Comprehensive expression analysis of hormone-like substances in the subcutaneous adipose tissue of the common bottlenose dolphin Tursiops truncatus.

Marine mammals possess a specific subcutaneous fat layer called blubber that not only insulates and stores energy but also secretes bioactive substances. However, our understanding of its role as a secretory organ in cetaceans is incomplete. To exhaustively explore the hormone-like substances produced in dolphin subcutaneous adipose tissue, we performed seasonal blubber biopsies from captive female common bottlenose dolphins (Tursiops truncatus; N = 8, n = 32) and analyzed gene expression via transcriptomics. Analysis of 186 hormone-like substances revealed the expression of 58 substances involved in regulating energy metabolism, tissue growth/differentiation, vascular regulation, immunity, and ion/mineral homeostasis. Adiponectin was the most abundantly expressed gene, followed by angiopoietin protein like 4 and insulin-like growth factor 2. To investigate the endocrine/secretory responses of subcutaneous adipose tissue to the surrounding temperature, we subsequently compared the mean expression levels of the genes during the colder and warmer seasons. In the colder season, molecules associated with appetite suppression, vasodilation, and tissue proliferation were relatively highly expressed. In contrast, warmer seasons enhanced the expression of substances involved in tissue remodeling, immunity, metabolism, and vasoconstriction. These findings suggest that dolphin blubber may function as an active secretory organ involved in the regulation of metabolism, appetite, and tissue reorganization in response to changes in the surrounding environment, providing a basis for elucidating the function of hormone-like substances in group-specific evolved subcutaneous adipose tissue.

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.

Draft genome sequencing and assembly of Risso's dolphin, Grampus griseus.

The Risso's dolphin (Grampus griseus) is one of the migratory marine mammals and they have commonly dispersed in tropical and temperate seas. It is a least concerned species in the IUCN red list of threatened species. However, their population size and factors affecting their population structure are unknown. Due to the wide distribution of this species, their populations might be genetically stable and less structured. To support genetic studies on dolphins and other marine mammals, we assembled the draft genome of Risso's dolphin that was found in Japan. The tissue samples were used to extract high molecular DNA and subjected to sequencing by Illumina HiSeq X, Oxford Nanopore MinION, and Bionano Saphyr. The assembled hybrid genome was 75.9% of complete eukaryotic BUSCOs and the genome size was 2.256 Gb with 2.042 Mb of scaffold N50. De novo assembly of this genome by Bionano Saphyr recovered 2.036 Gb total genome map length and structural variations. The gene structures of this draft genome were identified by BRAKER2, and 9947 genes were recovered. The data will be useful for future studies of cetaceans.

Selection of a reference gene for studies on lipid-related aquatic adaptations of toothed whales (Grampus griseus).

Toothed whales are one group of marine mammals that has developed special adaptations, such as echolocation for predation, to successfully live in a dynamic aquatic environment. Their fat metabolism may differ from that of other mammals because toothed whales have acoustic fats. Gene expression in the metabolic pathways of animals can change with respect to their evolution and environment. A real-time quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for studying the relative expressions of genes. However, since the accuracy of RT-qPCR data is totally dependent on the reference gene, the selection of the reference gene is an essential step. In this study, 10 candidate reference genes (ZC3H10, FTL, LGALS1, RPL27, GAPDH, FTH1, DCN, TCTP, NDUS5, and UBIM) were initially tested for amplification efficiency using RT-qPCR. After excluding DCN, the remaining nine genes, which are nearly 100% efficient, were selected for the gene stability analysis. Stable reference genes across eight different fat tissue, liver, and muscle samples from Grampus griseus were identified by four algorithms, which were provided in Genorm, NormFinder, BestKeeper, and Delta CT. Finally, a RefFinder comprehensive ranking was performed based on the stability values, and the nine genes were ranked as follows: LGALS1 > FTL > GAPDH > ZC3H10 > FTH1 > NDUS5 > TCTP > RPL27 > UBIM. The LGALS1 and FTL genes were identified as the most stable novel reference genes. The third-ranked gene, GAPDH, is a well-known housekeeping gene for mammals. Ultimately, we suggest the use of LGALS1 as a reliable novel reference gene for genomics studies on the lipid-related aquatic adaptations of toothed whales.

Another polymorphic mitochondrial genome of Grampus griseus and phylogeny of family Delphinidae.

