Olseni Infection Research Articles

Spatio-Temporal Variation in Perkinsus olseni Infection Intensity in Manila Clam Ruditapes philippinarum in Anmyeondo and Cheonsu Bay Tidal Flats on the West Coast of Korea

Spatio-Temporal Variation in Perkinsus olseni Infection Intensity in Manila Clam Ruditapes philippinarum in Anmyeondo and Cheonsu Bay Tidal Flats on the West Coast of Korea

Anti Perkinsus olseni Monoclonal Antibody Generation Using Hypnospores as Antigens

Antibodies are valuable for the detection, identification, and diagnosis of pathogens in many fields, including aquaculture. In this study, we aimed to produce monoclonal antibodies (mAbs) against the parasite Perkinsus olseni, isolated from the clam Paphia undulata in Thailand. The mAbs were characterized using dot blotting and immunohistochemistry techniques in order to identify their class and their ability to detect the hypnospores and zoospores of P. olseni. Immunoreactions between the obtained mAbs and P. olseni produced black or grey spots according to the concentration of parasites detected by the mAbs. The quantitative detection thresholds of hypnospores for the two most strongly reactive mAbs were 105 cells/mL (producing grey spots) to 107 cells/mL (producing black spots), and more than 107 cells/mL (grey spots) for zoospores. Both mAbs showed immunoreactivity at the surface of trophozoite cells of P. olseni in infected clam tissues as assessed by immunohistochemistry analysis, indicating that they could be used for the detection and identification of P. olseni infection in commercial Pa. undulata production.

Effect of the alveolate parasite Perkinsus olseni infection on sexual maturation and spawning efficiency of the clam Ruditapes decussatus.

The effect of Perkinsus olseni infection on the reproduction ability of clams has been underestimated so far. Although some studies found evidence of reduction of egg production and delay in gonad maturation after infection, the total effect of the infection is still unclear. In this study, Ruditapes decussatus clams from a naïve population were injected with two different doses of P. olseni parasites, a low dose leading to a light infection and a high dose leading to a heavy infection. Clams were maintained during 2 months for maturation, and at the end of the experiment, the spawning was induced, the number of larvae release and mortality were evaluated. During the maturation period, infection level, gonadal stage, condition index, gross biochemical composition and oxidative status of progenitors were evaluated at days 0, 30 and 60 post-injection. The effects of P. olseni infection on clams showed alterations on biochemical parameters, namely lipid peroxidation, a significant mortality and a delayed gonad maturation, with a greater effect in the highly infected individuals. The reproductive capacity of the clams was impaired in both infected groups showing a lower production and a higher mortality rate of larvae. Finally, this study indicates that the production of natural beds with a high prevalence of P. olseni could be compromised by a deregulation of the natural reproduction cycle and a decrease in larvae production by infected animals, probably due to a combination of lower egg production and lower lipid reserves in larvae from infected clams.

Evaluation of the Ruditapes decussatus immune response after differential injected doses of Perkinsus olseni

Ruditapes decussatus is a highly appreciated clam species, with a high demand, especially in European countries. However, natural populations of this species have been affected by over-exploitation, abiotic factors and also by diseases mainly caused by bacteria, virus and protozoans. Within the latter group of pathogens, Perkinsus spp. has been attracting attention for causing relevant mortality rates on bivalves around the world. From all species of this genus, Perkinsus olseni is one of the most impactful for R. decussatus, as it may cause chronic infection with fatal consequences under adverse conditions. Therefore, in this study, naïve clams were injected with two doses of P. olseni in order to better understand the response of R. decussatus against this parasite. So, one of the main goals of this experiment was to evaluate the differential response of the clams at gene expression level after injection and also to monitor the possible mortalities related to parasite. Results indicated that the methods applied for injection and detection of parasite load are effective. Furthermore, altogether, R. decussatus gene expression studies indicate differences on expression of some of the addressed genes among distinct levels of infection over time, which require further studies to confirm if these genes could be used as molecular markers of P. olseni infection. According to the results obtained, Adiponectin-C1q seems to be a promising gene to be used as molecular marker of chronic infection in claḿs haemolymph.

