2025 Volume 60 Issue 3 Pages 103-112
Abalone asfa-like virus (AbALV) has been strongly implicated as the causative agent of amyotrophia in Haliotis spp. However, investigations targeting AbALV in abalone hatcheries have been very limited. The present study continuously monitored this virus in a hatchery rearing black abalone H. discus discus. In surveys of 0-year-old juvenile abalone, amyotrophia with high mortality was confirmed from April to June or July, and copy number of AbALV in the foot muscle remained high during this period. From late July to August, AbALV copy number decreased along with a decline in mortality, supporting the conclusion that AbALV is the causative agent of amyotrophia. Additionally, the present study first detected AbALV in early February, followed by mortality in March, suggesting occurrence of amyotrophia in early spring around March. In the survey of 1-year-old abalone, AbALV copy number increased from September to December. Additionally, mortality and AbALV copy number were much lower than those in 0-year-old juveniles from April to July. During seed production in the hatchery, AbALV was frequently detected in wild broodstock. However, AbALV was not detected in fertilized eggs after washing, nor in swimming larvae that developed from those washed eggs.
Abalone amyotrophia is known to cause atrophy of foot muscle, formation of abnormal cell masses in the nervous tissues, and high mortality in juveniles of large abalone species (Haliotis discus discus, H. discus hannai, H. madaka) during the seasonal increase in water temperature from spring to summer (Nakatsugawa et al., 1988; Momoyama et al., 1999; Nakatsugawa, 2000). The occurrence of these characteristic abnormal cell masses in the nervous tissues has been reported from many abalone hatcheries in Japan when high mortality of juvenile abalone was recognized, suggesting that this disease occurs in various regions of this country (Nakatsugawa, 2000).
Nakatsugawa (1990) reproduced the disease by injection of 0.22 μm-filtered homogenates from moribund abalone, indicating that abalone amyotrophia is an infectious disease caused by a viral pathogen. Countermeasures designed for this presumed viral pathogen, such as UV irradiation of intake water, rearing in isolation from disease-experienced groups, and washing of fertilized eggs, also proved to be effective (Nakatsugawa, 2000; Okada et al., 2000; Shibata et al., 2002).
Recently, Matsuyama et al. (2020) conducted comprehensive nucleotide analyses for a fraction containing the disease agent, and identified a novel Abalone asfa-like virus (AbALV) as the most likely pathogen causing abalone amyotrophia. Koch’s postulates have not been fulfilled for AbALV, due to the lack of any appropriate cell lines for virus isolation and propagation. However, Matsuyama et al. (2021a) successfully established infection in four abalone species using rearing seawater from AbALV-infected individuals and confirmed the presence of AbALV in infected abalone through immunohistochemical staining with an AbALV specific antibody. This almost conclusively confirms that AbALV is the causative agent of amyotrophia. Currently, based on genomic comparison and transmission electron microscopic observations, classification of AbALV into the family Asfaviridae is proposed (Matsuyama et al., 2023).
Data on the epidemiology of amyotrophia in abalone hatcheries have accumulated based on histological signs of the disease (Nakatsugawa, 1990; Nakatsugawa and Momoyama, 1999; Nakatsugawa, 2000; Momoyama, 2000; Nakatsugawa et al., 2000; Shibata et al., 2002). However, information on AbALV, the presumed causative agent of this disease, in hatcheries remains limited, except for that presented by Aikawa et al. (2022), in which AbALV was detected during the occurrence of high mortality among juvenile black abalone. We expected the AbALV specific qPCR assays developed by Matsuyama et al. (2020) and Matsuyama et al. (2023) to provide novel information to further characterize the disease. Accordingly, we conducted continuous surveys of AbALV infection and amyotrophia among juvenile black abalone H. discus discus reared in a hatchery where AbALV and its associated mortality were previously reported by Aikawa et al. (2022). Additionally, we also examined AbALV among broodstock, gametes, fertilized eggs, and hatched larvae in the hatchery, to assist in establishing effective control measures for the disease during seed production.
