Histological study of seventeen organs from dugong (Dugong dugon).

Background: Dugongs are marine mammals with a crescent-shaped tail fluke and a concave trailing margin that belong to the family Dugongidae., They are distributed widely in the warm coastal waters of the Indo-Pacific region. Importantly, the population of dugongs has decreased over the past decades as they have been classified as rare marine mammals. Previous studies have investigated the habitat and genetic diversity of dugongs. However, a comprehensive histological investigation of their tissue has not yet been conducted. This study provides unique insight into the organs of dugongs and compares them with other mammal species. Methods: Tissue sections were stained with Harris's hematoxylin and eosin Y. The histological structure of 17 organ tissues obtained from eight systems was included in this study. Tissue sections were obtained from the urinary system (kidney), muscular system (striated skeletal muscle and smooth muscle), cardiovascular system (cardiac muscle (ventricle), coronary artery, and coronary vein), respiratory system (trachea and lung), gastrointestinal system (esophagus, stomach, small intestine, liver, and pancreas), reproductive system (testis), lymphatic system (spleen and thymus), and endocrine system (pancreas). Results: While most structures were similar to those of other mammal species, there were some differences in the tissue sections of dugongs when compared with other mammalian species and manatees. These include the kidneys of dugongs, which were non-lobular and had a smooth, elongated exterior resulting in a long medullary crest, whereas the dugong pyloric epithelium did not have overlying stratified squamous cells and was noticably different from the Florida manatee. Discussion: Histological information obtained from various organs of the dugong can serve as an essential foundation of basal data for future microanatomical studies. This information can also be used as high-value data in the diagnosis and pathogenesis of sick dugongs or those with an unknown cause of death.

The first study of genetic diversity and population structure of Indo-Pacific bottlenose dolphin (Tursiops aduncus) and pantropical spotted dolphin (Stenella attenuata) in the Thai Andaman Sea based on ISSR.

Background and Aim: The Indo-Pacific bottlenose dolphin, Tursiops aduncus, and the pantropical spotted dolphin, Stenella attenuata, are protected marine mammals in Thailand; however, knowledge regarding the populations of both species in Thai seas is minimal. We aimed to reveal the genetic diversity and population structure of two species, T. aduncus, and S. attenuata, based on inter-simple sequence repeats (ISSRs). Materials and Methods: Samples of stranded T. aduncus (n = 30) and S. attenuata (n = 23) found along Thai Andaman Sea coasts from 1998 to 2018 were used in this study. A total of 17 and 16 ISSR primers that produced clear and polymorphic bands were selected for T. aduncus and S. attenuata, respectively. Results: The highest percentages of polymorphic bands for T. aduncus and S. attenuata were 93.750% and 92.857%, respectively. Phylogenetic dendrograms indicated that the population of each species was clustered into three groups. This outcome was consistent with the genetic population structure, as both suggested three genetic clusters (DK = 3). Genetic diversity analysis revealed that the average Shannon's information index (I) was 1.926 ± 0.066 for T. aduncus and 1.714 ± 0.090 for S. attenuata, which indicate a high level of genetic variation. Further, low fixation index (F) values were observed for T. aduncus and S. attenuata at -0.231 ± 0.024 and -0.312 ± 0.042, respectively, suggesting that inbreeding is unlikely to have occurred for both species over the past decades. Conclusion: At least three genetic clusters of both species were found in the Thai Andaman Sea, and the diversity indices of each species indicated that these species are not at a critical level for extinction. However, monitoring their population status should be prioritized to observe any future changes in the level of diversity.

High genetic differentiation of Indo-Pacific humpback dolphins (Sousa chinensis) along the Asian Coast of the Pacific Ocean.

