Introduction

Manila clams, Ruditapes philippinarum, form the basis of one of the most important molluskan fisheries in the coastal waters in Japan. In addition, researchers have pointed to their ability to filter large quantities of water as being of benefit to improving water quality [1–3]. However, the annual catch of Manila clams has decreased since the mid-1980s because of a decrease in the population size of several Manila clam beds [4]. The primary cause of this decrease remains unclear, despite numerous studies. Although a variety of conservation measures (e.g., restricted harvest, expansion of conservation areas, and cultivation and soil dressing of intertidal flats) have been implemented to recover the resource, there has been little success to date [4–8].

Recent research suggests that infection with protozoan Perkinsus species may be playing a major role in the decrease in Manila clam abundance. This is based on detection of high infection levels of Perkinsus species in several Japanese and Korean populations of Manila clams that have suffered declines in population size [9–11]. Perkinsus species are parasitic protozoans that infect mollusks and belong to Alveolata [12, 13]. They generally have two phases in their life cycles: an asexual propagation phase in the host tissue and a zoosporulation phase in seawater [14]. Uninucleate trophozoites propagate by repeated cell divisions in host tissue and, when exposed to anaerobic conditions after the death of the host, they enlarge and transform into prezoosporangia and subsequently into zoosporangia, in which infective zoospores are generated and released into the surrounding seawater.

Within the genus Perkinsus, two species, P. olseni and P. honshuensis, are known to infect Manila clams in Japan [11, 15, 16]. The two species are also known to co-infect Manila clams [11, 15]. Umeda and Yoshinaga [11] demonstrated that P. olseni was dominant in all Manila clam populations they examined in Japan; the prevalence and infection intensity of P. olseni ranged from 45 to 100 % and from 102 to 107 cells/g soft tissue, respectively, and those of P. honshuensis ranged from 0 to 40 % and from 102 to 104 cells/g soft tissue, respectively. This suggests that the impact of P. olseni is larger than that of P. honshuensis, which likely has smaller effect on Manila clam populations. Shimokawa et al. [17] demonstrated that a cultured strain of P. olseni became lethal to juvenile Manila clams when infection intensities exceeded 107 cells/g soft tissue at 22 °C. Waki et al. [18] challenged Manila clams held at 22 °C with wild Perkinsus species (unidentified but presumed to be P. olseni) isolated from naturally infected wild Manila clams and demonstrated that the infection was lethal. A number of recently deceased juveniles were infected with >107 Perkinsus trophozoite cells/g soft tissue. Interestingly, however, the maximum infection intensity of Perkinsus species was ~106 cells/g soft tissue in Manila clam populations with the highest infection levels, such as those in Ariake Bay [11, 19–21]. Moreover, Yoshinaga et al. [21] reported few negative effects of Perkinsus infection on the physiological condition of commercially sized Manila clams naturally infected at 103–107 cells/g soft gill tissue. Thus, the results of experimental studies are often inconsistent with observations in naturally infected Manila clams, particularly with respect to the pathogenicity of Perkinsus infection. However, it is reasonable to assume that the pathogenicity of the infection is influenced by ambient conditions and host size. For example, Waki et al. [18] reported that approximately 80 % of experimentally challenged juveniles died within 48 days at 22 °C, whereas mortality was not observed until 49 days after challenge in adults. Such inconsistencies may be explained by differences in pathogenicity under different experimental conditions and in hosts of different sizes. We conducted a challenge experiment at a range of temperatures using adult and juvenile Manila clams to determine the effect of temperature and host size on the pathogenicity of infection with P. olseni.

Materials and methods

Manila clams

We obtained Perkinsus-free [22, 23] Manila clams (32- to 41-mm shell length) of commercial size from Akkesi Bay, Hokkaido Prefecture. The clams were subsequently used to produce P. olseni prezoosporangia. We obtained uninfected juvenile Manila clams (3- to 6-mm shell length) and adult Manila clams (18- to 22-mm shell length) produced in a Perkinsus-free hatchery at the Fisheries Research Institute, Oita Prefectural Agriculture, Forestry and Fisheries Research Center, located in Bungotakada City, Oita Prefecture. These clams were used in the challenge experiment. Prior to the challenge, we tested 50 juveniles and 50 adult Manila clams for Perkinsus infection using the RFTM method (see below) and found no evidence of infection.