The first survey was conducted from April 13 to August 31, 2021, on 0-year-old black abalone produced in the autumn of 2020 at the Kanagawa Sea Farming Association in Kanagawa, Japan. The abalone were reared in a 10-ton indoor tank supplied with sand-filtered seawater, with water being completely exchanged once or twice per hour. Since the UV irradiator (dose of 2.6 × 105 μW·s/cm2, FLONLIZER, Chiyoda Kohan) for the sand-filtered seawater at this facility was frequently found to be non-operational, the sand-filtered seawater was assumed to be non-sterilized in this study. Temperature of the intake water was monitored every day by Kanagawa Fisheries Technology Center. Artificial food (Halios EX, Feed One) and natural marine algae were fed every day. The tank was cleaned to remove feces, remaining feed and dead individuals once a day, and the number of dead individuals was recorded daily. During the survey period, the rearing tank was changed to another 10-ton indoor tank after the estimation of total surviving individuals on June 11, as described below.
Since the total number of abalone reared in the tank at the start of the survey was not counted, the total number of surviving individuals was estimated on June 11 based on the average weight of 300 haphazardly selected individuals and the total weight of all surviving individuals. The number of surviving individuals each day from April 5 to June 10 were then retrospectively calculated based on this estimate and the daily recorded number of deaths up to that point. After June 11, the daily number of surviving individuals was determined based on the June 11 estimate and the daily recorded deaths thereafter. Based on these estimated daily numbers of surviving individuals, the cumulative survival rate over the study period was calculated using the Kaplan-Meier method.
For examination of juvenile abalone, twenty individuals were haphazardly sampled approximately every two weeks from April 13 to August 31, 2021. After grossly inspecting shell deformity (Fig. 1A) and conchiolin deposit (Fig. 1B) which were reported in association with amyotrophia (Nakatsugawa, 1991), whole soft bodies were preserved in 70% ethanol. In order to exclude the possibility of detection of AbALV attaching to the external body surface, an internal part of the foot muscle (approximately 20 mg) was excised and used for DNA extraction and the subsequent qPCR assay.
Additionally, five individuals with abnormal behaviors associated with the disease (Momoyama et al., 1999), such as turning over on the tank bottom or crawling up in the light period, were collected on May 12, June 21, July 26 and August 31. Their soft bodies were fixed in Davidson’s fixative, and fixed tissues were dehydrated in an ethanol series and embedded in paraffin after replacement with G-NOX. Five μm sections were prepared and stained with hematoxylin and eosin. The sections were observed for abnormal cell masses in the nervous tissues, a characteristic of abalone amyotrophia (Momoyama et al., 1999) and hyperplasia in the gills associated with AbALV infection (Matsuyama et al., 2021a).
Second survey of 0-year-old abalone in 2022To investigate the infection status of AbALV from an earlier time of year than the first survey, and to compare with the results of the first survey, the second survey was conducted from January 11 to August 31, 2022, on 0-year-old abalone produced in the autumn of 2021 at the same hatchery. The juvenile abalone settled on corrugated plastic boards were maintained in a 10-ton indoor tank from January 11 to April 5. For examination, 20 individuals were sampled on January 11, February 11 and March 19, and after gross inspection of the shell symptoms, these individuals were preserved in 70% ethanol for the subsequent AbALV qPCR. Due to the difficulty in counting small individuals, mortality was not examined until the following tank transfer on April 5.
Since the growing juvenile abalone leave the plastic boards to move to the bottom of the rearing tanks, they were collected and transferred to another 10-ton tank on April 5. The rearing conditions of abalone were the same as those in the first survey of 0-year-old abalone in 2021. From April 5 to August 12, 20 individuals were sampled approximately every month, and preserved in 70% ethanol for the subsequent AbALV qPCR after gross inspection of shell symptoms.
Since the total number of abalone transferred to the tank was not counted, the total number of surviving individuals was estimated on June 10 in the same way as the first survey. Then, the daily number of surviving individuals before and after June 10 were estimated as described above, and these estimates were used to calculate the cumulative survival rate over the study period using the Kaplan–Meier method. As in the first survey above, after estimating the number of surviving individuals on June 10, the rearing tank was changed to another 10-ton indoor tank.
Survey of 1-year-old abaloneIn order to monitor AbALV infection beyond August, when black abalones reached approximately one year of age, 981 individuals were haphazardly selected from the tank of the second survey, transferred to a 1-ton outdoor tank on August 31, 2022, and examined until September 26, 2023. The rearing conditions were almost the same as above, with an exception in tank cleaning frequency (once in 1 to 3 d).
