Mass mortality of pearl oyster (Pinctada fucata (Gould)) in Japan in 2019 and 2020 is caused by an unidentified infectious agent

Animals

Details and localities of the pearl oysters, Pinctada fucata (Gould), used in each infection test are shown in Table 1 and Fig. 1. One-year-old, healthy pearl oysters were obtained from Ishikawa or Kanagawa Prefecture, where pearls are not cultivated (Fig. 1, Table 1). These oysters were used as recipients and negative control donors in the infection tests. The pearl oysters from Ishikawa Prefecture that were used as recipients in Test 2 showed slight pigmentation on the nacre in 2 of the 10 individuals observed, although the nature of pigmentation was much darker than those typically found in the oysters affected by summer atrophy and rather similar to that of black-spot shell disease. For the donors, shells exhibiting atrophy in the soft body were obtained from a pearl oyster farmer in Mie Prefecture (Table 1). Prior to the start of each experiment, 5 or 10 individuals were randomly sampled from each batch to examine atrophy of the soft body and nacre abnormality. Oysters of each experimental group were reared in the same 65-L tank and different groups were separately reared in different 65-L tanks. The tanks were filled with 56 L running seawater (approximately 250 mL/min) filtered with a one-μm pore size filter and maintained at 23–25 °C under the natural photoperiod in the laboratory. For food, 500 mL of cultured diatoms (Chaetoceros neogracile, approximately 5 × 10⁵ cells/mL), was added to each aquarium five times per week.

Scoring degree of disease

Although the infection tests were performed using 1-year-old adult oysters in this study, the mortality by summer atrophy of shells over 1-year-old is low. Consequently, severity of the disease in the infection tests could not be assessed by mortality. The atrophy of the soft body is characteristic of the disease. However, the atrophy occurs only transiently at different times in different individual oysters, and shells must be opened when the atrophy is most severe, to determine the severity of the disease. This is intrinsically difficult. On the other hand, observations of the valves of spontaneously diseased oysters suggest that the deposition of brown pigment and loss of nacre luster are also characteristic of this disease. These nacre abnormalities remain long after the oysters recover from the disease, so that the shells can be opened at any arbitrary timing to determine the severity of the disease. Therefore, in this study, the degree of disease severity was determined using abnormalities in the nacre.

The extent of abnormalities in the nacre was quantified with a subjective index as shown in Fig. 3. For the infection tests, the scores for the two valves of an individual were totaled for each pearl oyster, and the sum of the scores of oysters in one group was divided by the number of tested oysters in the group to obtain the mean shell score of the group. For the investigation of the nacre of farmed oysters that was affected by spontaneously occurring summer atrophy, the sum of the scores of valves for each group of oysters was divided by the number of observed valves to obtain the mean shell score of the group. This is because the two valves of a stored individual oyster had usually fallen apart and reconstruction of an individual by two valves was difficult.

Investigation of spontaneously occurring disease in farmed oysters

Atrophy of the soft body and abnormalities in the nacreous layer were investigated in cultured and wild pearl oysters. Cultured oysters were obtained from the areas where summer atrophy is occurring, and wild, disease-free oysters were from the areas where the disease has not been reported (Table 2). We also observed the nacreous layer of stored valves of cultured pearl oysters. These oysters had been collected in Mie prefecture before June 2019, prior to the first outbreak of summer atrophy.

Test 1: cohabitation and injection challenge using adult pearl oysters as the infection source

In the cohabitation test, five diseased 3-year-old oysters and 14 healthy oysters were reared in different net cages set in a 65-L tank under the conditions described above so that the diseased and healthy oysters are not in contact with each other. For the negative control, 19 healthy oysters were reared similarly in another 60-L tank. In the injection test, the hemolymph of 15 diseased oysters was collected from the adductor muscles. The hemolymph was pooled and centrifuged at 1,200×g for 10 min to obtain the supernatant, which was used as the inoculum. This inoculum was injected into the adductor muscles of 14 healthy pearl oysters (100 μl/individual) with 23 × 1/4 gauge needles. For the negative control, 14 healthy oysters were inoculated with hemolymph into the adductor muscles using supernatant prepared from three healthy oysters by the above method. Experiments were performed at two different water temperatures, 25 °C and 22–23 °C. That is, a total of eight test groups were set (Table 2). The mortality was monitored during the experiment. Since the abnormality on the nacre seemed to take several days to appear and mortality sometimes occurred early in the experiments, the shell scores were determined only on surviving specimens 27 days after the start of each experiment.

