Antillean manatee (Trichechus manatus manatus Linnaeus, 1758) Tongue Morphology and Adaptive Herbivorous Implications.

Morphological study of the tongue is an interesting way of understanding evolutionary processes associated with feeding habits. Therefore, the aim of the present study was to describe the tongue morphology of the Antillean manatee and to understand possible morphological relationships with its way of capturing food. Macroscopic dissections and light and scanning electron microscopy analyses of seven manatee tongues were performed. The tongue in Antillean manatees is a muscular and robust organ, divided into apex, body, and root. It is firmly adhered to the floor of the oral cavity. Lingual papillae were distributed over the entire tongue surface. They were identified as filiform papillae concentrated in the apex. Fungiform papillae were present on the apex and lateral regions. Foliate papillae were located on the dorsolateral portion of the root. Lentiform papillae were located across the dorsal tongue surface. The mucosa was lined by a keratinized stratified squamous epithelium presenting compound tubuloacinar glands and taste buds in the foliate papillae. The tongue of the Antillean manatee is similar to other Sirenia species, both of which share a completely herbivorous diet.

Cytochemical staining of leukocytes and platelets in the Florida manatee (Trichechus manatus latirostris): identification of a bilobed monocyte similar to other members of the Paenungulata.

Manatees (Antillean-, Amazonian, and African-) and dugongs belong to the Order Sirenia, and when combined with elephants and rock hyraxes, form the Paenungulata. A bilobed mononuclear cell has previously been identified in elephants and rock hyraxes, but not in manatees and dugongs, with cytochemical staining identifying these cells as bilobed monocytes in elephants. The objective of this study was to characterize leukocytes (white blood cells, WBC) and platelets in blood films of Florida manatees (Trichechus manatus latirostris; n = 8) using one routine hematological (Wright-Giemsa) and eight cytochemical stains: alkaline phosphatase (ALP), α-naphthyl butyrate esterase (ANBE), chloroacetate esterase (CAE), Luna, myeloperoxidase (MPx), periodic acid-Schiff (PAS), Sudan black B (SBB), and toluidine blue (TB). Heterophils and lymphocytes comprised most of the WBC, with low numbers of eosinophils, basophils, and monocytes. Additionally, 1-3% of the WBC were bilobed mononuclear cells. Bilobed mononuclear cell proportions were similar to rock hyraxes, but lower than elephants (approximate range 20-60%). Heterophils and eosinophils were positive for MPx, ALP, SBB, and PAS, with heterophils also being positive for CAE. Most of the lymphocytes were positive for ANBE and they were variably positive for CAE. Monocytes and bilobed mononuclear cells had similar cytochemical staining reactions (variably positive for all stains, except Luna and TB), supporting a monocytic origin, like elephants. Platelets were ANBE- and PAS-positive. Luna stain was useful for identifying eosinophils and TB was uninformative. This study provides new information on the morphological features and cytochemical staining characteristics of WBC and platelets and will aid in obtaining accurate hematological data of Florida manatees.

Evolution of an extreme hemoglobin phenotype contributed to the sub-Arctic specialization of extinct Steller's sea cows.

The extinct Steller's sea cow (Hydrodamalis gigas; †1768) was a whale-sized marine mammal that manifested profound morphological specializations to exploit the harsh coastal climate of the North Pacific. Yet despite first-hand accounts of their biology, little is known regarding the physiological adjustments underlying their evolution to this environment. Here, the adult-expressed hemoglobin (Hb; α2ß/δ2) of this sirenian is shown to harbor a fixed amino acid replacement at an otherwise invariant position (ß/δ82Lys→Asn) that alters multiple aspects of Hb function. First, our functional characterization of recombinant sirenian Hb proteins demonstrates that the Hb-O2 affinity of this sub-Arctic species was less affected by temperature than those of living (sub)tropical sea cows. This phenotype presumably safeguarded O2 delivery to cool peripheral tissues and largely arises from a reduced intrinsic temperature sensitivity of the H. gigas protein. Additional experiments on H. gigas ß/δ82Asn→Lys mutant Hb further reveal this exchange renders Steller's sea cow Hb unresponsive to the potent intraerythrocytic allosteric effector 2,3-diphosphoglycerate, a radical modification that is the first documented example of this phenotype among mammals. Notably, ß/δ82Lys→Asn moreover underlies the secondary evolution of a reduced blood-O2 affinity phenotype that would have promoted heightened tissue and maternal/fetal O2 delivery. This conclusion is bolstered by analyses of two Steller's sea cow prenatal Hb proteins (Hb Gower I; ζ2ε2 and HbF; α2γ2) that suggest an exclusive embryonic stage expression pattern, and reveal uncommon replacements in H. gigas HbF (γ38Thr→Ile and γ101Glu→Asp) that increased Hb-O2 affinity relative to dugong HbF. Finally, the ß/δ82Lys→Asn replacement of the adult/fetal protein is shown to increase protein solubility, which may have elevated red blood cell Hb content within both the adult and fetal circulations and contributed to meeting the elevated metabolic (thermoregulatory) requirements and fetal growth rates associated with this species cold adaptation.