For examination of AbALV by qPCR, 20 individuals were sampled on September 7, while 10 individuals were sampled approximately every month from November 21 to the end of the survey. Observation for shell symptoms and tissue sampling for qPCR were conducted in the same way as in the above surveys.
Cumulative survival over the survey period was calculated from the initial stocking number and the daily count of dead individuals using the Kaplan-Meier method.
Examination of AbALV during seed production of black abaloneThe presence of AbALV in broodstock, gametes from the broodstock, fertilized eggs and developed larvae were examined at three seed production dates in this hatchery: August 30, September 9 and October 17, 2022.
For each seed production date, wild male and female abalone used for broodstock were separately placed in 500 L tanks supplied with UV-irradiated sand-filtered seawater. Since the males released sperm first, seawater containing suspended sperm in the male tank was transferred to the female tank to induce spawning. After confirming that the females had spawned, a few liters of the sperm-suspended seawater were further added to the female tank, and the mixture was stirred manually to facilitate fertilization. The fertilized eggs were left undisturbed for five minutes to allow them to settle at the bottom of the tank. The supernatant was then discarded, fresh sand-filtered seawater was added to resuspend the fertilized eggs, and the supernatant was discarded again. After repeating this washing process three times, the washed fertilized eggs were then transferred to a separate 500 L larval rearing tank supplied with sand-filtered seawater.
To examine AbALV infection, foot muscle mucus of broodstock was individually collected before spawning induction using cotton swabs according to Matsuyama et al. (2021b). These swabs were stored in DNA extraction buffer (180 μL of Buffer ATL and 20 μL of Proteinase K solution (QIAGEN)) at −20°C until DNA extraction.
For gamete samples, 500 mL of seawater suspending sperm was mixed with the same volume of 99.5% EtOH and stored at 4°C. After a few days, precipitated sperm was recovered and further preserved in 99.5% EtOH as sperm samples. Spawned eggs on the tank bottom were collected by a glass pipet and stored in 99.5% EtOH. Sperm samples were collected once on each seed production date on August 30 and October 17, while the egg sample was collected once only on August 30. For DNA extraction and the subsequent qPCR, 20 mg of the preserved sperm pellet or approximately 500 eggs were used as one batch.
Fertilized eggs on the tank bottom were collected by a glass pipet before and after the washing process. For collection of 1-day veliger stage larvae, 1 L of the larval rearing tank water was retrieved by a plastic container, and larvae settled at the bottom were collected by a glass pipet. During each seed production date, these samples were collected three times from the same tank, and each sample was separately preserved in 99.5% EtOH. Approximately 500 eggs or larvae were used as one batch, and subjected to the subsequent qPCR.
Quantitative PCR detection of AbALVFor all qPCR assays, DNA was extracted with a QIAamp DNA mini kit (QIAGEN) and the total amount of the extracted DNA was quantified with SimpliNano (Biochrom).
The present study utilized two different qPCR assays targeting the p72 capsid gene of AbALV. For the samples from the first survey of 0-year-old abalone in 2021, the qPCR assay developed by Matsuyama et al. (2020) was employed. In brief, 1 μL of extracted DNA was added to a qPCR mixture consisting of 5.0 μL of THUNDERBIRD SYBR qPCR Mix (TOYOBO), 0.3 μL of 10 μM primers (Q-ASFV-like-F: 5′-CCC GGA GCG ACC TAC AGA A-3′ and Q-ASFV-like-R: 5′-GCA TTC CGA CAG CAT CAC AG-3′), and 3.4 μL of molecular-grade distilled water (Invitrogen). The qPCR was conducted using CFX-Connect Real-Time System (Applied Biosystems). The thermal cycle condition was one cycle of pre-denaturation at 95°C for 60 s and 40 cycles of denaturation step at 95°C for 15 s, and an annealing/extension step at 60°C for 60 s during which the amplification signal was detected. In each reaction, serial dilutions of a plasmid containing the target region of AbALV (1.25 × 107 to 1.25 × 101 copy/μL), kindly provided by Dr. Matsuyama, were also applied to generate a standard curve for estimation of virus copy numbers. Samples with estimated copy numbers less than 1.25 × 101 copy/μL were evaluated as negative, otherwise, copy number per 1 ng of total amount of extracted DNA was calculated. All DNA samples were analyzed in duplicate, and the average copy number was calculated.
