Distinct immune responses of juvenile and adult oysters (Crassostrea gigas) to viral and bacterial infections

Green et al. Vet Res (2016) 47:72 DOI 10.1186/s13567-016-0356-7 Open Access RESEARCH ARTICLE Distinct immune responses of juvenile and adult oysters (Crassostrea gigas) to viral and bacterial infections Timothy J. Green1,2, Agnes Vergnes1, Caroline Montagnani1* and Julien de Lorgeril1 Abstract Since 2008, massive mortality events of Pacific oysters (Crassostrea gigas) have been reported worldwide and these disease events are often associated with Ostreid herpesvirus type 1 (OsHV-1). Epidemiological field studies have also reported oyster age and other pathogens of the Vibrio genus are contributing factors to this syndrome. We undertook a controlled laboratory experiment to simultaneously investigate survival and immunological response of juvenile and adult C. gigas at different time-points post-infection with OsHV-1, Vibrio tasmaniensis LGP32 and V. aestuarianus. Our data corroborates epidemiological studies that juveniles are more susceptible to OsHV-1, whereas adults are more susceptible to Vibrio. We measured the expression of 102 immune-genes by high-throughput RT-qPCR, which revealed oysters have different transcriptional responses to OsHV-1 and Vibrio. The transcriptional response in the early stages of OsHV-1 infection involved genes related to apoptosis and the interferon-pathway. Transcriptional response to Vibrio infection involved antimicrobial peptides, heat shock proteins and galectins. Interestingly, oysters in the later stages of OsHV-1 infection had a transcriptional response that resembled an antibacterial response, which is suggestive of the oyster’s microbiome causing secondary infections (dysbiosis-driven pathology). This study provides molecular evidence that oysters can mount distinct immune response to viral and bacterial pathogens and these responses differ depending on the age of the host. Introduction The Pacific oyster, Crassostrea gigas forms the basis of an important global aquaculture industry with one of the largest annual productions of any marine animals. The Pacific oysters is cultivated on all continents, except Antarctica [1]. Oysters are typically reared in the open environment and are therefore vulnerable to the adverse impacts of disease. Historically, C. gigas were chosen in many countries for aquaculture because they were nonnative and naturally resistant to many of the protozoan parasites (Bonamia spp. and Marteilia spp.) that have decimated aquaculture production of indigenous oyster species [2, 3]. Sporadic mortality events of adult C. gigas have occurred in many countries over the last four decades [4–6]. These mortality events were often correlated *Correspondence: 1 IFREMER, IHPE, UMR 5244, Univ. Perpignan Via Domitia, CNRS, Univ. Montpellier, 34095 Montpellier, France Full list of author information is available at the end of the article with elevated seawater temperatures and physiological stresses associated with maturation and spawning [reviewed by 5, 6]. No specific pathogen was routinely isolated from these sporadic mortality episodes of C. gigas [reviewed by 6]. Surveys often isolated viruses belonging to Ostreid herpesvirus type I (OsHV-1) and bacteria belonging to the Vibrio genus [7, 8]. V. splendidus-related strains have been isolated from moribund oysters during mortality events [9] and have been widely described regarding their pathogenicity to bivalves. V. aestuarianus has been detected in epidemiologic surveys in oysters and the environment since 2001 [8]. OsHV-1 has been detected in mass mortality outbreaks in hatcheries and in epidemiological surveys since 1993 [10]. Since 2008, mass mortality episodes of C. gigas have dramatically affected juvenile oysters with high intensities and a wide geographic distribution [7, 11]. This syndrome has been associated with the presence of a particular genotype of Ostreid herpesvirus type 1 (OsHV-1), © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Green et al. Vet Res (2016) 47:72 Page 2 of 11 Europe [27] allowing us to compare their transcriptomic patterns. We made sure to use oysters from a multiparental breeding program to avoid emphasizing any genetic impact on a potential variability of susceptibility on viral and bacterial infections. As mentioned earlier, adult and juvenile animals also share similar life traits of life regarding our trait of interest (i.e. pathogen exposure and defense systems) as they were all grown in hatchery structures and never faced mortality events. Prior to experimentation, juvenile and adult C. gigas were tested for OsHV-1 and its variants by qPCR according to [28]. No viral DNA was detected using real time PCR analyses on a sample of 10 individual juvenile and adult animals. termed μVar [12]. Mortality associated with this variant was first detected in Europe in 2008 [12], but a closely related genotype has since been associated with mortality of C. gigas in New Zealand and Australia in 2010–2011 [13, 14]. In Europe, co-detection of the variant and different Vibrio species, including Vibrio tasmaniensis LGP32 and V. aestuarianus have been reported during mortality events of C. gigas [7]. These Vibrio species are also considered important pathogens in bivalve aquaculture and may be a contributing factor in the mass mortality episodes [8, 15]. Host physiology and ontogeny is another key determinant in the mass mortality episodes of C. gigas. Younger age classes of C. gigas are more susceptible to OsHV-1 infection [16, 17], whereas older age classes are reported to be more susceptible to V. aestuarianus [8]. Thus, determining how host development influences the immunological response and survival of C. gigas is a major goal in understanding the recent mass mortality episodes associated with OsHV-1 and different Vibrio species. The immunological response of C. gigas against pathogenic viruses and Vibrio bacteria are well documented [18–25]. However, most of these studies focused on a single developmental stage and type of pathogen. To date there has not been a controlled laboratory study to simultaneously investigate the effect of oyster age on the immunological response and survival of C. gigas infected with three different pathogens. In this study, we address this question by using high-throughput RT-qPCR to measure the immunological response of juvenile and adult C. gigas at different time-points post-infection with OsHV-1, Vibrio tasmaniensis LGP32 and V. aestuarianus. Ostreid herpe (...truncated)