mortality events in the production of Crassostrea gigas oysters in France [6].

The circulating hemocytes and a variety of molecular effectors provide the first line of defense against potential pathogens. Hemocytes participate directly in pathogen elimination by first phagocytosis and encapsulation and second, they produce humoral components including lysosomal enzymes, aminopeptidases, lectins and antimicrobial molecules that contribute to destroy pathogens [13,14]. Recently, it was shown that pathogenic V. splendidus strain affected M. edulis hemocyte functions by inhibiting cell adhesion and disrupting acidic vacuole formation [15,16].

The initial step of the innate system is the detection and recognition of foreign invaders. Unique and characteristic molecules present at the surface of microorganisms, such as lipopolysaccharides (Gram-negative bacteria) or peptidoglycans (Gram-positive bacteria), known as pathogen-associated molecular patterns (PAMP) can be recognized by hemocytes. The host cell recognizes the PAMP through lectins and membrane bound receptors, like toll-like receptors, which are referred as pathogen recognition receptors (PRR) [17]. Different types of lectins (C-type lectin, sialic acid binding lectin, fucolectin, galectin) have been characterized in Mytilus galloprovincialis [18]. The diversity of C-type lectin sequences may answer to the variety of pathogens. Therefore, C-type lectins are considered as PRR by some authors [[19], [20], [21]]. Also, many C1q-domain-containing (C1qDC) proteins can be classified as specialized class of pattern recognition proteins through the expanding interaction properties of the trimeric globular domain gC1q [20,22]. In addition, toll-like receptors (TLR-2 homolog and CgToll-1) have been identified in hemocytes of Mya arenaria and C. gigas, respectively [23,24] and the concept that TLRs recognize specific molecular patterns in various pathogens has been established [25]. Different molecules are involved in the TLR pathway: MyD88 is a Toll/IL-1 receptor (TIR) domain-containing adaptor modulating TLR pathway by interacting with IL-1 receptor associated kinase (IRAK). IRAK associates with TRAF6 and then activates NF-κB pathway [25,26].

Once activated by interaction between PAMPs and PRR, hemocytes display chemotactic and chemokinetic reactions and carry out cell-mediated defense reactions such as phagocytosis and activation of a variety of cytotoxic reactions like release of lysozomal enzymes and antimicrobial peptides (AMPs) [27,28]. For example, TLR pathway regulates chemokine and antimicrobial release in the hemolymph [29]. Antimicrobial peptides known as cysteine-rich peptides, can destroy bacteria by destabilizing their membrane permeability [30]. In mussels, four groups of AMPs (defensins, mytilins, myticins and mytimycins) have been identified and characterized [31]. These AMPs have specific and complementary antimicrobial activities. Defensins and myticins are more active against Gram-positive bacteria than against the Gram-negative. Mytimicins are exclusively anti-fungal [32]. Mytilins act both on Gram-negative and Gram-positive bacteria, including vibrios [31,33].

Associated with the phagocytic activity, the NADPH oxidase as well as nitric oxide (NO) synthase are activated leading then to the production of reactive oxygen species (ROS) and NO enabling the oxidation of the foreign invaders [[34], [35], [36], [37], [38]]. Excessive production of toxic radicals may lead to several negative effects such as lipid peroxidation, DNA damage, loss of cellular function, and ultimately apoptosis and necrosis [39]. To protect themselves from damage caused by toxic radicals, organisms use antioxidants, such as superoxide dismutases (SOD) and gluthatione peroxidase (GPx) to eliminate these free radicals by converting them to less toxic compounds [40].