For the samples from the second survey of 0-year-old abalone, the survey of 1-year-old abalone, and the samples from seed production, the TaqMan probe qPCR assay developed by Matsuyama et al. (2023) was employed. In brief, 1 μL of extracted DNA was added to a qPCR mixture consisting of 5.0 μL of KAPA PROBE FORCE qPCR Master Mix (Roche), 0.2 μL of 10 μM primers (p72-qF: 5′-CCC ATC CAA CAC TCA TTC TC-3′ and p72-qR: 5′-AGC CGA ACA TCT TCA TTA AAC C-3′), 0.2 μL of the 10 μM fluorescent probe ([FAM]-CGG CAC TAA ATG GTC CAC AAA CAC CAA-[BHQ1]) and 3.4 μL of molecular-grade distilled water. The qPCR was conducted using the same thermal cycler as above. The thermal cycle condition was one cycle of pre-denaturation at 98°C for 3 min and 40 cycles of denaturation step at 95°C for 10 s and an annealing/extension step at 60°C for 30 s, during which the amplification signal was detected. Quantification of AbALV copy numbers and determination of positive/negative detection in the assay were followed as described above.
Shell length of the examined abalone was 16.3 ± 1.3 mm (mean ± standard deviation (S.D.)) at the initial sampling on April 13 and 17.0 ± 1.6 mm (mean ± S.D.) at the final sampling on August 31 (Fig. 2A).
The prevalence of AbALV between April 13 and July 20 ranged from 90–100%, and then declined to 35% on August 31. The geometric mean of AbALV copy number/ng of extracted DNA was 1.5 × 101 on April 13, and then increased to 3.8 × 103 on May 11. A sudden decline of the copy number was observed twice from June 22 to July 6, and from July 20 to August 4. At the end of the survey on August 31, all of the examined individuals had less than 10 copy/ng of extracted DNA (Fig. 2B).
Based on the daily mortality count, the sampling number and the number of surviving individuals measured on June 11, the total number of abalone at the start of the survey on April 13 was retrospectively estimated to be 64,611. High mortality of juvenile abalone was first observed from May 3 to May 8, and further observed from May 16 to May 21, and May 24 to June 12. From early July, daily mortality had almost subsided, and the cumulative survival at the end of the survey was 35.6% (Fig. 2B).
Shell symptoms, such as shell deformity (Fig. 1A) and conchiolin deposit (Fig. 1B), were found in less than 25% of examined abalone up until May 25, but were found in more than half of the examined abalone on June 8 and 22. In July and August, 35-50% of abalone showed these signs (Fig. 2C).
For histology, five individuals were sampled on May 12, June 21, July 26 and August 31. An abnormal cell mass in the nervous tissues within the foot muscle (Fig. 3A) was observed in one individual on May 12, June 21 and July 26, and in two individuals on August 31. The characteristic gill symptoms of AbALV, such as hydrocephalic voids in the epithelial cell layer and uneven surfaces (Fig. 3B), were observed in all individuals on May 12, July 26 and August 31, and in four of five individuals sampled on June 26.
At the start of the survey, the temperature of the intake water was 15.8°C, and reached the highest temperature of 27.3°C on August 6. The water temperature from May 3 to June 12, when the decline in cumulative survival rate was marked, ranged from 17.4°C to 21.3°C (Fig. 2D).
Second survey of 0-year-old abalone in 2022Shell length of the examined abalone was 7.3 ± 1.3 mm (mean ± S.D.) at the initial sampling on January 11 and 21.9 ± 2.1 mm (mean ± S.D.) at the final sampling on August 12 (Fig. 4A).
