Biological characteristics of pregnancy in captive Yangtze finless porpoises revealed by urinary metabolomics†.

The Yangtze finless porpoises (Neophocaena asiaeorientalis a.) are an endemic and critically endangered species in China. Intensive captive breeding is essential for understanding the biology of critically endangered species, especially their pregnancy characteristics, knowledge of which is crucial for effective breeding management. Urine metabolomics can reveal metabolic differences, arising from physiological changes across pregnancy stages. Therefore, we used the urinary metabolomic technology, to explore urinary metabolite changes in pregnant Yangtze finless porpoises. A total of 2281 metabolites were identified in all samples, which including organic acids and derivatives (24.45%), organoheterocyclic compounds (20.23%), benzenoids (18.05%), organic oxygen compounds (7.73%), and phenylpropanoids and polyketides (6.48%). There were 164, 387, and 522 metabolites demonstrating differential abundance during early pregnancy, mid pregnancy, and late pregnancy, respectively, from the levels observed in nonpregnancy. The levels of pregnenolone, 17α-hydroxyprogesterone, and tetrahydrocortisone were significantly higher during all pregnancy stages, indicating their important roles in fetal development. The differential metabolites between nonpregnancy and pregnancy were mainly associated with amino acid and carbohydrate metabolism. Moreover, metabolic activity varied across pregnancy stages; steroid hormone biosynthesis was predominant in early pregnancy, and amino acid biosynthesis and carbohydrate metabolism were predominant in mid pregnancy and late pregnancy, respectively. Our results provide new insights into metabolic characteristics in the Yangtze finless porpoises' urine during pregnancy, and indicate that the differential levels of urine metabolites can determine pregnancy in Yangtze finless porpoises, providing valuable information for the husbandry and management of pregnant Yangtze finless porpoises in captivity.

Variations in echolocation click characteristics of finless porpoise in response to day/night and absence/presence of vessel noise.

Small odontocetes produce echolocation clicks to feed and navigate, making it an essential function for their survival. Recently, the effect of vessel noise on small odontocetes behavior has attracted attention owing to increase in vessel activities; however, the effects of the surrounding environmental factor, vessel noise, and day/night on echolocation click characteristics have not been well studied. Here, we examined the effects of vessel noise and day/night on variations in echolocation clicks and click trains parameters. Passive acoustic monitoring of on-axis echolocation clicks produced by free-ranging finless porpoises (Neophocaena asiaeorientalis sunameri) was performed at two sites in Japan, Seto Inland Sea and Mikawa Bay, in June-September 2021 and March-August 2022, using A-tag and SoundTrap 300HF. Generalized Linear Model was used to elucidate the effect of vessel noise, day/night, and surrounding environmental factors (water temperature, synthetic flow velocity, and noise level) on echolocation click and click train parameters. Echolocation click and click train parameters were strongly affected by day/night, whereas the absence/presence vessel noise did not exhibit statistically significant influence. Particularly, -3 dB bandwidth was wider, click duration was shorter, and inter-click intervals in a train were shorter at night, which may facilitate information processing at night, thereby compensating for the lack of visual information. The interaction between day/night and the absence/presence of vessel noise affected the source level of finless porpoises, with higher levels observed in the absence of vessel noise during the daytime compared to other conditions at the site with low vessel traffic. Overall, these results suggest that echolocation clicks by finless porpoise were likely to fluctuate to adapt with surrounding complex environmental conditions, especially day/night.

Cetaceans as Bioindicators to Assess Alkylphenol Exposure and Hormone-Disrupting Effects in the South China Sea.