Production of prezoosporangia

We produced prezoosporangia by incubating the tissues of Manila clams injected with the cultured trophozoites of a strain of P. olseni. The trophozoite strain was purchased from the American Type Culture Collection (ATCC#: PRA-181) and routinely cultured in Perkinsus broth medium (ATCC medium 1886; PBM) at 25 °C. After culture for 7 days, the trophozoites were concentrated to 1.0 × 107 cells/ml by centrifugation (120 × g, 5 min) and injected into uninfected Manila clams as described below.

Two holes (2 mm diameter) were drilled into the shells of 40 Manila clams near the anterior and posterior adductor muscles. Into each of the anterior and posterior adductor muscles, 100 μl of the concentrated trophozoite suspensions was injected (2.0 × 106 cells/individual), through the holes. The injected Manila clams were maintained unfed in a recirculating seawater aquarium equipped with a sand biological filter with aeration at 22 °C for 26 days. At the end of the experiment, the gill and mantle tissue was removed from each of the injected Manila clams and cut into fine pieces. The tissues (20 g) were divided into four tubes containing 50 ml Ray’s fluid thioglycollate medium (RFTM) supplemented with 500 iu/ml penicillin G potassium and 500 μg/ml streptomycin sulfate, and incubated at 25 °C for 4 days to transform trophozoites into prezoosporangia [24]. The incubated tissues containing prezoosporangia were dissociated in seawater by dissolving with 0.25 % trypsin for 90 min at 22 °C, then washed with filtered seawater by centrifugation (300 × g, 5 min) according to Casas et al. [25]. Prezoosporangia were isolated from the pellets consisting of prezoosporangia and Manila clam tissue by serial filtration through 200-, 100-, and 50-μm mesh sieves. The prezoosporangia and tissue residues trapped on the 50-μm sieve were collected and resuspended in 20 ml of filtered seawater. The density of prezoosporangia in this suspension was determined using a Burker-Turk hemocytometer. Last, we obtained 4.7 × 107 prezoosporangia cells from the tissues for use in the challenge experiment. Uninjected Manila clams were maintained in another aquarium separate from the injected Manila clams and processed in the same way as the injected Manila clams. The residual tissues of uninjected Manila clams trapped on a 50-μm sieve were used as a negative control in the challenge experiment.

Challenge experiment

We challenged juvenile and adult Manila clams by immersing them in suspensions of prezoosporangia. All the obtained prezoosporangia were incubated in 40 l seawater in an aquarium for 4 days (25 °C, salinity 30), then divided into two aquaria, each containing 20 l seawater. Juvenile (N = 620) and adult Manila clams (N = 620) were immersed in one of the two aquaria for 24 h (juveniles and adults held separately). The timing of the immersion was determined based on our preliminary observation that the highest daily zoospore release ratio occurred between the 4th and 5th day during incubation. The zoospore release ratio was calculated by collecting triplicate ~400 μl samples of seawater daily from the bottom of aquaria. The number of prezoosporangia and zoosporangia in each sample were counted under an inverted light microscope. The negative controls consisted of juvenile and adult Manila clams (N = 620 of each) that were immersed (life stages held separately) in the residual tissues of uninjected Manila clams suspended in 40 l seawater for 24 h.