AbALV was not detected in any individual at the initial sampling on January 11, 2022, However, from February to July, AbALV was detected in all individuals, except for two individuals on April 4. The prevalence at the final sampling on August 12 was 75%. The mean copy/ng of extracted DNA exceeded 7.0 × 102 on March 19, then declined to 1.9 × 102 on April 4. After transfer to a 10-ton tank, the mean copy/ng of extracted DNA increased to higher than 2.0 × 102 on May 10 and June 7, and reached 4.5 × 103 on July 4. On August 12, the mean copy number/ng of extracted DNA declined to 1.3 (Fig. 4B).
Based on the daily mortality count, the sampling number and the number of surviving individuals measured on June 10, the number of individuals transferred to the 10-ton tank on April 4 was retrospectively estimated to be 30,052. Moderate mortality continued from April 4 to July 24. Since then, mortality diminished until the end of the survey on August 31. The cumulative survival rate at the end of the survey was 62.8% (Fig. 4B).
Shell abnormalities were first observed on April 4, and were found in 20% of individuals until June 7. Subsequently, the rate of this abnormality decreased in July (Fig. 4C).
The temperature of the intake water was 13.2°C at the start of the survey on January 11, and reached a maximum temperature of 26.3°C on August 27. The water temperature from April 4 to the end of July 25, when a decline in cumulative survival rate occurred slightly earlier, ranged from 14.4°C to 25.4°C (Fig. 4D).
Survey of 1-year-old abaloneShell length of the examined abalone was 23.9 ± 2.2 mm (mean ± S.D.) at the initial sampling on September 7, 2022 and 34.4 ± 4.3 mm (mean ± S.D.) at the final sampling on September 26, 2023 (Fig. 5A).
AbALV was detected from five of 20 examined individuals at the initial sampling on September 7, and was then detected from all examined individuals until February 17. From March 27, AbALV prevalence gradually declined. Although AbALV was detected in all individuals on June 26, it was no longer detected on August 29. At the final sampling on September 26, AbALV was detected in four of ten individuals (Fig. 5B).
The mean copy number of AbALV increased from September 7 to December 19, and then decreased. From April 24, the mean copy number remained lower than 2 copy/ng of extracted DNA until the end of the experiment (Fig. 5B).
During the survey period, cumulative survival continuously declined, but the marked mortality observed in 0-year abalone was not observed from April to July. After moderate mortality on June 30 and 31, significant mortality was observed on August 28 (Fig. 5B), which was possibly caused by invasion of a lethal phytoplankton Karenia mikimotoi into the facility (data not shown).
Shell symptoms were observed in less than 40% of examined individuals from September 7 to April 24, and in more than 50% of examined individuals from March 19 to September 26, except for 20% on August 29 (Fig. 5C).
The temperature of the intake water was 24.5°C at the beginning of the survey on September 1. This temperature dropped to the lowest of 13.9°C on February 22, and then increased to the highest of 28.2°C on August 12 (Fig. 5D). The water temperature ranged from 14.0°C to 20.1°C between November 21 and January 24, when the mean AbALV copy/ng of extracted DNA exceeded 10.
Examination of AbALV during seed production of black abaloneAbALV was detected in the body mucus of most adult abalone used for broodstock at all of the seed production dates (Table 1). When combining qPCR results of all broodstock examined across the three seed production dates, AbALV prevalence in males was significantly higher than that in females (Chi-square test, p < 0.01).
| Date of seed production | Sex | Numbers of examined | Shell length (mm) (mean ± SD) | Whole weight (g) (mean ± SD) | AbALV prevalence (Range of detected AbALV copy number*) |
|---|---|---|---|---|---|
| August 30 | male | 10 | 124.3 ± 7.9 | 252.0 ± 63.3 | 100.0% |
| (7.9–625.5, (32.7)) | |||||
| female | 12 | 123.3 ± 8.9 | 247.5 ± 69.4 | 66.7% | |
| (1.1–292.8 (33.8)) | |||||
| September 9 | male | 14 | 117.0 ± 11.0 | 203.4 ± 57.5 | 64.3% |
| (1.2–1,198.0 (50.5)) | |||||
| female | 15 | 120.1 ± 11.5 | 222.1 ± 63.3 | 33.3% | |
| (4.6–5,864.6 (85.1)) | |||||
| October 17 | male | 18 | 121.3 ± 9.4 | 210.2 ± 56.5 | 88.9% |
| (2.1–90.8 (13.8)) | |||||
| female | 21 | 124.5 ± 9.2 | 238.2 ± 61.8 | 61.9% | |
| (0.4–184.1 (4.2)) |
AbALV was not detected in eggs before fertilization, although only one sample on August 30 was examined. AbALV was detected in both sperm samples collected on August 30 and October 17 (Table 2). Before washing, AbALV was detected in fertilized eggs from two of three seed production dates, but not detected in any batches of fertilized eggs after washing. Also, AbALV was not detected from veliger larvae that developed from washed fertilized eggs (Table 2).