Alkylphenols (APs) represent one of the highest exposure levels among endocrine disrupting chemicals (EDCs) in the South China Sea (SCS) due to their extensive use as plastic additives. The concerns about EDCs, including APs, have been reiterated since the surge in plastic waste from the COVID-19 response, but far less is known about the response of AP loadings in the SCS to emerging public policies and activities, including the COVID-19 pandemic. Here, we used cetaceans as bioindicators for monitoring two major APs, 4-nonylphenol (4-NP) and 4-tert-octylphenol (4-t-OP), in nine stranded cetacean species (n = 110) in the SCS between 2004 and 2021. Prior to the COVID-19, APs loads showed decreasing temporal trends for finless porpoises and humpback dolphins, most likely due to China's restrictions on AP use or a shift in dominant prey species. Unexpectedly, AP loads continued to decline after the COVID-19 outbreak, probably due to a temporal-lag response of marine AP fluxes to the pandemic. The health risk assessments based on hormone biomarkers and toxicity thresholds suggest the potential adverse effects of APs on cetaceans, while recent declines in APs, though limited, may mitigate the detrimental impacts.

Toothed whales use distinct vocal registers for echolocation and communication.

Echolocating toothed whales (odontocetes) capture fast-moving prey in dark marine environments, which critically depends on their ability to generate powerful, ultrasonic clicks. How their supposedly air-driven sound source can produce biosonar clicks at depths of >1000 meters, while also producing rich vocal repertoires to mediate complex social communication, remains unknown. We show that odontocetes possess a sound production system based on air driven through nasal passages that is functionally analogous to laryngeal and syringeal sound production. Tissue vibration in different registers produces distinct echolocation and communication signals across all major odontocete clades, and thus provides a physiological basis for classifying their vocal repertoires. The vocal fry register is used by species from porpoises to sperm whales for generating powerful, highly air-efficient echolocation clicks.

Ecological impacts of unsustainable sand mining: urgent lessons learned from a critically endangered freshwater cetacean.

Sand mining, which has tripled in the last two decades, is an emerging concern for global biodiversity. However, the paucity of sand mining data worldwide prevents understanding the extent of sand mining impacts and how it affects wildlife populations and ecosystems, which is critical for timely mitigation and conservation actions. Integrating remote sensing and field surveys over 14 years, we investigated mining impacts on the critically endangered Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) in Dongting Lake, China. We found that sand mining presented a consistent, widespread disturbance in Dongting Lake. Porpoises strongly avoided mining sites, especially those of higher mining intensity. The extensive sand mining significantly contracted the porpoise's range and restricted their habitat use in the lake. Water traffic for sand transportation further blocked the species's river-lake movements, affecting the population connectivity. In addition, mining-induced loss of near-shore habitats, a critical foraging and nursery ground for the porpoise, occurred in nearly 70% of the water channels of our study region. Our findings provide the first empirical evidence of the impacts of unregulated sand extractions on species distribution. Our spatio-temporally explicit approach and findings support regulation and conservation, yielding broader implications for sustainable sand mining worldwide.

Blubber fatty acid compositions in different geographic populations of finless porpoise in Chinese waters: implications for thermal adaptation.

The stratification of blubber fatty acids (FAs) is a widely used strategy in marine mammals and can be influenced by many factors, including their diet, environmental temperature, and physiological status. There are 3 distinct finless porpoise species/subspecies in Chinese coastal and inland waters in a wide range from 20°N to 40°N. The biochemical stratification of the blubbers of finless porpoises in different regions may provide valuable information for understanding their environmental adaptations. The FA compositions of 4 geographic populations of finless porpoise (FP) collected from the Bohai Gulf, East China Sea, South China Sea, and Yangtze River were therefore analyzed and compared. The blubber FA compositions of finless porpoises were dominated by ΣMUFAs, followed by ΣSFAs and ΣPUFAs, and were generally consistent with those of other cetaceans. The blubber of finless porpoises was significantly stratified with increasing levels of ΣMUFAs and decreasing levels of ΣSFAs and ΣPUFAs from the inner to the outer layers. The 3 marine populations shared more similarities in their FA compositions and stratifications compared to the freshwater porpoises, particularly regarding the PUFAs in the inner layers, which might have been mainly influenced by the diet compositions of marine and freshwater porpoises. Contrary to what was expected, in the 3 marine populations, the SFA and MUFA levels showed opposite correlations (negative and positive, respectively) with habitat temperatures, possibly indicating an adaptation mechanism in finless porpoises characterized by a trade-off between the insulation and fluidity properties of the blubber through the adjustment of the compositions and gradients of MUFAs and SFAs across the blubber depth in response to environmental temperature/latitude changes.