We maintained the challenged and control clams in floating baskets (30 × 15 × 15 cm L × W × D for adults and 15 × 15 × 15 cm for juveniles) divided among four 1-ton recirculating seawater aquaria equipped with a biological filter and a temperature controller. Twelve baskets were prepared for each of the four treatment groups (challenged juveniles, challenged adults, control juveniles, and control adults). Fifty Manila clams were placed in each basket. Each recirculating aquarium accommodated 12 baskets (four treatments in triplicate). At the time the Manila clams were introduced into the aquaria, water temperature and salinity were 25 °C and 30 ppt, respectively. Within 1 day after the introduction, the water temperature in each aquarium was gradually changed to the appropriate target temperature (18, 23, 28, or 30). The clams in each aquarium were fed with 10 ml of a commercially produced diatom (cell density, approximately 600,000 cells/l; Sunculture, Nissin Marintech, Aichi, Japan). One clam was sampled from each basket at 0, 1, 3, 5, 7, 12, or 14 dpc (days post challenge), and then every 7 days until 42 dpc. The experiment with juvenile clams was terminated 15 and 26 dpc for challenged and control groups, respectively, at 30 °C; 22 and 30 dpc for challenged and control groups, respectively, at 28 °C; 32 dpc for both groups at 23 °C; and 43 dpc for both groups at 18 °C. The experiment with adult clams was terminated 34, 38, 42, and 44 dpc for both the challenged and control groups maintained at 30, 28, 23, and 18 °C, respectively. All surviving Manila clams were also sampled at the time the experiment was terminated. Each individual was weighed (soft tissue weight) and measured (shell length) at the time of sampling or soon after death. The entire soft tissue of each individual was then used to determine the infection intensity by the RFTM method. We plotted Kaplan–Meier survival curves to assess survival in each experimental basket.

Ray’s fluid thioglycollate medium (RFTM) assay

We used the RFTM method to quantify infection intensity and prezoosporangia production [26, 27]. The whole soft tissue of each Manila clam was cultured in 5 ml RFTM supplemented with antibiotics for 4–7 days at 25 °C. After the incubation, the tissue was pelleted by centrifugation (300 × g, 5 min) and lysed in 2 M sodium hydroxide solution at 60 °C until the tissue was dissolved. Prezoosporangia in the dissolved tissue were collected by centrifugation (300 × g, 5 min), washed 2 times in deionized water, and counted in 96-well plates (following dilution and staining with Logol’s solution).

Results

Zoospore production

During incubation in seawater, 42 % of the zoosporangia released zoospores. The release of zoospores occurred between 3–7 days after transfer from RFTM to seawater and peaked on the 5th day. During the period when Manila clams were exposed to the suspension, approximately 25 % of zoosporangia released zoospores.

Challenge experiment

Challenged Manila clams began to die earlier and had significantly lower survival than controls, regardless of the developmental stage (juvenile or adult) or temperature with the exception of the adult stage held at 18 °C. However, we also observed mortality in control groups at most temperatures (Table 1 and Fig. 1). The difference in survival was greater in juveniles than in adults and was apparent earlier in the experiment. As temperature increased, we were able to detect a significant difference in survival at an earlier stage.

Table 1 Time to first mortality [days post challenge (dpc)] in the triplicate groups for each treatment group and time to a significant increase in mortality relative to control treatments
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Fig. 1
figure 1figure 1

Kaplan–Meier survival curves and infection intensities in regularly sampled juvenile (left panels) and adult (right panels) Manila clams maintained at 30 (a), 28 (b), 23 (c), and 18 °C (d). The numbers 1 and 2 represent the survival curves and 3 and 4 represent the infection intensities of juveniles and adults, respectively. In 1 and 2, open and closed circles represent challenged and control groups, respectively. In 3 and 4, open circles represent infection intensities in the regularly sampled Manila clams, with the solid line representing the geometric mean infection intensity. The crosses in 4 represent the infection intensities in recently deceased individuals

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All challenged juveniles and adults became infected with P. olseni. The infection intensity in 12 juveniles and 12 adults examined soon after the challenge (three juveniles and three adults from each of the four temperature groups) varied considerably and ranged from 103 to 105.5 and 102 to 106 cells/g soft tissue, respectively (Fig. 1). The geometric mean infection intensity was between 104 and 105 cells/g soft tissue in both juveniles and adults. Although the infection intensity gradually increased with time in all challenged groups, the geometric mean infection intensity peaked at ~107 cells/g soft tissue in juveniles and only ~106 cells/g soft tissue in adults. As temperature increased, we observed an earlier increase in the geometric mean infection intensity in both life stages (Fig. 1). At the end of the experiment, the infection intensity in challenged juveniles and adults ranged from 103 to 109 and 102 to 107 cells/g soft tissue, respectively (Fig. 1).

