Masaaki Kashiwagi

Phylogeography and the maternal origin of the tetraploid form of the Japanese spined loach, Cobitis biwae, revealed by mitochondrial DNA analysis

The Japanese spined loach Cobitis biwae includes the tetraploid form, which has 96 chromosomes, as well as the diploid form, which has 48 chromosomes. In the present study, we analyzed the mitochondrial DNA (mtDNA) to examine the genetic relationships among 82 populations of diploid–tetraploid complexes of C. biwae. Restriction fragment length polymorphism (RFLP) analysis of the ND1 region on 202 individuals revealed that C. biwae contains three genetically divergent major groups corresponding to geographical proximities (the Eastern, Western, and Kochi groups). Phylogenetic analyses (neighbor-joining, NJ and maximum-parsimony, MP) of a part of the cytochrome b gene sequence (748 bp) in 31 individuals supported the three major groups recognized by RFLP, and indicated considerable genetic differentiation between the Western group and the other two groups (average, 15.2%). The genetic relationship and distribution pattern of the three major groups hypothesized two major dispersions of C. biwae during the middle Miocene: first, the ancestor of the Eastern and Kochi groups had spread from the West through wide regions of present-day Honshu and Shikoku Islands, and following that period, the ancestor of the Western group with a different mtDNA composition, probably resulting from the mtDNA introgression from C. takatsuensis, moved into western Japan and pushed the former north eastward. All tetraploid form populations were included in the Western group and treated as a monophyletic cluster with low genetic divergence. It is notable that two diploid populations geographically adjacent to the tetraploid range were genetically closely related to the tetraploid forms. This result suggests that these diploid populations were directly related to the maternal origin of the tetraploid form.

Daily spawning and development of sensitivity to gonadotropin and maturation-inducing steroid in the oocytes of the bambooleaf wrasse, Pseudolabrus japonicus

The cycle of oocyte development of the bambooleaf wrasse, Pseudolabrus japonicus, was studied to elucidate the endocrinological mechanism of oocyte maturation in a marine teleost. A single female reared with two males spawned every day for 17 days in captivity, indicating that this species is a daily spawner. Ovarian histology revealed that germinal vesicle migration of the largest oocytes progressed from 12:00 to 3:00 h, and germinal vesicle breakdown (GVBD) was completed at 6:00 h. Ovulation and spawning occurred between 6:00 and 9:00 h. The effectiveness of human chorionic gonadotropin (HCG) and 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P), which is one of the most potent steroidal inducers of GVBD in bambooleaf wrasse oocytes, in inducing final oocyte maturation was examined at eight different times of the day. The responsiveness of the oocyte to HCG and steroid differed at different times of the day. The GVBD could be induced by HCG but not 17,20β-P at 9:00 h. Between 12:00 and 18:00 h, not only HCG but also 17,20β-P induced GVBD. Both GVBD and ovulation spontaneously occurred between 0:00 and 6:00 h without any hormonal treatment. These results clearly showed that the oocyte of the bambooleaf wrasse possessed a diurnal maturation cycle. Responsiveness of oocytes to HCG appeared earlier than responsiveness to 17,20β-P. This suggests that sensitivity to 17,20 β-P is induced by gonadotropic hormone (GTH).

Endocrine Control of Diurnal Oocyte Maturation in the Kyusen Wrasse, Halichoeres poecilopterus

Abstract The present study examined diurnal cycles of oocyte development and maturation in the kyusen wrasse, Halichoeres poecilopterus, and investigated the sensitivity of oocytes to maturation-inducing hormone (MIH) and gonadotropic hormone (GTH). Female fish were sampled at fixed intervals throughout the day, revealing that final oocyte maturation and ovulation were completed by 6:00 hr, and that spawning occurred daily between 6:00 and 9:00 hr. In vitro experiments showed that the steroids 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) were equally potent and highly effective inducers of germinal vesicle breakdown (GVBD) in kyusen wrasse oocytes. Additionally, circulating levels of 17,20β-P and 20β-S increased around the time of GVBD and ovulation, suggesting that 17,20β-P and 20β-S act as MIHs in the kyusen wrasse. Moreover, In vitro experiments clearly showed that kyusen wrasse oocytes had a daily developmental cycle of GTH and MIH sensitivity, and that oocytes that completed vitellogenesis acquired GTH-induced maturational competence. An endogenous GTH surge likely occurs between 12:00 and 15:00 hr, and this daily pre-maturational GTH surge probably controls the diurnal maturation cycles of kyusen wrasse oocytes.

Genetic relationships among the Japanese and Korean striated spined loach complex (Cobitidae : Cobitis) and their phylogenetic positions

The “striata complex,” a group of spined loaches included in the genus Cobitis and characterized by a striped coloration pattern on the lateral midline, is distributed in rivers in northeastern Asia to western Japan. The complex comprises 2 continental species (Cobitis tetralineata and Cobitis lutheri) and 3 Japanese races of species rank (large race, middle race, and small race), the small race further comprising 6 local forms of subspecific rank (Tokai form, Biwa form, Yodo form, Sanyo form, San-in form, and Kyushu form). Previous karyological studies have revealed that the large race is an allotetraploid, the others being diploid. In this study, mitochondrial (mt) DNA analyses were conducted for 30 diploid populations of the Cobitis striata complex from Japan and Korea to examine: (1) their phylogenetic relationships and the position of the complex among the major lineages of Cobitis; and (2) the genetic relationships among the Japanese and Korean populations. The results, based on cytochrome b sequences (724 base pairs) analyzed with those of the main lineage of European and Japanese Cobitis, indicated that the striata complex should be considered as a monophyletic group, which evolved in northeastern Asia. Initially considered as a subspecies of Cobitis taenia, widely distributed from Europe to Asia, the striata complex does not have a sister-relationship with the former. Although the Korean species C. tetralineata was formerly believed to be closely related to the middle race in Japan, and a second continental species, C. lutheri, closely related to the Kyushu or San-in forms of the small race in Japan, the trees resulting from the present study revealed that the two Korean species were clustered with each other and separated from all Japanese races.