Population-level laterality in foraging finless porpoises.

Laterality has been reported in many vertebrates, and asymmetrical cerebral hemisphere function has been hypothesized to cause a left-bias in social behavior and a right-bias in feeding behavior. In this paper, we provide the first report of behavioral laterality in free-ranging finless porpoises, which seems to support the aforementioned hypothesis. We observed the turning behavior of finless porpoises in Omura Bay, Japan, using land-based and unmanned aerial system observations. We found a strong tendency in finless porpoises to turn counterclockwise with their right side down when pursuing and catching fish at the surface of the water. Our results suggest that this population of finless porpoises shows consistent right-biased laterality. Right-biased laterality has been observed in various foraging cetaceans and is usually explained by the dominance of the right eye-left cerebral hemisphere in prey recognition; however, right-biased laterality in foraging cetaceans may have multiple causes.

Contextual impacts on individual and synchronous breathing rate variations in three captive odontocete groups.

Breathing rates are often collected both in the wild and in captivity to inform on cetaceans' internal state. However, few studies have investigated the effect of various factors on this breathing rate. We investigated the variations of individual and synchronous breathing rates depending on individual features (species, sex, age), displayed behavior, social parameters (social grouping), and environmental parameters (diurnal variation, presence of enrichment, unusual events, and presence of visitors in three groups of captive odontocetes (Yangtze finless porpoises, Neophocaena asiaeorentalis asiaeorientalis, East-Asian finless porpoises, Neophocaena asiaeorentalis sunameri, bottlenose dolphins, Tursiops truncatus). Both individual and synchronous breathing rates were the highest when animals engaged in energetic or social behaviors. Individual breathing rate decreased but synchronous breathing rate increased with the presence of enrichment. Both rates increased during unusual events (e.g., pool cleaning, presence of a diver in the pool, noise, transport) and when public was present for Yangtze finless porpoises. Finally, synchronous breathing rate increased for Yangtze finless porpoises when experiencing social separation. We suggest that individual and synchronous breathing rates are useful parameters to measure, both in wild and captive animals, to obtain information on their arousal/stress state. However, these rates should be interpreted with caution and should be used together with other parameters to allow more accurate inferences.

Mitochondrial genomics reveals the evolutionary history of the porpoises (Phocoenidae) across the speciation continuum.

Historical variation in food resources is expected to be a major driver of cetacean evolution, especially for the smallest species like porpoises. Despite major conservation issues among porpoise species (e.g., vaquita and finless), their evolutionary history remains understudied. Here, we reconstructed their evolutionary history across the speciation continuum. Phylogenetic analyses of 63 mitochondrial genomes suggest that porpoises radiated during the deep environmental changes of the Pliocene. However, all intra-specific subdivisions were shaped during the Quaternary glaciations. We observed analogous evolutionary patterns in both hemispheres associated with convergent evolution to coastal versus oceanic environments. This suggests that similar mechanisms are driving species diversification in northern (harbor and Dall's) and southern species (spectacled and Burmeister's). In contrast to previous studies, spectacled and Burmeister's porpoises shared a more recent common ancestor than with the vaquita that diverged from southern species during the Pliocene. The low genetic diversity observed in the vaquita carried signatures of a very low population size since the last 5,000 years. Cryptic lineages within Dall's, spectacled and Pacific harbor porpoises suggest a richer evolutionary history than previously suspected. These results provide a new perspective on the mechanisms driving diversification in porpoises and an evolutionary framework for their conservation.

Evoked-potential audiogram variability in a group of wild Yangtze finless porpoises (Neophocaena asiaeorientalis asiaeorientalis).

