Association of seasonal changes in circulating cortisol concentrations with the expression of cortisol biosynthetic enzymes and a glucocorticoid receptor in the blubber of common bottlenose dolphin.

Cortisol is secreted from the adrenal cortex in response to stress, and its circulating levels are used as robust physiological indicators of stress intensity in various animals. Cortisol is also produced locally in adipose tissue by the conversion of steroid hormones such as cortisone, which is related to fat accumulation. Circulating cortisol levels, probably induced by cold stress, increase in cetaceans under cold conditions. However, whether cortisol production in subcutaneous adipose tissue is enhanced when fat accumulation is renewed during the cold season remains unclear. Therefore, in this study, we examine the effect of environmental temperature on the expression of cortisol synthesis-related enzymes and a glucocorticoid receptor in the subcutaneous fat (blubber) and explore the association between these expressions and fluctuations in circulating cortisol levels in common bottlenose dolphins (Tursiops truncatus). Skin biopsies were obtained seasonally from eight female dolphins, and seasonal differences in the expression of target genes in the blubber were analyzed. Blood samples were collected throughout the year, and cortisol levels were measured. We found that the expressions of cytochrome P450 family 21 subfamily A member 2 (CYP21A2) and nuclear receptor subfamily 3 group C member 1 (NR3C1), a glucocorticoid receptor, were increased in the cold season, and 11 beta-hydroxysteroid dehydrogenase type 1 (HSD11B1) showed a similar trend. Blood cortisol levels increased when the water temperature decreased. These results suggest that the conversion of 17-hydroxyprogesterone to cortisol via 11-deoxycortisol and/or of cortisone to cortisol is enhanced under cold conditions, and the physiological effects of cortisol in subcutaneous adipose tissue may contribute to on-site lipid accumulation and increase the circulating cortisol concentrations. The results obtained in this study highlight the role of cortisol in the regulation of the blubber that has developed to adapt to aquatic life.

Blubber biopsy in common bottlenose dolphins using a novel biopsy puncher: Evaluation of the impact on living individuals and possibility of applications in cetacean research.

Biopsy has recently become a preferred protocol for sampling the skin and blubber of many cetacean species, although it is desirable to collect as minimally invasive as possible. Here, the effect of biopsy sampling on the captive common bottlenose dolphins was evaluated by analyzing the process of wound healing and changes in hematological and blood biochemical parameters after biopsy using a puncher developed to collect up to the inner layer of the dolphin blubber. Results showed that the wounds caused by biopsy were closed in as early as 1 day and completely covered with the epidermis within 5-11 days. Blood fibrinogen, which generally increases due to a wound-induced inflammatory response or activation of the coagulation system, was significantly elevated after the biopsy indicating ongoing tissue repair, while other parameters did not exhibit significant differences. Furthermore, histological observation and RNA extraction of samples were performed to investigate the versatility of this method to cetacean research. Histological examination revealed three distinct layers of the blubber in the biopsy samples. Moreover, total RNA extracted from biopsy samples exhibited sufficient quality and quantity for gene expression analyses. Overall, the puncher utilized in our study represents a valuable and minimally invasive tool for investigating various aspects of small cetacean studies.

Non-invasive age estimation based on faecal DNA using methylation-sensitive high-resolution melting for Indo-Pacific bottlenose dolphins.

Age is necessary information for the study of life history of wild animals. A general method to estimate the age of odontocetes is counting dental growth layer groups (GLGs). However, this method is highly invasive as it requires the capture and handling of individuals to collect their teeth. Recently, the development of DNA-based age estimation methods has been actively studied as an alternative to such invasive methods, of which many have relied on used biopsy samples. However, if DNA-based age estimation can be developed from faecal samples, age estimation can be performed entirely non-invasively. We developed an age estimation model using the methylation rate of two gene regions, GRIA2 and CDKN2A, measured through methylation-sensitive high-resolution melting (MS-HRM) from faecal samples of wild Indo-Pacific bottlenose dolphins (Tursiops aduncus). The age of individuals was known through conducting longitudinal individual identification surveys underwater. Methylation rates were quantified from 36 samples collected from 30 individuals. Both gene regions showed a significant correlation between age and methylation rate. The age estimation model was constructed based on the methylation rates of both genes which achieved sufficient accuracy (after LOOCV: MAE = 5.08, R2 = 0.33) for the ecological studies of the Indo-Pacific bottlenose dolphins, with a lifespan of 40-50 years. This is the first study to report the use of non-invasive faecal samples to estimate the age of marine mammals.

