Apneustic anesthesia ventilation improves pulmonary function in anesthetized bottlenose dolphins (Tursiops truncatus).

Introduction: Use of mechanical ventilation during general anesthesia is a necessary practice in the anesthetization of small cetaceans as spontaneous ventilation fails to provide adequate gas exchange. Currently available methods of ventilation do not account for the intermittent breathing strategy of representative species within this infraorder of fully aquatic mammals and may have a significant effect on cardiac and respiratory physiology. Methods: To understand the impact of mechanical ventilation on cardiopulmonary function in one small species of cetacean, the bottlenose dolphin (Tursiops truncatus), we compared controlled mechanical ventilation (CMV) to a novel ventilation method known as apneustic anesthesia ventilation (AAV). AAV simulates the normal inspiratory breath-hold pattern of dolphins. Ten anesthetic procedures (dental procedure, n = 9; bronchoscopy, n = 2) were performed on nine dolphins (age range: 10-42 years; mean = 32 years; median = 37 years; female = 3, 40%; male = 6, 60%). In a cross-over study design, dolphins were instrumented and randomly assigned to AAV or CMV as the initial mode of ventilation, then switched to the alternate mode. Baseline cardiopulmonary data were collected and again after 30 min on each mode of ventilation. Cardiac index, stroke volume index, systemic vascular resistance, alveolar dead space, alveolar-arterial oxygen tension gradient, arterial oxygen content, oxygen delivery index, and dynamic respiratory system compliance index were calculated at each of the four time points. Results: During AAV, dolphins had higher arterial oxygen tension, higher mean airway pressure, reduced alveolar dead space ventilation and lower alveolar-arterial oxygen difference. Cardiovascular performance was not statistically different between the two modes. Discussion: Our study suggests AAV, which more closely resembles the conscious intermittent respiratory pattern phenotype of dolphins, improves ventilation and pulmonary function in the anesthetized dolphin. Future studies should evaluate the cardiopulmonary effects of neutral buoyancy and cardiopulmonary sparing drug protocols to reduce the need for hemodynamic support of current protocols.

Novel processing and staining methodology of bottlenose dolphin (Tursiops truncatus) teeth for age determination.

Age determination of bottlenose dolphins (Tursiops truncatus) is a critical tool in understanding both individual and population health. There are many methods of aging bottlenose dolphins including analysis of teeth, pectoral flipper radiographs, and epigenetics. The most common and oldest method for aging toothed cetaceans is the counting of growth layer groups (GLGs) in the teeth. Current techniques have technical and repeatability challenges. Therefore, a processing technique that results in better resolution of GLGs is needed. This study compares different decalcifications and different histochemical staining techniques. Decalcification was done using 10% EDTA, Kristensen's decalcification, and Rapid Decalcification Solution (RDO). Following decalcification and routine processing, GLGs were assessed using Hematoxylin and Eosin (H&E), hematoxylin, Giemsa, Wright-Giemsa, Toluidine Blue (T-Blue), Masson's Trichrome, and Congo Red staining techniques. Decalcification with Kristensen's and staining with Masson's Trichrome and Congo Red were determined to best highlight GLGs. This processing and staining was then applied to a sample population of 102 bottlenose dolphins that were evaluated independently and blindly by two observers. Of the 102 dolphin samples, 13 (12.7%) were unable to age due to no clear distinction or distortion between GLGs.

First description of Rhodococcus equi infection in common bottlenose dolphin (Tursiops truncatus).

Rhodococcus equi is a terrestrial bacterium and a common pathogen in foals (Equus caballus), in which causes pneumonia. This report describes for the first time the infection caused by R. equi in a common bottlenose dolphin (Tursiops truncatus) stranded in the Calabrian coast, Italy. The post mortem examination of the animal revealed lesions in lung and colon. The animal was also positive to dolphin morbillivirus. The histological study showed lesions attributable to R. equi infection, such as pyogranulomatous bacterial pneumonia and chronic granulomatous colitis. Whole genome sequencing of the isolated strain confirmed its identification as R. equi.