In 1741, shipwrecked naturalist Georg Wilhelm Steller made detailed observations of large marine mammals grazing on seaweed in the shallow waters surrounding a remote island in the North Pacific Ocean. Within thirty years, these 'Steller's sea cows' had been hunted to extinction. Unlike their remaining tropical relatives ­ dugongs and manatees ­ Steller's sea cows were specialized to cold, sub-Arctic environments. Measuring up to 10 meters long, they were much larger than other sea cow species. This, along with having very thick skin, helped them to reduce heat loss. Previous work showed that the hemoglobin protein ­ which binds to and carries oxygen around mammalian bodies ­ of Steller's sea cows had a decreased affinity for oxygen, resulting in greater delivery of oxygen to organs and tissues. It was thought that this could be an adaptation to fuel heightened metabolic heat production in cold conditions. Studies of ancient DNA also identified the substitution of a single building block in the Steller's sea cow hemoglobin protein that is not present in other mammals and was suspected to underlie this modification. To determine how this unique substitution affects Steller's sea cow hemoglobin function ­ and whether it contributed to their ability to live in cold environments ­ Signore et al. generated hemoglobin proteins of Steller's sea cows, dugongs and Florida manatees. Testing their biochemical properties showed that this single exchange profoundly alters multiple aspects of how the Steller's sea cow hemoglobin works. Alongside reducing hemoglobin's oxygen affinity, the Steller's sea cow substitution also makes the protein more soluble, potentially increasing the level of hemoglobin within red blood cells. Additionally, it eliminates hemoglobin sensitivity to a molecule involved in oxygen binding ­ known as DPG ­ saving energy by no longer requiring production of this molecule. Furthermore, the same substitution makes hemoglobin less sensitive to changes in temperature, which would have helped to safeguard the delivery of oxygen to cool limbs and other extremities, reducing costly heat loss. Together, these changes in hemoglobin would have helped the Steller's sea cow to more efficiently transport oxygen around the body. Importantly, generating and testing Steller's sea cow pre-natal hemoglobins suggested this substitution may have also helped to enhance the fetal growth rate of these immense marine mammals by improving gas exchange between the mother and fetus. Signore et al. have revealed how a mutated form of hemoglobin allowed an extinct mammal to adapt to an extreme environment. Similar methods could be used to understand the physiological attributes of other extinct animals. In the future, this increased understanding of hemoglobin mutations could aid the development of human hemoglobin substitutes for therapeutic uses.

Fossil Sirenia from the Pleistocene of Qatar: new questions about the antiquity of sea cows in the Gulf Region.

One of the largest and least documented populations of dugongs (Dugong dugon) resides in the coastal waters of the United Arab Emirates, and waters surrounding Saudi Arabia, Bahrain, and Qatar. The archaeological record of dugongs in the Gulf Region is abundant, but little is known about their fossil record in the region. Here we report an isolated sirenian rib fragment from the Futaisi Member of the Fuwayrit Formation near the town of Al Ruwais, in northern Qatar. The Fuwayrit Formation is a marine Pleistocene deposit exposed onshore in Qatar and the United Arab Emirates. Based on the correlative dating of the basal Futaisi Member with other onshore platforms, the rib fragment is approximately 125 ka. We propose that this isolated rib (likely the first rib from the right side) belongs to Dugongidae, with strong similarities to extant Dugong. We cannot, however, eliminate the possibility that it belongs to an extinct taxon, especially given its similarities with other fossil dugongid material from both Qatar and elsewhere in the world. Aside from reflecting the presence of Gulf seagrass communities in the Pleistocene, this occurrence also suggests that different (and potentially multiple) lineages of sirenians inhabited the Gulf Region in the geologic past.