Hearing is considered the primary sensory modality of cetaceans and enables their vital life functions. Information on the hearing sensitivity variability within a species obtained in a biologically relevant wild context is fundamental to evaluating potential noise impact and population-relevant management. Here, non-invasive auditory evoked-potential methods were adopted to describe the audiograms (11.2-152 kHz) of a group of four wild Yangtze finless porpoises (Neophocaena asiaeorientalis asiaeorientalis) during a capture-and-release health assessment project in Poyang Lake, China. All audiograms presented a U shape, generally similar to those of other delphinids and phocoenids. The lowest auditory threshold (51-55 dB re 1 µPa) was identified at a test frequency of 76 kHz, which was higher than that observed in aquarium porpoises (54 kHz). The good hearing range (within 20 dB of the best hearing sensitivity) was from approximately 20 to 145 kHz, and the low- and high-frequency hearing cut-offs (threshold > 120 dB re l µPa) were 5.6 and 170 kHz, respectively. Compared with aquarium porpoises, wild porpoises have significantly better hearing sensitivity at 32 and 76 kHz and worse sensitivity at 54, 108 and 140 kHz. The audiograms of this group can provide a basis for better understanding the potential impact of anthropogenic noise.

Investigating the distribution of the Yangtze finless porpoise in the Yangtze River using environmental DNA.

Determining the distribution of the Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis, YFP) in the Yangtze River has to date relied on traditional visual and counting methods, but such field surveys are time-consuming and expensive. Analyses using environmental DNA (eDNA) to investigate the presence and range of endangered aquatic species have proven to be more economical and effective detection methods, and are a non-invasive approach to sampling. A challenge of relying on eDNA for YFP monitoring is that the Yangtze River is characterized by high turbidity and a strong current. Here, we used an eDNA-based approach to estimate the presence of YFP at 18 sites in the Yangtze River in August 2017 and at an additional 11 sites in January 2018. At each sampling site, we filtered six 1 L water samples with 5 µm pore size filter paper and quantified the amount of YFP eDNA in each water sample using quantitative real-time polymerase chain reaction (qPCR). In addition, YFP eDNA was successfully detected in locations where we visually observed YFP, as well as in locations where YFP were not observed directly. We found that our eDNA-based method had higher detection rates than traditional field survey methods. Although YFP was visually observed in the Yangtze River in winter, water samples collected during the summer contained significantly higher YFP eDNA than winter water samples. Our results demonstrate the potential effectiveness of eDNA detection methods in determining the distribution of YFP in the Yangtze River.

Porpoise click classifier (PorCC): A high-accuracy classifier to study harbour porpoises (Phocoena phocoena) in the wild.

Harbour porpoises are well-suited for passive acoustic monitoring (PAM) as they produce highly stereotyped narrow-band high-frequency (NBHF) echolocation clicks. PAM systems must be coupled with a classification algorithm to identify the signals of interest. Here, the authors present a harbour porpoise click classifier (PorCC) developed in matlab, which uses the coefficients of two logistic regression models in a decision-making pathway to assign candidate signals to one of three categories: high-quality clicks (HQ), low-quality clicks (LQ), or high-frequency noise. The receiver operating characteristics of PorCC was compared to that of PAMGuard's Porpoise Click Detector/Classifier Module. PorCC outperformed PAMGuard's classifier achieving higher hit rates (correctly classified clicks) and lower false alarm levels (noise classified as HQ or LQ clicks). Additionally, the detectability index (d') for HQ clicks for PAMGuard was 2.2 (overall d' = 2.0) versus 4.1 for PorCC (overall d' = 3.4). PorCC classification algorithm is a rapid and highly accurate method to classify NBHF clicks, which could be applied for real time monitoring, as well as to study harbour porpoises, and potentially other NBHF species, throughout their distribution range from data collected using towed hydrophones or static recorders. Moreover, PorCC is suitable for studies of acoustic communication of porpoises.

Trace elements accumulation in the Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) - A threat to the endangered freshwater cetacean.