Helicobacter delphinicola infection and the risk of gastric disease in common bottlenose dolphin.

Gastritis and gastric ulcers are well-recognized conditions in cetaceans; bacteria of the genus Helicobacter are considered the primary cause of these diseases. Dolphins have been shown to be susceptible to infection by at least 2 gastric species of Helicobacter, H. cetorum and H. delphinicola, both of which are closely related to the human pathogen H. pylori. In the present study, we evaluated the carriage rate and relationship to gastric disease of H. cetorum and H. delphinicola, based on a study population of 82 dolphins maintained at 21 facilities in Japan. Of these 82 dolphins, 79 (96.3%) and 45 (54.9%) were positive for H. cetorum and H. delphinicola, respectively; H. delphinicola infection was significantly associated with chronic gastric diseases (odds rate: 5.9; 95% CI: 2.1-16.9), but no such association was detected for H. cetorum. Of the 21 facilities, 20 (95%) and 11 (55%) housed H. cetorum- and H. delphinicola-positive dolphins, respectively, and our study suggested that the transmission between dolphins occurs quickly within pools. These findings indicate that methods will need to be established to prevent the transmission of Helicobacter infections within facilities housing dolphins.

Yawn-like behavior in captive common bottlenose dolphins (Tursiops truncatus).

Yawning is an involuntary action that begins with a slow opening of the mouth with inhalation, followed by a maximum gaping phase, and ends with a short exhalation and the closing of the mouth. A wide variety of vertebrate species, including humans, yawn. Here, we report underwater yawn-like behavior in three captive common bottlenose dolphins, inferred from 119-h of observations. Five cases of yawn-like behavior were selected out of 2045 open-mouth behaviors, after removing intentional open-mouth behaviors. Yawn-like behaviors were chosen that had a mouth open-close duration ratio of ≤ 1 (duration of Phase 3, the period of mouth closing after maximum opening, divided by the duration of Phase 1, the period of mouth opening from start to maximum opening). Naïve human evaluators selected "yawn-like" behaviors. All five cases of yawn-like behavior occurred during inactive periods, similar to human yawns. In three of the five cases, inactivity levels significantly decreased within 4 min after the yawn-like behavior; therefore, yawn-like behavior in dolphins may increase their arousal level in drowsy states. Thus, the yawn-like behavior of dolphins, without breathing, is similar to yawning in terrestrial animals, including humans.

Helicobacter delphinicola sp. nov., isolated from common bottlenose dolphins Tursiops truncatus with gastric diseases.

Gastritis and gastric ulcers are well-recognized symptoms in cetaceans, and the genus Helicobacter is considered as the main cause. In this study, we examined the gastric fluid of captive common bottlenose dolphins Tursiops truncatus with gastric diseases in order to isolate the organisms responsible for diagnosis and treatment. Four Gram-negative, rod-shaped isolates (TSBT, TSH1, TSZ, and TSH3) with tightly coiled spirals with 2-4 turns and 2-6 bipolar, sheathed flagella, were obtained from gastric fluids of common bottlenose dolphins with gastric diseases. Phylogenetic analysis, based on 16S rRNA, atpA, and 60 kDa heat-shock protein (hsp60) genes, demonstrated that these isolates form a novel lineage within the genus Helicobacter. Analyses of 16S rRNA, atpA, and hsp60 gene sequences showed that isolate TSBT was most closely related to H. cetorum MIT99-5656T (98.5% similarity), H. pylori ATCC 43504T (76.7% similarity), and H. pylori ATCC 43504T (78.0% similarity), respectively. Type strains of Helicobacter showing resistance to 2% NaCl have not been reported previously; however, these novel isolates were resistant to 2% NaCl. Culture supernatant of some isolates induced intracellular vacuolization in mammalian cultured cells. These data, together with the different morphological and biochemical characteristics of the isolates, reveal that these isolates represent a novel species for which we propose the name Helicobacter delphinicola sp. nov. with type strain TSBT (= JCM 32789T = TSD-183T). Future studies will confirm whether H. delphinicola plays a role in lesion etiopathogenesis in cetaceans.

