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.

Directional and amplitude characteristics of pulsed call sequences in captive free-swimming Pacific white-sided dolphins (Lagenorhynchus obliquidens).

We investigated the directional properties and gain control of a pulsed call sequence that functions as a contact call in Pacific white-sided dolphins (Lagenorhynchus obliquidens). The pulsed call sequences were stereotyped patterns composed of three or more pulsed call elements and were collected from two dolphins, separated into adjacent pools, and allowed to swim freely. Eight hydrophones and an overhead camera were used to determine the positions and directions of the participants. The mean peak frequency and source levels were 8.4 ± 4.4 (standard deviation)-18.7 ± 12.7 kHz and 160.8 ± 3.8 to 176.4 ± 7.9 dB re 1 µPa (peak-to-peak), respectively, depending on the element types. The elements were omnidirectional, with mean directivity index of 0.9 ± 3.4 dB. The dolphins produced sequences, regardless of their relative position and direction to the lattice, leading to the adjacent pool where the conspecific was housed. They increased the amplitude by 6.5 ± 4.6 dB as the distance from the caller to an arbitrary point in the adjacent pool doubled. These results suggest that callers broadcast pulsed call sequences in a wide direction to reach dispersed conspecifics. However, they can adjust the acoustic active space by controlling the source levels.

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.

Interacting effects of vessel noise and shallow river depth elevate metabolic stress in Ganges river dolphins.

In riverine 'soundscapes', complex interactions between sound, substrate type, and depth create difficulties in assessing impacts of anthropogenic noise pollution on freshwater fauna. Underwater noise from vessels can negatively affect endangered Ganges river dolphins (Platanista gangetica), which are 'almost blind' and rely entirely on high-frequency echolocation clicks to sense their environment. We conducted field-based acoustic recordings and modelling to assess acoustic responses of Platanista to underwater noise exposure from vessels in the Ganga River (India), which is now being transformed into a major waterway. Dolphins showed enhanced activity during acute noise exposure and suppressed activity during chronic exposure. Increase in ambient noise levels altered dolphin acoustic responses, strongly masked echolocation clicks, and more than doubled metabolic stress. Noise impacts were further aggravated during dry-season river depth reduction. Maintaining ecological flows, downscaling of vessel traffic, and propeller modifications to reduce cavitation noise, could help mitigate noise impacts on Ganges river dolphins.

Pulsed call sequences as contact calls in Pacific white-sided dolphins (Lagenorhynchus obliquidens).

Pacific white-sided dolphins are a group-living species and appear to exchange "contact calls" to maintain group cohesion. The aim of this study was to find and characterize their contact calls. Calls were recorded from two females at Osaka Aquarium KAIYUKAN (OAK) and three females at Izu-Mito Sea Paradise (IMSP). Because they often produced pulsed calls consecutively, a "pulsed call sequence" was defined as three or more successive pulsed calls occurring within 325 ms, which was calculated using a bout analysis. The pulsed call sequences increased during separation periods and decreased during reunions and were used for vocal exchange, suggesting that the sequences are contact calls in Pacific white-sided dolphins. Most of the pulsed call sequences were classified into unique types; several stereotyped, repeated patterns were found. One sequence type was found at OAK and the two dolphins shared the type; they exchanged sequences with type matching. On the other hand, three sequence types were found in IMSP and the three dolphins shared all of the types; however, each dolphin preferentially used different types and frequently exchanged with their own favorite types but not with type matching. These results suggest that the sequence type may function as an individual and/or group identity.

A wild Indo-Pacific bottlenose dolphin adopts a socially and genetically distant neonate.

Alloparental behaviour and adoption have been reported in many mammals and birds. Such behaviours are energetically costly, and their causes and functions remain unclear. We observed the adoption behaviour of a wild Indo-Pacific bottlenose dolphin (Tursiops aduncus) near Mikura Island, Japan. A calf was seen with its mother on six observation days. Following the mother's death, the calf was observed with a sub-adult female on all 18 observation days from May to September 2012. On three days, the calf was observed swimming with this female in the suckling position and milk was seen leaking from the female's mammary slit. A five-year dataset revealed no significant social or kin relationships between the biological mother and allomother, indicating that kinship and social relationships did not play an important role in the observed adoption.

Individuality embedded in the isolation calls of captive beluga whales (Delphinapterus leucas).