Perkinsus olseni and other parasites and abnormal tissue structures in New Zealand Greenshell™ mussels (Perna canaliculus) across different seasons

The New Zealand Greenshell™ mussel (Perna canaliculus) supports the largest aquaculture industry in the country. However, summer mortality events and potential disease outbreaks may threaten the growth of this industry. As an approach to gauging potential threats through the seasons, a detailed histopathological examination was conducted on 256 adult cultured mussels collected from a farm between April 2018 to September 2019, which covered the austral autumn, winter, spring and summer seasons. Histological sections followed by confirmatory in situ hybridization (ISH) resulted in the identification of Perkinsus olseni at an overall prevalence of 56%. Other parasites and pathogens were identified by histology: apicomplexan parasite X (APX) (78%), copepods (Pseudomyicola spinosus or Lichomolgus uncus) (1%), Microsporidium rapuae (1%), intracellular microcolonies of bacteria (IMCs) (2%) and bacilli and cocci bacteria (4%) in gills, mantle, gonads, digestive epithelium and digestive tubules. There was a significant association between P. olseni and APX infection in mussels. This is the first report on seasonal variations of P. olseni and APX in New Zealand Greenshell™ mussel. There was a significant association between seasons and the presence of P. olseni and APX in mussels. A significant positive association between the brown material accumulation and parasites (P. olseni and APX) and between haemocytosis and P. olseni infections were recorded. A significant association between presence of parasites and health condition (healthy and unhealthy) of mussels was observed. Moreover, a significant association between digestive tubule deterioration (large lumen, with a thin epithelial wall) and P. olseni infection was noted. Therefore, this study provides information regarding the infections of potential parasites and pathogens for the first time in P. canaliculus, their seasonal variations and host-parasite interactions within a commercial farm.

Negative Impacts of Perkinsus olseni Infection in Manila Clam Ruditapes philippinarum Observed from Tidal Flats in Anmyeondo Island on the West Coast of Korea During Post-Spawning Period

Negative Impacts of Perkinsus olseni Infection in Manila Clam Ruditapes philippinarum Observed from Tidal Flats in Anmyeondo Island on the West Coast of Korea During Post-Spawning Period

Genetic variability in Ruditapes decussatus clam combined with Perkinsus infection level to support founder population selection for a breeding program.

Clam farmers worldwide face several challenges, including irregular seed supply and high mortalities due to pathogenic organisms such as Perkinsus olseni. In Europe, there is a high unmet consumer demand for native clam species such as Ruditapes decussatus. The high market value of R. decussatus makes the culture of this species potentially more attractive than that culture of the alien species Ruditapes philippinarum. Thus, there is a market opportunity in breeding and producing R. decussatus at an industrial scale. A selective breeding program to improve R. decussatus performance will be carried out in Portugal; and the first critical step to develop such a breeding program is the establishment of a founder population. In this study, intra- and interpopulation genetic diversity was assessed using 13 microsatellite markers in eight natural beds located in Portugal, Spain and Italy. Also, allele and genotypic frequencies of each microsatellite locus were assessed discriminating between clams infected and non-infected by P. olseni. All locations showed similar values for several genetic diversity parameters. Analyses of population differentiation (FST, Bayesian clustering and AMOVAs) revealed five genetically differentiated regions: Rías Altas and Rías Baixas (NW Spain), North/Central Coast of Portugal, Gulf of Cadiz and Adriatic Sea. Significant differences in the allelic and genotypic frequency distribution between infected clams and non-infected ones at four microsatellite loci are reported suggesting that resistance to the disease could have a genetic basis. Moreover, a positive or negative relationship between the frequency of certain alleles and the parasite infection was inferred. Further studies should confirm the potential use of those alleles as genetic markers for P. olseni infection. Integrating results of genetic diversity within and between populations and Perkinsus infection levels, a founder population for a R. decussatus breeding program is proposed, composed by individuals from Barallobre (Rías Altas), Pontevedra or Cangas (Rías Baixas), Óbidos (North/Central Coast of Portugal), Algarve (Gulf of Cadiz) and Venice (Adriatic Sea).