Preventive efficacy of sodium hypochlorite against water mold infection on eggs of chum salmon Oncorhynchus keta

To protect chum salmon eggs from water mold infection during incubation, the eggs were treated daily with sodium hypochlorite solution (NaOCl) at 10 mg/L residual chlorine concentration for 15 min during their developmental period from fertilization to eyed stages. The number of infected eggs and number of eyed eggs were observed on day 23 of incubation. The percentage of infected eggs to total eggs was significantly lower with NaOCl treatment (1.8∶33.4%) than in the control (11.3∶59.3%, P<0.01). The percentage of eyed eggs to total eggs was significantly higher with NaOCl (85.9∶98.6%) than in the control (66.1∶97.5%, P<0.01). The antifungal activity of NaOCl resulted in improving egg survival. Accordingly, NaOCl is a useful antifungal agent against water mold infection on chum salmon eggs.

Diurnal Spermatogenesis and Spawning in the Secondary Male of a Protogynous Wrasse, Pseudolabrus japonicus (Teleostei, Labridae)

Abstract The bambooleaf wrasse, Pseudolabrus japonicus, exhibits diandric protogyny, and the secondary male performs pair spawning with a female who enters his territory. We examined diurnal spermatogenesis and spawning in the secondary male of a protogynous wrasse. In captivity, a single secondary male spawned daily over one month between 06:00 and 09:00 from October to November. Number of B-type spermatogonia and spermatocytes showed the lowest level at 00:00, increased gradually thereafter, peaked at 15:00, and decreased rapidly from 21:00 to 00:00. Spermatid number did not change significantly throughout the day. The number of spermatozoa increased gradually from 18:00, reached a maximum at 06:00, just prior to spawning, and thereafter decreased markedly at 09:00, after spawning. These results clearly showed that spermatogonial proliferation and meiosis occurred between 00:00 and 15:00, and spermiation occurred between 18:00 and 06:00. Thus, the secondary male of bambooleaf wrasse exhibits a diurnal rhythm of spermatogenesis and spermiation.

Extent of chum salmon egg softening caused by sodium hypochlorite as an antifungal agent

Salmonid eggs are highly sensitive to physical shock during the period from fertilization to eyed stages. In hatcheries, once eggs are set into incubating trays, they are not moved even in cases where some of them die during those stages. Therefore, if there is no effective prophylactic and/or therapeutic treatments applied to the incubating eggs, an aquatic fungus infects the dead eggs, spreads rapidly, forms fungal clumps engulfing the adjoining live eggs, and then often causes high egg mortality due to oxygen deficiency. Malachite green is quite an effective antifungal agent, but in Japan the Pharmaceutical Affairs Law has prohibited the use of it since 2003. An effective alternative antifungal agent is needed. The antifungal activities of sodium hypochlorite (NaOCl), a food additive, have been investigated, and these have suggested that NaOCl would be a promising agent to replace malachite green. However, further research is required to make it fit for practical use because it may bring about the undesirable effects resulting in egg softening. The softening seems to be a major reason why NaOCl has not been discussed as a suitable drug for fishery use, though the actual harm of softening is not clear. Under these circumstances, Kashiwagi et al. indicated that eggs of rainbow trout Oncorhynchus mykiss did not soften with NaOCl treatment at 10 mg/L with daily 15-min exposure. It has been known that the same treatment with NaOCl is effective in controlling fungal infections on the eggs of chum salmon Oncorhynchus keta, and maintaining a higher percentage of their eying. The purpose of this study is to determine the extent of chum salmon egg softening caused by NaOCl treatment at the same dose. The experiments were carried out at the Tsukidate Salmon Hatchery, Miyagi Prefecture, Japan. Unfertilized eggs were stripped from four adult females caught in Hasama river. The eggs from each female were separately fertilized in situ with sperm squeezed from several adult males, and transported to the hatchery. The eggs of each female were divided into two trays each containing approximately 500 eggs, and then separately set into the two tanks for NaOCl treatment and the untreated control, receiving water with a flow rate of 20 L/min. Water temperatures and dissolved oxygen ranged from 8.0 to 14.5°C and 12.2– 16.0 mg/L, respectively, during the incubation. Commercial grade NaOCl (Wako Pure Chemical, Osaka, Japan) was used for the treatment that started from the next day of fertilization (incubation). NaOCl of 2.6 mL/min was administered into incubation water by a pump (Masterflex Micorprocessor Peristaltic Pump 7524-40; Cole-Parmar, Vernon Hills, IL, USA). Target chlorine concentration of 10 mg/L was achieved 10 min following the initiation of treatment and maintained during a 15-min period. The residual chlorine concentration was checked with a DPD (N, N-diethyl-pphenylenediamine) test kit (Pocket Colorimeter 4670-00; Hack, Loveland, CO, USA). On day 23 after fertilization, as the eggs developed into the eyed stage at which time the eyes became clearly visible, the treatments were terminated. Thirty eggs at the eyed stage were randomly picked from each tray and the hardness of them measured. The egg diameter ranged from 6.3 to 7.9 mm in diameter (mean 7.2 mm). To measure the hardness, the *Corresponding author: Tel: 81-59-231-9526. Fax: 81-59-231-9540. Email: motoi@bio.mie-u.ac.jp Received 18 October 2005. Accepted 10 April 2006. FISHERIES SCIENCE 2007; 73: 1205–1207