Occurence of Perkinsus sp. in two clam species (Ruditapes philippinarum and R. decussatus) from the Ebro Delta, Spain

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1 Bull. Eur. Ass. Fish Pathol., 28(1) 2008, 1 Occurence of Perkinsus sp. in two clam species (Ruditapes philippinarum and R. decussatus) from the Ebro Delta, Spain L.M. Elandaloussi*, N. Carrasco, A. Roque, M. Fernández-Tejedor and D. Furones IRTA-Sant Carles de la Ràpita, Crta. Poble Nou, Km 5,5, Sant Carles de la Ràpita, Spain. Abstract Distribution of Perkinsus sp. was investigated in two commercial clam species: Ruditapes decussatus and R. philipppinarum collected from the Ebro delta, Spain. Clams of commercial size were collected from September 2005 to August 2006 and Perkinsus prevalence and infection intensity were determined using the Ray s fluid thioglycollate medium (RFTM) diagnostic method. The prevalence of Perkinsus was relatively high all year around ranging from 20% to 100 % with an average of 70.5% and 79.1% of infected R. decussatus and R. philippinarum, respectively. Perkinsus prevalence was associated with Perkinsus infection intensity. Infection intensity was relatively low in all samples ranging from 216 to cells g -1 wet tissue or 622 to cells clam -1. No obvious seasonality in Perkinsus occurrence could be observed. In addition, no significant correlation between infection intensity and either seawater temperature or salinity could be determined. Introduction Spain is the leading producer and consumer of bivalves in Europe and the second producer worldwide. Currently, Catalonia is the second production area in Spain ( and most of the production of bivalves is concentrated in the two bays of the delta of the Ebro River. The bays of the Ebro Delta (Fangar and Alfacs, NW Mediterranean Sea) are very productive coastal areas in comparison with the oligotrophic Western Mediterranean. Primary production in the bays is an order of magnitude greater than in the adjacent open sea, and these embayments support important shellfish culture and fisheries. Protozoan parasites of the genus Perkinsus have been associated with mass mortalities of commercially important bivalves and have been demonstrated to infect a wide range of marine bivalves around the world (Andrew, 1966; DaRos & Canzonier, 1985; Goggin & Lester, 1995; Casas et al., 2004). To date, knowledge about perkinsosis along the Catalonian coast is very scarce (Sagrista et al., 1995; 1996) although Perkinsus-induced mortality has been reported in 1990 (Santmarti et al., 1995). Perkinsus infection and clam mortality are believed to intensify with high temperature, whereas low salinity areas are characterised by null or low infection levels (Andrew & Ray; 1988; Krantz & Jordan, 1996; Villalba et al., 2005). For this reason, Perkinsusassociated mortalities are usually reported at the end of the summer. *Corresponding author s laurence.elandaloussi@irta.es

2 Bull. Eur. Ass. Fish Pathol., 28(1) 2008, 2 The Ebro River forms a delta of approximately 320 km 2. The delta is arrow shaped and contains two distinct shallow-water embayments divided by the river: Fangar at the north and Alfacs at the South (Camp & Delgado, 1987). Both bays are characterised by seasonal variation in the salinity of the water (from 10 to 39 ppt), due to freshwater input from agricultural activities (mainly rice culture) and high summer temperature (26-32 C) of the seawater. Ruditapes philipinarum, the Manila clam, is mainly farmed in the Fangar bay, whereas the Alfacs bay is a natural bed for the carpet-shell clam R. decussatus. Farming ground in Fangar bay consists of sand, whereas the natural bed ground in Alfacs bay is constituted of mud substrate. Ray s Fluid Thioglycolate Medium (RFTM) diagnosis of clams collected from September 2005 to August 2006 is reported in this study. Infection intensity and prevalence of Perkinsus in the Manila clam, R. philippinarum and carpet-shell clam, R. decussatus from the Ebro Delta were determined. Key parameters, such as temperature and salinity of the seawater, were also recorded in order to relate the presence of the parasite to environmental conditions. Results presented in this study confirm that sampling scheme for the surveillance programme should be all year around and not only during the summer months. Materials and methods Clam (Bivalve) sampling and study area characteristics Fangar and Alfacs bays are located on the Ebro Delta, Catalonia, NE Spain. Alfacs, the southern bay of the Delta, extends 50 km 2 in area, at a maximum depth of 6.5 m. Fangar bay is smaller and very shallow with a maximum depth of 4 m and communicates with the sea through an opening 1 km wide (Camp & Delgado, 1987). Samples of R. decussatus from a wild stock and R. philippinarum from cultured beds were obtained monthly or bimonthly from Alfacs and Fangar bays, respectively. Temperature and salinity of the surface water were recorded weekly from September 2005 to August The shell length of clams was measured using callipers. From each sample, a minimum of 30 individuals was processed for the RFTM assay and determination of the condition index. Condition index The condition index of clams was calculated as described by Almeida et al. (1999). Briefly, 30 clams were shucked from their shells, and the tissue and shell were weighted separately to obtain the total weight as the tissue weight plus the shell weight. In order to evaluate the health status of each sample, the mean condition index (tissue weight/total weight x 100) was determined. Prevalence and infection intensity of Perkinsus Perkinsus prevalence (percentage of infected clams) and Perkinsus infection intensity were determined by the RFTM diagnostic method. All reagents were acquired from Sigma, Spain. For each sample, 30 whole clams were individually incubated in 20 ml fluid thioglycollate medium supplemented with 500 U ml -1 penicillin G and 500 μg ml -1 Streptomycin sulphate, at room temperature for 7-10 days, in the dark (Ray, 1966, Almeida et al., 1999). After incubation, RFTM was