Development of an analytical method to exclude the effect of decomposition on carbon and nitrogen stable isotope ratios using muscle samples collected from stranded narrow-ridged finless porpoise (Neophocaena asiaeorientalis).

Carbon and nitrogen stable isotope ratios (i.e., δ13 C and δ15 N values) in soft tissues of cetaceans are reported to change on decomposition. Care should be taken when determining the values using samples collected from dead-stranded cetaceans. We have developed a new analytical method to exclude the effect of decomposition on δ13 C and δ15 N values using muscles from narrow-ridged finless porpoises (Neophocaena asiaeorientalis). METHODS: The muscles from 53 fresh (bycatch and live-stranded individuals) and 213 dead-stranded individuals were analyzed by isotope ratio mass spectrometry combined with an elemental analyzer. The carbon and nitrogen content was compared between fresh and dead-stranded samples to investigate any decrease in the content with decomposition. In dead-stranded samples, the effect of changes in the carbon and nitrogen content and the C:N ratio (carbon/nitrogen) on δ13 C and δ15 N values was predicted by the generalized additive model (GAM). RESULTS: The carbon and nitrogen content (mean ± SE) in the muscles of fresh samples was 0.462 ± 0.0019 mg/mg and 0.147 ± 0.0005 mg/mg, respectively, while the content of dead-stranded samples was lower: carbon: 0.441 ± 0.0033 mg/mg and nitrogen: 0.135 ± 0.0012 mg/mg. When using the GAM, knowledge of the carbon and nitrogen content and the C:N ratio improved the prediction precision. CONCLUSIONS: Prediction by the GAM showed changes in δ13 C and δ15 N values with a change in carbon and nitrogen content. The effect of decomposition can be excluded in statistical analysis of stranded samples by using samples with carbon and nitrogen content and C:N ratio equivalent to those of fresh samples or by using the carbon and nitrogen content and C:N ratio as an explanatory variable in the GAM.

Plasma metabolomic analysis in mature female common bottlenose dolphins: profiling the characteristics of metabolites after overnight fasting by comparison with data in beagle dogs.

The present study was aimed at determining the characteristics of plasma metabolites in bottlenose dolphins to provide a greater understanding of their metabolism and to obtain information for the health management of cetaceans. Capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) and liquid chromatograph-time-of-flight mass spectrometry (LC-TOFMS) were conducted on plasma samples after overnight fasting from three common bottlenose dolphins as well as three beagle dogs (representative terrestrial carnivores) for comparison. In total, 257 and 227 plasma metabolites were identified in the dolphins and the dogs, respectively. Although a small number of animals were used for each species, the heatmap patterns, a principal component analysis and a cluster analysis confirmed that the composition of metabolites could be segregated from each other. Of 257 compounds detected in dolphin plasma, 24 compounds including branched amino acids, creatinine, urea, and methylhistidine were more abundant than in dogs; 26 compounds including long-chained acyl-carnitines and fatty acids, astaxanthin, and pantothenic acid were detected only in dolphins. In contrast, 25 compounds containing lactic acid and glycerol 3-phosphate were lower in dolphins compared to dogs. These data imply active protein metabolism, differences in usage of lipids, a unique urea cycle, and a low activity of the glycolytic pathway in dolphins.

Long-term monitoring of circulating progesterone and its relationship to peripheral white blood cells in female false killer whales Pseudorca crassidens.

Long-term monitoring of circulating progesterone levels in three captive female false killer whales, Pseudorca crassidens, was conducted to characterize their reproductive events and to reveal the relationship between their estrous cycles or pregnancies and peripheral white blood cell (WBC) counts. Blood samples were collected at 2-3-day intervals or on a weekly-to-monthly basis for up to 10 years, from 2006 to 2017. In two mature females (initial body lengths of 4.22 and 4.07 m), some cyclic progesterone elevations were detected during the study period; the estimated mean (± SE) estrous cycle length was 40.5 ± 0.7 days (n=12). The seasonality of ovulation, estimated from the elevation of progesterone levels, varied among individuals or years, and ovulation did not occur every year. The third female (3.26 m) showed progesterone elevations, despite irregular cycles after sexual maturity, and became pregnant. The progesterone levels during pregnancy ranged from 7.3 to 42.2 ng/ml, and the gestation period lasted for 14 months until parturition. The mean WBC counts during estrous cycles were the lowest before the progesterone levels began to increase and then gradually increased toward the luteal phase. The WBC counts were significantly higher during pregnancy than before and were particularly high in early pregnancy. To the best of our knowledge, this is the first report of the relationship between the estrous cycle or pregnancy and WBC counts in cetaceans.

