Like a virginica... touched for the very first time.

Identification of potential general markers of disease resistance in American oysters, Crassostrea virginica through gene expression studies.

Nikapitiya et al., (2014).

The American oyster, Crassostrea virginica, is an economically and ecologically important bivalve species in the Atlantic coasts of North America and the Gulf of Mexico. Development of intensive harvesting has led to reduced water quality, disease and habitat degradation, and therefore, significant declines of this species. During the larval and juvenile stages, oysters are susceptible to bacterial pathogens and have been described as the cause of mass mortality events in this species. Several Vibrio species can cause extensive bivalve larval mortality in hatcheries in many shellfish growing regions within hours of exposure. Probably the most well known bacterium is Roseovarius crassostreae, also a causative agent of juvenile oyster disease, being coined as Roseovarius oyster disease (ROD). This bacterium affects the juvenile stages of oysters (< 25 mm in shell length), leading to high seasonal mortality rates. However, adult oysters are susceptible to disease also; the protistan parasite Perkinsus marinus is a major cause of mortality in adult C. virginica. Several approaches are used to manage oyster diseases; careful site selection, frequent monitoring of shellfish condition and water quality to name a few. Knowledge of environmental conditions that can trigger disease outbreaks affecting host susceptibility help schedules the timing of deployment and harvesting. Several lines of oysters resistant to disease have been produced by traditional selective breeding practices, however, little is known about the mechanisms underlying the disease resistance.

Recently, cDNA microarray technology and transcriptomic analyses have been used to characterise gene expression patterns in tissues of Crassostrea gigas and C. virginica exposed to several environmental stressors; bacteria, protozoans, hypoxia, pesticides and hyperthermia. Analysis of gene expression patterns in resistant and susceptible oysters (RSOs) in response to these challenges could provide potential biomarkers and would be a useful tool in environmental monitoring and disease management. Previous work (McDowell et al., 2014) provided a list of gene candidates that could be associated with disease resistance or susceptibility to ROD and were used in this study. By identifying genes involved in the early responses of oysters to the protozoan parasite, P. marinus and by focusing on several novel and previously annotated candidate genes, Nikapitiya et al., (2014) were attempting to provide insight into the immune responses of RSOs to a variety of pathogenic challenges and ultimately, identification of biomarkers associated with disease in C. virginica.

Donated bacterial and parasitic isolates were cultured overnight at 28 °C in 10 mL YP3 broth with agitation, following centrifugation and re-suspension. Suspensions were diluted to the required dose to be used in three challenge experiments; Roseovarius crassostreae, Vibrio tubiashii and Perkinsus marinus. Depending on the bacterium that the C. virginica was being exposed to, varied methods were used during the three challenges regarding concentration, exposure and RNA extraction time. RNA isolation and cDNA synthesis was performed by selecting random samples and then being diluted and kept at - 20 °C until used for gene expression studies. To evaluate the gene expression, quantitative real time-PCR (qRT-PCR) was performed in duplicate for each gene. For the R. crassostreae challenge, a t-test was used to compare levels of fold-changes in gene expression relative to unchallenged controls between RSOs. Log-rank survival analysis was used to analyse differences in survival curves between oyster families. Differences in cumulative percent mortality between susceptible and control oysters at selected time points were also determined using Pearson's chi-squared test with Bonferroni corrections. Two-way ANOVA was used to determine effect of time and treatment on gene expression. Independent sample t-test was performed to compare levels of gene expression for each gene between controls and experimentally challenged oysters at each time point. For the P. marinus challenge experiment, multivariate analysis of variance (MANOVA) was performed for at all-time points to determine relationships between levels of gene expression for all genes and P. marinus spore numbers in hemolymph.

The overall results (far too many to mention in this review) of this study has led to the identification of candidates for general markers (applicable to multiple diseases) of disease resistance; serine protease inhibitor (SPIs). Alongside this, biomarkers that may be associated with an unsuccessful immune response in oysters, has been determined by the inability of the oysters showing high levels of expression of heat shock protein 70 (HSP70) and histone H4 (HIS4). Nikapitiya et al., (2014) tried to overcome the complexity in oyster responses to environmental and pathogenic challenges as well as biological variability through several strategies. SPIs are important in oyster immune defences, probably by direct inhibition of pathogen proteases. Patterns of gene expression of HSP70 and HIS4, showing that oysters with the highest levels of expression of these genes on day one after P. marinus challenge suffered the highest P. marinus load at the end of the challenge, confirm previous findings suggesting that these molecules, in particular HSP70, may be associated with disease susceptibility, and could be used as biomarkers of stress and/or an unsuccessful immune response. The results from the R. crassostreae challenge, showing significantly higher levels of HSP70 in susceptible oysters than in resistant oysters, as well as the results from the V. tubiashii challenge in adult oysters, showing no significant changes in HSP70 or HIS4 expression in oysters able to clear this bacterial infection, supports this hypothesis.

The end result of Nikapitiya et al’s., (2014) study provides further information on the role of several molecules in immune responses in oysters and providing further evidence supporting the potential use of SPIs as a general marker of resistance to bacterial and parasitic diseases, and simultaneously, the use of HSP70 and HIS4 as markers of disease susceptibility in oysters. I believe that this study will provide the foundations in how the culture of American oysters will be regulated for future stocks by identifying markers for selection and insights into immune responses in oysters.

References

McDowell, I.C., Nikapitiya, C., Aguiar, D., Lane, C.E., Istrail, S., & Gomez-Chiarri, M. (2014) 'Transcriptome of American oysters, Crassostrea virginica, in response to bacterial challenge: insights into potential mechanisms of disease resistance' PLoS ONE 9(8): e105097. doi:10.1371/journal.pone.0105097

Nikapitiya, C., McDowell, I.C., Villamil, L., Munoz, P., Sohn, S., & Gomez-Chiarri, M. (2014) 'Identification of potential general markers of disease resistance in American oysters, Crassostrea virginica through gene expression studies' Fish & Shellfish Immunology 41: 27-36.