Are Juvenile Manila Clam Ruditapes philippinarum Free from Perkinsus olseni Infection in Korean Waters?

As a suspension feeder, Manila clam Ruditapes philippinarum (A. Adams and Reeve 1850) plays a crucial role in the coastal soft bottom ecosystem in the temperate region, linking the benthic primary production to the upper trophic level. Manila clam density on tidal flats on the west coast of Korea has been declining for the past decades, and infection by the protozoan parasite Perkinsus olseni (Lester and Davis 1981) is one of the major causes for the decline. Recent studies carried out in Japan revealed that P. olseni induces mortalities of the juveniles in their natural habitats, which may lead to the recruitment failure and subsequent decline in the clam population. In this study, we surveyed P. olseni infection in juvenile Manila clam occurring on two tidal flats on the Taean coast. Ray’s fluid thioglycollate medium assay (RFTM) revealed that P. olseni infection was not limited to the adult clams, and the juvenile and small-sized clams are also infected by P. olseni. As young as four-month-old juveniles from Jugyo tidal flat were infected by P. olseni, with the prevalence (i.e., percentage of the infected individuals) of 75.0% and the intensity of 7.77 × 105 cells g−1 wet tissue weight (WT). The adult Manila clams (SL > 30 mm) from Jugyo tidal flat showed a prevalence of 96.0%, and the intensity as 5.80 × 10 cells g−1 WT. The observed infection prevalence and intensity of the juvenile are somewhat comparable to those of the adult clams, suggesting that a high level of P. olseni infection in the juveniles may lead to mortality and a long term decline in the clam population density.

Enhanced growth, gonad maturation, and low-level parasite infection in juvenile Manila clam Ruditapes philippinarum cultured in subtidal cages on the south coast of Korea

The growth performance of intertidal marine bivalves is often enhanced when they are transplanted to the subtidal environment due to extended feeding time. In this study, juvenile Manila clams Ruditapes philippinarum were transplanted to subtidal suspended cages (SSC) to examine enhanced growth and reproductive activity. Juvenile clams (11.6 mm in shell length, SL) were placed in cages and suspended at 2 m depth, and shell and somatic growth, gonad maturation, and pathology were monitored. Over 103 days of the experiment, shell and somatic growth of SSC clams were significantly greater than those of the control clams cultured at the intertidal bottom (p < 0 .05). At the end of the experiment, SSC clams reached 29.0 mm in SL, whereas the intertidal bottom cage (IBC) clams only reached 18.0 mm. In late July, after 44 days in the subtidal zone, the first spawning clam was observed, and 80% of SSC clams were actively engaged in spawning by late August. In contrast, spawning in IBC was not observed until late September, and most (25%) remained sexually immature. The protozoan parasite P. olseni was detected after 30 days in the intertidal zone, increasing from 1.6 × 104 cells g−1 tissue wet weight in July to 1.30 × 106 cells g−1 tissue wet weight in September, while P. olseni infection in the SSC clams was not observed until September at much lower intensity (6.6 × 104 cells g−1 tissue wet weight). High mortality of IBC clams occurred in late July, preceded by a dramatic increase in P. olseni infection in August, suggesting that the parasitic infection was, in part, responsible for the mortalities. We speculated that prolonged feeding time in the SSC enhanced growth and gonad maturation and that the high level of parasite infection in the IBC reduced growth and reproduction further.

Perkinsus olseni in the short neck yellow clam, Paphia malabarica (Chemnitz, 1782) from the southwest coast of India.

Parasites of the genus Perkinsus predominantly infect bivalves, and two species among them, P. olseni and P. marinus, are notifiable to OIE. P. olseni infections are known to cause extensive damage to wild as well as farmed bivalves globally with enormous implications to its fishery. Consequent to the initiation of a surveillance programme for aquatic animal diseases in India, Perkinsus infections were observed in many species of bivalves. The present paper describes P. olseni infections in the short neck yellow clam, Paphia malabarica from the southwest coast of India. Diagnosis of the parasite was carried out using Ray's Fluid Thioglycollate Medium culture, histology, in-situ hybridisation and molecular taxonomy. Pathology of infection and development of zoospores is also described. This forms the first report of a P. olseni infection in P. malabarica. High prevalence and intensity of infection of Perkinsus in clams raises concerns, as clam reserves in this geographical area sustain fisheries and the livelihoods of local fishing communities.