Distribution of calcium-binding proteins immunoreactivity in the bottlenose dolphin entorhinal cortex.

Introduction: The entorhinal cortex has been shown to be involved in high-level cognitive functions in terrestrial mammals. It can be divided into two main areas: the lateral entorhinal area (LEA) and the medial entorhinal area (MEA). Understanding of its structural organization in cetaceans is particularly important given the extensive evidence for their cognitive abilities. The present study describes the cytoarchitectural and immunohistochemical properties of the entorhinal cortex of the bottlenose dolphin (Tursiops truncatus, Montagu, 1821), perhaps the most studied cetacean species and a paradigm for dolphins and other small cetaceans. Methods: Four bottlenose dolphins' entorhinal cortices were processed. To obtain a precise overview of the organization of the entorhinal cortex we used thionin staining to study its laminar and regional organization, and immunoperoxidase technique to investigate the immunohistochemical distribution of three most commonly used calcium-binding proteins (CBPs), calbindin D-28k (CB), calretinin (CR) and parvalbumin (PV). Entorhinal cortex layers thickness were measured, morphological and morphometric analysis for each layer were conducted and statistically compared. Results: Six layers in both the LEA and MEA were identified. The main difference between the LEA and the MEA is observed in layers II and III: the neurons in layer II of the LEA were denser and larger than the neurons in layer II of MEA. In addition, a relatively cell-free zone between layers II and III in LEA, but not in MEA, was observed. The immunohistochemical distribution of the three CBPs, CB, CR and PV were distinct in each layer. The immunostaining pattern of CR, on one side, and CB/PV, on the other side, appeared to be distributed in a complementary manner. PV and CB immunostaining was particularly evident in layers II and III, whereas CR immunoreactive neurons were distributed throughout all layers, especially in layers V and VI. Immunoreactivity was expressed by neurons belonging to different morphological classes: All CBPs were expressed in non-pyramidal neurons, but CB and CR were also found in pyramidal neurons. Discussion: The morphological characteristics of pyramidal and non-pyramidal neurons in the dolphin entorhinal cortex are similar to those described in the entorhinal cortex of other species, including primates and rodents. Interestingly, in primates, rodents, and dolphins, most of the CBP-containing neurons are found in the superficial layers, but the large CR-ir neurons are also abundant in the deep layers. Layers II and III of the entorhinal cortex contain neurons that give rise to the perforant pathway, which conveys most of the cortical information to the hippocampal formation. From the hippocampal formation, reciprocal projections are directed back to the deep layer of the entorhinal cortex, which distributes the information to the neocortex and subcortical area. Our data reveal that in the dolphin entorhinal cortex, the three major CBPs label morphologically heterogeneous groups of neurons that may be involved in the information flow between entorhinal input and output pathways.

Vascularization of the gastrointestinal tract of the bottlenose dolphin (Tursiops truncatus, Montagu 1821).

Odontocetes primarily rely on fish, cephalopods, and crustaceans as their main source of nutrition. In the digestive system, their polygastric complex exhibits similarities to that of their closest terrestrial relatives such as cows, sheep, and giraffes, while the entero-colic tract shares similarities with terrestrial carnivores. The morphology, caliber, and structure of the odontocete intestine are relatively constant, and, since there is no caecum, a distinction between the small and large intestine and their respective subdivisions is difficult. To address this issue, we used the intestinal vascularization pattern, specifically the course and branching of the celiac artery (CA) and the cranial and caudal mesenteric arteries (CrMA and CdMA). A series of pictures and dissections of 10 bottlenose dolphins (Tursiops truncatus) were analyzed. Additionally, we performed a cast by injecting colored polyurethane foam in both arteries and veins to measure the caliber of the arteries and clarify their monopodial or dichotomous branching. Our results showed the presence of multiple duodenal arteries (DAs) detaching from the CA. The CrMA gave origin to multiple jejunal arteries, an ileocolic artery (ICA), and, in six cases, a CdMA. In four specimens, the CdMA directly originated from the abdominal aorta. The ICA gave rise to the mesenteric ileal branches (MIB) and mesenteric anti-ileal branches and the right colic arteries (RCA) and the middle colic arteries. From the CdMA originated the left colic and cranial rectal arteries (LCA and CrRA). The measurements revealed a mixed monopodial and dichotomous branching scheme. The analysis of the arteries and their branching gave us an instrument, based on comparative anatomy, to distinguish between the different intestinal compartments. We used the midpoint of anastomoses between MIB and RCA to indicate the border between the small and the large intestine, and the midpoint of anastomoses between LCA and CrRA, to tell the colon from the rectum. This pattern suggested an elongation of the duodenum and a shortening of the colic tract that is still present in this species. These findings might be related to the crucial need to possess a long duodenal tract to digest prey ingested whole without chewing. A short aboral part is also functional to avoid gas-producing colic fermentation. The rare origin of the CdMA on the CrMA might instead be a consequence of the cranial thrust of the abdominopelvic organs related to the loss of the pelvic girdle that occurred during the evolution of cetaceans.