Seasonal changes in circulating gonadal steroid levels and physiological evidence for the presence of intrinsic circannual reproductive cycles in captive finless porpoises Neophocaena asiaeorientalis from the western Inland Sea, Japan.

We monitored annual fluctuations of gonadal steroid levels in three sexually mature captive finless porpoises (Neophocaena asiaeorientalis; two males and one female) from two different facilities over 56-91 months. Two animals (one male and one female) were held in an indoor tank with a sunroof (facility A) and the other male was held in an indoor tank without a sunroof (facility B). Water temperatures in both facilities reflected seasonal changes during the study period with a minor difference in the fluctuation pattern. Testosterone levels of the male in facility A were higher from spring to summer every year and exhibited a 12-month cycle. The female showed estrus cycles in 1-month intervals from summer to winter, excluding 2 anestrus years. In contrast, the period of higher testosterone levels of the male in facility B gradually initiated earlier over the years under a constant photoperiod (11.5L:12.5D) and exhibited a 9-month cycle during the first 52 months. After changing the light conditions to a natural photoperiod, its testosterone levels were high from early spring to summer for 3 consecutive years and exhibited a 12-month cycle. Our results showed that under a constant artificial photoperiod, the male in facility B failed to recognize the seasonal changes of a natural external environment, resulting in a 9-month, free-running hormone cycle.

Spontaneous ejaculation in a wild Indo-Pacific bottlenose dolphin (Tursiops aduncus).

Spontaneous ejaculation, which is defined as the release of seminal fluids without apparent sexual stimulation, has been documented in boreoeutherian mammals. Here we report spontaneous ejaculation in a wild Indo-Pacific bottlenose dolphin (Tursiops aduncus), and present a video of this rare behavior. This is the first report of spontaneous ejaculation by an aquatic mammal, and the first video of this behavior in animals to be published in a scientific journal.

Molecular identification of novel alpha- and gammaherpesviruses from cetaceans stranded on Japanese coasts.

Herpesviral infections have been documented in some cetaceans; however, they have not yet been identified in species in the western North Pacific. In the present study, 178 tissue samples from 76 stranded cetacean individuals were tested for the presence of herpesviruses. Herpesvirus genomic DNA fragments surrounding the DNA polymerase gene were amplified in samples from four individuals. TA cloning and direct sequencing of these DNA fragments revealed the presence of two novel alphaherpesviruses, and two novel gammaherpesviruses in the four cetacean individuals. The alphaherpesviruses were associated with the lung tissue of a false killer whale (Pseudorca crassidens), and with the mucus of a melon-headed whale (Peponocephala electra). The gammaherpesviruses were found in the lymph tissues of a Stejneger's beaked whale (Mesoplodon stejnegeri) and a sperm whale (Physeter macrocephalus). The phylogenetic tree using amino acid sequences of the DNA polymerase gene supported the inclusion of the novel viruses identified here in a single monophyletic group containing alphaherpesviruses from other Atlantic cetacean species. Conversely, the novel gammaherpesviruses formed an independent clade distant from other known cetacean gammaherpesviruses.

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.

Natriuretic peptides in cetaceans: identification, molecular characterization and changes in plasma concentration after landing.