INTRODUCTION: Species with fission-fusion social systems tend to exchange individualized contact calls to maintain group cohesion. Signature whistles by bottlenose dolphins are unique compared to the contact calls of other non-human animals in that they include identity information independent of voice cues. Further, dolphins copy the signatures of conspecifics and use them to label specific individuals. Increasing our knowledge of the contact calls of other cetaceans that have a fluid social structure may thus help us better understand the evolutionary and adaptive significance of all forms of individually distinctive calls. It was recently reported that one type of broadband pulsed sounds (PS1), rather than whistles, may function as individualized contact calls in captive belugas. The objective of this study was to assess the function and individual distinctiveness of PS1 calls in an isolation context. Recordings were made from five captive belugas, including both sexes and various ages. RESULTS: PS1 was the predominant call type (38 % in total) out of five broader sound categories. One sub-adult and three adults had individually distinctive and stereotyped pulse repetition pattern in PS1; one calf showed no clear stereotyped pulse repetition pattern. While visual inspection of the PS1 power spectra uncovered no apparent individual specificity, statistical analyses revealed that both temporal and spectral parameters had inter-individual differences and that there was greater inter-individual than intra-individual variability. Discriminant function analysis based on five temporal and spectral parameters classified PS1 calls into individuals with an overall correct classification rate of 80.5 %, and the most informative parameter was the average Inter-pulse interval, followed by peak frequency. CONCLUSION: These results suggest that belugas use individually distinctive contact calls in an isolation context. If belugas encode signature information in PS1 calls, as seen in bottlenose dolphins, the pulse repetition pattern may be the carrier, as it is individually stereotyped and appears to require vocal development. This idea is supported by the finding that the average inter-pulse interval is the most powerful discriminator in discriminant analysis. Playback experiments will elucidate which parameters are perceived as individual characteristics, and whether one of the parameters functions as a signature.

Post-conflict affiliation as conflict management in captive bottlenose dolphins (Tursiops truncatus).

Post-conflict affiliation between former opponents or between one of the former opponents and bystanders might have the function of conflict management, which reduces the costs associated with aggressions. One of the suggested functions of post-conflict affiliation is decreased renewed aggressions directed from aggressors to victims. However, the effect of post-conflict affiliation on renewed aggressions by victims has not been investigated. We examined whether post-conflict affiliations decreased the number of renewed aggressions initiated by winners or losers in captive bottlenose dolphins. Both winners and losers initiated renewed aggressions. However, these aggressions decreased after post-conflict affiliation between former opponents, initiated by bystanders to winners, initiated by losers to bystanders, and initiated by bystanders to losers. Post-conflict affiliation between former opponents is suggested to function as reconciliation. Post-conflict affiliation initiated by losers to bystanders is suggested to function as the protection of losers. Post-conflict affiliations initiated by bystanders to one of former opponents are suggested to function as both appeasement and protection of the opponent who affiliates with bystanders.

Sound variation and function in captive Commerson's dolphins (Cephalorhynchus commersonii).

Commerson's dolphin (Cephalorhynchus commersonii), one of the smallest dolphin species, has been reported to produce only narrow-band high-frequency (NBHF) clicks and no whistles. To clarify their sound repertoire and examine the function of each type, we analysed the sounds and behaviour of captive Commerson's dolphins in Toba Aquarium, Japan. All recorded sounds were NBHF clicks with peak frequency >110kHz. The recorded click-trains were categorised into four types based on the changing pattern of their Inter-click intervals (ICI): Decreasing type, with continuously decreasing ICI during the last part of the train; Increasing type, with continuously increasing ICI during the last part; Fluctuating type, with fluctuating ICI; and Burst-pulse type, with very short and constant ICI. The frequency of the Decreasing type increased when approaching an object newly introduced to the tank, suggesting that the sound is used for echolocation on approach. The Burst-pulse type suddenly increased in front of the object and was often oriented towards it, suggesting that it was used for echolocation in close proximity to the object. In contrast, the Increasing type was rarely recorded during approach, but increased when a dolphin approached another dolphin. The Increasing and Burst-pulse types also increased when dolphins began social behaviours. These results suggest that some NBHF clicks have functions other than echolocation, such as communication.

Captive bottlenose dolphins (Tursiops truncatus) spontaneously using water flow to manipulate objects.

