Brucella infection in rough-toothed dolphin (Steno bredanensis) with severe orchitis stranded on the Pacific coast of Japan.

In the western North Pacific, prominent granulomatous testes have been detected in many Brucella-infected common minke whales (Balaenoptera acutorostrata), but there have been no reports in toothed cetaceans. We found severe orchitis with granulomatous lesions in a rough-toothed dolphin (Steno bredanensis) stranded on the Pacific coast of Japan in 2011. Histopathological examination revealed leukocyte infiltration of the lesions. DNA from the lesion was analyzed by PCR and it showed molecular biological similarities with those of Brucella-infected common minke whales and Brucella ceti of sequence-type 27 (ST27). These results suggest that the type of Brucella ceti that infected the dolphin was ST27, which may have caused severe orchitis. This study adds to our understanding of Brucella infections in marine mammals.

Functional anatomy of shoulder muscles in the Pacific white-sided dolphin (Lagenorhynchus obliquidens).

Most intrinsic muscles of the forelimb in dolphins are either degenerated or lost; however, the muscles around the shoulder joint are well preserved. We dissected the forelimbs of Pacific white-sided dolphins and constructed a full-scale model of the flipper to compare and examine their movements following dissection. The humerus was oriented at approximately 45° ventrally from the horizontal plane of the dolphin and 45° caudally from the frontal plane. This maintains the neutral position of the flipper. The deltoideus and pectoralis major muscles were inserted into the body of the humerus, and the flipper was moved in the dorsal and ventral directions, respectively. A large tubercle, known as the common tubercle, was observed at the medial end of the humerus. Four muscles were inserted into the common tubercle: the brachiocephalicus, supraspinatus, and cranial part of the subscapularis, which laterally rotated the common tubercle. Subsequently, the flipper swung forward, and its radial edge was lifted. Conversely, the medial rotation of the common tubercle caused by the coracobrachialis and the caudal part of the subscapularis was accompanied by backward swinging of the flipper and lowering of the radial edge. These findings suggest the function of the flipper as a stabilizer or rudder is caused by the rotation of the humerus's common tubercle.

Notes on stomach contents of pygmy and dwarf sperm whales (Kogia spp.) from around Japan.

The diets of pygmy (Kogia breviceps) and dwarf (K. sima) sperm whales in Japanese waters are poorly known. We report new information on the diets of these two species from these waters based on identifiable hard-part remains recovered from the stomach contents of 29 whales (11 pygmy and 18 dwarf sperm whales) that stranded between 1991 and 2021; those of a further two dwarf sperm whales were empty. The cephalopod (and secondarily fish and crustacean) component of the diets of these 29 whales, based on analysis of identifiable stomach-content remains, is described. The main prey includes cephalopods, represented by 1556 identifiable lower beaks (and 1483 upper beaks), crustaceans (represented by heavily digested, unidentifiable remains), and fishes (as represented by 92 otoliths). Identified prey comprises 30 species from 16 cephalopod families and 5 families from 5 fish orders. Oceanic cephalopods are the main prey of both whale species, particularly Enoploteuthis (Paraenoploteuthis) chunii and Chiroteuthis (Chirothauma) picteti. Prey diversity index values (Shannon-Weaver's diversity index H') are 2.41 for the pygmy sperm whale and 2.66 for the dwarf sperm whale. Although the main cephalopod component in the diets of these two whale species is similar, Pianka's index (0.40), a measure of niche overlap, is not that high, and may be influenced by differences in prey dominance in different feeding areas.

Comparative analysis of the genetic structures of Kogia spp. populations in the western North Pacific.