| Date of seed production | Sperm | Unfertilized egg | Fertilized egg | Larvae | |
|---|---|---|---|---|---|
| before wash | after wash | ||||
| positive/sample (*copy /ng DNA) | positive/sample (copy /ng DNA) | positive/sample (copy /ng DNA) | positive/sample (copy /ng DNA) | positive/sample (copy /ng DNA) | |
| August 30 | 1/1 | 0/1 | 0/3 | 0/3 | 0/3 |
| (1.5) | (0) | (0) | (0) | (0) | |
| September 9 | **N.E. | N.E. | 3/3 | 0/3 | 0/3 |
| (384–5,634) | (0) | (0) | |||
| October 17 | 1/1 | N.E. | 3/3 | 0/3 | 0/3 |
| (0.5) | (1.7-9.3) | (0) | (0) | ||
It has been known that amyotrophia causes high mortality of 0-year-old juvenile abalone during the period when water temperatures rise above 13°C, and that the disease subsides as the temperature reaches 25°C (Nakatsugawa, 2000). In the first survey of 0-year-old abalone in 2021, high mortality as well as abnormal cell masses in the foot nervous tissues and shell symptoms reported in amyotrophia were observed from April to June, when the water temperature ranged between 17.4°C to 21.3°C. In the second survey in 2022, although the cumulative mortality from April to August was lower than that of the first survey, a decline in the cumulative mortality and shell symptoms were observed from April to July when the water temperature ranged between 14.4°C to 25.4°C. The water temperature range during the period of mortality in this survey, along with the observed histopathological changes and shell lesions, were consistent with those reported in previous studies on amyotrophia (Nakatsugawa, 1991; Momoyama et al., 1999; Nakatsugawa, 2000). Therefore, the high mortality observed from April to June in 2021 and from April to July in 2022 among the 0-year-old abalone at this hatchery was considered to be primarily caused by amyotrophia.
In both surveys, AbALV copy numbers in the foot muscles during the mortality period appeared higher than those in the other periods. Additionally, along with subsiding of the mortality when water temperature was above 25°C, the copy number of AbALV drastically decreased. These correlations between fluctuating AbALV copy number and the occurrence and timing of mortality endorse the proposal of Matsuyama et al. (2020) that AbALV is the causative agent of amyotrophia in juvenile abalone. It is noted, however, that the abnormal cell masses around the nervous tissues, which have been identified as a characteristic of amyotrophia in previous studies, were observed in only five of 20 specimens in this survey. In contrast, gill symptoms, such as hydrocephalic voids in the epithelial cell layer and uneven surfaces as reported by Matsuyama et al. (2021b), were observed at a high frequency in 19 out of 20 specimens. This raises the possibility that the symptoms associated with amyotrophia may have changed compared to those seen in the past.
Igari et al. (2005) presumed that the transmission of amyotrophia occurred after mid-February in a Kagoshima hatchery. Similarly, in the second survey of the present study, AbALV was first detected on February 11. Since AbALV was not detected at the initial sampling on January 11, it is suggested that AbALV may have invaded the tank between January 11 and February 11. However, there is also a possibility that AbALV had already entered by January 11 but its amplification in abalone was suppressed due to low water temperature, preventing detection by qPCR. Another possibility is that infection did not establish in black abalone at their growth stage on January 11. Therefore, to accurately determine the timing of AbALV invasion, it is necessary to analyze the intake water for AbALV using qPCR.
Following the first detection of AbALV on February 11, the copy number increased up to March. Although cumulative survival rate was not calculated from January to April as described above, unusually high mortality was observed at the daily tank cleaning time in March (Suzuki and Harada, personal communication). In March, the water temperature exceeded 13°C, which is the threshold for amyotrophia (Nakatsugawa, 2000). Therefore, deaths during this period may have been caused by AbALV. So far, an outbreak of the disease before April has not been documented, but attention to amyotrophia in abalone hatcheries should be paid earlier than April, if the water temperature is in the range for the occurrence of the disease.