3 Bull. Eur. Ass. Fish Pathol., 28(1) 2008, 3 TEMPERATURE TEMPERATURE ALFACS BAY FANGAR BAY Figure1. Monthly changes of surface water temperature ( C) and salinity (ppt) during the sampling period. discarded by centrifugation (10 min at 1500 g) and samples were digested in 20 ml of 2M NaOH at 60 C for 3h. The samples were then washed three times by centrifugation (10 min at 1500 g) with filtered seawater and stained with Lugol s iodine solution. The number of hypnospores of 3 subsamples was counted using a hemocytometer (Neuebauer chamber). Infection intensity of each clam was calculated by the RFTM assay as the total body burden and expressed either as the total number of Perkinsus clam -1 or number of Perkinsus g -1 of tissue (Almeida et al., 1999). Data analysis Temperature (ºC) Salinity (ppt) Temperature (ºC) Salinity (ppt) Data analysis of results from infection intensity experiments was performed using SALINITY SALINITY the computer program GraphPad Prism (GraphPad software). A two-tailed Student s t-test was used to compare differences between infection intensity of clams collected in Fangar bay with those from Alfacs bay. The level of statistical significance was set at p<0.05. Calculation of the Spearman rank correlation coefficient (rs) was used to estimate the association between Perkinsus infection intensity and either infection prevalence or seawater temperature and salinity. The last two parameters used for correlation analyses were calculated as the mean temperature and salinity of the two previous weeks from the date of each sampling. Results Clam characteristics A total of 520 clams of commercial size were collected from September 2005 to August 2006, with a mean shell length of 37.4 ± 1.2 mm for R. decussatus and 38.8 ± 0.3 mm for R. philippinarum and a mean wet tissue weight of 2.01 ± 0.14 g for R. decussatus and 2.70 ± 0.06 g for R. philippinarum (Table 1). Condition index varied from to with a mean of for R. decussatus and from to with a mean of for R. philippinarum (Table 1). No obvious correlation was observed between the infection intensity and size of the clams, as well as the condition index. Environmental conditions The temperature and salinity of surface water from September 2005 to August 2006 are plotted in Figure 1. Water temperature varied from 8.0 C (February, both bays) to 31.9 C (July, Alfacs Bay) and salinity from 12.8 ppt