Dolphins are aquatic animals free from gravity, and this may have imposed significant changes in their cardiovascular status and its hormonal regulation compared with terrestrial animals. This study molecularly characterized two major cardiovascular hormones, atrial and B-type natriuretic peptides (ANP and BNP) and measured their changes in dolphin plasma concentrations in relation to the cardiovascular status of the animal. We initially identified ANP and BNP in three species of dolphins (Lagenorhynchus obliquidens, Phocoenoides dalli and Tursiops truncatus). ANP precursors are highly conserved in most mammals, but dolphin BNP precursors were more variable. In molecular phylogenetic analyses, dolphin ANP and BNP precursors grouped with those of artiodactyls, particularly to the camel peptides. The chromatographic characterization of tissue and plasma molecular forms using specific radioimmunoassays showed that the predominant ANP and BNP in the atrium are prohormone and mature peptide, respectively, whereas mature ANP and BNP are circulating in the dolphin blood. A mass spectrometric analysis showed that atrial BNP consists of 26 amino acids, rather than the 32-amino-acid form detected in other mammals. Finally, changes in plasma ANP and BNP concentrations were examined in captive bottlenose dolphins (Tursiops truncatus) after their pool was drained. Plasma ANP and BNP concentrations did not change after landing, unlike terrestrial mammals. Plasma angiotensin II and cortisol concentrations did not change either, showing minor stress after landing. Since landed dolphins show a different cardiovascular status on land than terrestrial mammals, plasma ANP and BNP concentrations seem to reflect the cardiovascular status characteristic of dolphins.

Bioaccumulation, temporal trend, and geographical distribution of synthetic musks in the marine environment.

Bioaccumulation of synthetic musks in a marine food chain was investigated by analyzing marine organisms at various trophic levels, including lugworm, clam, crustacean, fish, marine mammal, and bird samples collected from tidal flat and shallow water areas of the Ariake Sea, Japan. Two of the polycyclic musks, HHCB and AHTN, were the dominant compounds found in most of the samples analyzed, whereas nitro musks were not detected in any of the organisms, suggesting greater usage of polycyclic musks relative to the nitro musks in Japan. The highest concentrations of HHCB were detected in clams (258-2730 ng/g lipid wt.), whereas HHCB concentrations in mallard and black-headed gull were low, and comparable with concentrations in fish and crab. These results are in contrast to the bioaccumulation pattern of polychlorinated biphenyls; for which a positive correlation between the concentration and the trophic status of organisms was found. Such a difference in the bioaccumulation is probably due to the metabolism and elimination of HHCB in higher trophic organisms. Temporal trends in concentrations of synthetic musks were examined by analyzing tissues of marine mammals from Japanese coastal waters collected during 1977-2005. HHCB concentrations in marine mammals have shown significant increase since the early 1990s, suggesting a continuous input of this compound into the marine environment. Comparison of the time trend for HHCB with those for PCBs and PBDEs suggested that the rates of increase in HHCB concentrations were higher than the other classes of pollutants. To examine the geographical distribution of HHCB, we have analyzed tissues of fish, marine mammals, and birds collected from several locations. Synthetic musks were not detected in a sperm whale (pelagic species) from Japanese coastal water and in eggs of south polar skua from Antarctica. While the number of samples analyzed is limited, these results imply a lack of long-range transportation potential of synthetic musks in the environment.

Population differentiation in the Pacific white-sided dolphin Lagenorhynchus obliquidens inferred from mitochondrial DNA and microsatellite analyses.

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 (pi=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.

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.

Genetic structure of a Japanese allotetraploid loach of the genus Cobitis (Osteichthyes, Cobitidae).

The Japanese allotetraploid spined loach of the genus Cobitis "yamato complex" sensu SAITOH et al. (2000), distributed in Western Japan, originated from hybridization between C. biwae on the maternal side and C. striata (Kyushu form) on the paternal side. Mitochondrial (mt) and nuclear DNA were analyzed in order to determine the genetic relationships among 15 populations spanning the entire range of the yamato complex. PCR-RFLP analysis of the ND1 mtDNA gene indicated that the yamato complex contains two divergent types of mtDNA: type A, consisting of one haplotype observed only in the Fukagawa River and type B consisting of 12 haplotypes found in the entire area. Phylogenetic analysis based on the cytochrome b mtDNA gene corroborated RFLP analysis, and indicated that type A was closely related to a different species, C. biwae (Kochi group) and C. striata (large race), rather than type B. The results of RAPD analysis on the Fukagawa River individuals, where types A and B sympatrically existed suggested that no reproductive isolation occurs between them. The existence of two distinct mtDNA types within the yamato complex suggest either multiple maternal origin at the speciation (tetraploidization) time or mtDNA introgression from other species afterwards.