Yoshihiro Fujise

Persistent organochlorines in minke whale (Balaenoptera acutorostrata) and their prey species from the Antarctic and the North Pacific.

Persistent organochlorines such as polychlorinated biphenyls (PCBs), DDTs, chlordane compounds (CHLs), hexachlorocyclohexanes (HCHs) and hexachlorobenzene (HCB) were determined in the blubber of minke whale and its diet collected from the Antarctic and the North Pacific Oceans. Residue levels of these compounds (except HCB) in minke whale from the Antarctic were apparently lower than those from the North Pacific. This is due to the lower levels of these pollutants in the southern hemisphere than in the northern hemisphere and the specific feeding habit of the minke whale from the Antarctic which feeds on lower trophic organisms, primarily euphausiids. The north-south difference for HCB residue levels was small, reflecting its dispersible nature through long-range atmospheric transport. Compositions of DDT and CHL compounds in minke whale from the Antarctic were similar to those from the North Pacific. However, the composition of HCH isomers was different between the Antarctic and the North Pacific as was observed in their diet, suggesting a larger or on-going usage of lindane in the southern hemisphere countries. In minke whale from the Antarctic, the elevated level of PCBs residues was noted during a period of 1984 to 1993, implying a continuous discharge of PCBs in the southern hemisphere. A similar discharge was also suggested in the North Pacific, while a decreasing contamination by DDTs was apparent.

Using trace elements in skin to discriminate the populations of minke whales in southern hemisphere

Concentrations of 12 trace elements (V, Cr, Mn, Cu, Zn, Se, Rb, Sr, Cd, Cs, Ba, and Hg) were determined in liver and skin tissues of minke whales from various regions within the Antarctic Ocean. Cd concentrations in livers of southern minke whale were apparently higher than those in cetaceans from other regions, while Hg concentrations were lower. There were significant positive correlations between body length and concentrations of Cd and Hg in the liver. The concentrations of all trace elements in the skin were lower than those in other cetaceans reported previously. Significant positive correlations between liver and skin were found for Cr, Mn, Cu, Zn, Rb, Cd, and Cs, implying that the concentrations of these trace elements in the skin reflect those of internal organs. Large interannual variation of the accumulation pattern of trace elements in the skin was observed for the southern minke whales from Area V. There were significant differences in the skin element concentrations among Areas III, IV, and V, especially for males. Also, discriminant analysis between geographically two different groups collected during 1995/1996 austral summer season, based on the concentrations of trace elements in the skin, allowed for a correct classification of 90% of these minke whales. These results suggest that measurement of trace elements in skin samples could provide valuable information on the status of contamination and possible geographic differences in the accumulation levels in southern minke whales.

Identification of novel cytochrome P450 1A genes from five marine mammal species

Marine mammals, being endangered by the chronic exposure of hydrophobic environmental contaminants as an assorting result of global pollution, are especially focused as indicators for organochlorine pollution. The use of contaminant-induced xenobiotic metabolizers, particularly P450 (CYP) 1A, in marine mammals can be effective as potential biomarkers of the contaminant exposure and/or toxic effects. In this study, we identified the first marine mammalian CYPs. Six novel CYP1A cDNA fragments were cloned from the livers of marine mammal species, minke whale (Balaenoptera acutorostrata), dalls porpoise (Phocoenoides dalli), steller sea lion (Eumetopias jubatus), largha seal (Phoca largha), and ribbon seal (Phoca fasciata) by the method of reverse transcription/polymerase chain reaction (RT/PCR); two distinct fragments were from steller sea lion and one fragment each was obtained from the other species. Five of the fragments, one from each species, were classified in the subfamily of CYP1A1, and the other fragment cloned from steller sea lion was designated CYP1A2. Degenerate PCR primers were used to amplify the fragments from liver cDNAs. The deduced amino acid sequences of these fragment CYP1As showed identities ranging from 50.0 to 94.3% with other known vertebrate CYPs in the subfamily of CYP1A, including those from fish, chicken, and terrestrial mammals. The isolated fragments were used to construct a molecular phylogeny, along with other vertebrate CYP1A cDNAs cut down in size to the corresponding region of 265 bp in which those newly determined fragments were cloned. This phylogenetic analysis by the maximum parsimony method using the PHYLIP program suggests two distinct evolutional pathways for aquatic mammalian CYP1As, compatible to a conservative taxonomy. Pinniped genes are clustered together with dog gene, forming a carnivore group, and cetaceans form another branch. Identification of CYP1A genes in marine mammals will be an introductory step to provide new insights into the metabolic or toxicological functions of CYP1As in these animals.

