Motoi Yoshioka

Effects of acute stress on plasma cortisol, sex steroid hormone and glucose levels in male and female sockeye salmon during the breeding season

In the sockeye salmon (Onchorynchus nerka), as well as a number of migratory salmonid species, the plasma level of cortisol, the main stress hormone in fish, increases during the spawning period, and then fish die after the spawning. The response of cultured individuals of this species to an artificial acute stress during the spawning period was investigated by measuring levels of cortisol, sex steroid hormones and glucose in blood plasma. In addition, physiological changes in hormone and glucose levels were studied in wild individuals caught in the lake at different spawning stages of the natural condition. The fish cultured and matured in the research pond were confined in a small net and their blood was collected at 3, 5, 15 or 30 min after the start of confinement. Wild sockeye salmon were caught when they gathered in the mouth of their maternal river and their blood was collected just before and after ovulation and also after the artificial removal of eggs and sperm in pre-ovulatory individuals. Acute stress increased cortisol and glucose levels in males. However, in females, the initial cortisol level in the plasma was higher than that in males and did not change significantly with the confinement time. Testosterone levels in both sexes, 11-ketotestosterone levels in males and plasma 17α-progesterone levels in females decreased with the confinement time. In the natural condition, cortisol levels were higher in females than in males and did not change until the last sampling. In males, the cortisol levels increased from the time of spawning until just before death. Glucose levels in males and females increased whereas cortisol levels in females did not change. It is concluded that during the breeding season, male sockeye salmon respond to confinement stress with elevated cortisol levels and decreased sex steroid levels, while female sockeye salmon show only the latter response. It is postulated that female sockeye salmon are not refractory to stress in the breeding period, because their sex steroid levels decrease in response to stress. In females, cortisol levels probably reach maximum levels before blood samples are taken.

POPULATION STRUCTURE OF FINLESS PORPOISES (NEOPHOCAENA PHOCAENOIDES) IN COASTAL WATERS OF JAPAN BASED ON MITOCHONDRIAL DNA SEQUENCES

Abstract To clarify population structure of finless porpoises (Neophocaena phocaenoides) in Japan, we examined mitochondrial DNA sequences of 174 animals. All individuals except for a female were collected in 5 geographically discrete coastal waters where Japanese porpoises are mainly distributed: Sendai Bay–Tokyo Bay, Ise–Mikawa Bays, Inland Sea–Hibiki Nada, Omura Bay, and Ariake Sound–Tachibana Bay. We analyzed 345 base pairs of the control region for all animals and detected 10 haplotypes. Two of those were shared by animals from > 1 area, whereas the other 8 were each found only in 1 area. The most common haplotype in Ise–Mikawa Bays and Ariake Sound–Tachibana Bay was not found at other locations. Analysis of the frequency distribution of haplotypes quantified genetic differentiation, and measurements of gene flow indicated limited dispersal of animals among locations. We conclude that finless porpoises in each of the 5 locations belong to distinct populations.

Diurnal and annual changes in serum cortisol concentrations in Indo-Pacific bottlenose dolphins Tursiops aduncus and killer whales Orcinus orca.

Until present, fundamental studies on cortisol secretory patterns have not been conducted in cetaceans. The objectives of this study were: (1) to examine diurnal changes in serum cortisol concentrations in Indo-Pacific bottlenose dolphins Tursiops aduncus and killer whales Orcinus orca, (2) to investigate annual cortisol changes in killer whales, and (3) to investigate the relationship between cortisol and sex steroids (testosterone and progesterone) concentrations in killer whales. Diurnal changes in serum cortisol concentrations were investigated at various intervals in the two species. In Indo-Pacific bottlenose dolphins, serum cortisol levels exhibited the same episodic fluctuations for 24 h as did diurnal terrestrial mammals: cortisol levels were lower at 18:00 h and higher in the early morning. In killer whales, cortisol concentrations continued to decrease until 18:00 h, after which they fluctuated, and then increased in the next morning. Annual changes in cortisol levels were investigated by collecting blood samples every two weeks from two male killer whales and a pregnant female one twice per day (during 09:00-10:00 and 16:00-17:00 h) throughout a one-year period. Regarding sera collected during 09:00-10:00 h from the female, cortisol concentrations showed cyclic changes having about 4-month intervals. In males, cortisol showed higher concentrations in winter and lower concentrations during the summer season. There was a negative correlation between cortisol and progesterone levels in the female and a negative correlation was also observed between cortisol and testosterone in male no. 2. In the female and male no. 1, cortisol levels during 09:00-10:00 h were significantly higher than those during 16:00-17:00 h, and their data are considered to support observations regarding diurnal changes in cortisol levels in the two cetacean species.