We observed a significant difference in survival only after the geometric mean infection intensity reached ~106 cells/g soft tissue in any of the treatment groups, except the adult groups held at 18 °C (Table 1 and Fig. 1). After reaching this infection level, survival began to decrease rapidly, other than in challenged adults held at 23 °C. In this latter group, survival began to decrease 2 weeks after the geometric mean infection intensity reached ~106 cells/g soft tissue at 14 dpc.

We were able to quantify the infection intensity in adults soon after death, as their soft tissue remained viable for some time. The intensities ranged from 105 to 107 cells/g soft tissue and exceeded 106 cells/g soft tissue in a number of individuals (36 of 42 adults) (Fig. 1). In contrast, we were unable to determine infection intensity in juveniles after death as the soft tissue was quickly lysed. We found no evidence of P. olseni infection in any control juveniles. However, we observed low levels of infection in adult control groups 14 dpc (Fig. 2). The prevalence of infection at the end of the experiment was 6.1, 7.7, 7.8, and 4.3 % in control adults held at 30, 28, 23, and 18 °C, respectively. The intensity of infection appeared to increase over time. The maximum infection intensity in control adults was ~101 and ~103 cells/g soft tissue at 30 and 28 °C, respectively, and 105 cells/g soft tissue at both 23 and 18 °C. These levels were significantly lower than in challenged adults.

Fig. 2
figure 2

Infection intensities in control adults infected with P. olseni. Open triangles, squares, diamonds, and circles represent infection intensities in the regularly sampled surviving adults maintained at 30, 28, 23, and 18 °C, respectively. Closed triangles and closed squares represent recently deceased adults held at 30 and 28 °C, respectively

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Discussion

We obtained prezoosporangia from the gill and mantle tissue, the primary sites of P. olseni infection in Manila clams [28]. Approximately 40 % of the prezoosporaniga developed into zoosporangia and released zoospores. Furthermore, all challenged Manila clams were infected with P. olseni. Shimokawa et al. [17] conducted a challenge experiment using prezoosporangia obtained from the whole tissue of Manila clams injected with P. olseni trophozoites. However, this approach suffers from a decrease in zoospore release rates caused by contamination of seawater with residual Manila clam tissue. We minimized contamination by using only gill and mantle tissue.

We observed mortality in challenged juveniles and adults when the geometric mean infection intensity reached ~106 cells/g soft tissue. Moreover, the majority of recently moribund, challenged adults were infected with 106–107 cells/g soft tissue. Our results suggest that P. olseni infection can be lethal when the infection intensities exceed ~106 cells/g soft tissue in juvenile and adult Manila clams. Interestingly, some surviving Manila clams had much higher infection intensities than 106 cells/g soft tissue, suggesting that there is variability among individuals in the sensitivity to infection. Shimokawa et al. [17] reported that hatchery-raised juvenile Manila clams (3- to 10-mm shell length) challenged with P. olseni began to die when the infection intensity exceeded ~107 cells/g soft tissue in the majority of recipient Manila clams at 22 °C. In other challenge experiments using wild juvenile Manila clams (3- to 15-mm shell length) and wild Perkinsus isolates from naturally infected wild Manila clams, mortality was observed at infection intensities above 106 cells/g soft tissue [18]. Despite differences in rearing conditions, the history and size of the host, and the source of P. olseni, the combined evidence from these studies and our own suggests that an infection intensity of >106 cells/g soft tissue can be lethal to Manila clams.