Manatee cognition in the wild: an exploration of the manatee mind and behavior through neuroanatomy, psychophysics, and field observations.

Cognition refers to the mechanisms for acquiring, processing, storing, and acting on information, all of which are critical to understanding the behavior of animals. These mechanisms are poorly known in manatees, especially how they are expressed in the wild. To expand our understanding of manatee cognition, we gathered information from behavioral experimentation in the laboratory, neuroanatomical research, controlled field studies, integrated laboratory and field measurement, and natural history observations (published reports, written surveys, and interviews with knowledgeable observers). Laboratory research, both neuroanatomical and behavioral, provided the most empirical data, primarily on sensory/perceptual capacities. Inferences from these data and narratives from surveys and interviews illuminated possibilities for higher order cognition. Evidence from field measurements was sparse, although substantial amounts of information have been collected from tracking data and to a lesser extent vessel impact studies, which can be used to infer cognitive attributes. Manatees are tactile-auditory specialists with complementary visual and chemosensory abilities. They demonstrate learning characteristics typical of vertebrates. Movement tracking data plus direct observations suggest that they have good spatial cognition, indicated by their ability to traverse complicated water networks and memory for foraging and warm water sites. They engage in a wide range of play-like, object manipulation, and mimetic behaviors, which suggests cognitive capacities beyond basic associative learning. Understanding manatee cognition beyond the laboratory will be necessary for conservation of manatees as they face challenges such as habitat degradation and threats from water-borne vessel traffic. There is a clear need for more direct research in natural settings.

Total evidence time-scaled phylogenetic and biogeographic models for the evolution of sea cows (Sirenia, Afrotheria).

Molecular phylogenetic studies that have included sirenians from the genera Trichechus, Dugong, and Hydrodamalis have resolved their interrelationships but have yielded divergence age estimates that are problematically discordant. The ages of these lineage splits have profound implications for how to interpret the sirenian fossil record-including clade membership, biogeographic patterns, and correlations with Earth history events. In an effort to address these issues, here we present a total evidence phylogenetic analysis of Sirenia that includes living and fossil species and applies Bayesian tip-dating methods to estimate their interrelationships and divergence times. In addition to extant sirenians, our dataset includes 56 fossil species from 106 dated localities and numerous afrotherian outgroup taxa. Genetic, morphological, temporal, and biogeographic data are assessed simultaneously to bring all available evidence to bear on sirenian phylogeny. The resulting time-tree is then used for Bayesian geocoordinates reconstruction analysis, which models ancestral geographic areas at splits throughout the phylogeny, thereby allowing us to infer the direction and timing of dispersals. Our results suggest that Pan-Sirenia arose in North Africa during the latest Paleocene and that the Eocene evolution of stem sirenians was primarily situated in the Tethyan realm. In the late Eocene, some lineages moved into more northern European latitudes, an area that became the source region for a key trans-Atlantic dispersal towards the Caribbean and northern-adjacent west Atlantic. This event led to the phylogenetic and biogeographic founding of crown Sirenia with the Dugongidae-Trichechidae split occurring at the Eocene-Oligocene boundary (~33.9 Ma), temporally coincident with the onset of dropping global sea levels and temperatures. This region became the nexus of sirenian diversification and supported taxonomically-rich dugongid communities until the earliest Pliocene. The Dugonginae-Hydrodamalinae split occurred near Florida during the early Miocene (~21.2 Ma) and was followed by a west-bound dispersal that gave rise to the Pacific hydrodamalines. The late middle Miocene (~12.2 Ma) split of Dugong from all other dugongines also occurred near Florida and our analyses suggest that the Indo-Pacific distribution of modern dugongs is the result of a trans-Pacific dispersal. From at least the early Miocene, trichechid evolution was based entirely in South America, presumably within the Pebas Wetlands System. We infer that the eventual establishment of Amazon drainage into the South Atlantic allowed the dispersal of Trichechus out of South America no earlier than the mid-Pliocene. Our analyses provide a new temporal and biogeographic framework for understanding major events in sirenian evolution and their possible relationships to oceanographic and climatic changes. These hypotheses can be further tested with the recovery and integration of new fossil evidence.