Impact of Perkinsus olseni infection on a wild population of Manila clam Ruditapes philippinarum in Ariake Bay, Japan.

Many studies have addressed the production decline of Manila clam, Ruditapes philippinarum, in Japan, but infection of clams with Perkinsus olseni has received scarce attention. To evaluate the impact of P. olseni, infection levels and host density of a wild, unexploited clam population were monitored monthly or bimonthly on a tidal flat from June 2009 to January 2013. Real-time PCR analysis discriminating P. olseni and Perkinsus honshuensis detected only P. olseni in tested clams. The prevalence of P. olseni was 100% or nearly 100% in 7 cohorts throughout the study period, except in newly recruited clams. Infection intensity remained low and seldom reached 106 cells/g wet tissue in newly recruited clams until the month of September. Infection intensity reached 106 cells/g in autumn and remained high at 104-106 cells/g until each cohort of clams disappeared. Clam density began to decrease in the autumn when the infection intensities reached ca. 106 cells/g. Density was relatively stable in winter, increased in spring and decreased again in clams aged 1 year or older during summer and autumn in the following years. Survival of clams experimentally infected with P. olseni at ca. 106 cells/g and placed in a cage in the tidal flat for 1 or 2 months was significantly lower than survival of uninfected control clams. Our results suggested that heavy infection with P. olseni was a major cause of the clam density decrease, although other environmental and biological factors also may have contributed to the decline in density. In addition, uninfected clams were deployed in cages for 1-2 months from June 2010 to January 2013 and prevalence and infection intensity were monitored. Parasite transmission and infection progression increased in summer and autumn.

High parasite burden increases the surfacing and mortality of the Manila clam (Ruditapes philippinarum) in intertidal sandy mudflats on the west coast of Korea during hot summer

BackgroundOver the past few decades, mass mortality events of Manila clams have been reported from several tidal flats on the west coast of Korea during hot summers. During such mortality events, once clams simultaneously surface, they fail to re-burrow, perishing within a week. The present study aimed to identify the possible causes of the mass mortality of this clam species by investigating the Perkinsus olseni parasite burden and immune parameters of surfaced clams (SC) and normal buried clams (NBCs) when sea water or sediment temperature in the study area varied from 25 °C to 34 °C from late July through mid-August 2015.ResultsWe collected 2 groups of clams distributed within a 10-m2 area when a summer clam mortality event occurred around Seonyu-do Island on the west coast of Korea in 2015. The clams were collected 2 days after they surfaced on the sediment and still looked healthy without any gaping. The clams were transported to the laboratory, and we compared P. olseni infection intensity and cell-mediated hemocyte parameters between the NBCs and SCs. SCs showed significantly higher levels of P. olseni burden, lower condition index, and lower levels of cell-mediated immune functions than those of NBCs.ConclusionsOur study suggests that high P. olseni infection weakens Manila clams’ resistance against thermal stress, causing them to surface. We surmise that the summer mass mortality of Manila clams on the west coast of Korea is caused by the combined effects of high P. olseni infection levels and abnormally high water temperature stress.

Transcriptomic profile of Manila clam (Ruditapes philippinarum) haemocytes in response to Perkinsus olseni infection