Optimization and validation of a micro-QuEChERS method for phthalates detection in small samples of cetacean blubber.

In this study, an innovative method was developed to detect and quantify phthalates in fresh cetacean blubber. An adaptation of the ammonium formate QuEChERS method was used and adapted as a micro-extraction for small quantities of samples. Significantly, this technique utilized minimal quantities of reagents and salts, with the additional implementation of rigorous Quality Assurance/Quality Control protocols to further reduce background contamination. To ensure the reliability of this method, comprehensive validation procedures were conducted, with a specific focus on two widely studied cetacean species: the common bottlenose dolphin (Tursiops truncatus) and the short-finned pilot whale (Globicephala macrorhynchus). Determination coefficients (R2) for matrix-matched calibration were >0.93 with limits of quantifications (LOQ) of the method in the range of 5-10 ng/g. Mean recovery values were between 40 and 100 %. This novel methodology holds particular relevance for environmental research studies, offering the capability to detect emerging contaminants with minimal sample requirements. This aspect is particularly valuable in investigations that involve free-ranging animals and rely on biopsy sampling. It allows for the assessment of contaminant levels in healthy individuals within wild populations, enhancing our understanding of ecological impacts and potential conservation measures.•A micro-extraction adaptation of the ammonium formate QuEChERS method was developed and applied to a small quantity of fresh cetacean blubber to detect phthalates.•Small quantities of reagents and salts were used, and additional Quality Assurance/ Quality Control procedures were taken to further minimize background contamination.•Method validation was carried out for two cosmopolitan and extensively studied cetacean species: the common bottlenose dolphin (Tursiops truncatus) and the short-finned pilot whale (Globicephala macrorhynchus).

Electric boat underwater radiated noise and its potential impact on species of conservation interest.

Electric boats are thought to be noiseless, but in-situ measurements are generally rare. The Underwater Radiated Noise (URN) of 8-m Trimaran Pontoon Boat with two outboard electric engines was measured in the Miramare Marine Protected Area (Trieste, Italy), together with the URN of a fibreglass 5-m boat, with a outboard gasoline engine, for comparisons. International standards and guidelines for shallow waters were considered. URN were provided in one-third octave band and in narrow band spectra. The electric boat produced a low input of underwater noise at low frequencies. Given a low-frequency hearing sensitivity, the listening space reduction (LSR) was lower when generated by the electric than by combustion engine boat for the brown meagre, a local Teleost fish. No difference was found for the bottlenose dolphin LSR although continuous, tonal, high frequency components generated by the electric boat are expected to be highly detrimental for the bottlenose dolphin.

Establishment and characterization of a novel lung cell line derived from the common bottlenose dolphin.