The two Kogia species, the pygmy sperm whale (K. breviceps) and the dwarf sperm whale (K. sima), have similar morphological and biological features as well as diets. Both species are deep divers, and both have wide distributions from tropical to warm-temperate zones. Although K. breviceps is larger than K. sima, there are few reports of habitat differentiation between the two species. The distribution of K. breviceps is concentrated in higher-latitudes, and this species dives deeper than K. sima. We investigated whether these two species differ in their population structures in the western North Pacific. Using stranded specimens from Japan, we compared the population genetic patterns of the two Kogia species using mtDNA control region variation (941 bp). In total, 34 K. breviceps samples and 54 K. sima samples from stranded individuals around Japan were successfully sequenced. Thirty haplotypes were detected in K. breviceps and 34 in K. sima, indicating high genetic diversity for both. Almost all these haplotypes are unique to the western North Pacific, but did not constitute distinct phylogeographic clades within either species. We detected differences between the species in the shape of haplotype networks and in the potential time of population expansion, indicating that the western North Pacific population of the two biologically similar species could have different population demographies. This may reflect differences in evolutionary histories and in the details of their ecological niches.

Directly Reprogrammed Neurons as a Tool to Assess Neurotoxicity of the Contaminant 4-Hydroxy-2',3,5,5'-tetrachlorobiphenyl (4'OH-CB72) in Melon-Headed Whales.

Whales accumulate high levels of environmental pollutants. Exposure to polychlorinated biphenyls (PCBs) and their metabolites (OH-PCBs) could be linked to abnormal behavior, which may lead to mass stranding of marine mammals. Whales may thus suffer from adverse effects such as neuronal dysfunction, yet testing the neurotoxicity of these compounds has never been feasible for these species. This study established neurons chemically reprogrammed from fibroblasts of mass stranded melon-headed whales (Peponocephala electra) and used them for in vitro neurotoxicity assays. Exposure to 4-hydroxy-2',3,5,5'-tetrachlorobiphenyl (4'OH-CB72), a metabolite of PCBs, caused apoptosis in the reprogrammed neurons. Transcriptome analysis of 4'OH-CB72-treated whale neurons showed altered expressions of genes associated with oxidative phosphorylation, chromatin degradation, axonal transport, and neurodegenerative diseases. These results suggest that 4'OH-CB72 exposure may induce neurodegeneration through disrupted apoptotic processes. A comparison of the results with human reprogrammed neurons revealed the specific effects on the whale neurons. Our noninvasive approach using fibroblast-derived neurons is useful for hazard and risk assessments of neurotoxicity in whales.

Disseminated Toxoplasmosis in a Narrow-Ridged Finless Porpoise (Neophocaena asiaeorientalis) with Transplacental Embryonal Transmission.

We describe a case of systemic toxoplasmosis in a female adult narrow-ridged finless porpoise (Neophocaena asiaeorientalis) found in May 2018 inside a gillnet set in the Ariake Sound, southern Japan. The main lesions observed were lymphoplasmacytic and focally necrotizing encephalitis, necrotizing to granulomatous adrenalitis, myocarditis, and inflammation in the intestinal wall, associated with protozoal tissue cysts and tachyzoites. Additionally, the individual had a 5.6 mm (crown-rump length) early-stage embryo in the left uterine horn, which had multifocal necrotizing lesions with intralesional tissue cysts and tachyzoites in the parenchyma. Immunohistochemistry and PCR and sequencing of the internal transcribed spacer 1 region confirmed a Toxoplasma gondii infection. Further genotyping revealed an atypical type II genotype with a type I pattern for the Apico locus. Narrow-ridged finless porpoises are an endangered coastal species already facing various anthropogenic threats. Toxoplasmosis, especially with its ability to transmit to an early-stage embryo, should be considered an emerging threat to this vulnerable species.

Anthropogenic and natural organohalogen compounds in melon-headed whales (Peponocephala electra) stranded along the Japanese coastal waters: Temporal trend analysis using archived samples in the environmental specimen bank (es-BANK).