4 Bull. Eur. Ass. Fish Pathol., 28(1) 2008, 4 Sampling date Clam species Site of sampling Shell length (mm) Wet tissue weight (g) Condition Index Perkinsus prevalence (%) No Perkinsus clam 1 26/09/2005 Rd Alfacs 35.1 ± ± ± ± /10/2005 Rp Fangar 37.3 ± ± ± ± /11/2005 Rp Fangar 40.1 ± ± ± ± /11/2005 Rd Alfacs 37.0 ± ± ± ± /12/2005 Rd Alfacs 41.2 ± ± ± ±471 7/02/2006 Rp Fangar 38.6 ± ± ± ± /02/2006 Rd Alfacs 39.5 ± ± ± ± /03/2006 Rp Fangar 40.3 ± ± ± ± /04/2006 Rd Alfacs 37.4 ± ± ± ± /05/2006 Rd Alfacs 31.8 ± ± ± ± /05/2006 Rp Fangar 39.6 ± ± ± ± /06/2006 Rp Fangar 37.6 ± ± ± ± /07/2006 Rd Alfacs 39.7 ± ± ± ± /08/2006 Rp Fangar 38.3 ± ± ± ± 174 Rp= R. philippinarum; Rd= R. decussatus Table 1. Mean shell length, wet tissue weight and condition index of manila and carpet-shell clams and Perkinsus infection prevalence and intensity determined from the RFTM assay during the sampling period. Data are means ± SE.

5 Bull. Eur. Ass. Fish Pathol., 28(1) 2008, 5 Figure 2. Monthly mean Perkinsus infection intensity of manila and carpet-shell clams determined by the RFTM assay. Infection intensity is expressed as the number of Perkinsus per g of clam ± standard deviation of the mean. (November, Fangar Bay) to 38.9 ppt (February, Fangar Bay). From mid October to mid January, salinity of the surface layer dropped 3 times below 20 ppt in Fangar bay. In contrast, no drop of salinity below 20 ppt was recorded for the Alfacs bay. Prevalence of Perkinsus Table 1 summarises the survey results of Perkinsus infection in the two species of clams tested in both bays of the Ebro delta. The prevalence of Perkinsus was relatively high all year around (over 56.7%) except for the carpet-shell clams collected in Alfacs bay in May 2006 that showed a lower prevalence (20.0%). Prevalence of 100% was observed in February for the clams (R. philippinarum) collected in Fangar and in July for the clams (R. decussatus) collected in Alfacs bay. An average of 70.5 and 79.1% of infected R. decussatus and R. philippinarum, respectively, were found during the sampling period. Infection intensity of Perkinsus Figure 2 shows the seasonal changes in the infection intensity of Perkinsus sp. as the mean number of Perkinsus per g wet tissue in both clams. Infection intensity was relatively low in all sampling periods ranging from 216 to cells g -1 wet tissue corresponding to 622 to cells clam -1. A peak of infection intensity was observed in October in R. philippinarum collected from Fangar bay. Mean infection intensity was higher in R. philippinarum (6652 cells g -1 wet tissue) collected in Fangar bay than in R. decussatus (1006 cells g -1 wet tissue) collected in Alfacs bay. The observed difference in infection intensities was found statistically significant with a p value of Correlation between infection intensity and infection prevalence, seawater temperature and salinity Seasonal variation of Perkinsus infection intensity and prevalence, seawater temperature and salinity are showed in Figure 3. The seasonal variation of infection intensity matched that of the prevalence in both R. decussatus and R. philippinarum. These two variables were significantly correlated (rs = ; p = and rs =0.8571; p = , respectively). In contrast, no significant correlation between infection intensity and either seawater temperature or salinity could be determined (p>0.05). Discussion This paper presents the first report of prevalence and infection intensity of Perkinsus sp. in the Ebro Delta, Spain. Perkinsus occurence has been reported in Spain in

6 Bull. Eur. Ass. Fish Pathol., 28(1) 2008, 6 Figure 3. Seasonal variation of Perkinsus infection intensity in R. decussatus (A) and R. philippinarum (B) and prevalence, seawater temperature and salinity.