Risso's dolphin (Grampus griseus Cuvier, 1812) is the only species of genus Grampus and a cosmopolitan marine inhabitant. Here, we report a polymorphic complete mitochondrial genome of G. griseus. The size of the total mitochondrial genome was 16,386 bp in length and contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a control region. 37 single nucleotide polymorphic sites (SNPs) were identified compared to the references. Based on the available total mitochondrial dolphin genomes' phylogenetics, G. griseus has formed a clade with 0.1415 distance, sister to the following species of the subfamily Globicephalinae and the taxonomy of Orcinus orca still needs further investigations.

Towards non-invasive heart rate monitoring in free-ranging cetaceans: a unipolar suction cup tag measured the heart rate of trained Risso's dolphins.

Heart rate monitoring in free-ranging cetaceans to understand their behavioural ecology and diving physiology is challenging. Here, we developed a simple, non-invasive method to monitor the heart rate of cetaceans in the field using an electrocardiogram-measuring device and a single suction cup equipped with an electrode. The unipolar suction cup was placed on the left lateral body surface behind the pectoral fin of Risso's dolphins (Grampus griseus) and a false killer whale (Pseudorca crassidens) in captivity; their heart rate was successfully monitored. We observed large heart rate oscillations corresponding to respiration in the motionless whales during surfacing (a false killer whale, mean 47 bpm, range 20-75 bpm; Risso's dolphins, mean ± s.d. 61 ± 15 bpm, range 28-120 bpm, n = 4 individuals), which was consistent with the sinus arrhythmia pattern (eupneic tachycardia and apneic bradycardia) observed in other cetaceans. Immediately after respiration, the heart rate rapidly increased to approximately twice that observed prior to the breath. Heart rate then gradually decreased at around 20-50 s and remained relatively constant until the next breath. Furthermore, we successfully monitored the heart rate of a free-swimming Risso's dolphin. The all-in-one suction cup device is feasible for field use without restraining animals and is helpful in further understanding the diving physiology of free-ranging cetaceans. This article is part of the theme issue 'Measuring physiology in free-living animals (Part II)'.

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.

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 plasma levels of thyroid hormones and the effects of the hormones on cellular ATP content in common bottlenose dolphin.

In general, thyroid hormones (THs) stimulate cellular metabolism by inducing ATP utilization that collaterally leads to thermogenesis. However, in cetaceans, TH functions and the contribution of THs to cold adaptation are not fully understood. To investigate the role of THs in metabolism of cetaceans, seasonal changes in circulating levels in THs were investigated in the common bottlenose dolphin Tursiops truncatus that were monitored under two different conditions for two years, with routine measurements of body temperature (BT), water temperature (WT) and air temperature (AT). The effects of THs on ATP synthesis were determined using cultured cells. Blood samples were collected from the species kept in different conditions at the Taiji Whale Museum located in the temperate zone and at Okinawa Expo Park in the subtropical zone. Circulating levels in total thyroxine (T4) for the dolphins at both aquaria and total 3,5,3'-tri-iodothyronine (T3) levels in dolphins at Taiji were measured by enzyme-linked immunoassay methods, respectively, and average concentrations were compared among seasons. To confirm the effects of THs on ATP synthesis, T3 or T4 was administrated to cultured kidney cells from the same species and cellular ATP contents were quantified at 0, 24, 48, 96 and 192 h after administration. BT of common bottlenose dolphins in each aquarium was measured for health check by chance in Taiji and every morning in Okinawa. WT in pools and AT were also measured every morning. Circulating T4 levels in autumn and winter were lower than those in spring and summer in dolphins in Taiji where WT and AT varied greatly from season to season. T4 levels showed a small difference between spring and autumn in dolphins in Okinawa with warmer WT and AT in smaller amplitude ranges than in Taiji. Total T3 level in Taiji was highest in spring and lowest in autumn as T4 levels, but not significant. The BT of dolphins in Taiji was also lower in autumn and winter compared with those in spring and summer, whereas the BT of dolphins in Okinawa fell in autumn but rose in summer, albeit to a lesser extent than in Taiji. Cellular ATP was increased by administration of both T3 and T4 compared to control. Collectively, these results suggest that the cellular metabolic activities regulated by THs may be enhanced in dolphins exposed to increasing surrounding temperature for lipolysis and reduced in dolphins exposed to colder conditions for fat accumulation.

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.