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Advanced Perkinsus Marinus Infections in Crassostrea Ariakensis Maintained Under Laboratory Conditions

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Advanced Perkinsus Marinus Infections in Crassostrea Ariakensis Maintained Under Laboratory Conditions W&M ScholarWorks VIMS Articles Virginia Institute of Marine Science 2006 Advanced Perkinsus Marinus Infections In Crassostrea Ariakensis Maintained Under Laboratory Conditions JA Moss Virginia Institute of Marine Science EM Burreson Virginia Institute of Marine Science Kimberly S. Reece Virginia Institute of Marine Science Follow this and additional works at: https://scholarworks.wm.edu/vimsarticles Part of the Marine Biology Commons Recommended Citation Moss, JA; Burreson, EM; and Reece, Kimberly S., Advanced Perkinsus Marinus Infections In Crassostrea Ariakensis Maintained Under Laboratory Conditions (2006). Journal Of Shellfish Research, 25(1), 65-72. 10.2983/0730-8000(2006)25[65:APMIIC]2.0.CO;2 This Article is brought to you for free and open access by the Virginia Institute of Marine Science at W&M ScholarWorks. It has been accepted for inclusion in VIMS Articles by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. Journal of Shellfish Research, Vol. 25, No. 1, 65–72, 2006. ADVANCED PERKINSUS MARINUS INFECTIONS IN CRASSOSTREA ARIAKENSIS MAINTAINED UNDER LABORATORY CONDITIONS JESSICA A. MOSS, EUGENE M. BURRESON AND KIMBERLY S. REECE* Virginia Institute of Marine Science, The College of William and Mary, Gloucester Point, VA 23062 ABSTRACT The Suminoe oyster, Crassostrea ariakensis, has been under investigation since the early 1990s for potential use in restoring the commercial harvest or for aquaculture of oysters in the Chesapeake Bay, USA. Initial studies focusing on C. ariakensis documented a significant level of tolerance to the protozoan parasite Perkinsus marinus, a pathogen found in almost all reaches of the Bay and widely acknowledged as one of the main reasons for the decline in the eastern oyster, Crassostrea virginica, harvest since the late 1980s. Crassostrea ariakensis was demonstrated to acquire P. marinus, however infection intensities, as measured using Ray’s thioglycollate medium assay indices, generally were found to be light. As part of a series of experiments to study potential impacts on the Chesapeake Bay region of pathogens found in C. ariakensis in Asia, a challenge experiment was conducted to study the pathogenicity of Perkinsus olseni to C. ariakensis. During this study, we observed the acquisition of moderate and heavy infection intensities of P. marinus in triploid C. ariakensis oysters being maintained in the laboratory. Results suggest that there may be some risk of mortality from P. marinus if C. ariakensis is held under stressful conditions at least in hatchery or laboratory settings. KEY WORDS: Perkinsus marinus, Crassostrea ariakensis, PCR, RFTM, oyster INTRODUCTION olseni was found during a recent survey of C. ariakensis popula- tions in Asia (Moss & Reece 2005). Prior to the start of the chal- The eastern oyster, Crassostrea virginica (Gmelin, 1791), an lenge experiment, C. ariakensis were initially determined to be integral part of the economy and ecology of the Chesapeake Bay, Perkinsus sp.-free, based on molecular diagnostics using PCR (Ca- has been in a severe state of decline in recent decades. Two patho- sas et al. 2002). The oysters acquired detectable levels of the gens, Haplosporidium nelsoni (Haskin, Stauber & Mackin, 1966) parasite, however, after being held for about 8 weeks in the labo- and Perkinsus marinus (Mackin, Owen & Collier, 1950), the para- ratory aquaria. We used the infected oysters in our challenge study, sites responsible for the diseases known as MSX and dermo re- despite baseline P. marinus infection prevalence, to examine the spectively, have contributed significantly to the decimation of the potential for C. ariakensis oysters to become coinfected with mul- oyster populations in Chesapeake Bay since the 1950s (Sinder- tiple Perkinsus species. We report here our observations regarding mann 1990). the P. marinus infection levels observed in C. ariakensis during The decline in oyster production in Virginia led to the forma- this challenge experiment. tion of a number of panels in the early 1990s to make recommen- dations on restoring oyster populations. One option currently being MATERIALS AND METHODS seriously considered is the introduction and use of a nonnative oyster in Chesapeake Bay. A Virginia Institute of Marine Science Experimental Design (VIMS) study conducted in 1996 focusing on the Pacific oyster, Crassostrea gigas (Thunberg, 1793), documented lower disease On January 24, 2005, 120 market sized (∼75 mm shell height) susceptibility in C. gigas than in the native eastern oyster, C. triploid C. ariakensis were received from the Aquaculture Genetics virginica; however, growth rates of the Pacific oyster were equal and Breeding Technology Center hatchery at VIMS. The animals to or inferior