Ruditapes philippinarum, one of the major contributors to the global mollusc production, is susceptible to perkinsosis, a disease caused by the protozoan Perkinsus olseni (Alveolata, Perkinsidae). Understanding the molecular response of Manila clam defence cells (haemocytes) to this parasite is essential to head towards controlling this disease. Here, we assembled the Manila clam haemocyte transcriptome from healthy and P. olseni infected clams collected from wild and experimental conditions (in vivo and in vitro) using RNA-seq. The Manila clam de novo transcriptome consists of 33,079 transcripts, of which 7300 were significantly annotated (E-value <1.0E−5) with the NCBI non-redundant (nr) protein database. Among these annotated transcripts, many were functionally linked to signalling, cell proliferation, cell adhesion, immune system (e.g. immune response, immune effector process, antigen processing) and response to stress and stimuli. Our study integrates previous transcriptomic information of R. philippinarum haemocytes with present resources, including a notable amount of new transcripts with annotation description expressed after challenge with P. olseni in different infection scenarios, many of them linked to stress-related processes. A preliminary analysis of differentially expressed genes (DEGs) was performed with the sequences from the pool samples of control and challenged haemocytes obtained under similar experimental conditions. A higher number of DEGs were detected in in vitro than in vivo stimulated haemocytes, denoting the more acute and different in vitro challenges with P. olseni trophozoites, zoospores and extracellular products. Most in vitro DEGs were down-regulated and associated with immune and stress response, likely reflecting the immune-suppression occurring at the onset of infection, while DEGs in the in vivo infected haemocytes were mostly related to metabolic process. The preliminary DEG analysis on haemocyte response provides some insight into the molecular mechanism of Manila clam defence cells against perkinsosis. Statement of relevanceHigh Manila clam mortality rates have been reported due to perkinsosis caused by Perkinsus olseni, however, the molecular mechanisms of Ruditapes philippinarum response against perkinsosis are still not fully understood. Our study provided new data to enrich the R. philippinarum haemocyte transcriptome databases from previous reports, and preliminary information on haemocyte response against perkinsosis is provided.

급성 폐사 바지락 (Ruditapes philippinarum) 으로부터 Perkinsus olseni의 확산 기작

This study was conducted in order to elucidate the dissemination mechanism of P. olseni using field and laboratory experiments. For this purpose, we quantified the level of P. olseni infection in buried (healthy) and surfaced (gapped) R. philippinarum from a clam bed on Wi-do Island on the west coast of Korea. In addition, the levels of internal and released P. olseni cells from artificially infected (and later dead) R. philippinarum were monitored for 8 days using the RFTM-2 M NaOH lysis method. Our results indicate that P. olseni cells in buried R. philippinarum was <TEX>$2,655,625{\pm}1,536,936cells/clam$</TEX>; the level in gapped R. philippinarum was considerably lower, <TEX>$28,203{\pm}24,889cells/clam$</TEX> (p < 0.05). In the laboratory experiment, the P. olseni cells remained in the host tissue 2 days after death was approximately 50% lower than the level of infection measured in living clams. The level dropped to 20% 4 days after death and to 1.5% 6 days after death; eight days after death, P. olseni cells were undetectable since the R. philippinarum flesh had completely decomposed. The level of released cells on the day of death was only 0.05% of the internal level in live R. philippinarum; however, the level increased to 2.3% 5 days after death then gradually decreased and no released cells were detected 8 days after death. Therefore, our laboratory experiment suggest that the low level of P. olseni infection observed in gapped R. philippinarum at Wi-do Island could be caused by lysis of the most of P. olseni cells during the decomposition of dead R. philippinarum tissues. Until the end of decomposition of R. philippinarum, 6.68% of the total amount of P. olseni was released within 8 days. Our study showed that the amount of P. olseni cells from dead host is a considerably higher level than naturally released from healthy R. philippinarum, suggesting that death of the host plays an important role in the dissemination of P. olseni.

여름철 산란기에 있어 바지락 번식량의 공간적 변이와 기생 원생생물 Perkinsus olseni 감염이 바지락 번식에 미치는 영향

Spatial variation in the reproductive effort of Manila clam Ruditapes philippinarum is often closely associated with variation in the seawater temperature and food availability, which determines gonad maturity and the quantity of gamates produced during spawning. Previous studies also have reported that severe infection by the protozoan parasite Perkinsus olseni exerts a negative impact on clam reproduction, retarding gonad maturation or decreasing the reproductive effort. In the present study, we investigated impacts of P. olseni infection on the reproductive condition of Manila clam during a spawning season. Histology revealed that 54% of female clams in Wando off the south coast were in spawning, while only 10% of the female from Gomso and 0% of the female from Seonjaedo in Gyeonggi bay off the west coast were engaged in spawning at the end of May in 2004. Ray's fluid thioglycollate media (RFTM) assay was applied to assess P. olseni infection and indicated that the infection intensity in Wando (3,608,000 ± 258,000 cells/g wet tissue) was significantly higher than the levels in Gomso (1,305,000 ± 106,000 cells/g wet