The Indo-Pacific humpback dolphin (Sousa chinensis) is a vulnerable marine mammal species that inhabits shallow, coastal waters from Southeast China, southward throughout Southeast Asia, and westward around the Bay of Bengal to eastern India. Polymorphic microsatellites are useful for elucidating ecological and population genetics-related questions. Here, 18 new polymorphic microsatellites were developed from S. chinensis genomic DNA by Illumina MiSeq sequencing. Population genetic analyses were conducted on 42 S. chinensis individuals from three geographic locations, including the Xiamen Bay of China, the Western Gulf of Thailand, and Andaman Sea. Our microsatellite data revealed a strong and significant population structure among the three sampling regions (overall F ST = 0.371, p = .001). Pairwise mutual information index also demonstrated high levels of genetic differentiation between different region pairs (values range from 0.272 to 0.339, p < .001). Moreover, Structure analysis inferred three genetic clusters, with the high assignment probabilities of 95.92%, 99.47%, and 99.68%, respectively. Principal coordinate analysis plots of individuals divided entire genotypes into three clusters, indicating high level of genetic differentiation. Our results indicated the strong genetic structure in S. chinensis populations is a result of geographic distances. Other factors such as environmental variables, anthropogenic interference, and social behavior may also have contributed to population differentiation.

Relationship of stranded cetaceans in Thai territorial waters to global populations: Mitochondrial DNA diversity of Cuvier's beaked whale, Indo Pacific finless porpoise, pygmy sperm whale, and dwarf sperm whale.

Cetaceans inhabit oceans throughout the world. Four specific odontocetes, namely Cuvier's beaked whale (Ziphius cavirostris), Indo Pacific finless porpoise (Neophocaena phocaenoides), pygmy sperm whale (Kogia breviceps), and dwarf sperm whale (Kogia sima), have occasionally been found stranded along Thailand's coastal waters (the Andaman Sea and the Gulf of Thailand). Although shared haplotypes of each species for many locations have been found, and some species have revealed genetic structure through haplotype networks, cetaceans in Thai waters have never been investigated in terms of comparing haplotypes to those that have existed before. Herein, we have illustrated the matrilineally phylogeographic relationships among worldwide populations through Bayesian Phylogenetic tree computations using Markov Chain Monte Carlo (MCMC) and Median-Joining Networks (MJNs). Unique haplotypes of the control region mitochondrial DNA of Thai odontocetes were found for all species. Moreover, a high degree of worldwide haplotype diversity (hd) above 0.8 among the four species was detected, while the lowest degree of nucleotide diversity (π) was observed in the Indo Pacific finless porpoise (1.12% ± 0.184%). An expansion of the effective female population size worldwide of three odontocete species was detected using Bayesian Skyline Reconstruction, but this did not include the Indo Pacific finless porpoise. Because Thai seas are located within the Indo Polynesian province, where this biodiversity hotspot exists, we speculate that these odontocetes may also inhabit specific habitats within the Malay Peninsula and Thailand's territorial waters. Therefore, closer attention and monitoring of these cetacean populations will be necessary for future conservation efforts.

Microsatellite Polymorphism and the Population Structure of Dugongs (Dugong dugon) in Thailand.

The dugong (Dugong dugon) is an endangered species of marine mammals, so knowledge of genetic diversity of these populations is important for conservation planning within different habitats. In this study, six microsatellite markers were used to assess the genetic diversity and population structure of 77 dugongs from skin samples of stranded animals collected from 1994-2019 (69 from Andaman Sea and 8 from the Gulf of Thailand). Our results found that dugongs in the Andaman Sea had higher genetic variation than those in the Gulf of Thailand. Populations in Trang, Satun, and some areas of Krabi had highest diversity compared to other regions of Thailand. Bayesian genetic clustering analysis revealed that dugongs in Thailand consist of five genetic groups. Moreover, dugongs in the middle and lower Andaman Sea presented the greatest gene flow compared to other regions. However, based on calculation of inbreeding coefficients (Fis value = 0.239), dugong populations in the Sea of Thailand are experiencing some levels of inbreeding, and so may warrant special protections. These results provide important information for understanding the genetic status of dugongs that can lead to improved management and conservation of this endangered species.

Life Expectancy in Marine Mammals Is Unrelated to Telomere Length but Is Associated With Body Size.

Marine mammals vary greatly in size and lifespan across species. This study determined whether measures of adult body weight, length and relative telomere length were related to lifespan. Skin tissue samples (n = 338) were obtained from 23 marine mammal species, including four Mysticeti, 19 Odontoceti and one dugong species, and the DNA extracted to measure relative telomere length using real-time PCR. Life span, adult body weight, and adult body length of each species were retrieved from existing databases. The phylogenetic signal analysis revealed that body length might be a significant factor for shaping evolutionary processes of cetacean species through time, especially for genus Balaenoptera that have an enormous size. Further, our study found correlations between lifespan and adult body weight (R 2 = 0.6465, p < 0.001) and adult body length (R 2 = 0.6142, p ≤0.001), but no correlations with relative telomere length (R 2 = -0.0476, p = 0.9826). While data support our hypothesis that larger marine mammals live longer, relative telomere length is not a good predictor of species longevity.