Cetaceans are specialized marine mammals with a unique respiratory system adapted for diving behavior. Furthermore, respiratory diseases are commonly observed in these mammals. Nevertheless, much of their respiratory physiology remains unknown due to the limited supply and poor quality of their biological samples for research. In this study, we established a novel lung cell line, dLu, derived from the common bottlenose dolphin (Tursiops truncatus), which can prove useful in cetacean research, including for understanding the pathogenesis of respiratory diseases in cetaceans. The cells were cultured in a simple medium consisting of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The morphology of the cells was fibroblast-like. dLu was produced by transfecting the simian virus 40 large T antigen into primary cultured cells. Although dLu exhibited approximately 80 cell divisions, it was unable to achieve complete immortalization, as the cells stopped proliferating beyond this number. dLu cells expressed toll-like receptor 3 but not toll-like receptor 4. Immunostimulation with poly(I:C) altered the gene expressions of interferon beta 1 and tumor necrosis factor alpha in dLu cells. In summary, dLu established in this study is a novel cetacean cell resource that can be easily cultured and is a useful in vitro tool in cetacean research, particularly for studying host immune responses in the lungs.

Isolation and Characterization of Cetacean Cell-Derived Extracellular Vesicles.

Cetaceans are of scientific interest because they are good candidates as environmental bioindicators. However, in vivo research is arduous and in vitro studies represent a rarely used valid alternative. Extracellular vesicles (EVs) are membrane-bound structures playing roles in cell-to-cell communication. Despite being a promising investigative tool in different fields of science, EVs have been poorly studied in cetaceans. To fill this gap, we describe the preliminary characterization of EVs isolated from a bottlenose dolphin and a Cuvier's beaked whale cell line. EVs have been isolated with ultracentrifugation (UC) or size exclusion chromatography (SEC) and characterized with nanoparticle tracking analysis (NTA), Western blotting (WB), and scanning transmission electron microscopy (STEM). UC and SEC allowed the isolation of mainly small EVs (<200 nm). A higher number of particles were isolated through UC compared to SEC from both cell lines. At WB, all EVs expressed the EV-markers CD9 and integrin-ß. Only EVs isolated with UC were positive for TSG101. In conclusion, we isolated for the first time EVs from a bottlenose dolphin and a Cuvier's beaked whale cell line using two different techniques. Further studies on cell-derived EVs will be useful to deepen our knowledge on cetacean pathophysiology and health status assessment.

Systemic Erysipelas Outbreak among Free-Ranging Bottlenose Dolphins, San Diego, California, USA, 2022.

We diagnosed fatal Erysipelothrix rhusiopathiae sepsis in 3 stranded bottlenose dolphins (Tursiops truncatus) during summer 2022, in San Diego, California, USA. The previously undetected disease in this relatively small, regional population of dolphins most likely indicates an environmental or biological change in the coastal ocean or organisms.

Vessels Disturb Bottlenose Dolphin Behavior and Movement in an Active Ship Channel.

Although the Port of Corpus Christi, Texas, has become a top oil exporter, it is unknown if local dolphins are disturbed by high year-round vessel traffic. A shore-based digital theodolite and automatic identification system receiver were used to record data to assess common bottlenose dolphin (Tursiops truncatus) behavioral states and movement patterns in the Corpus Christi Ship Channel (CCSC) in relation to vessel traffic. Multinomial logistic regression and generalized additive models were applied to analyze the data. Vessels were present within 300 m of dolphins during 80% of dolphin observations. Dolphins frequently foraged (40%), traveled (24%), socialized (15%), and milled (14%), but rarely oriented against the current (7%) or rested (1% of observations). Season, time of day, group size, vessel type, vessel size, and number of vessels were significant predictors of dolphin behavioral state. Significant predictors of dolphin movement patterns included season, time of day, group size, calf presence, vessel type, and vessel numbers. The CCSC is an important foraging area for dolphins, yet the high level of industrial activity puts the dolphins at risk of human-related disturbance and injury. There is a crucial need to monitor the impact of increased anthropogenic influences on federally protected dolphins in the active CCSC, with broad application to dolphins in other ports.

MORBIDITY AND MORTALITY PATTERNS OF INDIAN RIVER LAGOON COMMON BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS TRUNCATUS) 2002-2020.