7 Bull. Eur. Ass. Fish Pathol., 28(1) 2008, 7 Galicia (Figueras et al., 1992), Balearic islands (Casas et al., 2004) and on the western Mediterranean coast (Sagrista et al., 1995; 1996). RFTM is the most widely used method for Perkinsus detection albeit it does not distinguish Perkinsus to the species level. However, ultrastructural observation of Perkinsus infecting the clams R. decussatus and R. philippinarum from the region of the Ebro delta suggested that the species encountered in the Mediterranean coast of Spain was P. olseni (Sagrista et al., 1995; 1996). The monthly prevalence of infected clams from the Ebro delta was usually over 50%. The relatively high prevalence observed could be related to the age of clams used in this study. No juvenile clams were included in this study with the size of clams surveyed ranging from to mm. Perkinsus infection has been previously reported to be related to the age/ size of clams. Choi and Park (1997) demonstrated that no clams smaller than 15 mm in shell length exhibited Perkinsus infection and an infection prevalence of 100% was found for those longer than 20 mm. Mean infection intensity as determined by the RFTM assay was found significantly higher in R. philippinarum (6652 cells g -1 wet tissue) collected in Fangar bay than in R. decussatus (1006 cells g -1 wet tissue) collected in Alfacs bay. Albeit a differential susceptibility of these two species to the parasite could explain this result, it is also likely that the higher population density in the commercial beds of R. phillipinarum than in the natural beds of R. decussatus resulted in a higher disease transmission. Host organism s density has been previously positively correlated with infection intensity (Andrew, 1965; Da Ros & Canzonier, 1985). Nevertheless, it cannot be excluded that the different environmental conditions encountered in both bays could influence the level of infection. A significant association between infection intensity and prevalence was observed. This result is in agreement with several studies showing that Perkinsus prevalence matches well with infection intensities in terms of number of Perkinsus cells g -1 tissue (Choi & Park, 1997; Liang et al., 2001; Park & Choi, 2001). Temperature and salinity are believed to be the major environmental factor controlling P. marinus prevalence and infection intensity (Burreson & Ragone Calvo, 1996). In this study, measured seawater salinity varied from 12.8 to 38.9 ppt and a relatively high prevalence was observed all year around. High temperature values in summer together with salinity values over 15 ppt in the Ebro delta would have been expected to be conducive for Perkinsus proliferation. Unexpectedly, the prevalence and infection intensity were low in R. philipinarum collected in August from Fangar when seawater temperature reached 29 C and salinity was 35 ppt. This result agrees with Ngo and Choi (2004) study showing that Perkinsus prevalence and infection intensity in Manila clams from Korea was amongst the lowest in August. Similarly to this study, Park and Choi (2001) reported relatively high prevalence and infection intensity of Perkinsus sp. in R. philippinarum from West and South coast of Korea where salinity fluctuated from to ppt. In addition, prevalence of % and high infection intensity was observed in winter with temperature as low as 3.1 C. Contrary to P. marinus that appeared

8 Bull. Eur. Ass. Fish Pathol., 28(1) 2008, 8 to have a seasonal pattern and which geographical distribution is believed to be controlled by temperature and salinity (Ragone Calvo et al., 2001), previous reports of Perkinsus sp. distribution (Park & Choi, 2001; Ngo & Choi, 2004) do not corroborate this hypothesis. The results presented in this study suggest that Perkinsus sp. from the Ebro delta has a wider range of salinity tolerance than P. marinus and that temperature and salinity might not be the major factors controlling its distribution. There were no obvious seasonal trends in the results presented here; prevalence and intensity of infection were higher in certain months in Fangar bay (October 2005 and February 2006) and others in Alfacs bay (September 2005 and April 2006). For example, whereas a peak of infection could be observed in Fangar bay in February 2006 (8490 Perkinsus g -1 wet tissue), infection intensity was found low in Alfacs bay (561 Perkinsus g -1 wet tissue). In addition, no association was found between infection intensity and seawater temperature or salinity. The absence of seasonality in Perkinsus occurrence highlights the need to survey for Perkinsus prevalence and infection intensity all year around and not only during the summer-autumn months. In addition, on the basis of this work, the lack of correlation between infection intensity and seawater temperature and salinity does not permit predicting the degree of infection based on these environmental parameters. Disease progression is likely to result from a combination of factors affecting the host, the pathogen or their interaction. Stressing factors such as hypoxia, nutrient limitation, pollution or spawning stages of the bivalve would influence the physiological status of the host and therefore its resistance to diseases. In conclusion, Perkinsus was found in the natural beds of R. decussatus and in the cultivated R. philippinarum of the Ebro delta. Infection intensity could not be correlated with seawater temperature and salinity and Perkinsus infection was detected all year around. Acknowledgements The authors would like to thank Ms Cristina Lleti and the staff of IRTA-Sant Carles de la Ràpita for their help with sampling and histological preparations. N. Carrasco also thanks INIA for providing research predoctoral fellowship. References Almeida M, Berthe F, Thebault A & Dinis MT (1999). Whole clam culture as a quantitative diagnostic procedure of Perkinsus atlanticus (Apicomplexa, Perkinsea) in the clam Ruditapes decussatus. Aquaculture 177, Andrew JD (1965). Infection experiments with Dermocystidium marinum in Chesapeake bay. Cheasapeake Science 6, Andrew JD (1966). History of Perkinsus marinus, a pathogen of oysters in Chesapeake Bay Journal of Shellfish Research 15, Andrew JD & Ray SM (1988). Epizootiology of the disease caused by the oyster pathogen Perkinsus marinus and its effects on the oyster industry. American Fisheries Special Publication 18, Burreson EM & Ragone Calvo LM (1996). Epizootiology of Perkinsus marinus disease of oysters in Chesapeake Bay, with emphasis on data since Journal of Shellfish Research 15,

