183Ž. 2000 215±221 www.elsevier.nlrlocateraqua-online

Prevalence of marinus ž/Dermo in Õirginica along the Connecticut shoreline

John Karolus ), Inke Sunila, Stacey Spear, John Volk Connecticut Department of Agriculture, Bureau of Aquaculture, 190 Rogers AÕenue, P.O. Box 97, Milford, CT 06460, USA

Received 9 May 1999; received in revised form 25 August 1999; accepted 8 September 1999

Abstract

The prevalence of Perkinsus marinus in the eastern Crassostrea Õirginica along the Connecticut shoreline was surveyed during a period of 2 years from 1996 to 1997. The survey was conducted in shallow and deep waters. Fifty-four samples involving 1430 were tested. Using Ray's Fluid Thioglycollate Medium, the parasite was found at all sampling locations with a prevalence range from 10% to 100%. The weighted prevalence values were significantly greater in shallow waters compared to deep waters in 1996 Ž.t-test, P-0.05 . However, in 1997, the difference between the two areas was not significant. The results demonstrated P. marinus had established enzootics throughout the Connecticut shoreline. Oyster aquaculture practices of transplanting natural seed from shallow waters to approved areas found in deeper waters had probably resulted in comparable weighted prevalence values between these areas. q 2000 Elsevier Science B.V. All rights reserved.

Keywords: Perkinsus marinus; Dermo; Connecticut

1. Introduction

Perkinsus marinus Ž.Dermo was identified in the late 1940s as the cause of extensive oyster mortalities in the . Along with Ž.MSX ,

) Corresponding author. Tel.: q1-203-874-0696; fax: q1-203-783-9976; e-mail: [email protected]

0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S0044-8486Ž. 99 00306-3 216 J. Karolus et al.rAquaculture 183() 2000 215±221 both parasites have caused hundreds of millions of dollars in losses in the Chesapeake and Delaware BaysŽ. Ewart and Ford, 1993 . SindermannŽ. 1969 reported the 's presence in oysters in 1968. However, FordŽ. 1996 reported that a number of surveys, as well as occasional sampling by several laboratories, had never found conclusive evidence of the pathogen north of New Jersey before 1991. Ford concluded the parasite's presence must have been scattered and sparse to have been missed by the survey work she reported in 1996. In early May 1992, very light infections were detected in 65±70% of oysters from Oyster Bay, located on the north shore of Long Island, NY and from the Connecticut shore of Long Island Sound. Known infection patterns indicate the pathogen must have been present in a substantial number of oysters the year before to have caused such a prevalence in 1992Ž. Ford, 1996 . Brousseau Ž. 1996 reported the presence of Dermo in Westport, Bridgeport and Milford, CT during the time from September 1993 to December 1994. The majority of the infections at all sites were classified as light. In 1996, she found an increase in prevalence and weighted prevalence at two of these sites. According to FordŽ. 1996 , Dermo had a prevalence of 75%±100% and weighted prevalence of 2.5 or more in many Long Island Sound locations by late summer of 1995. Based on the literature results and with the occurrence of a depletion of natural oyster seed from some economically important oyster beds in 1996, we decided to examine the oyster population for the presence of Dermo to determine if there was a continued increase in the prevalence or intensity of infection along the Connecticut shoreline. Oysters sampling sites were categorized as follows:Ž. 1 shallow waters where the oyster population was primarily seed;Ž. 2 deeper water locations classified as approved according to the National Shellfish Sanitation Program and Interstate Shellfish Sanita- tion ConferenceŽ. 1997 .

2. Materials, methods and techniques

A map of the sampling sites is shown in Fig. 1. In 1996, 20 oysters were sampled from each station. In 1997, 30 oysters were used from each location. The shallow water sites were intertidal, as well as subtidal with the water being 2.5 m or less deep. The deep water locations were areas approved for market harvest, with depths from 2.5±12 m. Testing was done with 2±4 year oysters. Ray's Fluid Thiogylocollate Medium containing 2% NaCl and supplemented with a final concentration of 50 units of penicillin G and 50 mg of streptomycin sulfate per ml of medium was usedŽ Bushek et al., 1994. . Approximately 0.5 cm3 of labial palps, mantle, digestive glands and rectalranal tissue were removed. The tissues were incubated in the medium for 5±12 days at room temperature. The intensity of the infection in the oyster was rated using the Mackin scaleŽ. Ray, 1954 . Numerical values were given after the microscopic examina- tion of the tissues. A value of zero was given if no hypnospores were found up to a value of five when the infection was considered heavy. The values for each animal were averaged. These averaged values for all the animals in the sample are averaged to determine the weighted prevalence value. J. Karolus et al.rAquaculture 183() 2000 215±221 217 sampling locations along the Connecticut coast. P. marinus Fig. 1. 218 J. Karolus et al.rAquaculture 183() 2000 215±221