Mortality patterns in cetaceans are critical to understanding population health. Common bottlenose dolphins (Tursiops truncatus truncatus) inhabiting the Indian River Lagoon (IRL), Florida have been subjected to four unusual mortality events (UMEs), highlighting the need to evaluate morbidity and mortality patterns. Complete gross examinations were conducted on 392 stranded dolphins and histopathological analyses were conducted for 178 animals (2002-2020). The probable causes of mortality were grouped by etiologic category: degenerative, metabolic, nutritional, inflammatory (infectious and noninfectious disease), and trauma. Probable cause of mortality was determined in 57% (223/392) of cases. Inflammatory disease (infectious/noninfectious) and trauma were the most common. Inflammatory disease accounted for 41% of cases (91/223), with the lungs (pneumonia) most commonly affected. Trauma accounted for 36% of strandings (80/223). The majority of trauma cases were due to anthropogenic activities (entanglement, fishing gear or other debris ingestion, and propeller strikes), accounting for 58% of trauma cases (46/80). Natural trauma (prey-associated esophageal obstruction or asphyxiation, shark bites, and stingray interactions) accounted for 12% of all cases (26/223), and trauma of undetermined origin was identified in 4% of cases (8/223). Starvation or inanition (nutritional) were the probable cause of mortality in 17% of cases and peaked during the 2013 UME (61% of cases). Degenerative and metabolic etiologies accounted for 5% of cases. This study represents the most comprehensive evaluation of morbidity and mortality patterns in IRL dolphins. Because IRL dolphins are routinely exposed to anthropogenic threats and have endured multiple UMEs, these baseline data are critical to the conservation and management of this population.

Determination of water balance maintenance in Orcinus orca and Tursiops truncatus using oxygen isotopes.

The secondary adaptation of Cetacea to a fully marine lifestyle raises the question of their ability to maintain their water balance in a hyperosmotic environment. Cetacea have access to four potential sources of water: surrounding salt oceanic water, dietary free water, metabolic water and inhaled water vapour to a lesser degree. Here, we measured the 18O/16O oxygen isotope ratio of blood plasma from 13 specimens belonging to two species of Cetacea raised under human care (four killer whales Orcinus orca, nine common bottlenose dolphins Tursiops truncatus) to investigate and quantify the contribution of preformed water (dietary free water, surrounding salt oceanic water) and metabolic water to Cetacea body water using a box-modelling approach. The oxygen isotope composition of Cetacea blood plasma indicates that dietary free water and metabolic water contribute to more than 90% of the total water input in weight for cetaceans, with the remaining 10% consisting of inhaled water vapour and surrounding water accidentally ingested or absorbed through the skin. Moreover, the contribution of metabolic water appears to be more important in organisms with a more lipid-rich diet. Beyond these physiological and conservation biology implications, this study opens up questions that need to be addressed, such as the applicability of the oxygen isotope composition of cetacean body fluids and skeletal elements as an environmental proxy of the oxygen isotope composition of present and past marine waters.

New insights into the pathogenesis and transmission of Brucella pinnipedialis: systemic infection in two bottlenose dolphins (Tursiops truncatus).

IMPORTANCE: Brucella spp. are zoonotic pathogens that can affect both terrestrial and marine mammals. Brucella ceti has been identified in various cetacean species, but only one sequence type (ST27) has been reported in humans. However, it is important to conduct surveillance studies to better understand the impact of marine Brucella species on marine mammals, a typically understudied host group. Here, we describe a systemic infection by two related strains of Brucella pinnipedialis (ST25) in a couple of live-stranded bottlenose dolphins, with more severe lesions in the younger animal. Furthermore, B. pinnipedialis was first detected in milk from a female cetacean that stranded with its offspring. Our study reveals novel insights into the epidemiology and pathological consequences of B. pinnipedialis infections in cetaceans, emphasizing the crucial importance of ongoing surveillance and accurate diagnosis to understand the impact of this pathogen on marine mammal populations.

Trace elements and lipidomic datasets of stranding samples in the blubber of Turpsios truncatus from the Yucatan coast: Association with biological features.