Karyotypes of Manatees: New Insights into Hybrid Formation (Trichechus inunguis × Trichechus m. manatus) in the Amazon Estuary.

Great efforts have been made to preserve manatees. Recently, a hybrid zone was described between Trichechus inunguis (TIN) and the Trichechus manatus manatus (TMM) in the Amazon estuary. Cytogenetic data on these sirenians are limited, despite being fundamental to understanding the hybridization/introgression dynamics and genomic organization in Trichechus. We analyzed the karyotype of TMM, TIN, and two hybrid specimens ("Poque" and "Vitor") by classical and molecular cytogenetics. G-band analysis revealed that TMM (2n = 48) and TIN (2n = 56) diverge by at least six Robertsonian translocations and a pericentric inversion. Hybrids had 2n = 50, however, with Autosomal Fundamental Number (FNA) = 88 in "Poque" and FNA = 74 in "Vitor", and chromosomal distinct pairs in heterozygous; additionally, "Vitor" exhibited heteromorphisms and chromosomes whose pairs could not be determined. The U2 snDNA and Histone H3 multi genes are distributed in small clusters along TIN and TMM chromosomes and have transposable Keno and Helitron elements (TEs) in their sequences. The different karyotypes observed among manatee hybrids may indicate that they represent different generations formed by crossing between fertile hybrids and TIN. On the other hand, it is also possible that all hybrids recorded represent F1 and the observed karyotype differences must result from mechanisms of elimination.

Survey for Leptospira and Brucella in Amazonian manatees, Amazon river dolphins, and a tucuxi in the Brazilian Amazon.

Leptospirosis and brucellosis are zoonotic diseases with global distributions that represent severe hazards to humans and animals. We investigated exposure to Leptospira spp. and Brucella spp. in samples from Amazonian manatees Trichechus inunguis, Amazon river dolphins Inia geoffrensis, and a tucuxi Sotalia fluviatilis. The animals were free-ranging or undergoing in situ rehabilitation in the mid-Solimões River region, Brazilian Amazon. Serum samples from 19 Amazonian manatees were tested by microscopic agglutination test, Rose Bengal test, and 2-mercaptoethanol Brucella agglutination test. Antibodies against Leptospira spp. were detected in 63% of the manatees tested and serovar Patoc was considered the infecting serovar in all positive samples. Titers were generally low, indicating chronic exposure, but higher titers indicative of an active infection were detected in 3 animals. Anti-Brucella spp. antibodies were not detected. Tissue and/or body fluid samples from 12 Amazon river dolphins, a tucuxi, and 2 Amazonian manatees were investigated by multiplex PCR and bacteriology for Leptospira spp. and Brucella spp. All samples were negative. However, Enterococcus faecalis was isolated from uterine fluid, lymph node, and lung of 3 Amazon river dolphins. Bacillus spp. were isolated from milk and synovial fluid from 2 Amazon river dolphins and from a milk sample from 1 Amazonian manatee. Knowledge of the pathogens present in Amazonian manatees, Amazon river dolphins, and tucuxis is of great relevance to species conservation and environmental health. Although no clinical signs were noted, further research is needed to elucidate the clinical relevance of infection by Leptospira sp. serovar Patoc in Amazonian aquatic mammals.

Novel insights on aquatic mammal MHC evolution: Evidence from manatee DQB diversity.

