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.

Study of trace elements in stranded green turtles (Chelonia mydas), hawksbill turtles (Eretmochelys imbricata), and olive ridley turtles (Lepidochelys olivacea) in Gulf of Thailand and Andaman Sea.

The purpose of this study was to survey and compare the amounts of elements in the serum of stranded sea turtles from the Gulf of Thailand and the Andaman Sea. The sea turtles from the Gulf of Thailand had Ca, Mg, P, S, Se, and Si concentrations significantly higher than those in sea turtles from the Andaman Sea. The Ni and Pb concentrations of sea turtles from the Gulf of Thailand was higher, but not significantly so, than in sea turtles from the Andaman Sea. Rb was detected only in sea turtles from the Gulf of Thailand. This may have been related to the industrial activities in Eastern Thailand. The concentration of Br in the sea turtles from the Andaman Sea were significantly higher than those in sea turtles from the Gulf of Thailand. The higher serum concentration of Cu in hawksbill (H) and olive ridley turtles (O) than in green turtles may be due to hemocyanin, as an important component in the blood of crustaceans. The higher Fe concentration in the serum from green turtles than for H and O may be due to chlorophyll, which is an important component of chloroplasts in eel grass. Co was not found in the serum of green turtles but was found in the serum of H and O. The monitoring of important elements in sea turtles may be used as a tool to assess the levels of pollution in marine ecosystems.

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.

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.

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.

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.

What the skull and scapular morphology of the dugong (Dugong dugon) can tell us: sex, habitat and body length?

The dugong (Dugong dugon, Müller) is an endangered marine mammal species. We examined the relationship between sex, habitat and body length based on the skull and scapular morphology and morphometrics of 81 dugong samples in Thailand. A total of 58 parameters from the skull and scapula (25 from the cranium, 23 from the mandible and 10 from the scapula) as well as tusks were used in this study. Data were analyzed by univariate analysis, followed by discriminant analysis and multivariate linear regression. Here we show, 100% and 98.5% accuracy rates for sexing using large tusks and the skull, respectively. Scapular morphology using the caudal border tubercle and coracoid process showed 91.30% and 96.15% accuracy rates for identifying males and females. Skull morphometrics could categorize dugong habitat, i.e. living in the Andaman Sea or Gulf of Thailand, with 100% accuracy. Moreover, our model could be used to estimate body length with coefficient of determination (R 2) of 0.985. The results of our study showed that skull morphology and morphometric measurements could be used as a tool for sex identification, location identification and estimation of body length. But scapular morphology is the best tool for sex identification in dugongs.

Osteoarthritis in two marine mammals and 22 land mammals: learning from skeletal remains.

The occurrence of osteoarthritis (OA) in marine mammals is still questionable. Here we investigated the prevalence of OA in marine (dolphin and dugong) and terrestrial mammals (Asian elephant, Asiatic buffalo, camel, cat, cattle, deer, dog, domestic goat, horse, human, hyena, impala, lion, Malayan tapir, Assam macaque, mule, pig, rabbit, red kangaroo, sheep, tiger and waterbuck). Skeletal remains obtained from five institutes were used as subjects; a total of 45 different parts (locations) of bones were observed for OA lesions. The prevalence of OA was reported as number of OA lesions/total number of bones. Our results revealed that the presence of OA in marine species (dolphin and dugong) was 2.44% and 3.33%, respectively. In dolphins, the highest OA occurrence was on the left and right humeral trochlea, with 13.68% and 12.63%, respectively, while the highest number of OA lesions in dugongs was on the lumbar vertebrae (8.79%). No significant difference (P > 0.05) in the prevalence of OA between sexes in dolphins and dugongs was observed, but we found a significant difference (P < 0.05) in 24 bone locations of human bones, which had the highest OA prevalence (48.93%), followed by dogs (3.94%). In conclusion, OA can occur in marine mammals, similar to terrestrial mammals, even though their natural habitat is the ocean.

Elemental classification of the tusks of dugong (Dugong dugong) by HH-XRF analysis and comparison with other species.

The elemental composition was investigated and applied for identifying the sex and habitat of dugongs, in addition to distinguishing dugong tusks and teeth from other animal wildlife materials such as Asian elephant (Elephas maximus) tusks and tiger (Panthera tigris tigris) canine teeth. A total of 43 dugong tusks, 60 dugong teeth, 40 dolphin teeth, 1 whale tooth, 40 Asian elephant tusks and 20 tiger canine teeth were included in the study. Elemental analyses were conducted using a handheld X-ray fluorescence analyzer (HH-XRF). There was no significant difference in the elemental composition of male and female dugong tusks, whereas the overall accuracy for identifying habitat (the Andaman Sea and the Gulf of Thailand) was high (88.1%). Dolphin teeth were able to be correctly predicted 100% of the time. Furthermore, we demonstrated a discrepancy in elemental composition among dugong tusks, Asian elephant tusks and tiger canine teeth, and provided a high correct prediction rate among these species of 98.2%. Here, we demonstrate the feasible use of HH-XRF for preliminary species classification and habitat determination prior to using more advanced techniques such as molecular biology.

Elemental Analysis of Bone, Teeth, Horn and Antler in Different Animal Species Using Non-Invasive Handheld X-Ray Fluorescence.

Mineralized tissues accumulate elements that play crucial roles in animal health. Although elemental content of bone, blood and teeth of human and some animal species have been characterized, data for many others are lacking, as well as species comparisons. Here we describe the distribution of elements in horn (Bovidae), antler (Cervidae), teeth and bone (humerus) across a number of species determined by handheld X-ray fluorescence (XRF) to better understand differences and potential biological relevance. A difference in elemental profiles between horns and antlers was observed, possibly due to the outer layer of horns being comprised of keratin, whereas antlers are true bone. Species differences in tissue elemental content may be intrinsic, but also related to feeding habits that contribute to mineral accumulation, particularly for toxic heavy metals. One significant finding was a higher level of iron (Fe) in the humerus bone of elephants compared to other species. This may be an adaptation of the hematopoietic system by distributing Fe throughout the bone rather than the marrow, as elephant humerus lacks a marrow cavity. We also conducted discriminant analysis and found XRF was capable of distinguishing samples from different species, with humerus bone being the best source for species discrimination. For example, we found a 79.2% correct prediction and success rate of 80% for classification between human and non-human humerus bone. These findings show that handheld XRF can serve as an effective tool for the biological study of elemental composition in mineralized tissue samples and may have a forensic application.

Determining comparative elemental profile using handheld X-ray fluorescence in humans, elephants, dogs, and dolphins: Preliminary study for species identification.

Species identification is a crucial step in forensic anthropological studies. The aim of this study was to determine elemental profiles in bones from four mammal species, to be used for species discrimination. Human, elephant, dog, and dolphin bones were scanned by X-ray fluorescence (XRF); the differences in elemental profiles between species were determined using discriminant analysis. Dogs had the greatest number of elements (23), followed by humans (22) and elephants (20). Dolphins had the lowest number of elements (16). The accuracy rate of species identification in humans, elephants, dogs, and dolphins was 98.7%, 100%, 94.9%, and 92.3%, respectively. We conclude that element profiles of bones based on XRF analyses can serve as a tool for determining species.