Influence of reproductive condition of the Manila clam Ruditapes philippinarum on hemocyte parameters during early post-spawning period

In marine bivalve aquaculture, mortality is a major problem for sustainability of the industry. Mortalities usually result from complex interactions in which the physiological status is suspected to play a major role. In many bivalves, spawning appears as a critical period. Spawning is an energy demanding process potentially resulting in immunodepression and higher susceptibility to pathogenic infection. In an effort to understand the actual impacts of spawning on the cellular defense system (i.e., hemocytes) of the Manila clam Ruditapes philippinarum, we collected clams on a one-time sampling during early post-spawning period. We then applied, for the first time, a histology–flow cytometry combined approach to these animals. Hemocyte parameters, including concentration, sub-populations, mortality, phagocytosis capacity and reactive oxygen species production were analyzed along with gender, condition index as well as the tissue protein and carbohydrate levels. Among the 200 clams analyzed, 60.5% were spent, 24% spawning, 12% resting and 3.5% ripe and ready for spawning. No gender differences could be observed among all parameters and no correlation was determined between the protozoan parasite Perkinsus olseni infection intensity and the immune parameters. CI and tissue protein level of spawning and post-spawning (spent and resting) clams were significantly lower than those of pre-spawning (ripe) clams. The concentration of hemocytes tended to decline from ripe to spent clams, partly because of a concomitant increase of cell mortality and, most of all, a decrease in the quantity of circulating granulocytes. The decline in circulating granulocytes also resulted in a reduced phagocytosis capacity (% of cells able to phagocytise). At the animal scale, these observations support a depression of the global immune defense of spawning and post-spawning clams. However, at the cellular scale, remaining circulating cells did not display signs of stress nor altered metabolism. Indeed, phagocytosis, as the number of beads ingested per cell, was normal and no oxidative stress could be detected. Further studies will be necessary to determine if these hemocytes already reached their limit of stress tolerance, and to potentially help develop immunoprotective food additives for the aquaculture industry.

Correlation between perkinsosis and growth in clams Ruditapes spp.

Perkinsosis is one of the most widespread diseases affecting commercially important species of molluscs globally. We examined the impact of Perkinsus spp. on shell growth at the individual scale in 2 clam species: Ruditapes decussatus from Mundaka Estuary (Spain) and R. philippinarum from Arcachon Bay (France). At Arcachon, 2 contrasting sites in terms of environment and Perkinsus olseni presence were chosen: Arguin (disease-free) and Ile aux Oiseaux (infected site). We monitored the dynamics of perkinsosis over the course of the experiment at Mundaka and Ile aux Oiseaux. Prevalences were high (>70%), and intensities were around 105 cells g-1 wet gills at Ile aux Oiseaux, and 106 cells g-1 at Mundaka. No significant differences in prevalence or intensity were observed over time. A 2 yr field growth experiment of tagged-recaptured clams was performed to determine individual clam growth rate, condition index (CI), and Perkinsus spp. infection intensity. Clams were collected at Ile aux Oiseaux and transplanted to Arguin. The growth rate was always significantly and negatively correlated with Perkinsus spp. infection, and positively correlated with CI. CI and Perkinsus spp. infection explained 19% and 7% of the variability of the growth rate at Mundaka and Ile aux Oiseaux, respectively. In experimental clams at Arguin, P. olseni infection explained 26% of the variability of the growth rate at the lower tidal level. Our results suggest that at a concentration of between 105 and 106 cells g-1, perkinsosis affects the physiological functions of the clams, highlighted by its impact on the growth rate.