The low diversity in marine mammal major histocompatibility complex (MHC) appears to support the hypothesis of reduced pathogen selective pressure in aquatic systems compared to terrestrial environments. However, the lack of characterization of the aquatic and evolutionarily distant Sirenia precludes drawing more generalized conclusions. Therefore, we aimed to characterize the MHC DQB diversity of two manatee species and compare it with those reported for marine mammals. Our results identified 12 and 6 alleles in T. inunguis and T. manatus, respectively. Alleles show high rates of nonsynonymous substitutions, suggesting loci are evolving under positive selection. Among aquatic mammals, Pinnipeda DQB had smaller numbers of alleles, higher synonymous substitution rate, and a dN/dS ratio closer to 1, suggesting it may be evolving under more relaxed selection compared to fully aquatic mammals. This contradicts one of the predictions of the hypothesis that aquatic environments impose reduced pathogen pressure to mammalian immune system. These results suggest that the unique evolutionary trajectories of mammalian MHC may impose challenges in drawing ecoevolutionary conclusions from comparisons across distant vertebrate lineages.

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.

Study of inorganic elements in different organs and tissues of Amazonian manatee (Trichechus inunguis) from Brazil.

Multielement concentrations (P, S, Cl, K, Ca, Cr, Mn, Fe, Ni, Cu, Zn, Rb, and Rh) and total mercury (T-Hg) were analyzed in different organs and tissues of Amazonian manatee (Trichechus inunguis). Samples of 27 T. inunguis specimens, maintained in the collection of the Amazonian Center for the Research and Preservation of Aquatic Mammals, were used, situated in an area highly impacted by gold mining in the northern region of the Brazilian Amazon. Samples of aquatic plants used as food by the animals were also analyzed. The elements S, Cl, K, Cr, and Mn accumulated mainly in the musculature, while Fe and Cu were more concentrated in the liver. Trace elements, such as rubidium (Rb) and rhodium (Rh), not previously reported in the organs of animals of the family Trichechidae, were also identified. The averages for T-Hg in the skin, muscle, encephalon, liver, kidney, and lung samples were, respectively, 0.1540 ± 0.1332, 0.0593 ± 0.1044, 0.0517 ± 0.0467, 0.0486 ± 0.0543, 0.0237 ± 0.0336, and 0.0013 ± 0.0032 µg.g-1. The values obtained for the vibrissae samples were below the limit of quantification, which allows for the conclusion that this tissue cannot be used as a contamination marker. It was observed that even when kept in a conservation breeding site, these animals were exposed to non-essential trace elements. Differences in the accumulation of elements were observed between the different organs and tissues analyzed. The presence of contaminants in animals that live in a preservation center, even at low levels, deserves attention.

Manatee Trichechus manatus latirostris calf mortality in Florida: a retrospective review of pathology data from 2009-2017.

High maternal investment and extended inter-calving intervals in Florida manatees Trichechus manatus latirostris make calf survivorship critical to overall population growth. However, detailed patterns of causes of mortality in calves have not been reported and state agency statistics report portions of perinatal mortality based on body length rather than actual cause of death (COD). The objectives of this study were to categorize COD based on necropsy data and geographical location in Florida for 1209 manatee calf carcasses (<236 cm total length) examined between January 2009 and December 2017 and to describe factors contributing to calf mortality. Results indicated COD was attributed to natural causes (47%, n = 573), cold stress syndrome (38%, n = 457), watercraft injury (13%, n = 155), or other human-related causes (2%, n = 24). Natural causes were the leading COD for small calves <151 cm, with death due to stillbirth or dystocia most frequent (48%, n = 273/573). Enteric trematodiasis contributed to a large proportion of deaths from natural causes in large calves within the southwest region of Florida, with an increasing annual trend. Brevetoxicosis contributed substantially to natural causes within the southwest region exclusively and was commonly comorbid with enteric trematodiasis. Cold stress syndrome was the leading cause of death for large calves (151-235 cm), with the Atlantic region having the highest proportion of cases. Watercraft injury was a sustained threat to large calves, especially within the southwest region. This report provides details on specific health threats and patterns of mortality among manatee calves.

Diet composition of the African manatee: Spatial and temporal variation within the Sanaga River Watershed, Cameroon.