Several terrestrial animals and delphinids manipulate objects in a tactile manner, using parts of their bodies, such as their mouths or hands. In this paper, we report that bottlenose dolphins (Tursiops truncatus) manipulate objects not by direct bodily contact, but by spontaneous water flow. Three of four dolphins at Suma Aqualife Park performed object manipulation with food. The typical sequence of object manipulation consisted of a three step procedure. First, the dolphins released the object from the sides of their mouths while assuming a head-down posture near the floor. They then manipulated the object around their mouths and caught it. Finally, they ceased to engage in their head-down posture and started to swim. When the dolphins moved the object, they used the water current in the pool or moved their head. These results showed that dolphins manipulate objects using movements that do not directly involve contact between a body part and the object. In the event the dolphins dropped the object on the floor, they lifted it by making water flow in one of three methods: opening and closing their mouths repeatedly, moving their heads lengthwise, or making circular head motions. This result suggests that bottlenose dolphins spontaneously change their environment to manipulate objects. The reason why aquatic animals like dolphins do object manipulation by changing their environment but terrestrial animals do not may be that the viscosity of the aquatic environment is much higher than it is in terrestrial environments. This is the first report thus far of any non-human mammal engaging in object manipulation using several methods to change their environment.

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.

Echolocation signals of Heaviside's dolphins (Cephalorhynchus heavisidii).

Field recordings of echolocation signals produced by Heaviside's dolphins (Cephalorhynchus heavisidii) were made off the coast of South Africa using a hydrophone array system. The system consisted of three hydrophones and an A-tag (miniature stereo acoustic data-logger). The mean centroid frequency was 125 kHz, with a -3 dB bandwidth of 15 kHz and -10 dB duration of 74 µs. The mean back-calculated apparent source level was 173 dB re 1 µPa(p.-p.). These characteristics are very similar to those found in other Cephalorhynchus species, and such narrow-band high-frequency echolocation clicks appear to be a defining characteristic of the Cephalorhynchus genus. Click bursts with very short inter-click intervals (up to 2 ms) were also recorded, which produced the "cry" sound reported in other Cephalorhynchus species. Since inter-click intervals correlated positively to click duration and negatively to bandwidth, Heaviside's dolphins may adjust their click duration and bandwidth based on detection range. The bimodal distribution of the peak frequency and stable bimodal peaks in spectra of individual click suggest a slight asymmetry in the click production mechanism.

Fine-scale analysis of synchronous breathing in wild Indo-Pacific bottlenose dolphins (Tursiops aduncus).

We quantitatively analysed synchronous breathing for dyads in Indo-Pacific bottlenose dolphins at Mikura Island, Tokyo, Japan. For most cases, we observed dyads swimming in the same direction (97%), in close proximity (i.e., less than 1.5m) and with their body axes parallel as they breathed synchronously. Moreover, the pairs engaged in identical behaviour before and after the synchronous breathing episodes. These results suggest that the dolphins synchronize their movements, and that synchronous breathing is a component of "pair-swimming", an affiliative social behaviour. Same sex pairs of the same age class frequently engaged in synchronous breathing for adults and subadults, as well as mother-calf and escort-calf pairs. The distance between individuals during synchronous breathing for mother-calf pairs was less than for other pairs. The distance observed between individuals for female pairs was less than for male pairs. The time differences between each exhale for each of the two dolphins involved in synchronous breathing episodes for female pairs were smaller than for male pairs, and time differences for adult pairs were smaller than subadult pairs. These results suggest that age and sex class influenced the characteristics of this behaviour.

Does body contact contribute towards repairing relationships? The association between flipper-rubbing and aggressive behavior in captive bottlenose dolphins.

Bottlenose dolphins exhibit complex social affiliations that may be shaped by interactions among individuals. Affiliative body contact among dolphins may repair deteriorated relationships or reduce tension within the group following aggressive interactions. We investigated the time-series association between one type of contact behavior (flipper-rubbing) and aggression by continuous observation of three captive bottlenose dolphins. For all three dolphin pairs, the elapsed time to aggressive events was significantly greater following flipper-rubbing. In two dolphin pairs comprised of a young male and an adult female, one-zero score of inter-opponent flipper-rubbing was higher for 10 min following aggressive bouts (post-AG periods) than for the same length of control (Ctrl) periods. For all three focal pairs, one-zero score of third-party rubbing was higher for post-AG than Ctrl periods. Neither the direction of rubbing nor the identity of the partner that approached prior to rubbing showed any significant tendencies. Flipper-rubbing may contribute to restore friendly relationships between former opponents or reduce conflicts in at least juvenile-adult female associations. Our results also give preliminary suggestions of the functions of third-party flipper-rubbing among bottlenose dolphins, including tension easing by the third party, or displacement as a result of aggressive interactions.