Pathological and serological evidence of Brucella-infection in baleen whales (Mysticeti) in the western North Pacific

Abnormal testes and uterus were observed in 13 males (33%) and one female (3%) out of 40 common minke whales (Balaenoptera acutorostrata) in the western North Pacific. Similar lesions were found in testis and ovary, respectively, in one male (2%) and female (2%) out of 43 Brydes whales (Balaenoptera edeni) in the western North Pacific. Grossly, granular lesions with caseation and calcification were main pathological signs, and they were restricted to reproductive organs of mature whales. Chronic purulent or granulomatous orchitis was observed by microscopic analysis. Antibodies to Brucella species were detected in the serum samples of 15/40 (38%) of common minke whales and 4/43 (9%) of Brydes whales. Neither pathological nor serological change was found in the examined sperm whales (Physeter macrocephalus) in the western North Pacific and Antarctic minke whales (Balaenoptera bonaerensis). These results strongly suggest that Brucella infection was involved in two species of baleen whales (Mysticeti) in the North Pacific.

Age-related accumulation of heavy metals in bone of the striped dolphin, Stenella coeruleoalba

Concentration levels of metals (Fe, Mn, Zn, Cu, Pb, Ni, total Hg, methyl Hg and Se) in whole bone of the striped dolphin, Stenella coeruleoalba, and their variations with growth and sex were investigated. The metal concentrations characteristically changed with bone growth. Accumulation of Fe, Mn, Zn and Cu depended upon the metabolic turnover connected with ossification whilst, for the accumulation of Pb, Ni, Cd and Hg, the age or exposure time was a dominant factor. However, Pb and Ni were markedly accumulated during the suckling period and probably transferred from mother to calf via milk. The Cd concentration rapidly increased during the late period of the suckling stage, indicating higher absorption efficiency and accumulation of Cd through the digestive tract of the calf than those of mature dolphin. The transfer of total Hg to calf via milk was negligible, and its concentration increased with age until about 20 years, while methyl Hg reached a plateau at about 10 years. A significant positive correlation (r = 0·798, p < 0·01) was found between the concentrations of total Hg and Se in the bone, and the molar ratio depended upon that of the blood. Furthermore, the concentrations of Fe and Cd in the bone were higher in the female than in the male, while Pb and Ni were lower in the female; they are probably due to parturition and lactation.

Production of cloned sei whale (Balaenoptera borealis) embryos by interspecies somatic cell nuclear transfer using enucleated pig oocytes.

In this study, we examined the feasibility of using subzonal cell injection with electrofusion for interspecies somatic cell nuclear transfer (iSCNT) to produce sei whale embryos and to improve their developmental capacity by investigating the effect of osmolarity and macromolecules in the culture medium on the in vitro developmental capacity. Hybrid embryos produced by the electrofusion of fetal whale fibroblasts with enucleated porcine oocytes were cultured in modified porcine zygote medium-3 to examine the effects of osmolarity and fetal serum on their in vitro developmental capacity. More than 66% of the whale somatic cells successfully fused with the porcine oocytes following electrofusion. A portion (60~81%) of the iSCNT whale embryos developed to the two- to four-cell stages, but no embryos were able to reach the blastocyst stage. This developmental arrest was not overcome by increasing the osmolarity of the medium to 360 mOsm or by the addition of fetal bovine or fetal whale serum. Our results demonstrate that sei whale-porcine hybrid embryos may be produced by SCNT using subzonal injection and electrofusion. The pig oocytes partly supported the remodeling and reprogramming of the sei whale somatic cell nuclei, but they were unable to support the development of iSCNT whale embryos to the blastocyst stage.