Prior reports concluded that the maximum infection intensity was ~106 cells/g soft tissue in Manila clam populations on the coast of Ariake Bay in Japan [19, 20]. Perkinsus infection appears to have had a significant effect on the survival of wild Manila clams in the area, likely because the infection intensity is close to the experimental lethal level. Researchers have tested for Perkinsus infection throughout Japan and documented a number of incidences in wild Manila clam populations [11, 21, 22, 29–32]. However, the impact of these infections is unclear as the reports only document prevalence or infection intensity in the gills. Thus, these data cannot be compared with the experimental lethal infection intensity. Moreover, the study periods were short and there was no consideration to the temporal change in infection intensity. Additional research into the temporal changes in Perkinsus infection and wild Manila clam abundance is needed to determine the impact of the parasite on wild Manila clam populations.

In general, juveniles died earlier than adults following the challenge with P. olseni. Moreover, the peak geometric mean infection intensity was much higher in juveniles than adults (107 versus 106 cells/g soft tissue, respectively). We speculate that the propagation of trophozoites was more rapid in juveniles than in adults, resulting in earlier death in the juveniles. Furthermore, adult clams appear to be able to limit the propagation of trophozoites to levels around 106 cells/g soft tissue. Regardless, persistence of infection at the lethal level still resulted in mortality in adults.

Increases in temperature were associated with a more rapid increase in the rate of mortality. Peyre et al. [33] demonstrated that in vitro propagation of P. olseni trophozoites was promoted by increasing temperature within a range of 4–28 °C. Umeda et al. [34] concluded that the optimal temperature for the in vitro propagation of P. olseni was between 28 and 34 °C. Our results also suggest that trophozoite propagation is more rapid at 28 and 30 °C than at 18 and 23 °C. Increasing temperature is known to stimulate hemocyte immune activity in Manila clams within the range 8–21 °C [35]. However, further studies are needed to determine the nature of the interaction between trophozoite propagation and the immune response of hosts at high temperatures. Interestingly, we observed higher mortality in control groups held at higher temperature, suggesting that thermal stress alone is sufficient to cause mortality in Manila clams.

Only one challenged adult died in the group that was held at 18 °C, even though the infection intensity exceeded 106 cells/g soft tissue in several individuals within this group. This suggests that P. olseni infection may have less of an effect on adults at this temperature. Lester and Davis [36] reported that blacklip abalones Haliotis ruber infected with P. olseni died at 20 °C, whereas abalones held at 15 °C encapsulated P. olseni trophozoites and killed them. Similarly, the mortality of cockles, Anadara trapezia, was higher in a challenged group maintained at 27–30 °C than in a group maintained at 20 °C [37]. Taken together, these results suggest that the pathogenicity and propagation of P. olseni is suppressed by low temperature.

We observed infection with P. olseni in some control adults. Zoospores are thought to be the primary source of infection with P. olseni [14]. However, trophozoites in the feces and pseudofeces of infected Manila clams may also be a source of infection [38]. We speculate that the control clams were infected via zoospores released from zoosporangia formed in the tissue of dead hosts and trophozoites released from live hosts. However, because the infection intensity remained low in control clams (<105.2 cells/g soft tissue), we do not believe infection contributed to the death of the control adults. The absence of infection in control juveniles may be explained by the lower filtration rate of juveniles, which decreases the probability of invasion by the parasite. Previous reports suggest that the absence of Perkinsus infection in juveniles smaller than 15- or 19-mm shell length in highly infected populations of Manila clams in Korea and Spain can be explained by their low filtration rates [39, 40].

In contrast to our results, Yoshinaga et al. [21] demonstrated that Perkinsus infection intensity did not affect tolerance to high water temperature stress, clearance rate, or boring activity in naturally infected wild large adults (34- to 44-mm shell length). In light of our current observations, the adults in the prior study may have been of sufficient size that they were less sensitive to Perkinsus infection.

In conclusion, P. olseni infection had a greater impact on juvenile survival than adult survival in Manila clams. Furthermore, increased temperature resulted in a more rapid decrease in survival. Our results suggest that Perkinsus infection can be a major cause of mortality among wild Manila clams in heavily infected populations, particularly in juveniles during periods of high temperature.