Table 1 Prevalence and weighted prevalence of P. marinus from shallow waters in 1996 Town Date Location Weighted Prevalence prevalenceŽ. % XX Greenwich 05r09r96 41801.21 N:73835.74 W 0.8 80 XX Greenwich 05r09r96 41801.05 N:73834.52 W 0.6 80 XX Branford 05r28r96 41815.73 N:72845.63 W 1.2 100 XX Greenwich 07r11r96 41801.05 N:73834.52 W 1.6 100 XX Greenwich 07r11r96 41801.21 N:73835.74 W 2.5 100 XX Greenwich 08r22r96 41801.05 N:73834.52 W 2.5 100 XX Greenwich 08r22r96 41801.21 N:73835.74 W 2.8 100 XX Bridgeport 09r12r96 41808.50 N:73809.40 W 1.1 80 XX Bridgeport 09r12r96 41808.50 N:73809.40 W 2.2 100 XX Greenwich 09r19r96 41801.05 N:73834.52 W 2.6 100 XX Greenwich 09r19r96 41801.21 N:73835.74 W 3.0 100 XX Greenwich 11r19r96 41801.05 N:73834.52 W 2.1 100 XX Greenwich 11r19r96 41801.21 N:73835.74 W 2.2 100 XX Milford 11r20r96 41812.73 N:73803.14 W 1.3 100 XX Waterford 11r24r96 41819.00 N:728 10.63 W 0.01 10 XX Stonington 12r02r96 41821.02 N:71853.01 W 1.4 60 XX New Haven 12r18r96 41818.00 N:72854.00 W 1.3 100

For statistical analysis, the t-test used was the significance test on a difference between two independent sample means, population variance not knownŽ. Bahn, 1972 .

3. Results

The prevalenceŽ. percentage of infected oysters in a sample and weighted prevalence results for P. marinus are shown in Tables 1±4. P. marinus was found at all sampling

Table 2 Prevalence and weighted prevalence of P. marinus from deep waters in 1996 Town Date Location Weighted Prevalence prevalenceŽ. % XX Guilford 05r14r96 41816.00 N:72840.47 W 0.07 40 XX Milford 05r24r96 41810.85 N:73802.35 W 0.05 40 XX Westport 06r02r96 41804.50 N:73822.60 W 0.5 100 XX Madison 06r16r96 41816.10 N:73836.00 W 0.9 60 XX Norwalk 07r08r96 41805.16 N:73823.07 W 0.5 80 XX Norwalk 07r08r96 41803.88 N:73824.13 W 0.8 60 XX Westport 07r08r96 41803.01 N:73822.21 W 0.2 40 XX Madison 08r22r96 41816.02 N:72836.00 W 2.9 100 XX Guilford 09r29r96 41816.00 N:72809.47 W 2.3 100 XX Guilford 10r14r96 41816.05 N:72839.62 W 2.4 100 XX Milford 11r14r96 41810.53 N:73802.35 W 0.6 80 J. Karolus et al.rAquaculture 183() 2000 215±221 219

Table 3 Prevalence and weighted prevalence of P. marinus from shallow waters in 1997 Town Date Location Weighted Prevalence prevalenceŽ. % XX Greenwich 01r15r97 41801.21 N:73835.74 W 0.8 100 XX Greenwich 01r15r97 41801.05 N:73834.52 W 1.2 90 XX Waterford 03r04r97 41819.03 N:72810.63 W 0.2 30 XX Madison 04r04r97 41816.00 N:72836.00 W 0.8 50 XX Clinton 04r06r97 41815.97 N:72832.00 W 0.8 90 XX Guilford 04r06r97 41816.05 N:72839.62 W 0.3 70 XX Bridgeport 06r25r97 41809.08 N:73810.28 W 0.6 40 XX Greenwich 07r15r97 41801.21 N:73835.74 W 1.9 90 XX Bridgeport 08r29r97 41808.50 N:73809.40 W 2.2 100 XX Greenwich 09r15r97 41801.05 N:73834.52 W 0.7 80 XX Guilford 09r28r97 41816.05 N:72839.62 W 2.8 100 XX Clinton 10r14r97 41815.97 N:72832.00 W 2.1 100 XX Stratford 11r12r97 41809.94 N:73806.04 W 1.1 100 XX Bridgeport 11r12r97 41809.08 N:73810.28 W 0.6 60 XX Clinton 12r07r97 41816.05 N:72831.30 W 1.1 90 XX Clinton 12r07r97 41815.97 N:72832.84 W 1.4 100 XX Fairfield 12r06r97 41809.21 N:73814.00 W 0.1 10 X X Norwalk 12r17r97 41802.42 N:73825.25 W 2.0 90