As a freshwater cetacean with a population of only approximately 1000 individuals, the Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) is threatened by water pollution. However, studies of contaminants accumulated in the Yangtze finless porpoise remain limited. In this study, concentrations of 11 trace elements in different tissues sampled from 38 Yangtze finless porpoise individuals were determined. The elements V, Ni, Zn, and Pb were mostly accumulated in the epidermis, Cr, Mn, Cu, Se, and Hg were mostly accumulated in the liver, while As and Cd were mostly accumulated in the blubber and kidney, respectively. The results show that trace elements concentrations in the epidermis do not reliably indicate concentrations in internal tissues of the Yangtze finless porpoises. Positive correlations between different trace elements concentrations in tissues with the highest concentrations suggested the similar mechanism of metabolism or uptake pathway of those elements. Concentrations of As, Se, Cd, Hg, and Pb in the tissues with the highest concentrations were significantly positively correlated with the body length. Furthermore, significantly higher trace elements concentrations were measured in the reproductive organs of females (ovaries) than males (testis). However, no significant difference of trace elements concentrations between habitats was found. In consideration of higher Hg and Cd level in Yangtze finless porpoises compared to other small cetaceans, the potential risk of Hg (in particular) and Cd toxicity to Yangtze finless porpoises needs further attention.

Social responses of travelling finless porpoises to boat traffic risk in Misumi West Port, Ariake Sound, Japan.

Anthropogenic effects have created various risks for wild animals. Boat traffic is one of the most fatal risks for marine mammals. Individual behavioral responses of cetaceans, including diving behavior such as changing swimming direction and lengthing inter-breath interval, to passing boats is relatively well known; however, the social function of cetacean responses to boat traffic in a natural setting remains poorly understood. We focused on describing the behavioral responses of single and aggregated finless porpoises to boats passing at Misumi West Port, Ariake Sound, Japan, by using a drone characterized with a high-precision bird's-eye angle. During the study period, we collected 25 episodes of finless porpoise responses to boats passing by. A mean (± SEM) of 5.1 ± 1.0 individuals were observed for each episode. The primary response to passing boats was avoidance by dive, which implies boat traffic is a substantial disturbance to finless porpoises that travel along the seawater surface daily. The diving duration decreased significantly with an increase in the number of aggregated individuals. The diving and floating reaction times were 10.9 ± 2.3 s and 18.7 ± 5.0 s, respectively. There was no significant difference between the reaction times indicating that each individual was motivated to keep the group cohesion consistent when floating even after the risk had dissolved, which is comparable to the behavior of porpoises that dive when riskier conditions are present, such as when a boat approaches an aggregation. Our findings provide new insights on the sociality of finless porpoises even though there were limitations, like an inability to identify a specific individual. The drone enabled us to observe the social behavior of finless porpoises and other cetaceans at an unprecedented resolution, which may lead to a better understanding of the evolutionary diversity of intelligence and sociality and the bridge to human evolution.

Investigation on acoustic reception pathways in finless porpoise (Neophocaena asiaorientalis sunameri) with insight into an alternative pathway.

Sound transmission and reception are both vital components to odontocete echolocation and daily life. Here, we combine computed tomography (CT) scanning and finite element modeling to investigate the acoustic propagation of finless porpoise (Neophocaena asiaorientalis sunameri) echolocation pulses. The CT scanning and finite element method wave propagation model results support the well-accepted jaw-hearing pathway hypothesis and suggest an additional alternative auditory pathway composed of structures, mandible (lower jaw) and internal mandibular fat, with different acoustic impedances, which may also conduct sounds to the ear complexes. The internal mandibular fat is attached to the ear complex and encased by the mandibles laterally and anteriorly. The simulations show signals in this pathway initially propagate along the solid mandibles and are transmitted to the acoustically coupled soft tissue of the internal mandibular fat which conducts the stimuli posteriorly as it eventually arrives at ear complexes. While supporting traditional theories, this new bone-tissue conduction pathway might be meaningful to understand the hearing and sound reception processes in a wide variety of odontocetes species.

Click emission in Dall's porpoise Phocoenoides dalli, focusing on physical properties of tissues.