Test 2: injection challenge using juvenile pearl oysters as the infection source

Soft bodies were collected from 12 0-year-old juveniles from an affected group. They were pooled (0.41 g in total wet weight), chopped with scissors in two mL of autoclaved seawater, and centrifuged at 1,200×g for 10 min to obtain the supernatant as the inoculum. The inoculum for the negative control was prepared in the same way from healthy pearl oysters (0.43 g in total wet weight). Each inoculum was injected into the adductor muscles of 10 healthy oysters (100 μl/individual). The mortality was monitored, and the shell scores were determined on surviving individuals 18 days after the injections.

Test 3: filterability

Soft bodies were collected from 20 diseased 0-year-old juveniles, pooled (4.9 g in total wet weight), and cut into small pieces with scissors. The volume of the minced tissues was adjusted to 50 mL with autoclaved seawater and centrifuged at 1,200×g for 10 min to obtain the supernatant. The supernatant was then sequentially filtered through 0.8, 0.45, 0.22 and 0.1 μm filters (Millipore, Millipore Co., Tokyo, Japan). Part of each filtrate was saved and inoculated to the adductor muscles of seven healthy oysters (100 μl/individual). Thus, the total of 28 shells were used. For the positive control, diseased oysters (N = 5) and healthy oysters (N = 7) were reared in the same aquarium as in Test 1, since it had been known that the disease could be transmitted through cohabitation by the time when this test was conducted. Two negative control groups were also prepared. For one group, only healthy pearl oysters (N = 7) were reared in an aquarium. For the other, supernatant of the soft body (4.86 g) from a single healthy animal was prepared as described above and injected into the adductor muscles of seven healthy oysters (100 μl/individual) without filtration. Experiments were performed in duplicate in all test groups including positive and negative controls. The mortality was monitored, and the shell scores were determined on surviving individuals 38 days after the start of the experiment.

Test 4: serial passages

To further clarify whether the disease was transmissible, we performed an experiment to know whether the disease could be passed on repeatedly. The serial passages were conducted with three different intervals: 1 h, 5 and 10 days. The outline of the test is shown in Fig. 4. Soft bodies collected from 10 0-year-old oysters of an affected group were pooled (wet weight 4.0 g) and cut into small pieces with scissors. The volume of the minced tissues was adjusted to 6 mL with autoclaved seawater and centrifuged at 1,200×g for 10 min. The supernatant was filtered through a 0.1 um filter (Millipore). The filtrate was injected into the adductor muscles of 25 healthy oysters (100 μl/individual); this infection test was defined as the first passage. From the 25 oysters, five individuals were randomly selected at 1 h, 5 and 10 days after the inoculation, and the hemolymph was collected and pooled to prepare the inoculum for the second passage. The pooled hemolymph was centrifuged at 1,200×g for 10 min to obtain the supernatant as the second inoculum. The inoculum was injected into the adductor muscles of 12 healthy pearl oysters (100 μl/individual) in each test series (total N = 36). As for the third passage, five individuals were selected from each group at 1 h, 5 and 10 days after the second inoculation, and the hemolymph was collected and used as the inoculum. This procedure was repeated up to the 3rd passage for the 1-h interval passages, to the 4th passage for the 5-day interval passages, and to the 8th passage for the 10-day interval passages. The remaining oysters in each passage (10 individuals for the first passage and seven for the other passages) were kept for 3 weeks to observe shell abnormality. For negative controls, seven healthy oysters were similarly inoculated with supernatant of hemolymph that was prepared from five healthy animals. Serial passages were not conducted for the negative controls.

Test 5: chloroform sensitivity

This test was performed twice (Table 1). As the inoculum in the first test (Test 5-1), the supernatant of the hemolymph used for the infection of the first passage in Test 4 was used. In the second test (Test 5-2), the supernatant of the hemolymph used for the third passage in the 10-day interval passages in Test 4 was used. In both tests, the inoculum was divided into two equal aliquots, an equivalent volume of chloroform was added to one aliquot, and the mixture was shaken at 25 °C at 300 rpm for 10 min and then centrifuged at 1,200×g for 10 minutes to recover the aqueous layer as the chloroform-treated inoculum. The other was similarly shaken and centrifuged without adding chloroform and used as the non-treated inoculum. For the negative control, supernatant of the hemolymph (centrifuged at 1,200×g for 10 min) of a single healthy oyster was used as the inoculum. Each inoculum was injected into the adductor muscles of five and seven healthy pearl oysters in the Tests 5-1 and 5-2, respectively (100 μl/individual). The mortality was monitored during the experiments. The shell scores were determined on the surviving individuals 15 and 20 days after the injections in the two tests, respectively.