9 Bull. Eur. Ass. Fish Pathol., 28(1) 2008, 9 Camp J & Delgado M (1987). Hydrografia de las bahias del delta del Ebro. Investigacion Pesqueira 51, Casas SM, Grau A, Reece KS, Apakupakul K, Azevedo C & Villalba A (2004). Perkinsus mediterraneus n. sp., a protistan parasite of the European flat oyster Ostrea edulis from the Balearic Islands, Mediterranean Sea. Diseases of Aquatic Organisms 58, Choi K-S & Park K-I. (1997). Report on occurrence of Perkinsus sp in the manila clam Ruditapes philippinarum in Korea. Korean Journal of Aquaculture 10, Da Ros L & Canzonier WJ (1985). Perkinsus, a protistan threat to bivalve culture in the Mediterranean basin. Bulletin of the European Association of Fish Pathologists 5, Figueras A, Robledo JAF & Novoa B (1992). Occurence of Haplosporidium and Perkinsuslike infections in carpet-shell clams, Ruditapes decussatus (Linnaeus, 1758), of the Ria de Vigo (Galicia, NW Spain). Journal of Shellfish Research 11, Goggin CL & Lester RJG (1995). Perkinsus, a protistan parasite of abalone in Australia: A review. Marine & Freshwater Research 46, Krantz GE & Jordan SJ (1996). Management alternatives for protecting Crassostrea virginica fisheries in Perkinsus marinus enzootic and epizootic areas. Journal of Shellfish Research 15, Liang Y, Zhang X, Wang L, Yang B, Zhang Y & Cai C (2001). Prevalence of Perkinsus sp. in the Manila clam Ruditapes philippinarum along northern coast of Yellow Sea in China. Oceanologia et Limnologia Sinica 32, Ngo TT & Choi K-S (2004). Seasonal changes of Perkinsus and Cercaria infections in the manila clam Ruditapes philippinarum from Jeju, Korea. Aquaculture 239, Park K-I & Choi K-S (2001). Spatial distribution of the protozoan parasite Perkinsus sp. found in the Manila clams, Ruditapes philippinarum, in Korea. Aquaculture 203, Ragone Calvo LM, Wetzel RL & Burreson EM (2001). Development and verification of a model for the population dynamics of the protistan parasite, Perkinsus marinus, within its host, the eastern oyster, Crassostrea virginica, in Chesapeake Bay. Journal of Shellfish Research 20, Ray SM (1966). A review of the culture method for detecting Dermocystidium marinum, with suggested modifications and precautions. Proceedings of the National Shellfish Association 54, Sagrista E, Durfort M, & Azevedo C (1995). Perkinsus sp. (Phylum Apicomplexa) in Mediterranean clam Ruditapes semidecussatus: Ultrastructural observations of the cellular response of the host. Aquaculture 132, Sagrista E, Durfort M & Azevedo C (1996). Ultrastructural data on the life cycle of the parasite, Perkinsus atlanticus (Apicomplexa), on the clam, Ruditapes philippinarum, in the Mediterranean. Scientia Marina (Barcelona) 60, Santmarti MM, Garcia Valero J, Montes J, Pech A & Durfort M (1995). Seguimiento del protozoo Perkinsus sp., en las pobleciones de Tapes decussatus del delta del Ebro. In Actas del V Congreso Nacional de Acuacultura (F. Castelló & A Calderer, eds), p , S. Carlos de la Rápita. Universidad de Barcelona. Villalba A, Casas SM, Lopez C & Carballal MJ (2005). Study of perkinsosis in the carpet shell clam Tapes decussatus in Galicia (NW Spain). II. Temporal pattern of disease dynamics and association with clam mortality. Diseases of Aquatic Organisms 65,