The present study aimed to investigate the diet of African manatees in Cameroon to better inform conservation decisions within protected areas. A large knowledge gap on diet and seasonal changes in forage availability limits the ability to develop informed local management plans for the African manatee in much of its range. This research took place in the Sanaga River Watershed, which includes two protected areas in the Littoral Region of Cameroon: the Douala-Edea National Park and the Lake Ossa Wildlife Reserve. We analyzed 113 manatee fecal samples and surveyed shoreline emergent and submerged vegetation within the Sanaga River Watershed. We used microhistological analyses to determine the relative contribution of each plant species to African manatee diets and compared across locations and across seasons (wet vs. dry season). We found that the shoreline vegetation is diverse with over 160 plant species, unevenly distributed across space and season, and dominated by emergent vegetation mostly represented by the antelope grass (Echinochloa pyramidalis). We recorded a total of 36 plant species from fecal samples with a spatial and temporal distribution mostly reflecting that of the corresponding shoreline vegetation. African manatees appear to be primarily opportunistically feeding on available vegetation across the seasons and habitat. This work documents the current, but changing, state of plant availability in the Sanaga River Watershed and reports the African manatee diet in Cameroon for the first time. This information can play a critical role in successfully managing the species and these protected areas. If we wish to protect the African manatee and the aquatic ecosystems within the Sanaga River Watershed, we must understand how forage availability changes over time, especially as its waters become nutrient enriched, eutrophic, and exposed to invasive species of plants in a changing world.

Parallel Independent Losses of G-Type Lysozyme Genes in Hairless Aquatic Mammals.

Lysozyme enzymes provide classic examples of molecular adaptation and parallel evolution, however, nearly all insights to date come from chicken-type (c-type) lysozymes. Goose-type (g-type) lysozymes occur in diverse vertebrates, with multiple independent duplications reported. Most mammals possess two g-type lysozyme genes (Lyg1 and Lyg2), the result of an early duplication, although some lineages are known to have subsequently lost one copy. Here we examine g-type lysozyme evolution across >250 mammals and reveal widespread losses of either Lyg1 or Lyg2 in several divergent taxa across the mammal tree of life. At the same time, we report strong evidence of extensive losses of both gene copies in cetaceans and sirenians, with an additional putative case of parallel loss in the tarsier. To validate these findings, we inspected published short-read data and confirmed the presence of loss of function mutations. Despite these losses, comparisons of selection pressures between intact g- and c-type lysozyme genes showed stronger purifying selection in the former, indicative of conserved function. Although the reasons for the evolutionary loss of g-type lysozymes in fully aquatic mammals are not known, we suggest that this is likely to at least partially relate to their hairlessness. Indeed, although Lyg1 does not show tissue-specific expression, recent studies have linked Lyg2 expression to anagen hair follicle development and hair loss. Such a role for g-type lysozyme would explain why the Lyg2 gene became obsolete when these taxa lost their body hair.

Genomic evidence for the parallel regression of melatonin synthesis and signaling pathways in placental mammals.

Background: The study of regressive evolution has yielded a wealth of examples where the underlying genes bear molecular signatures of trait degradation, such as pseudogenization or deletion. Typically, it appears that such disrupted genes are limited to the function of the regressed trait, whereas pleiotropic genes tend to be maintained by natural selection to support their myriad purposes. One such set of pleiotropic genes is involved in the synthesis ( AANAT, ASMT) and signaling ( MTNR1A, MTNR1B) of melatonin, a hormone secreted by the vertebrate pineal gland. Melatonin provides a signal of environmental darkness, thereby influencing the circadian and circannual rhythmicity of numerous physiological traits. Therefore, the complete loss of a pineal gland and the underlying melatonin pathway genes seems likely to be maladaptive, unless compensated by extrapineal sources of melatonin. Methods: We examined AANAT, ASMT, MTNR1A and MTNR1B in 123 vertebrate species, including pineal-less placental mammals and crocodylians. We searched for inactivating mutations and modelled selective pressures (dN/dS) to test whether the genes remain functionally intact. Results: We report that crocodylians retain intact melatonin genes and express AANAT and ASMT in their eyes, whereas all four genes have been repeatedly inactivated in the pineal-less xenarthrans, pangolins, sirenians, and whales. Furthermore, colugos have lost these genes, and several lineages of subterranean mammals have partial melatonin pathway dysfunction. These results are supported by the presence of shared inactivating mutations across clades and analyses of selection pressure based on the ratio of non-synonymous to synonymous substitutions (dN/dS), suggesting extended periods of relaxed selection on these genes. Conclusions: The losses of melatonin synthesis and signaling date to tens of millions of years ago in several lineages of placental mammals, raising questions about the evolutionary resilience of pleiotropic genes, and the causes and consequences of losing melatonin pathways in these species.