to the native oyster in the Chesapeake Bay (Calvo et were held for 4 days inside a plastic mesh bag in a holding tank al. 1999). A 1998 field-based study on another Asian oyster, the prior to bringing them into the laboratory aquaria. The holding Suminoe oyster, Crassostrea ariakensis (Fujita, 1913), docu- tank was not covered and there was flow-through of nonfiltered mented rapid growth and survival in that species, as compared with York River water of approximately 9°C and 16 ppt salinity. On C. virginica, even when endemic diseases were prevalent (Calvo et removal from the holding tank, 20 C. ariakensis were immediately al. 2001). Although initial baseline samples of C. ariakensis in that sacrificed, and gill and mantle tissues were excised aseptically study revealed a 12% prevalence of P. marinus, all subsequent from each animal for DNA extraction. Genomic DNA of each samples collected after field deployment showed C. virginica with oyster was used in a PCR-based molecular diagnostic assay (Casas higher P. marinus prevalence than C. ariakensis. Additionally, et al. 2002) to examine the animals for the presence of DNA from Ray fluid thioglycollate medium (RFTM) (Ray 1952) diagnoses Perkinsus spp. parasites. The animals were subsequently held for showed several heavy P. marinus infections in C. virginica, how- 59 days in 10-gallon glass aquaria that were maintained at 20°C ever, only light infections were observed in C. ariakensis (Calvo et and 25 ppt salinity at a density of approximately 25 oysters per al. 2001). tank and were fed 0.1 g oyster−1 algal food daily (Reed Maricul- In this report we describe the first account of moderate to heavy ture, San Jose, CA). At the end of 59 days, on March 24, 2005, five P. marinus infections in C. ariakensis. The oysters in this study C. ariakensis were sacrificed and gill and mantle were excised were held in laboratory aquaria at VIMS during a challenge ex- aseptically from each animal to be used in Perkinsus spp. screen- periment conducted to examine the potential pathogenicity of Per- ing by PCR assays as described later. were (60 ס kinsus olseni Lester and Davis, 1981, to C. ariakensis, because P. On March 28, 2005, the remaining C. ariakensis (n used in a challenge experiment designed to evaluate the pathoge- nicity of P. olseni to C. ariakensis. Twenty C. ariakensis received *Corresponding author. E-mail: [email protected] single pallial cavity injections of 100 ␮L of 25 ppt sterile artificial 65 66 MOSS ET AL. seawater (SASW) through a notch in the shell, and 40 animals SSU Genes were inoculated with 105 log phase cultured P. olseni cells per To assure that PCR amplifiable DNA was present in all ex- gram body weight in a 100-␮L volume. The clonal P. olseni cul- tracted samples, genomic DNAs were tested using universal small ture isolated from a Japanese Venerupis philippinarum (Adams subunit (SSU) ribosomal RNA gene primers 16S-A (5Ј CCG AAT and Reeve, 1850) clam was obtained from Mr. Chris Dungan, TCG TCG ACA ACC TGG TTG ATC CTG CCA GT 3Ј) and Maryland DNR Cooperative Oxford Laboratory, Oxford Mary- 16S-B (5Ј GGA TCC AAG CTT GAT CCT TCT GCA GGT TCA land, (ATCC PRA-181). Thirty additional untreated, control C. CCTAC3Ј) (modified from Medlin et al. 1988) with an expected ariakensis were neither notched nor inoculated, but held in sepa- amplification product of approximately 1,800 bp. Each 25-␮L re- rate aquaria and otherwise maintained and fed in the same manner action contained 20 mM Tris-HCl (pH8.4), 50 mM KCl, 0.75 mM as the experimental animals for the duration of the experiment. MgCl , 0.1 mM of each dNTP, 0.5 ␮M each primer, 0.0125 U Those C. ariakensis inoculated with 25 ppt SASW were main- 2 ␮L−1 Taq polymerase, 0.2 mg mL−1 BSA and 0.5 ␮L genomic tained in one 10-gallon aquarium, and those inoculated with DNA (∼10–50 ng total). Amplifications were performed with an P. olseni were split evenly between two aquaria. All aquaria were initial denaturation of 94°C for 4 min, followed by 35 cycles at identical and each tank was covered with a plexiglass lid. 94°C for 30 sec, 45°C for 30 sec, 65°C for 2 min, with a final All aquaria environments were maintained at 20°C to 22°C and elongation of 65°C for 2 min. After amplification, 3 ␮LofPCR contained 25 ppt aerated, 1-␮m filtered York River water, two product were analyzed by agarose gel electrophoresis (2%), stained thirds of which was removed each week and replaced with an with ethidium bromide and visualized under UV light. Images equal volume of clean, 25 ppt, 1-␮m filtered York River water. were recorded with an Alpha Innotech FlourChem (SanLeandro, Animals were fed daily a single dose of approximately 0.1 g oys- CA) imaging system. ter−1 algal feed (Reed Mariculture). Aquaria were checked daily for oyster mortalities, and mori- Genus-specific Perkinsus sp. PCR Assay bund animals were removed. The cumulative mortality was calcu- lated for each sample as the sum of the mortalities that occurred Screening for Perkinsus sp.
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