Validation of high-throughput measurement system with microwave-assisted extraction, fully automated sample preparation device, and gas chromatography-electron capture detector for determination of polychlorinated biphenyls in whale blubber

Validation of a high-throughput measurement system with microwave-assisted extraction (MAE), fully automated sample preparation device (SPD), and gas chromatography-electron capture detector (GC-ECD) for the determination of polychlorinated biphenyls (PCBs) in minke whale blubber was performed. PCB congeners accounting for > 95% of the total PCBs burden in blubber were efficiently extracted with a small volume (20 mL) of n-hexane using MAE due to simultaneous saponification and extraction. Further, the crude extract obtained by MAE was rapidly purified and automatically substituted to a small volume (1 mL) of toluene using SPD without using concentrators. Furthermore, the concentration of PCBs in the purified and concentrated solution was accurately determined by GC-ECD. Moreover, the result of accuracy test using a certified material (SRM 1588b; Cod liver oil) showed good agreement with the NIST certified concentration values. In addition, the method quantification limit of total-PCB in whale blubbers was 41 ng g(-1). This new measurement system for PCBs takes only four hours. Consequently, it indicated this method is the most suitable for the monitoring and screening of PCBs in the conservation of the marine ecosystem and safe distribution of foods.

Relative Growth of the Skull of the Common Minke Whale Balaenoptera acutorostrata from the North Pacific in Comparison to Other Balaenoptera Species

Abstract. The allometric growth pattern of 666 common minke whales (Balaenoptera acutorostrata) collected in the North Pacific was investigated by comparing skull length and skull width with body length. For comparison, skull length and skull width in relation to body length were analyzed in 3 Balaenopteridae species (B. musclus, B. physalus, and B. borealis). The generalized linear model was adapted for evaluating the effects of sex and sexual maturity status on growth patterns. The skull proportion of large Balaenoptera whales (B. musculus and B. physalus) showed positive allometry, but that of the common minke whale showed negative allometry, despite being related species. Such differences in intraspecific growth patterns could be the result of adaptation driven by feeding strategy.

Mercury speciation and selenium in toothed-whale muscles

Mercury accumulates at high levels in marine mammal tissues. However, its speciation is poorly understood. The main goal of this investigation was to establish the relationships among mercury species and selenium (Se) concentrations in toothed-whale muscles at different mercury levels. The concentrations of total mercury (T-Hg), methylmercury (MeHg), inorganic mercury (I-Hg) and Se were determined in the muscles of four toothed-whale species: bottlenose dolphins (n=31), Rissos dolphins (n=30), striped dolphins (n=29), and short-finned pilot whales (n=30). In each species, the MeHg concentration increased with increasing T-Hg concentration, tending to reach a plateau. In contrast, the proportion of MeHg in T-Hg decreased from 90-100% to 20-40%. The levels of T-Hg and Se showed strong positive correlations. Se/I-Hg molar ratios rapidly decreased with the increase of I-Hg and reached almost 1 in all species. These results suggested that the demethylated MeHg immediately formed Se/I-Hg equimolar complex of mercury selenide (HgSe) in their muscles. In addition, an X-ray absorption fine structure analysis (XAFS) of a bottlenose dolphin muscle confirmed that the dominant chemical form of the Se/I-Hg equimolar complex was HgSe. HgSe was mainly localized in cells near the endomysium using electron probe microanalysis (EPMA). These results suggested that the demethylated MeHg finally deposits within muscle cells of bottlenose dolphin as an inert HgSe.

Amino acid sequence variations of signaling lymphocyte activation molecule and mortality caused by morbillivirus infection in cetaceans

Morbillivirus infection is a severe threat to marine mammals. Mass die‐offs caused by this infection have repeatedly occurred in bottlenose dolphins (Turiops truncatus) and striped dolphins (Stenella coeruleoalba), both of which belong to the family Delphinidae, but not in other cetaceans. However, it is unknown whether sensitivity to the virus varies among cetacean species. The signaling lymphocyte activation molecule (SLAM) is a receptor on host cells that allows morbillivirus invasion and propagation. Its immunoguloblin variable domain‐like (V) region provides an interface for the virus hemagglutinin (H) protein. In this study, variations in the amino acid residues of the V region of 26 cetacean species, covering almost all cetacean genera, were examined. Three‐dimensional (3D) models of them were generated in a homology model using the crystal structure of the marmoset SLAM and measles virus H protein complex as a template. The 3D models showed 32 amino acid residues on the interface that possibly bind the morbillivirus. Among the cetacean species studied, variations were found at six of the residues. Bottlenose and striped dolphins have substitutions at five positions (E68G, I74V, R90H, V126I, and Q130H) compared with those of baleen whales. Three residues (at positions 68, 90 and 130) were found to alternate electric charges, possibly causing changes in affinity for the virus. This study shows a new approach based on receptor structure for assessing potential vulnerability to viral infection. This method may be useful for assessing the risk of morbillivirus infection in wildlife.