The present study determined recent accumulation levels of polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane and its metabolites (DDTs), hexachlorocyclohexane isomers (HCHs), chlordane compounds (CHLs), hexachlorobenzene (HCB), polybrominated diphenyl ethers (PBDEs), hexabromocyclododecanes (HBCDDs), polychlorinated diphenyl ethers (PCDEs), methoxylated-PBDEs (MeO-PBDEs) and 2,3,3',4,4',5,5'-heptachloro-1'-methyl-1,2'-bipyrrole (Q1) in the blubber of melon-headed whales (Peponocephala electra) stranded along the Japanese coastal waters in 2015 and examined temporal trends of these organohalogen compound (OHC) levels by analyzing blubber samples of this species archived in the environmental specimen bank which were collected in 1982, 2001, 2002, 2006, 2010 and 2011. The median concentrations in melon-headed whales stranded recently were in the order of DDTs ≈ PCBs > HBCDDs > Q1 > CHLs > MeO-PBDEs > PBDEs > HCB > HCHs > PCDEs, indicating that considerable amounts of HBCDDs, in addition to DDTs and PCBs, have been transported to tropical and subtropical waters of the open ocean and pelagic whale species might be exposed to relatively high levels of these OHCs. Temporal trend analyses of OHC levels in the blubber of melon-headed whales revealed significant decrease for anthropogenic OCs such as DDTs, PCBs, HCB, HCHs and PCDEs, and significant increase for CHLs, PBDEs, HBCDDs, MeO-PBDEs and Q1 since 1982. Besides, the analyses from 2001 to 2015 showed no decreasing trends (unchanged) for some PCB congeners, p,p'-DDE, cis- and trans-nonachlors, Q1, BDE-47, -100 and -154, and significantly increasing trends for α-HBCDD and 6MeO-BDE47, suggesting their chronic exposure for this pelagic whale species.

Construction of an artificial system for ambrein biosynthesis and investigation of some biological activities of ambrein.

Ambergris, a sperm whale metabolite, has long been used as a fragrance and traditional medication, but it is now rarely available. The odor components of ambergris result from the photooxidative degradation of the major component, ambrein. The pharmacological activities of ambergris have also been attributed to ambrein. However, efficient production of ambrein and odor compounds has not been achieved. Here, we constructed a system for the synthesis of ambrein and odor components. First, we created a new triterpene synthase, "ambrein synthase," for mass production of ambrein by redesigning a bacterial enzyme. The ambrein yields were approximately 20 times greater than those reported previously. Next, an efficient photooxidative conversion system from ambrein to a range of volatiles of ambergris was established. The yield of volatiles was 8-15%. Finally, two biological activities, promotion of osteoclast differentiation and prevention of amyloid ß-induced apoptosis, were discovered using the synthesized ambrein.

Photogrammetric Three-dimensional Modeling and Printing of Cetacean Skeleton using an Omura's Whale Stranded in Hong Kong Waters as an Example.

The preparation of cetacean, in particular baleen whale, skeletons presents a great challenge due to their high lipid content and uncommon size. Documentation of the skeletal morphology is important to produce accurate and reliable models for both research and educational purposes. In this paper, we used a 10.8-meter long Omura's whale stranded in Hong Kong waters in 2014 as an example for the illustration. This rare and enormous specimen was defleshed, macerated, and sun-dried to yield the skeleton for research and public display. Morphology of each bone was then documented by photogrammetry. The complex contour of the skeleton made automated photoshoot inadequate and 3 manual methods were used on bones of different sizes and shapes. The captured photos were processed to generate three-dimensional (3D) models of 166 individual bones. The skeleton was printed half-size with polylactic acid for display purposes, which was easier to maintain than the actual cetacean bones with high residual fat content. The printed bones reflected most anatomical features of the specimen, including the bowing out rostral region and the caudal condylar facet that articulated with Ce1, yet the foramina on the parieto-squamosal suture, which are diagnostic character of Balaenoptera omurai, and an indented groove on the frontal bone at the posterior end of the lateral edge were not clearly presented. Extra photoshoots or 3D surface scanning should be performed on areas with meticulous details to improve precision of the models. The electronic files of the 3D skeleton were published online to reach a global audience and facilitate scientific collaboration among researchers worldwide.

Systemic Amyloid A Amyloidosis in Stejneger's Beaked Whales (Mesoplodon stejnegeri).