Deep neural networks based automated extraction of dugong feeding trails from UAV images in the intertidal seagrass beds.

Dugongs (Dugong dugon) are seagrass specialists distributed in shallow coastal waters in tropical and subtropical seas. The area and distribution of the dugongs' feeding trails, which are unvegetated winding tracks left after feeding, have been used as an indicator of their feeding ground utilization. However, current ground-based measurements of these trails require a large amount of time and effort. Here, we developed effective methods to observe the dugongs' feeding trails using unmanned aerial vehicle (UAV) images (1) by extracting the dugong feeding trails using deep neural networks. Furthermore, we demonstrated two applications as follows; (2) extraction of the daily new feeding trails with deep neural networks and (3) estimation the direction of the feeding trails. We obtained aerial photographs from the intertidal seagrass bed at Talibong Island, Trang Province, Thailand. The F1 scores, which are a measure of binary classification model's accuracy taking false positives and false negatives into account, for the method (1) were 89.5% and 87.7% for the images with ground sampling resolutions of 1 cm/pixel and 0.5 cm/pixel, respectively, while the F1 score for the method (2) was 61.9%. The F1 score for the method (1) was high enough to perform scientific studies on the dugong. However, the method (2) should be improved, and there remains a need for manual correction. The mean area of the extracted daily new feeding trails from September 12-27, 2019, was 187.8 m2 per day (n = 9). Total 63.9% of the feeding trails was estimated to have direction within a range of 112.5° and 157.5°. These proposed new methods will reduce the time and efforts required for future feeding trail observations and contribute to future assessments of the dugongs' seagrass habitat use.

Morphometric analysis of cervical vertebrae in some marine and land mammals.

Bones or skeletal remains can be used to answer a number of questions related to species, sex, age or cause of death. However, studies involving vertebrae have been limited as most were performed on skulls or long bones. Here, we have stated the hypothesis that the morphometry of cervical vertebrae can be used for species identification and body size estimation among marine and land mammals. The cervical vertebrae from eight and 14 species of marine and land mammals were used to collect morphometric data. Cluster dendrogram, principal component analysis, discriminant analysis and linear regression were used to analyse the data. The results indicate that, based on an index of C4 to C7, there were 13 out of 22 species for which identity could be correctly predicted in 100% of the cases. The correlations between cervical vertebrae parameters (height, width and length of centrum) in marine (average R2 =  0.87, p < .01) and land (average R2 =  0.51, p < .01) mammals were observed. These results indicate that vertebral morphometrics could be used for species prediction and verification of body weight in both marine and land mammals.

Feasibility of melting fingerprint obtained from ISSR-HRM curves for marine mammal species identification.

Currently, species identification of stranded marine mammals mostly relies on morphological features, which has inherent challenges. The use of genetic information for marine mammal species identification remains limited, therefore, new approaches that can contribute to a better monitoring of stranded species are needed. In that context, the ISSR-HRM method we have proposed offers a new approach for marine mammal species identification. Consequently, new approaches need to be developed to identify individuals at the species level. Eight primers of the ISSR markers were chosen for HRM analysis resulting in ranges of accuracy of 56.78-75.50% and 52.14-75.93% in terms of precision, while a degree of sensitivity of more than 80% was recorded when each single primer was used. The ISSR-HRM primer combinations revealed a success rate of 100% in terms of discrimination for all marine mammals included in this study. Furthermore, ISSR-HRM analysis was successfully employed in determining marine mammal discrimination among varying marine mammal species. Thus, ISSR-HRM analysis could serve as an effective alternative tool in the species identification process. This option would offer researchers a heightened level of convenience in terms of its performance and success rate. It would also offer field practice to veterinarians, biologists and other field-related people a greater degree of ease with which they could interpret results when effectively classifying stranded marine mammals. However, further studies with more samples and with a broader geographical scope will be required involving distinct populations to account for the high degree of intraspecific variability in cetaceans and to demonstrate the range of applications of this approach.

