The Eastern oyster: Crassostrea virginica

Abstract

A 3.2-kb fragment of Perkinsus marinus DNA was cloned and sequenced. A noncoding domain was identified and targeted for the development of a semiquantitative polymerase chain reaction (PCR) assay for the presence of P. marinus in eastern oyster tissues. The assay involves extracting total DNA from oyster hemolymph and using I g of that DNA as template in a stringent PCR amplification with oligonucleotide primers that are specific for the P. marinus 3.2-kb fragment. With this assay, we can detect 10 pg of total P. marinus DNA per 1 g of oyster hemocyte DNA with ethidium bromide (EtBr) staining of agarose gels, 100 fg total P. marinus DNA with Southern blot autoradiography, and 10 fg of total P. marinus DNA with dot- blot hybridizations. We have used the sensitivity of the PCR assay to develop a method for estimating the level of P. marinus DNA in oyster hemolymph and have successfully applied this technique to gill tissues. Our semiquantitative assay uses a series to essentially titrate the point at which a P. marinus DNA target is no longer amplified in a sample. We refer to this technique as dilution endpoint PCR. Using hemocytes obtained by withdrawing a 1-ml sample of hemolymph, this assay provides a nondestructive methodology for rapidly screening large numbers of adult oysters for the presence and quantification ofP. marinus infection levels. This technique is applicable to other tissues (gills) and could potentially be applied to DNA extracts of whole larvae or spat. The severe disease caused by the endoparasitic protozoan Perkinsus marinus (Apicomplexa: class Perkinsea; Levine, 1978) is a major cause of mortality in the eastern oyster (Crassostrea virginica) along the Gulf of Mexico and Atlantic coast, U.S.A. A review on the epizootiology of the disease has been published by Andrews (1988). In its vegetative form as a trophozoite, P. marinus can proliferate throughout the tissues of the oyster by multiple fission, budding, or both. Eventually, mature tropho- zoites enlarge and then undergo rapid reductive divisions to form a hypnospore that enters the water column and sporulates to release large numbers ofbiflagellated zoospores (1,000-2,000 per hypnospore; Perkins, 1966). These motile zoospores pre- sumably give rise to trophozoites once they infect an oyster, but neither the mechanism of infection is known, nor the oyster life stage that is the most susceptible to parasite entry. Although significant progress has been made in understanding this disease over the last 25 yr (Perkins, 1988), new methodologies need to be developed to address these gaps in our knowledge before coherent treatment and management strategies can be formu- lated. tions in oyster seed stocks, latent infections in overwintering oyster populations, the onset of infection in oyster larvae and spat, the presence of P. marinus in other marine organisms that may serve as secondary vectors or reservoirs, and the genetic structure of parasite field populations. In response to these needs, the present paper describes a semiquantitative PCR-based di- agnostic assay for P. marinus DNA that provides both a rapid and sensitive in vivo assay for the detection of P. marinus in oyster hemolymph and gill tissues.