Stejneger's beaked whales (Mesoplodon stejnegeri) are one of the lesser known species of mammals, with little information available on their population status or incidence of diseases. Recent pathologic investigations on stranded and bycaught wild cetaceans around Hokkaido, Japan, revealed an unusually high incidence of systemic amyloidosis in this species, warranting further investigation. The objective of this study was to further characterize the systemic amyloidosis of Stejneger's beaked whales by retrospective histopathologic analyses of tissues from animals that stranded in Japan between 1994 and 2018. Various tissues from 35 individuals were examined histologically with hematoxylin and eosin, Congo red, and immunohistochemistry for amyloid A (AA), in which 12 (34%) were diagnosed with systemic amyloidosis. The organs with the highest severity of amyloid deposition were the stomach and intestine. The type of amyloid was confirmed as AA of approximately 9 kDa by 2-dimensional gel electrophoresis with extracted amyloid from the liver and subsequent Western blotting with an antiserum against AA peptide. There were no statistically significant associations between amyloidosis and sex, body condition of the whales, or the presence of chronic inflammation. The high prevalence of this disease might be of concern for overall population numbers, and continued pathologic monitoring of stranded animals is necessary throughout its distributional range.

Serologic survey of Brucella infection in cetaceans inhabiting along the coast of Japan.

A serologic investigation of Brucella infection was performed in 7 species of cetaceans inhabiting along the coast of Japan. A total of 32 serum samples were examined by enzyme-linked immunosorbent assay (ELISA) using Brucella abortus and B. canis antigens. One serum sample from five melon-headed whales (Peponocephala electra) was positive for B. abortus. No serum sample showed positive for B. canis. The ELISA-positive melon-headed whale serum demonstrated a strong band appearance only against B. abortus antigens in Western blot analysis. Many detected bands were discrete, while some of them had a smeared appearance. The present results indicate that Brucella infection occurred in melon-headed whale population and the bacterial antigenicity is more similar to that of B. abortus than B. canis.

Description of a new species of beaked whale (Berardius) found in the North Pacific.

Two types of Berardius are recognised by local whalers in Hokkaido, Japan. The first is the ordinary Baird's beaked whale, B. bairdii, whereas the other is much smaller and entirely black. Previous molecular phylogenetic analyses revealed that the black type is one recognisable taxonomic unit within the Berardius clade but is distinct from the two known Berardius species. To determine the characteristics of the black type, we summarised external morphology and skull osteometric data obtained from four individuals, which included three individuals from Hokkaido and one additional individual from the United States National Museum of Natural History collection. The whales differed from all of their congeners by having the following unique characters: a substantially smaller body size of physically mature individuals, proportionately shorter beak, and darker body colour. Thus, we conclude that the whales are a third Berardius species.

Description of the karyotypes of Stejneger's beaked whale (Mesoplodon stejnegeri) and Hubbs' beaked whale (M. carlhubbsi)

Abstract The genus Mesoplodon (Cetacea: Odontoceti: Ziphiidae) is one of the few cetacean genera with the karyotype 2n = 42. The 2n = 42 karyotype of M. europaeus and M. carlhubbsi is largely consistent with the general cetacean karyotype 2n = 44, although other 2n = 42 karyotypes do not exhibit clear homologies with the general cetacean karyotype. Therefore, the chromosomes of Mesoplodon species may be the key to understanding cetacean karyological evolution. In the present study, the male karyotypes of M. stejnegeri and M. carlhubbsi were examined. In both species, the diploid number of the male karyotype was 42. Both species had the following characteristics: 1) a huge subtelocentric X chromosome with a large C-block; 2) a small metacentric Y chromosome; 3) nucleolus organizer regions (NORs) in the terminal regions of a large autosome and one or two small metacentric autosomes; 4) small metacentric autosomes; 5) large submetacentric and subtelocentric autosomes; 6) less accumulated C-heterochromatin in the centromeric region; and 7) heteromorphism in C-heterochromatin accumulation between homologues. Characteristics 1 and 3 are peculiar to only the karyotypes of Mesoplodon species, whereas characteristics 4, 5, 6, and 7 are also found in the species with the general cetacean karyotype 2n = 44.

