A novel Asfarvirus-like virus is proposed as the etiological agent responsible for mass mortality in abalone. The disease, called abalone amyotrophia, originally was recognized in the 1980s, but efforts to identify a causative agent were unsuccessful. We prepared a semi-purified fraction by nuclease treatment and ultracentrifugation of diseased abalone homogenate, and the existence of the etiological agent in the fraction was confirmed by a challenge test. Using next-generation sequencing and PCR-based epidemiological surveys, we obtained a partial sequence with similarity to a member of the family Asfarviridae. BLASTP analysis of the predicted proteins against a virus database resulted in 48 proteins encoded by the novel virus with top hits against proteins encoded by African swine fever virus (ASFV). Phylogenetic analyses of predicted proteins of the novel virus confirmed that ASFV represents the closest relative. Comparative genomic analysis revealed gene-order conservation between the novel virus and ASfV. In situ hybridization targeting the gene encoding the major capsid protein of the novel virus detected positive signals only in tissue from diseased abalone. The results of this study suggest that the putative causative agent should be considered a tentative new member of the family Asfarviridae, which we provisionally designate abalone asfa-like virus (AbALV). African swine fever virus (ASFV) is the causative agent of African swine fever (ASF). The virus causes a hemorrhagic fever with high mortality, with rates approaching 100% in domestic pigs 1. The virus infects domestic pigs and their relatives and ticks 2. ASF outbreaks had been recorded in Africa and Europe, but in recent years the disease has spread to China, Vietnam, Cambodia, Mongolia, Hong Kong, and Korea, becoming a threat to the swine industry worldwide 3. ASFV is a member of nucleocytoplasmic large DNA viruses (NCLDVs) with an average diameter of 200 nm. Although some related viruses, such as faustovirus 4 , kaumoebavirus 5 , and pacmanvirus 6 , have been reported, ASFV is the only member of the Asfarviridae family 7. In the present paper, we describe a virus likely to be the closest ASFV relative found to date; this novel virus was isolated as the presumptive causative agent of abalone amyotrophia. Mass mortalities of abalone have been reported since the early 1980s, during seed production in Japan. The disease was designated abalone amyotrophia because diseased abalone develop muscle atrophy in the mantle and foot 8. Diseased abalone show reduced ability to adhere to the substrate, and some diseased abalone exhibit incisions on the front margin of the shell and brown pigmentation inside of the shell 9. Histopathological evaluation has revealed the presence of abnormal cell masses that are produced extensively, primarily in the ganglion and peripheral nerve of the foot muscle 9. Cumulative mortality can reach 50% and higher 10. Abalone herpesvirus (AbHV) 11,12 and abalone shriveling syndrome-associated virus (AbSV) 13 also cause mortality ac...

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Effects of NH3 concentration in sea water and pH of sea water on the motility of spermatozoa obtained from testes were examined in the Japanese pearl oyster. Percent motility at 30 s after dilution increased with increasing NH3 concentration in sea water from 0.75-2.0 mM. When spermatozoa were diluted with sea water containing 0.75 mM NH3, which is widely used as the insemination fluid in the hatchery of this species, the percent motility increased with time elapsed after dilution, and peaked at 5 min. For spermatozoa diluted with sea water containing 2.0 mM NH3, the percent motility increased rapidly and peaked at 30 s. The pH of sea water increased with increasing NH3 concentration from 8.2 (0 mM NH3) to 9.9 (5.0 mM NH3). When spermatozoa were diluted with artificial sea water at various pH (buffered without NH3 at 6.0-10.0), only spermatozoa diluted with artificial sea water of pH 10.0 were motile, and the percent was considerably lower than those in ammonical sea water. These results indicate that sea water containing 2.0 mM NH3 is a suitable solution for evaluating sperm motility, and that NH3 and/or ammonium ions may activate sperm motility in this species.KEY WORDS: ammonical seawater, artificial fertilization, Japanese pearl oyster, sperm motility.

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To develop methods for large-scale cryopreservation of Japanese pearl oyster spermatozoa, post-thaw motility and fertility of spermatozoa cryopreserved in straw and in a ‰at-bottomed aluminum cup were compared. Spermatozoa diluted 1:19 with an extender comprising 10 methanol, 72 seawater, and 18 fetal bovine serum were placed in the straw (0.25, 1.0, and 2.0 mL) and in the cup (10 mL). The vessels were then cooled in LN vapor to -50°C at a cooling rate of -17.6 to -20.6°C/min, and immersed in LN. The percentages of motility of spermatozoa cryopreserved in 0.25 mL, 1.0 mL, 2 mL straws, and the cup were 35.2, 31.1, 23.2, and 31.8 , respectively, of the pre-cryopreserved spermatozoa. When 100 mL of semen (pre-cryopreserved or cryopreserved in the 3 kinds of straw) was introduced to 2.5 million eggs, the percentages of fertility were invariably high, but not signiˆcantly diŠerent from one another. When fresh spermatozoa with low motility were cryopreserved, thawed, and then introduced to eggs, the percentage of fertility increased in a time-dependent fashion, until 20 min after the commencement of insemination. There were no harmful eŠects on growth and feed intake ability of pearl oyster larvae when the contact time of gametes at fertilization had been prolonged from 5 min to 60 min.

To clarify factors reducing the motility and fertility of cryopreserved spermatozoa of the Japanese pearl oyster Pinctada fucata martensii, the structure of spermatozoa before and after cryopreservation was observed by scanning electron microscopy. Testicular spermatozoa were diluted with cryopreservation diluent (10% methanol + 18% fetal bovine serum + 72% sea water), and dispensed into 0.25-mL straws. The straws were cooled at a rate of approximately -20°C/min to -50°C, and subsequently immersed in liquid nitrogen. Percentage motility of spermatozoa before cryopreservation was 69.9 Ϯ 4.2%, and that of cryopreserved spermatozoa was 24.0 Ϯ 1.8%, respectively. In cryopreserved spermatozoa, the percentage that lacked or had a deformed flagellum was 56.6 Ϯ 3.9%, while in fresh spermatozoa this was 8.7 Ϯ 2.0%. In cryopreserved spermatozoa, the percentage of deformed acrosomes was 76.6 Ϯ 5.2%, while in fresh spermatozoa this was only 0.9 Ϯ 0.3%. Cryopreserved spermatozoa with a normal acrosome and flagellum were only 15.4 Ϯ 3.5% of those in fresh spermatozoa. These results indicate that lesion of the flagellum and deformation of the acrosome occurred through the cryopreservation procedure, and both types of damage lead to loss of the motility and fertility in thawed spermatozoa.