Dall's porpoise (Phocoenoides dalli) is one of most common North Pacific porpoise species, for which information on sound-emitting processes is limited. To evaluate the mechanism of click emission in the head of this porpoise, the distribution of acoustic impedance in head tissues was calculated using density and Young's modulus'which is a measure of linear resistance to linear compression. Two Dall's porpoise heads were examined: one for macroscopic dissection, and one for investigating the distribution of acoustic impedance calculated from CT-measured density, and Young's modulus measured by creep meter. Acoustic impedance increased from the dorsal bursae to the melon's emitting surface, with impedance matching observed at the boundary between the emitting surface and seawater, and was more similar in distribution to Young's modulus than it was to density. The distribution of acoustic impedance differed from that of harbor porpoise (Phocoena phocoena), despite similarities in the sound-producing organs in the heads of Dall's and harbor porpoises. A comparison of the physical properties of Dall's and harbor porpoise head tissues suggests that hypertrophic vestibular sacs and an oval emitting surface are common characteristics in Phocoenidae.

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.

Seasonal and diel patterns in cetacean use and foraging at a potential marine renewable energy site.

Marine renewable energy (MRE) developments often coincide with sites frequented by small cetaceans. To understand habitat use and assess potential impact from development, echolocation clicks were recorded with acoustic click loggers (C-PODs) in Swansea Bay, Wales (UK). General Additive Models (GAMs) were applied to assess the effects of covariates including month, hour, tidal range and temperature. Analysis of inter-click intervals allowed the identification of potential foraging events as well as patterns of presence and absence. Data revealed year-round presence of porpoise, with distinct seasonal and diel patterns. Occasional acoustic encounters of dolphins were also recorded. This study provides further evidence of the need for assessing temporal trends in cetacean presence and habitat use in areas considered for development. These findings could assist MRE companies to monitor and mitigate against disturbance from construction, operation and decommissioning activities by avoiding times when porpoise presence and foraging activity is highest in the area.

Genital interactions during simulated copulation among marine mammals.

Genitalia are morphologically variable across many taxa and in physical contact during intromission, but little is known about how variation in form correlates with function during copulation. Marine mammals offer important insights into the evolutionary forces that act on genital morphology because they have diverse genitalia and are adapted to aquatic living and mating. Cetaceans have a fibroelastic penis and muscular vaginal folds, while pinnipeds have a baculum and lack vaginal folds. We examined copulatory fit in naturally deceased marine mammals to identify anatomical landmarks in contact during copulation and the potential depth of penile penetration into the vagina. Excised penises were artificially inflated to erection with pressurized saline and compared with silicone vaginal endocasts and within excised vaginas in simulated copulation using high-resolution, diffusible iodine-based, contrast-enhanced computed tomography. We found evidence suggestive of both congruent and antagonistic genital coevolution, depending on the species. We suggest that sexual selection influences morphological shape. This study improves our understanding of how mechanical interactions during copulation influence the shape of genitalia and affect fertility, and has broad applications to other taxa and species conservation.

Behavioral laterality in Yangtze finless porpoises (Neophocaena asiaeorientalis asiaeorientalis).

The Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) is a critically endangered species with less than 1000 individuals expected to be left in the wild. While many studies have been conducted on laterality among several cetacean species, no studies investigating the Yangtze finless porpoise have been conducted. Using event sampling methods, several behaviors such as flipper-body touching, object touching, barrel-rolls, side swimming, and swimming direction were recorded from six captive porpoises (three males and three females). Analyses of 360 observations recorded over two months revealed that, at group level, porpoises showed laterality in swimming behaviors. Porpoises swam preferentially with their right pectoral fin upward and their left pectoral fin downward with a clockwise swimming direction and also displayed a consistent bias for a counterclockwise barrel-roll direction. No significant differences were reported for flipper use either during the interaction with conspecifics or with objects. The results from the current study provide novel insight into the cerebral asymmetry in a species previously ignored within the literature, thus improving our understanding on the extent of laterality in cetaceans and on the evolutionary history of hemispheric laterality for vertebrates in general.