Description of the karyotypes of Stejneger's beaked whale (Mesoplodon stejnegeri) and Hubbs' beaked whale (M. carlhubbsi).

The genus Mesoplodon (Cetacea: Odontoceti: Ziphiidae) is one of the few cetacean genera with the karyotype 2n = 42. The 2n = 42 karyotype of M. europaeus and M. carlhubbsi is largely consistent with the general cetacean karyotype 2n = 44, although other 2n = 42 karyotypes do not exhibit clear homologies with the general cetacean karyotype. Therefore, the chromosomes of Mesoplodon species may be the key to understanding cetacean karyological evolution. In the present study, the male karyotypes of M. stejnegeri and M. carlhubbsi were examined. In both species, the diploid number of the male karyotype was 42. Both species had the following characteristics: 1) a huge subtelocentric X chromosome with a large C-block; 2) a small metacentric Y chromosome; 3) nucleolus organizer regions (NORs) in the terminal regions of a large autosome and one or two small metacentric autosomes; 4) small metacentric autosomes; 5) large submetacentric and subtelocentric autosomes; 6) less accumulated C-heterochromatin in the centromeric region; and 7) heteromorphism in C-heterochromatin accumulation between homologues. Characteristics 1 and 3 are peculiar to only the karyotypes of Mesoplodon species, whereas characteristics 4, 5, 6, and 7 are also found in the species with the general cetacean karyotype 2n = 44.

Polybrominated diphenyl ethers (PBDEs) and their hydroxylated and methoxylated analogues in the blood of harbor, Dall's and finless porpoises from the Japanese coastal waters.

This study investigated the accumulation of polybrominated diphenyl ethers (PBDEs) and their hydroxylated and methoxylated analogues (OH-PBDEs and MeO-PBDEs) in the blood of harbor porpoises, Dall's porpoises, and finless porpoises stranded or bycaught in Japanese coastal waters and in the North Pacific Ocean. Moreover, we suggested the origins of these contaminants and the factors affecting their pattern of accumulation. Levels of PBDEs in Dall's porpoises were one order of magnitude greater than those in the other species. OH-PBDE and MeO-PBDE levels were comparable to those of PBDEs. However, no correlation was found between the levels of OH-PBDEs and PBDEs, whereas a strong correlation was found between that of OH-PBDEs and MeO-PBDEs (p < 0.001). 6OH-BDE47, reported compound biosynthesized by marine low-trophic level organisms, was the dominant congener. These results suggest that PBDEs found in these porpoise species derive from flame retardants, but OH-PBDEs and MeO-PBDEs are mainly of natural origins.

Toxic Identification and Evaluation of Androgen Receptor Antagonistic Activities in Acid-Treated Liver Extracts of High-Trophic Level Wild Animals from Japan.

Sulfuric acid-treated liver extracts of representative high-trophic level Japanese animals were analyzed by toxic identification and evaluation (TIE) with chemically activated luciferase expression (CALUX) and chemical analysis to elucidate androgen receptor (AR) antagonistic activities and potential contributions of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs). The activities were detected in striped dolphins (n = 5), Stejneger's beaked whales (n = 6), golden eagle (n = 1), and Steller's sea eagle (n = 1) with CALUX-flutamide equivalents (FluEQs) as follow: 38 (20-52), 47 (21-96), 5.0, and 80 µg FluEQ/g-lipid, respectively. The AR antagonism was detected in limited number of specimens at lower levels for finless porpoise, raccoon dog, and common cormorant. Theoretical activities (Theo-FluEQs) were calculated using the concentration of OCPs and PCBs and their IC25-based relative potency (REP) values. These total contribution to CALUX-FluEQ was 126%, 84%, 53%, 55%, and 44% for striped dolphin, Steller's sea eagle, Stejneger's beaked whale, finless porpoise, and golden eagle, respectively, and the main contributor was p,p'-DDE. However, most of the activities for raccoon dog (7.6%) and common cormorant (17%) could not be explained by OCPs and PCBs. This suggests other unknown compounds could function as AR antagonists in these terrestrial species.