Test 6: thermal stability

This test was performed twice (Table 1). In the first test (Test 6-1), the supernatant of the hemolymph used for the 7th passage in the 10-day interval passages in Test 4 was used as the inoculum. In the second test (Test 6-2), the inoculum was prepared from 0-year-old juveniles of an affected group (Table 1); soft bodies were collected from 10 juveniles, pooled (0.8 g in total wet weight), chopped with scissors in 5 mL of autoclaved seawater, and centrifuged at 1,200×g for 10 min to obtain the supernatant as the inoculum. For each experiment, the inoculum was dispensed into 1.5 mL tubes (1.0 mL/tube). The tubes were incubated for 30 min in heat blocks maintained statically at four different temperatures: 40, 50, 60 °C, or on ice. Subsequently, all tubes were rapidly cooled in an ice bath. For the negative controls, supernatant was prepared from hemolymph (Test 6-1) or macerated tissue (Test 6-2) of healthy animals and used without heat treatment. Each inoculum was injected into the adductor muscles of five healthy pearl oysters (100 μl/individual). The mortality was monitored during the experiments. The shell scores were determined on the surviving individuals 21 and 15 days after the injections in Tests 6-1 and 6-2, respectively.

Histopathology

Sampling for histopathology was conducted on days 5 and 10 after the inoculation of the first passage in the Test 4. For the 5-day samples, tissues were taken from the oysters used as donors for the 2nd passage for 5-day interval passages. For the 10-day samples, tissues were taken from the animals used as donors for the 2nd passage for 10-day interval passages. At each sampling, the mantle, gill, kidney, heart, adductor muscle, foot, and midgut (including the stomach and gonads) were dissected from five specimens after collecting the hemolymph. The tissues were fixed in Davidson’s solution (Bell & Lightner, 1988), dehydrated through an ethanol series and embedded in paraffin. Two sets of three μm sections were prepared from each tissue, stained with hematoxylin and eosin or May-Grunwald/Giemsa, and observed under a microscope.

Statistics

The mean shell scores were expressed as means ± standard error. The mortality and prevalence of nacre abnormalities in survivors were analyzed statistically with Fisher’s exact test, and the mean shell scores were tested by Mann–Whitney U-test or the Steel-Dwass test. A value of p < 0.05 was considered to be statistically significant.

Investigation of spontaneously occurring disease in farmed oysters

In the oysters obtained from pearl farmers in Mie and Ehime prefectures where summer atrophy occurred, nacre abnormalities were observed in 45.6∼81.6% of the oysters (Table 2). Cultured pearl oysters from Mie prefecture before the disease occurred, and wild pearl oysters from areas where pearl farming has never performed and the disease has not been recorded, did not show the nacre abnormalities that are seen in the disease.

Test 1: cohabitation and injection challenge using adult pearl oysters as the infection source

There were no significant differences in mortality between test groups (Table 3). Appearances of nacre abnormalities of the experimental groups were significantly higher than those of the corresponding negative controls, except for the cohabitation experiment at the low temperature (Table 3). Shell scores of the injection-challenged groups were significantly higher than those of the negative controls.

Test 2: injection challenge using juvenile pearl oysters as the infection source

No mortality was observed in the negative control group (Table 4). In the group injected with inoculum prepared from diseased pearl oysters, death of the experimental animals started on the 11^th day after inoculation, and 7 out of 10 individuals died by the end of the experiment. Six of the seven dead specimens exhibited shell abnormalities, and the mean shell score obtained for the seven dead individuals was 1.7 ± 0.4. The abnormalities on the nacre were also confirmed in all three surviving individuals, and the mean shell score for the three was 3.3 ± 0.7. No abnormalities were observed in the shells of the surviving individuals in the negative control group.

Test 3: filterability

The results are summarized in Table 5. Mortality was only found in the experimental and positive control groups. Nacre abnormalities were also mostly observed in the experimental and positive control animals, although slight abnormalities were observed in some control oysters. Thus, the disease was clearly reproduced only in the experimental and positive control groups including the experimental oysters injected with the hemolymph filtered through 0.1 μm filter.

Test 4: serial passages

Neither mortality nor nacre abnormalities were observed in the negative control group (Table 6). In the 1-h interval series, shell abnormalities were observed by the second passage, but no abnormalities were observed in the 3^rd passage. On the other hand, in the 5 and 10-day interval passages, the nacre abnormalities were observed up to the end of the experiments.

Test 5: chloroform sensitivity

In both experiments, the nacre abnormalities appeared similarly in the two experimental groups irrespective of the treatment of chloroform, whereas no abnormalities were found in the nacre of the control oysters (Table 7).

Test 6: thermal stability

No abnormalities in the nacreous layer were observed in the groups injected with the inoculums incubated at 50 and 60 °C in Test 6-1, and in the group injected with the inoculum treated with heat at 60 °C in Test 6-2 (Table 8).

Histopathology

In the oysters sampled on day 10, increased numbers of migrating cells were observed in the connective tissues in 4 out of 5 individuals, but no other significant histopathological changes were found in any of the observed specimens.