Feeding habits of Dall's porpoises (Phocoenoides dalli) in the subarctic North Pacific and the Bering Sea basin and the impact of predation on mesopelagic micronekton

Abstract We investigated the stomach contents of Dalls porpoises collected in pelagic waters spanning most of their range in the North Pacific and the Bering Sea. Analysis revealed the porpoises fed mainly on myctophid fishes in the subarctic North Pacific and on gonatid squids as well as myctophid fishes in the Bering Sea. Most of the prey items were mesopelagic micronekton, primarily fishes and squids that migrate vertically to shallower waters at night. Stomach content was greater during twilight hours, suggesting the porpoises foraged actively on myctophids at night in shallower waters. Stomach contents were strongly characterized by local mesopelagic prey fauna, and prey species selectivity was not apparent. The annual consumption by Dalls porpoises was estimated to be 2.0–2.8 million tons, or 4.7–6.5% of the biomass of mesopelagic fishes in the subarctic North Pacific, and may account for approximately 24–33% of the overall mortality of mesopelagic micronekton, especially myctophids. Myctophids are also common, but less important, prey of other subarctic predators. Dalls porpoises are likely the primary consumers of myctophids in the subarctic North Pacific. Since myctophids are the major component of the mesotrophic level, the trophic relationship between myctophids and Dalls porpoises is thought to be an important pathway of mass and energy in the pelagic food web in the subarctic North Pacific.

Population Differentiation in the Pacific White-sided Dolphin Lagenorhynchus obliquidens Inferred from Mitochondrial DNA and Microsatellite Analyses

Abstract We investigated genetic diversity and differentiation of the Pacific white-sided dolphin (Lagenorhynchus obliquidens) in Japanese coastal waters and offshore North Pacific by analyzing mitochondrial DNA and nuclear microsatellite variation. A total of 519 bp of the mitochondrial control region was sequenced and five microsatellite locus were genotyped for 59 individuals. A high level of haplotypic diversity (h=96.1%), moderate level of nucleotide diversity (π=1.65%) and average expected heterozygosity (HE=0.66–0.76) were within an extent of those reported for other odontocetes. Consistent genetic difference between the samples from Japanese coastal Pacific-Sea of Japan and offshore North Pacific was indicated by analyses of molecular variance (AMOVAs) based on mtDNA and microsatellite variations, comparison of genetic variabilities, and geographical distributions of mtDNA haplotypes and microsatellite alleles. This result suggests that Pacific white-sided dolphins in each of the above two areas belong to different populations between which gene flow has been severely restricted. The low genetic diversity and mtDNA genealogy of the population in Japanese coastal waters suggest that it originated from a small population that colonized the Sea of Japan or that experienced population reduction when this Sea was isolated from the North Pacific during a glacial period in the Late Pleistocene.

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.

Sex-specific Cortisol and Sex Steroids Responses in Stressed Sockeye Salmon during Spawning Period

Abstract Response to stress during the spawning period of sockeye salmon (Oncorhynchus nerka) was investigated by measuring cortisol, selected steroids and glucose levels in the plasma of cultured fish under acute confinement conditions. Fish were individually placed in nets and sacrificed to obtain blood samples after each confinement period: 0, 3, 5,15 and 30 min, respectively. In males, acute stress increased cortisol levels in the plasma at 15 min and testosterone, 11-ketotestosterone and glucose levels in the plasma within 3 min. In females, the cortisol level was higher than that in males and did not significantly change at 15 and 30 min. Testosterone levels in females decreased at 15 min in confinement. Progesterone, 17-hydroxyprogesterone and 17,20 β-dihydroxy-4-pregnen-3-one levels did not change with confinement time in females. To know the reproductive stage of the fish used in the stress experiment we collected blood and measured sex steroid hormones in different fish from the same pond one week and one day before the experiment. In these fish, the sex steroids in males and females changed with the pattern of breeding. Cortisol levels were higher in females than in males for one week. This study demonstrates that the cortisol level in females is higher than in males during their spawning period, and that females might be less sensitive to changes in the level of cortisol caused by acute stress than are the males.

Temporal and spatial trends of organotin contamination in the livers of finless porpoises (Neophocaena phocaenoides) and their association with parasitic infection status.

Organotins (OTs) are globally ubiquitous contaminants. Contamination of OTs, particularly butyltins (BTs), has been suspected to cause immunosuppressive effects leading to subsequent infectious diseases or opportunistic infection by pathogens in marine mammals. In the present study, we collected individuals from five different populations of finless porpoises (Neophocaena phocaenoides) from coastal areas of Japan (Seto Inland Sea, Ise Bay, Omura Bay, and Pacific coast) and Hong Kong, and measured OT concentrations in their livers. In addition, infection status of lung nematodes and liver trematodes was also observed to analyze the relationship between OT contamination and parasitic infection in finless porpoises. Hepatic OT concentrations were highest in the Hong Kong population, while levels in the Japanese populations were equivalent to those of other cetaceans from different locations. OT levels in Japan have been decreasing in the past few decades, while OT contamination has shown little improvement in Hong Kong. Observation of parasite infection in Seto Inland Sea porpoises revealed that 16 and 11 out of 22 individuals were infected by lung nematodes and liver trematodes, respectively. Additionally, a significant association between BT levels and parasitic infection status of lung nematodes was detected. Thus, the present study suggests that BTs could be a factor affecting parasitic infection, especially the presence or severity of lung nematodes in finless porpoises. Since chemical exposure may alter the susceptibility of organisms to infectious diseases, the interaction of chemical contamination with infectious diseases needs to be investigated in greater depth to understand the risk of population decline due to these factors in marine mammals.

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.