A case of stranded Indo-Pacific bottlenose dolphin (Tursiops aduncus) with lobomycosis-like skin lesions in Kinko-wan, Kagoshima, Japan.

Lobomycosis is a chronic fungal disease caused by the etiologic agent, Lacazia loboi, in the skin and subcutaneous tissues in humans and dolphins in tropical and transitional tropical climates. An Indo-Pacific bottlenose dolphin (Tursiops aduncus) stranded in Kagoshima, Japan, had severe skin lesions characterized by granulomatous reactions and hyperkeratosis that were similar to those of the lobomycosis, but no fungal organism was observed in the skin lesion. In this paper, we report a stranded Indo-Pacific bottlenose dolphin with lobomycosis-like lesions based on pathological examinations in Japan.

Pathological findings and probable causes of the death of Stejneger's beaked whales (Mesoplodon stejnegeri) stranded in Japan from 1999 and 2011.

One hundred and twenty stranding events of Stejneger's beaked whales were reported in Japan between 1999 and 2011. The purpose of this study is to introduce pathological data and to discuss probable causes of death for 44 Stejneger's beaked whales among them. The significant pathological findings were the pulmonary edema, parasitic granulomatous nephritis, emaciation, amyloidosis, suppurative bronchopneumonia and so on. The probable causes of death were categorized as noninfectious in 43 of the cases, which included drowning, starvation and secondary amyloidosis. One individual was diagnosed with septicemia, which was the only example of an infectious disease. Because we could not always perform advanced analyses, such as microbiology tests, biotoxin examinations or contaminant analyses, the finality of our findings may be impaired. However, the present study has broad implications on the causes of death of Stejneger's beaked whales of the seas around Japan, which are valuable for the future studies and for the detection of emerging diseases.

Halogenated phenolic contaminants in the blood of marine mammals from Japanese coastal waters.

Information on accumulation of halogenated phenolic contaminants in the blood of marine mammal is limited. The present study, we determined the residue levels and patterns of chlorinated and brominated phenolic contaminants (OH-PCBs, OH-PBDEs and bromophenols) in the blood collected from pinnipeds (northern fur seal, spotted seal, Steller sea lion and ribbon seal) and small cetaceans (harbor porpoise and Dall's porpoise) from Japanese coastal waters. Concentrations of PCBs and OH-PCBs found in pinnipeds were the same as in small cetaceans living in the same coastal area. However, significantly lower concentrations of brominated compounds (PBDEs, MeO-PBDEs, OH-PBDEs) were found in the blood of pinnipeds than the levels found in cetacean species which live same area (p < 0.05). This difference of accumulation pattern suggested pinnipeds have an enhanced capability to degrade organobromine compounds relative to cetaceans.

Accumulation of hydroxylated polychlorinated biphenyls (OH-PCBs) and implications for PCBs metabolic capacities in three porpoise species.

The present study investigated polychlorinated biphenyls (PCBs) and hydroxylated metabolites of PCBs (OH-PCBs) in blood from three porpoise species: finless porpoises (Neophocaena phocaenoides), harbor porpoises (Phocoena phocoena), and Dall's porpoises (Phocoenoides dalli). The porpoises were found stranded or were bycaught along the Japanese coast. Concentrations of OH-PCB were the highest in Dall's porpoises (58pgg(-1) wet wt), second highest in finless porpoises (20pgg(-1) wet wt), and lowest in harbor porpoises (8.3pgg(-1) wet wt). The concentrations in Dall's porpoises were significantly higher than the concentrations in finless porpoises and harbor porpoises (p<0.05 and p<0.01, respectively). There was a positive correlation between PCB and OH-PCB concentrations (r=0.67, p<0.001), suggesting the possible concentration-dependent induction of CYP enzymes. The three porpoise species may have exceptionally low metabolic capacities compared with other marine and terrestrial mammals, because low OH-PCB/PCB concentration ratios were found, which were 0.0016 for Dall's porpoises, 0.0013 for harbor porpoises, and 0.00058 for finless porpoises. Distinct differences in the OH-PCB congener patterns were observed for the three species, even though they are taxonomically closely related.