The Yucatan coastal zone is an area that contributes to many anthropogenic activities resulting in substantial contamination (metals, pesticides) in aquatic organisms. The dolphin is an excellent sentinel animal used in studying contamination in this area. Some substances found in dolphins have been identified as toxic causing alterations in the properties of membranes and produce lipid peroxidation especially heavy metals. The dataset presented here is associated with the research article paper entitled "Trace element and lipidomic analysis of bottlenose dolphin blubber from the Yucatan coast: Lipid composition relationships". In this article, we presented the trace element concentrations found in blubber and their comparison with other studies performed in mammal marine organisms. Lipidomic characterization of bottlenose dolphin blubber and their association with trace elements and the differences related to biological characteristics were presented. This data provides a correlation analysis between trace element concentrations, lipid species and body length and the lipid differences related to biological characteristics such as growth stage, stranding code, and the presence of stomach contents. We used Spearman correlation analysis to identify the association with body length, trace elements and lipids. Wilcoxon rank-sum test was used to determine differences in lipids related to stranding code (3: moderate decomposition, 4: advanced decomposition), growth stage (juveniles and adults) and whether they showed presence of stomach contents or not. The data indicates that Cr, Cd and Zn concentrations were higher compared to concentrations found in blubbler of T. truncatus from other studies (See Table 3). Cr, Co, As and Cd were found in higher concentration in larger organisms compared to smaller ones. The results of correlation between lipids and body length showed a decrease in some ceramides (CER, DCER, HCER), sterols (CE), glycerolipids (TAG, DAG) and phosphatidylethanolamines (LPE, PE) in larger dolphins (Table 4). Dolphins with advanced decomposition (code 4) showed lower concentrations of phosphatidylethanolamines (PE) compared with organisms with moderate decomposition (code 3). Organisms with empty stomachs showed higher concentrations of phosphoethanolamines suggesting a preferential metabolism of energy-rich lipids over structural lipids. The information in these datasets may contribute to understanding the potential associations of trace elements, lipids and their associations with biological characteristics.

Establishment and characterization of a novel kidney cell line derived from the common bottlenose dolphin.

Common bottlenose dolphin (Tursiops truncatus) is a well-known cetacean species that inhabits temperate and tropical seas worldwide. Limited supply and poor quality of samples hinder the investigation of the effects of various pathogens and environmental pollutants on this cetacean species. Cultured cells are useful for experimental studies; however, no cell lines derived from cetaceans are generally available. Therefore, in this study, we established a novel kidney cell line, TK-ST, derived from T. truncatus. Primary cells exhibited the morphological characteristics of epithelial and fibroblast cells, but their immortalization and passaging resulted in a predominantly epithelial cell morphology. TK-ST was immortalized using the large T SV40 antigen and human telomerase reverse transcriptase and exhibited long-term stable cell growth. TK-ST cells are generally cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37°C and 5% CO2 but can also be cultured in 5-20% fetal bovine serum and several other classical media commonly used for common animal cell culture. TK-ST cells were found to be susceptible to several viruses, including the dolphin morbillivirus (most important virus in cetaceans), and exhibited cytopathic effects, facilitating the replication of the dolphin morbillivirus. Furthermore, mRNA expression levels of cytokine genes were increased in TK-ST cells after stimulation with lipopolysaccharides and poly(I:C). Therefore, the novel TK-ST cell line derived in this study can potentially be used for further in vitro studies on cetaceans.

Entanglement in recreational fishing gear poses a threat to estuarine and coastal dolphins: Animal welfare and population level impacts should guide intervention decision making.

Derelict and active fishing gear poses a threat to marine wildlife. This study details Indo-Pacific bottlenose dolphin entanglements in recreational fishing gear in the Peel-Harvey Estuary, Western Australia between 2016 and 2022. Eight entanglements were recorded, three resulted in death. While concerning from an animal welfare perspective, the impact of entanglements on the viability of the local dolphin population was low. This is as majority of affected individuals were juvenile males. Should entanglements result in the loss of reproductive females, or impact their reproductive success, the population trajectory could quickly change. As such, management decision making should consider population level impacts as well as the welfare of entangled individuals. Government agencies, together with relevant stakeholders, should work together to maintain preparedness to respond to entanglements and take preventative action that lowers the risk of interactions with recreational fishing gear.

