Elsevier

Fish & Shellfish Immunology

Volume 29, Issue 6, December 2010, Pages 921-929
Fish & Shellfish Immunology

Microarray analysis of gene expression in eastern oyster (Crassostrea virginica) reveals a novel combination of antimicrobial and oxidative stress host responses after dermo (Perkinsus marinus) challenge

https://doi.org/10.1016/j.fsi.2010.07.035Get rights and content

Abstract

Dermo disease, caused by Perkinsus marinus, is one of the most severe diseases of eastern oysters, Crassostrea virginica. It causes serious mortalities in both wild and aquacultured oysters. Using existing expressed sequence tag (EST) resources, we developed a 12K in situ oligonucleotide microarray and used it for the analysis of gene expression profiles of oysters during the interactions between P. marinus and its oyster host. Significant gene expression regulation was found at day 30 post-challenge in the eastern oyster. Putative identities of the differentially expressed genes revealed a set of genes involved in several processes including putative antimicrobial defenses, pathogen recognition and uptake, anti-oxidation and apoptosis. Consistent with results obtained from previous, smaller-scale experiments, expression profiles revealed a large set of genes likely involved in an active mitigating response to oxidative stress and apoptosis induced by P. marinus. Additionally, a unique galectin from C. virginica, CvGal, which serves as a preferential receptor for P. marinus trophozoites, was found to be significantly down-regulated in gill tissue of oysters with both light and heavy infection, suggesting an attempt to control parasite uptake and proliferation in the later stages of infection. Potential histone-derived antimicrobial responses to P. marinus were also revealed in the gene expression profiles.

Introduction

The eastern oyster, Crassostrea virginica, is an economically important species. It is widely fished and cultured in the Gulf of Mexico and along the eastern coast of the United States [1]. Oysters also serve as a fundamental component of the aquatic ecosystem by creating habitat and removing phytoplankton and silt from the water column [2]. The eastern oyster has been used as a marine bivalve model to study the effects of environmental stressors [3] and as a bioindicator of estuarine pollution [4], [5]. However, eastern oyster populations have been threatened by disease as well as overfishing and habitat degradation [6], [7]. Epizootics of dermo disease have become one of the major threats to oysters because they result in massive mortalities in both wild and aquaculture populations [8].

Dermo disease (also known as Perkinsosis) is caused by Perkinsus marinus, a protozoan parasite belonging to the Alveolates [9], [10]. As infections develop in eastern oysters, P. marinus causes reduction in shell growth, decreased hemolymph protein concentrations and lysozyme activities, severe emaciation, inhibition of gonadal development and reproductive output, and ultimate death [11]. In spite of the devastating impact caused by the parasite [12], the mechanisms of pathogenicity and the physiological and defense responses of the host are still poorly understood [13].

One of the better studied aspects of host responses to P. marinus infection is the oxidative stress response. During infection, large quantities of cytotoxic oxidants are released, such as reactive oxygen species (ROS), which are responsible for eliminating some parasite species [14]. However, the ROS released in response to the parasite is converted to hydrogen peroxide (H2O2) by SOD, which can increase levels of other highly toxic species, such as hydroxyl radical (OH), or hydroxylchlorous acid [15]. These reactive intermediates cause oxidative stress in oysters. In mammalian systems, severe oxidative stress can cause cell death and necrosis while moderate oxidative stress can trigger apoptosis [16]. Apoptosis induced by P. marinus infection has been reported in the oyster [17], [18]. In order to reduce the damage caused by oxidative stress and apoptosis, the host can generate anti-oxidant and anti-apoptotic agents, such as metallothioneins, which display oxyradical scavenging capacity and can reduce oxidative stress [19], [20].

Additional avenues of research on oyster immune responses have focused on the roles of lectins in pathogen recognition and handling. Several lectins have been identified from Crassostrea gigas [21], but did not show an induction in synthesis following bacterial infection. More relevant to this study, Tasumi and Vasta [22] have reported that a unique C. virginica galectin, CvGal, serves as a preferential hemocyte receptor for P. marinus, providing passive access to the host tissues and circulatory system via phagocytosis.

Recent development and application of microarray technology has allowed rapid progress in understanding gene expression after various treatments in several finfish and shellfish species [23], [24], [25]. For oysters, a number of genome resources have been developed recently, including a large number of polymorphic markers [26], [27], [28], [29], construction of a framework genetic linkage map [30], construction of large-insert BAC libraries [31], initial analysis of expressed sequence tags (EST) [32], [33], [34], [35], [36], [37], and construction of a first-generation cDNA microarray [23]. The application of these resources is growing. For example, a cDNA microarray was used recently to study differences in tolerance to heat shock among selectively bred lines of Pacific oysters [38]. Here we report the development of a 12K oligonucleotide microarray for the eastern oyster and application of this array to study the host response at the transcriptional level of the eastern oyster after challenge by P. marinus. Our results serve to reinforce and extend previous observations of oxidative stress/apoptosis-driven host responses while providing novel observations of the broader innate immune response.

Section snippets

Experimental oysters, disease challenge and sampling

The eastern oyster strain, NEH, was employed in the challenge (Rutgers University). The cohort used in our challenge experiment was produced, and reared to 16 months of age, on Martha’s Vineyard, Massachusetts, where dermo is less prevalent than at the Rutgers rearing site in New Jersey. Shell height of the oysters ranged from 32.2 to 104.7 mm with a mean of 69.4 mm (±14.2 sd). Because P. marinus is present in Massachusetts, a Time 0 sample of 23 oysters was examined prior to the challenge. All

P. marinus infection

After 30 days, the infected oysters were divided into light infection (score = 0 to 2.0) and heavy infection (score = 3.67 to 4.67) groups based on infection intensity (Table 2). Both light infection and heavy infection groups had similar prevalence (percentage of oysters infected): 98% and 96% respectively. Both groups were successfully infected and each group developed a wide range of infection intensities by day 30 group (light infection 1.34, ±0.23 SD; heavy infection 3.96, ±0.11 SD) (Table

Discussion

Oysters, while possessing a robust innate immune system, are acutely susceptible to infection by P. marinus trophozoites. Large gaps remain in our understanding of P. marinus infection routes and host immunity. Strategies aimed at producing effective treatments for dermo disease must be informed by an understanding of broader host responses and host–parasite interactions points. Towards this end, we have created and utilized a 12K oyster microarray to study regulation of gene expression

Acknowledgements

This project was supported by a grant from NOAA Sea Grant Gulf Oyster Industry Program awarded to ZL, RW, and XG. I Burt, E Scarpa and others at HSRL provided assistance with challenge experiments.

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