Genetic diversity in a unique population of dugong (Dugong dugon) along the sea coasts of Thailand.

Dugong (Dugong dugon) populations have been shrinking globally, due in large part to habitat fragmentation, degradation and ocean pollution, and today are listed as Vulnerable by the IUCN. Thus, determining genetic diversity in the remaining populations is essential for conservation planning and protection. In this study, measures of inter-simple sequence repeat (ISSR) markers and mtDNA D-loop typing were used to evaluate the genetic diversity of 118 dugongs from skin samples of deceased dugongs collected in Thai waters over a 29-year period. Thirteen ISSR primers revealed that dugongs from the Andaman Sea and Gulf of Thailand exhibited more genetic variation in the first 12 years of the study (1990-2002) compared to the last decade (2009-2019). Dugongs from the Andaman Sea, Trang, Satun and some areas of Krabi province exhibited greater diversity compared to other coastal regions of Thailand. Eleven haplotypes were identified, and when compared to other parts of the world (235 sequences obtained from NCBI), five clades were apparent from a total 353 sequences. Moreover, dugongs from the Andaman Sea were genetically distinct, with a separate haplotype belonging to two clades found only in Thai waters that separated from other groups around 1.2 million years ago. Genetic diversity of dugongs in present times was less than that of past decades, likely due to increased population fragmentation. Because dugongs are difficult to keep and breed in captivity, improved in situ conservation actions are needed to sustain genetically healthy wild populations, and in particular, the specific genetic group found only in the Andaman Sea.

Genetic structure of the endangered Irrawaddy dolphin (Orcaella brevirostris) in the Gulf of Thailand.

The Irrawaddy dolphin (Orcaella brevirostris) is an endangered, small cetacean species which is widely distributed in rivers, estuaries, and coastal waters throughout the tropical and subtropical Indo-Pacific. Despite the extensive distribution of this species, little is known of individual movements or genetic exchange among regions in Thailand. Here, we evaluate the genetic diversity and genetic structure of O. brevirostris in the eastern, northern and western Gulf of Thailand, and Andaman Sea. Although phylogenetic relationships and network analysis based on 15 haplotypes obtained from 32 individuals reveal no obvious divergence, significant genetic differentiation in mitochondrial DNA (overall FST = 0.226, P < 0.001; ΦST = 0.252, P < 0.001) is apparent among regions. Of 18 tested microsatellite loci, 10 are polymorphic and successfully characterized in 28 individuals, revealing significant genetic differentiation (overall FST = 0.077, P < 0.05) among the four sampling sites. Structure analysis reveals two inferred genetic clusters. Additionally, Mantel analysis demonstrates individual-by-individual genetic distances and geographic distances follow an isolation-by-distance model. We speculate that the significant genetic structure of O. brevirostris in Thailand is associated with a combination of geographical distribution patterns, environmental and anthropogenic factors, and local adaptations.

Early divergence and differential population histories of the Indo-Pacific humpback dolphin in the Pacific and Indian Oceans.

The currently recognized Indo-Pacific humpback dolphin occurs in estuaries and surrounding shallow waters from the South China Sea to the Asian coast of the Indian Ocean. However, a recent study suggested that the humpback dolphin from the Bay of Bengal may represent a distinct phylogenetic species. In this study, we sequenced 915-bp mtDNA segments from five geographic populations in both Chinese and Thai waters; together with previously published sequences, these data revealed that the ancestral Indo-Pacific humpback dolphin might have split during the transition from the Oligocene to Miocene (23.45 Mya, 95% HPD: 16.65-26.55 Mya), and then dispersed along the Pacific and Indian Ocean coasts of Asia. Genetic differentiation was detected between most of the examined populations, except for only a few pairwise populations in the northern South China Sea. Genetic differentiation/distance between the humpback dolphins from the northern and southern South China Sea met the sub-species threshold value proposed for marine mammals, whereas that between the humpback dolphins in the Pacific and the Indian Ocean was above the species threshold. Bayesian inference of historic gene flow indicated low but constant northward gene flow along the Indian Ocean coast; however, there was a recent abrupt increase in gene flow in the Pacific region, likely due to the shortening coastline at the low stand of sea level. Our results revealed that the current taxonomic classification of Indo-Pacific humpback dolphins may not reflect their phylogeography.