Inter-group alliance dynamics in Indo-Pacific bottlenose dolphins (Tursiops aduncus).

The social intelligence hypothesis holds that complex social relationships are the major selective force underlying the evolution of large brain size and intelligence. Complex social relationships are exemplified by coalitions and alliances that are mediated by affiliative behavior, resulting in differentiated but shifting relationships. Male Indo-Pacific bottlenose dolphins in Shark Bay, Australia, form three alliance levels or 'orders', primarily among non-relatives. Strategic alliance formation has been documented within both first- and second-order alliances and between second-order alliances ('third-order alliances'), revealing that the formation of strategic inter-group alliances is not limited to humans. Here we conducted a fine-scale study on 22 adult males over a 6-year period to determine if third-order alliance relationships are differentiated, and mediated by affiliative interactions. We found third-order alliance relationships were strongly differentiated, with key individuals playing a disproportionate role in maintaining alliances. Nonetheless, affiliative interactions occurred broadly between third-order allies, indicating males maintain bonds with third-order allies of varying strength. We also documented a shift in relationships and formation of a new third-order alliance. These findings further our understanding of dolphin alliance dynamics and provide evidence that strategic alliance formation is found in all three alliance levels, a phenomenon with no peer among non-human animals.

Left or right, that is the question: use of egocentric frame of reference and the right-eye advantage for understanding gestural signs in bottlenose dolphins (Tursiops truncatus).

How do bottlenose dolphins visually perceive the space around them? In particular, what cues do they use as a frame of reference for left-right perception? To address this question, we examined the dolphin's responses to various manipulations of the spatial relationship between the dolphin and the trainer by using gestural signs for actions given by the trainer, which have different meanings in the left and right hands. When the dolphins were tested with their backs to the trainer (Experiment 1) or in an inverted position underwater (Experiments 2 and 3), correct responses from the trainer's perspective were maintained for signs related to movement direction instructions. In contrast, reversed responses were frequently observed for signs that required different sounds for the left and right hands. When the movement direction instructions were presented with symmetrical graphic signs such as " × " and "●", accuracy decreased in the inverted posture (Experiment 3). Furthermore, when the signs for sounds were presented from either the left or right side of the dolphin's body, performance was better when the side of the sign movement coincided with the body side on which it was presented than when it was mismatched (Experiment 4). In the final experiment, when one eye was covered with an eyecup, the results showed that, as in the case of body-side presentation, performance was better when the open eye coincided with the side on which the sign movement was presented. These results indicate that dolphins used the egocentric frame for visuospatial cognition. In addition, they showed better performances when the gestural signs were presented to the right eye, suggesting the possibility of a left-hemispheric advantage in the dolphin's visuospatial cognition.

Variation in foraging activity influences area-restricted search behaviour by bottlenose dolphins.

Area-restricted search (ARS) behaviour is commonly used to characterize spatio-temporal variation in foraging activity of predators, but evidence of the drivers underlying this behaviour in marine systems is sparse. Advances in underwater sound recording techniques and automated processing of acoustic data now provide opportunities to investigate these questions where species use different vocalizations when encountering prey. Here, we used passive acoustics to investigate drivers of ARS behaviour in a population of dolphins and determined if residency in key foraging areas increased following encounters with prey. Analyses were based on two independent proxies of foraging: echolocation buzzes (widely used as foraging proxies) and bray calls (vocalizations linked to salmon predation attempts). Echolocation buzzes were extracted from echolocation data loggers and bray calls from broadband recordings by a convolutional neural network. We found a strong positive relationship between the duration of encounters and the frequency of both foraging proxies, supporting the theory that bottlenose dolphins engage in ARS behaviour in response to higher prey encounter rates. This study provides empirical evidence for one driver of ARS behaviour and demonstrates the potential for applying passive acoustic monitoring in combination with deep learning-based techniques to investigate the behaviour of vocal animals.