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
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.
References (77)
- et al.
Temperature-dependent stress response in oysters, Crassostrea virginica: pollution reduces temperature tolerance in oysters
Aquat Toxicol
(2006) - et al.
A survey of oysters Crassostrea virginica from Tampa Bay, Florida: associations of internal defense measurements with contaminant burdens
Aquat Toxicol
(2000) - et al.
Relationships between tissue contaminants and defense-related characteristics of oysters (Crassostrea virginica) from five Florida bays
Aquat Toxicol
(2001) - et al.
Superoxide anion generation by Crassostrea virginica hemocytes as measured by nitrotetrazolium reduction
J Invertebr Pathol
(1992) - et al.
Apoptosis as a host defense mechanism in Crassostrea virginica and its modulation by Perkinsus marinus
Fish Shellfish Immunol
(2010) - et al.
Oyster metallothionein as an oxyradical scavenger: implications for hemocyte defense responses
Dev Comp Immunol
(1999) - et al.
Identification and tissue expression analysis of C-type lectin and galectin in the Pacific oyster, Crassostrea gigas
Comp Biochem Physiol B Biochem Mol Biol
(2008) - et al.
Expression analysis of the acute phase response in channel catfish (Ictalurus punctatus) after infection with a Gram-negative bacterium
Dev Comp Immunol
(2007) - et al.
Microarray analysis of gene expression in the blue catfish liver reveals early activation of the MHC class I pathway after infection with Edwardsiella ictaluri
Mol Immunol
(2008) - et al.
Genetic variation of wild and hatchery populations of the pacific oyster Crassostrea gigas assessed by microsatellite markers
J Genet Genomics
(2007)
Discovery of genes expressed in response to Perkinsus marinus challenge in Eastern (Crassostrea virginica) and Pacific (C. gigas) oysters
Gene
Single Nucleotide polymorphisms and their relationship to codon usage bias in the Pacific oyster Crassostrea gigas
Gene
Evaluation of methods using ray’s fluid thioglycollate medium for diagnosis of Perkinsus marinus infection in the eastern oyster, Crassostrea virginica
Annu Rev Fish Dis
Oxygen free radicals and metallothionein
Free Radic Biol Med
Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals
Biochim Biophys Acta
Increase in metallothionein produced by chemicals that induce oxidative stress
Toxicol Appl Pharmacol
Cytoplasmic metallothionein overexpression protects NIH 3T3 cells from tert-butyl hydroperoxide toxicity
J Biol Chem
Enhanced sensitivity to oxidative stress in cultured embryonic cells from transgenic mice deficient in metallothionein I and II genes
J Biol Chem
Inhibition of hydroxyl-radical-generated DNA degradation by metallothionein
Toxicol Lett
Suppression of gastric ulcer induced by stress and HCL-ethanol by intravenously administered metallothionein-II
Biochem Biophys Res Commun
Metallothionein protects retinal pigment epithelial cells against apoptosis and oxidative stress
Exp Eye Res
Human cathepsin S: chromosomal localization, gene structure, and tissue distribution
J Biol Chem
Molecular characterization and expression analysis of cathepsin L1 cysteine protease from pearl oyster Pinctada fucata
Fish Shellfish Immunol
TAJ, a novel member of the tumor necrosis factor receptor family, activates the c-Jun N-terminal kinase pathway and mediates caspase-independent cell death
J Biol Chem
Reduced expression of DRAM2/TMEM77 in tumor cells interferes with cell death
Biochem Biophys Res Commun
Calcium binding and conformational response in EF-hand proteins
Trends Biochem Sci
Characterization and expression analysis of EF hand domain-containing calcium-regulatory gene from disk abalone: Calcium homeostasis and its role in immunity
Fish Shellfish Immunol
Increasing genomic information in bivalves through new EST collections in four species: development of new genetic markers for environmental studies and genome evolution
Gene
A catalogue of selected species of living oysters (Ostreacea) of the world
Influence of simulated bivalve biodeposition and microphytobenthos on sediment nitrogen dynamics: a laboratory study
How habitat degradation through fishery disturbance enhances impacts of hypoxia on oyster reefs
Ecol Appl
Historical overfishing and the recent collapse of coastal ecosystems
Science
Growth and mortality of eastern oysters, Crassostrea virginica (Gmelin, 1791), and Pacific oysters, Crassostrea gigas (Thunberg, 1793) under challenge from the parasite, Perkinsus marinus
J Shellfish Res
Dermocystidium marinum infection in oysters
Mar Fish Rev NMFS
Perkinsus gen. n. and other new taxa in the protozoan phylum Apicomplexa
J Parasitol
Diseases and defense mechanisms in the Eastern Oyster Crassostrea virginica
Epizootiology of Perkinsus marinus disease of oysters in Chesapeake Bay, with emphasis on data since 1985
J Shellfish Res
Interactions of Perkinsus marinus with humoral factors and hemocytes of Crassostrea virginica
J Shellfish Res
Cited by (61)
A fas apoptotic inhibitory molecule from Ruditapes philippinarum: Investigation on molecular characterization and functional analysis
2020, Fish and Shellfish ImmunologyRegulation of apoptosis-related genes during interactions between oyster hemocytes and the alveolate parasite Perkinsus marinus
2018, Fish and Shellfish ImmunologyComparative immunogenomics of molluscs
2017, Developmental and Comparative Immunology