In the survey of 1-year-old abalone, the mean AbALV copy number did not exceed 100 copies/ng of extracted DNA, as observed in the surveys of 0-year-old abalone. However, the AbALV copy number increased from September to December. While it is possible that this increase was due to a new AbALV infection introduced into the facility, it is more likely that low levels of AbALV remaining in the surviving individuals gradually increased from September to December. Indeed, Nakatsugawa et al. (2000) demonstrated that 2-year-old abalone that had recovered from amyotrophia still carried AbALV. Therefore, although the therapeutic effect of heat treatment was reported by Nakatsugawa (1991), it is possible that such treatment does not completely eliminate AbALV infection.
In the water warming period of April to June 2023, AbALV copy numbers and mortality of 1-year-old abalone remained very low, compared with those in the surveys of 0-year-old abalone in the same period. This survey tested survivors from the second survey of 0-year-old abalone experiencing AbALV infection in the previous year, and our results lead us to suggest that most of the examined abalone may have an ability to suppress AbALV proliferation, genetically. Furthermore, no mass mortality suspected to be caused by amyotrophia was observed even in 0-year-old abalone at the facility during the same period in 2023 (data not shown), implying that some environmental factors may have also suppressed AbALV proliferation. However, since it has been reported that older individuals showed less mortality due to amyotrophia (Nakatsugawa and Momoyama, 1999; Okada et al., 2000; Matsuyama et al., 2021a), it is most likely that AbALV may not actively proliferate in abalone older than in 0-year. In the future, AbALV proliferation among abalone of different ages needs to be compared through experimental infection.
As described above, it is considered that there was neither significant proliferation of AbALV nor mass mortality caused by amyotrophia of 1-year-old abalone in this survey. However, over 40% mortality occurred even before the mass mortality caused by Karenia mikimotoi in late August. Additionally, shell symptoms were observed in more than half of the individuals from May to August. Therefore, further investigation is needed to examine the association of the mortality and shell symptoms observed in 1-year-old abalone with amyotrophia.
In this study, we also examined AbALV during seed production of black abalone in the hatchery. Nakatsugawa et al. (2000) suggested that wild individuals serving as broodstock were often infected with the causative agent of amyotrophia, and the present study found that wild individuals used for broodstock were highly contaminated with AbALV. Nakatsugawa et al. (2000) reported that occurrence of amyotrophia in juveniles cohabiting with female broodstock was significantly higher than when cohabiting with males. However, in the present study, AbALV prevalence was higher in males than in females. Although sex difference in AbALV prevalence among broodstock needs to be further reexamined using a larger sample size, abalone hatcheries should implement biosecurity measures to prevent the spread of AbALV from both wild male and female broodstock used for seed production, as recommended by Nakatsugawa et al. (2000) and Okada et al. (2000).
Nakatsugawa (2000) reported that a washing step for fertilized eggs was effective to prevent amyotrophia in juveniles. The present study also found that fertilized eggs and developed larvae were free from AbALV after the washing procedure, even though AbALV was detected from the broodstock, gametes and unwashed fertilized eggs. Although further studies are still needed to determine whether AbALV can infect fertilized eggs or swimming larvae, from our results, we recommend the washing procedure after fertilization for the production of AbALV-free abalone seeds.
As described above, prior to identification of the pathogen AbALV, knowledge regarding seasonality, temperature range, infection source and preventive measures of amyotrophia in hatcheries had accumulated. In this study, by examining AbALV, the presumed causative agent of amyotrophia using qPCR, we succeeded in supplementing previous findings from a pathogen perspective, and documented further detailed information, including the occurrence of amyotrophia in early spring, and AbALV proliferation in autumn.
Dr. Tomomasa Matsuyama (Fisheries Technology Institute, Japan Fisheries Research and Education Agency) kindly provided the qPCR positive control of AbALV and also gave technical suggestions for the present study. English of this manuscript was edited by Dr. Craig Hayward (UTas).