Age relationships with telomere length, body weight and body length in wild dugong (Dugong dugon).

The ability to estimate age and determine the growth status of free-ranging dugongs (Dugong dugon) is vital to providing insight into the basic biology of this endangered species. Currently, age estimation in dugong carcasses relies on counting dentin growth layer groups (GLGs) in tusks, but a disadvantage is they need to be intact. We explored whether measures of telomere length could be used as an alternative approach to age estimation in dugongs given that in other species, telomere length and age are inversely related. In this study, relative telomere length (rTL) was measured by qPCR in skin samples from 24 dugongs of varying ages determined by counts of GLGs. In addition, relationships between age by GLG counts and body weight and length and were examined. Our findings indicate that age estimated by GLGs was negatively correlated with telomere length using the logistic formula with a rate of telomere attrition of approximately 0.036 rTL/year between the ages of 5-20 years. By comparison, both body weight and length were positively correlated with GLG-based age, with growth rates of ~8.8 kg/year for weight and ~3.58 cm/year for length, respectively. After that, growth rates slowed substantially and then plateaued. The results suggest that physical maturity in dugongs occurs at 20 years of age and that measures of rTL might serve as a tool for age estimation in dugongs, living and deceased.

Pelvic bone morphometric analysis in the dugong (Dugong dugon).

The dugong (Dugong dugon Müller) is recognized as an endangered marine mammal. There is limited available anatomical data on the dugong's skeletal system, while what is available has not been well established due to the limited number of archived samples and limited access to them. Importantly, there are certain key questions that should be answered when examining the bones and/or remains of animals such as; what kind of bone is it?; what species does it belong to?; what sex was the animal?; how old was the animal? or how big was it?, etc. In this study, we have focused on the pelvic bone of the dugong by asserting the hypothesis that pelvic bone morphology is related to age, sex, and body size. Here, we have established certain morphometric data encompassing 8 parameters and 5 indexes to analyze the morphology of the pelvic bones obtained from 88 specimens (45 dugongs). We will present three main findings: (1) the pelvic bone in mature male subjects is larger than it is in female subjects, (2) a high rate of accuracy can be established for sex identification using morphometric data obtained from the pelvic bone, and (3) the pelvic bone has the highest degree of correlation with body length, followed by body weight and age. Notably, the important data on the pelvic bone of the dugong acquired in this study can be reliable and extremely useful in sex identification and body size estimation.

Optimal-rearing density for head-starting green turtles (Chelonia mydas Linnaeus, 1758).

While ex situ conservation programs of juvenile green turtles (Chelonia mydas Linnaeus, 1758), before release to natural habitats, have been conducted in several countries, the optimal-stocking density for husbandry has not yet been reported. The optimization of stocking density was the main purpose of this study. The 15-day-old post-hatching turtles (29.30 ± 0.05 g body weight) were reared in round fiberglass tanks at various stocking densities including 20 turtles/m3 (20TM), 40 turtles/m3 (40TM), 60 turtles/m3 (60TM), and 80 turtles/m3 (80TM), over an 8-week trial. Water quality, survival, growth performance, feed utilization, aggressive behavior, fecal digestive enzymes, and hematological parameters were compared between the treatments, and were used as indicators of a successful captive rearing program. The water quality across the four treatments was in the standard range, but a high-stocking density reduced the quality significantly. No mortality was observed in any treatment group. Superior growth and feed utilization were only observed with the 40TM treatment, relative to the others (P < 0.05). The turtles in this group had no aggressive behavior, as indicated by observing hind limb biting. This treatment manipulated the level of proteolytic activity of pepsin and trypsin in response to density stressor, but not amylase, lipase, and chymotrypsin. The 40TM treatment also maintained the hematological characteristics, indicating no negative effects on health status. Overall, the findings indicate that the captivity program of post-hatching turtles at 40 turtles/m3 is the preferred option in their head-started propagation, as well as in public displays in zoos or aquaria. Zoo Biol. 35:454-461, 2016. © Wiley Periodicals, Inc.