locations which spanned the entire Connecticut shoreline. In addition to location, Fig. 1 reveals the mean weighted prevalence results with the year of testing. The values for each year may represent both shallow and deep water sampling. Using the significance test, in 1996, the shallow water samples had significantly higher weighted prevalence values than the deep water samples Ž.ts5.4, P-0.05s1.706 . However, the differ- ence between the two areas was not significant in 1997 Ž.ts0.3, P)0.05s1.711 . During our study in 1996, many oyster meats appeared healthy by gross examination even though they were infected with P. marinus at a Mackin value of 3.0 or greater.

Table 4 Prevalence and weighted prevalence of P. marinus from deep waters in 1997 Town Date Location Weighted Prevalence prevalenceŽ. % XX Madison 05r28r97 41816.00 N:72836.00 W 0.5 90 XX Westport 07r03r97 41803.00 N:73823.46 W 0.2 20 XX Westport 08r27r97 41804.49 N:73822.61 W 2.0 100 XX Westport 09r24r97 41804.49 N:73819.79 W 0.9 90 XX Norwalk 12r17r97 41802.42 N:73825.25 W 0.3 40 XX Westport 12r17r97 41802.73 N:73822.21 W 1.3 90 XX Norwalk 12r17r97 41803.36 N:73825.12 W 1.7 90 XX Norwalk 12r17r97 41804.57 N:73823.28 W 1.7 90 220 J. Karolus et al.rAquaculture 183() 2000 215±221

4. Discussion

Our results indicated P. marinus was firmly entrenched in the Crassostrea Õirginica population along the Connecticut shoreline. As stated earlier, the shallow areas in 1996 had significantly higher weighted prevalence values than the deep water areas. The shallow water samples contained a number of natural or wild oyster seed areas. One would expect the shallow waters to have a higher temperature than the deep waters. The higher temperatures in addition to the salinity values for all sampling locations being above 15 ppt were conducive for Perkinsus proliferation. These shallow areas supply seed for planting in the deep water-growing areas. Hence, the continual restocking of infected oysters into approved market harvest areas could have resulted in the difference between weighted prevalence values for the two areas being not significant in 1997. Even with high weighted prevalence values, there had been no reported wide spread oyster mortalities in 1996. Some of the samples in the present article had approached weighted prevalence valuesŽ. 3.0 of the parasite, levels that were responsible for severe oyster mortalities in the Gulf of MexicoŽ. Mackin and Hopkins, 1961 . Severe epizootics of H. nelsoni occurred throughout Long Island Sound in 1997Ž. Sunila et al., 1999 . With the epizootics of H. nelsoni, it was impossible to assess the effect of P. marinus alone on the C. Õirginica population in Long Island Sound. The MSX parasite may have been present in low numbers in 1996. Unfortunately, testing for this parasite was not done in 1996. Infection of C. Õirginica with H. nelsoni resulted in oysters physio- logically stressed to the point of death. We believe this occurred at the towns listed in Fig. 1 where there was a decrease in the weighted prevalence from 1996 to 1997. The towns that had an increase in the weighted prevalence were sampling sites in deep waters where the prevalence of H. nelsoni had not caused significant oyster mortalities at the time of sampling. Therefore, surviving oysters had additional time to increase their Perkinsus parasite load. According to Brousseau et al.Ž. 1998 , wild oysters collected along the central Connecticut shoreline were largely uninfected. This was not in accordance with the results of this study. In fact, some of the highest weighted prevalence values were found from wild oysters in this region of the stateŽ Tables 2 and 3. . The state-wide presence of P. marinus and the epizootic presence of H. nelsoni make management strategies extremely difficult as Brousseau et al.Ž. 1998 suggested.

5. Conclusion

P. marinus has established enzootics throughout Connecticut's shoreline in Long Island Sound. One important routine, traditional oyster aquaculture practice along the Connecticut shoreline is removing natural seed from close to shore, shallow waters and transplanting the seed to approved areas found in deeper waters. This practice has resulted in weighted prevalence values of the parasite being comparable between the deeper, cooler waters and the shallow, warmer waters. Continued monitoring will be required to determine if the parasite will cause adverse physiological effects on the oyster population in the approved areas prior to development into a harvest-size product. J. Karolus et al.rAquaculture 183() 2000 215±221 221

References

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