Environmental factors contributing to the development of brown muscle disease and perkinsosis in Manila clams (Ruditapes philippinarum) and trematodiasis in cockles (Cerastoderma edule) of Arcachon Bay

AbstractThe aim of the present study was to identify environmental factors that could explain the distribution of different pathologies of commercially exploited intertidal bivalves in an Atlantic lagoon, Arcachon Bay. In particular, the role of the salinity gradient as a driver was explored. The Manila clam Ruditapes philippinarum experienced two severe pathologies, perkinsosis, which is due to a protozoan parasite, and brown muscle disease (BMD), the etiological agent of which remains unknown. Perkinsus olseni infection was very low in a small low‐salinity area but, at the scale of the entire lagoon, was more influenced by organic matter content in the sediment and by emersion time. BMD prevalence was also 2.6 times higher in the higher organic content area but was also negatively correlated with salinity. The sympatric cockle (Cerastoderma edule) was affected by eight trematode species. These parasites have a complex life cycle that generally involves three free‐living host species. The distribution of the different trematode communities was rather patchy within Arcachon Bay with no clear relationship with measured environmental factors. The dominance of particular trematode species could be due to the presence of other hosts involved in their life cycle, making it more difficult to detect a major environmental driver. This survey demonstrated that salinity is not a major factor explaining disease distribution in this temperate lagoon. This result has consequences for the research of ‘refuge areas’ (free of diseases) or ‘hot spots’ (heavy infection) for species of high economic value.

Temporal Variation ofPerkinsus olseniInfection Intensity in the Manila ClamRuditapes philippinarumin Gomso Bay, Off the West Coast of Korea

Interannual variation of Perkinsus olseni infection intensity in the adult Manila clam Ruditapes philippinarum in Gomso Bay, off the west coast of Korea, was monitored from 1999 to 2000. Infection intensity of P. olseni (i.e., total number of P. olseni cells in unit tissue weight) was determined using Ray's fluid thioglycollate medium assay and Choi's 2-M NaOH digestion. In Gomso Bay, P. olseni monthly infection prevalence ranged from 83 (April 2000)–100%. It was remarkable that of the 18 mo of sampling, the prevalence remained at 100 % in 12 sampling months. Infection intensity of P. olseni in Manila clam ranged 366,001 (July 1999)–2,235,325 cells/g wet tissue (October 1999). The infection intensity recorded in 1999 was significantly higher than the level measured in 2000, suggesting an interannual variation in the intensity (P < 0.05). A very high level of infection intensity observed during the fall (September, October, and November) coincided with a relatively low condition index and mass morality of clams in the bay. Our data suggest that the mass mortality of clams observed during late summer to mid fall in Gomso Bay could be, in part, explained by the high level of Perkinsus infection coupled with the poor physiological condition of clams during the postspawning season.

Study of perkinsosis in the grooved carpet shell clam Ruditapes decussatus in Galicia (NW Spain). III. The effects of Perkinsus olseni infection on clam reproduction

This study evaluated the effects of the protistan parasite Perkinsus olseni on the reproduction of the grooved carpet shell clam Ruditapes decussatus from Ria de Arousa, Galicia (NW Spain). Clams collected during a one-year period were processed by histology, and their histological sections were examined for gonadal stage, volume fraction of the gonadal components, and infection by P. olseni. R. decussatus had an annual cycle with four gonadal periods: rest (November–January), gametogenesis (February–March), ripeness (April–August) and reabsorption (September–October). The effects of the disease on the gonadal cycle occurred mainly during the gametogenesis and ripeness periods. P. olseni inhibited the gonadal development during the gametogenesis months, delaying the process until the ripeness period, and reduced the percentage of ripe individuals during the ripeness months. The inflammatory reaction caused by P. olseni infection affected the volume fraction (VF) of the gonadal components by increasing the VF occupied by hemocytes, and so reducing the VF of gametes, intrafollicular empty space, follicular walls, and connective tissue. However, no significant effect of infection on gonadal index, fecundity or spawning efficiency was obtained. Therefore, infected R. decussatus may recover from the delay of late gametogenesis, produce gametes and spawn without affecting spawning efficiency or fecundity.