Several studies have focused on understanding the molecular mechanisms of hemocytes challenged with different Vibrio, in particular V. splendidus LGP32 strain [20,24,27,[41], [42], [43], [44]]. Differential gene expression levels associated with immune responses (i.e. antimicrobial peptides and lysozyme genes) was found in M. galloprovincialis hemocytes exposed to V. splendidus LGP32 [20,27,44]. Furthermore, Araya et al. [43] identified differentially expressed immune genes in M. arenaria hemocytes exposed in vitro to V. splendidus LGP32 using a Suppression Subtractive Hybridization (SSH) approach. These authors found that ficolin, killer cell lectin-like receptor, natural resistance-associated macrophage protein 1 (Nramp-1), mitogen-activated protein kinases (MAPK), ferritin, heat shock proteins 90 (HSP90) and cathepsin exhibited similar gene expression patterns: an up-regulation at 1 h, followed by a down-regulation at 2 and 3 h following bacterial infection. Moreover, an exposition in vivo to V. splendidus LGP32 induces a down-regulation of Toll-like receptor 2 (TLR-2) and an up-regulation of interleukin 1 receptor-associated kinase 4 (IRAK-4) in hemocytes of M. arenaria [44]. Also, some immune related genes, which play an important role in pathogen recognition, destruction, elimination and detoxification (C1qDC, lysozyme, defensin, mytilin B, multidrug resistance associated protein, proteasome 26S, cyclooxygenase, SOD and GPx) were regulated in M. edulis hemocytes exposed to V. splendidus LGP32 strain [41]. These studies demonstrated that V. splendidus LGP32 has the capacity to regulate the expression of the genes involved in innate immunity of bivalve mollusks during the first hours of the bacterial challenge.

Understanding how the biological systems of mollusk respond to microbiological challenges is an opportunity to investigate and elucidate the basic physiological principles that rule the defense mechanisms in Mollusks [45] and is essential for both scientific and aquaculture researches. Next generation sequencing techniques, in particular RNA-seq technology, offer novel and rapid opportunities to describe the transcriptome and unravel molecular mechanisms involved in innate immune system of marine bivalves. Taking advantage of these new technologies, the goal of this study is to identify molecular actors involved in the early hemocyte's responses and provide new information on the molecular mechanisms by which the hemocytes defend themselves against the pathogens.

In our previous works, the pathogenicity of V. splendidus LGP32 for Mytilus edulis hemocyte was studied. We examined the kinetics of morphological (non-adherent hemocyte) and functional (phagocytic activity and oxidative burst activation) responses of M. edulis' hemocytes exposed to different strains of V. splendidus [41]. Also, in order to determine the transcriptome of M. edulis' hemocyte exposed to V. splendidus, we constructed and sequenced, using 454 pyrosequencing, a normalized cDNA library specific to M. edulis hemocytes unchallenged (control) and challenged with V. splendidus LGP32 strain during 2, 4 and 6 h [42]. This current study aims at identifying the regulated gene clusters during bacterial challenge using RNAseq technology. Expression of candidate genes playing a key role in the innate immunity were quantified using the multiplex DNA branched technology during the first hours of exposure to both V. splendidus LGP32 and 7SHRW strains.

Section snippets

Mussels and hemolymph collection

Adult blue mussels, Mytilus edulis (3–5 cm in shell length) were sampled from Prince Edward Island (Gulf of Saint Lawrence, Canada). Mussels were kept and maintained in a 300 l tank with re-circulating artificial seawater (Instant Ocean®) at a temperature of 16–17 °C and a salinity of 30 ppt. Animals were fed with Spat Formula (Innovative Aquaculture Products Ltd., Canada) every day.

Hemolymph (1–5 ml from each individual) was withdrawn from the adductor muscle using 3 ml syringes fitted with 25

Illumina sequencing and reads assembly

cDNAs from hemocytes of M. edulis exposed to V. splendidus LGP32 during 0 (control), 2, 4 and 6 h were sequenced with the Illumina HiSeq 2000 sequencing technology. A total of 141,241,930 nucleotide reads were generated with an average of 35,310,483 nucleotides reads for each condition (0, 2, 4 and 6 h). After a trimming step, all the generated reads were submitted to Velvet-Oases assemblies. A total of 149,296 assembled sequences were generated with an average length of 700 bp. The shortest

Conclusion

In this study, more than 149,200 assembled sequences has been annotated and molecular mechanisms were identified using KEGG reference pathways. Among the identified molecular pathways, immune related molecular mechanisms involving Toll-Like receptors (TLRs), transcription factors, cytokines, protease inhibitors, stress proteins and sequences encoding for proteins involved in cell adhesion, phagocytosis, oxidative stress, apoptosis and autophagy were identified and evaluated by both RNAseq and

Acknowledgements

The authors thank Dr. Frédérique Le Roux (IFREMER) for providing the bacterial strain LGP32. This program and the doctoral fellowship of Marion Tanguy were supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and Institute of Marine Science (University of Quebec at Rimouski), PEI Innovation and the Canadian Fund for Innovation.

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