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.

Osmoregulation and electrolyte balance in a fully marine mammal, the dugong (Dugong dugon).

Dugongs (Dugong dugon) are fully marine mammals that live independently of fresh water so must balance water and electrolytes in a hyperosmotic environment. To investigate osmoregulation, matched plasma and urine from 51 live wild dugongs were analysed for osmolality, major electrolytes (Na+, Cl-, K+), urea, creatinine, and glucose. Maximum urine osmolality (1468 mOsm kg -1) and Na+, K+, and Cl- concentrations (757, 131.3, 677 mmol L-1, respectively) indicate that dugongs are capable of concentrating urine above seawater and could potentially realise a net gain of free water from drinking seawater. However, mean urine osmolality of 925.4 (± 46.6) mOsm kg-1 suggests that mariposia is unlikely to be an important osmoregulatory mechanism. Dugongs may obtain enough preformed water from their seagrass diet and metabolic oxidation to maintain homeostasis. Mean plasma osmolality of 339.6 (± 1.8) mOsm kg-1 is higher than in the related manatees but within the range for fully marine cetaceans. Relatively high mean plasma Na+ (175.5 ± 1.7 mmol L-1) and K+ (6.9 ± 0.1 mmol L-1), as well as mean urinary Na+ (469.6 ± 22.5 mmol L-1) and K+ levels (32.5 ± 4.5 mmol L-1) may reflect a salt-rich seagrass diet. Pregnant females had higher mean plasma osmolality (355.3 ± 4.9 mmol L-1) than non-pregnant females and males (337.9 ± 1.7 mOsm kg-1), suggesting that fluid retention was not a feature of pregnancy. Further research on water intake and endocrinology will enhance our understanding of osmoregulation in dugongs.

ESOPHAGEAL MEASUREMENT OF CORE BODY TEMPERATURE IN THE FLORIDA MANATEE (TRICHECHUS MANATUS LATIROSTRIS).

Cold-stress syndrome (CSS) is one of the leading natural threats to free-ranging Florida manatees (Trichechus manatus latirostris). Cold water exposure below the species' acceptable physiologic range is a frequent occurrence for manatees during cold weather months causing CSS-induced systemic illness and significant annual mortality. Although CSS is a commonly presented condition at manatee rehabilitation facilities, the core body temperatures in CSS manatees are currently unknown due to the lack of clinically applicable and accurate temperature measurement methodologies. Our objective was to establish a clinically applicable measurement methodology of core body temperature in manatees. A novel, minimally invasive temperature technique to obtain esophageal temperature by placing a temperature sensor through an oro-gastric tube was compared to current oral and nasal methods in 20 clinically healthy manatees. Results identified the esophageal measurement as the best performing and most precise temperature methodology. The superior performance of esophageal temperature measurements differed significantly from both nasal and oral measurements, while nasal and oral measurements did not differ when compared with each other. The esophageal measurements were consistent with manatee core body temperature, facilitating generation of a reference interval for core body temperature in healthy manatees (35.0-35.8 C). Four CSS medical cases were evaluated with the newly validated esophageal temperature method, facilitating diagnosis of hypothermia. The application of this temperature measurement technique to CSS manatees in field or rehabilitation settings will help in understanding CSS pathophysiology, improve medical assessments during rehabilitation, and contribute to conservation efforts.

As, Cr, Hg, Pb, and Cd Concentrations and Bioaccumulation in the Dugong Dugong dugon and Manatee Trichechus manatus: A Review of Body Burdens and Distribution.

The death of dozens of manatees Trichechus manatus recently in Tabasco, Mexico, has captured international attention. Speculation about possible causes include water and food contamination by metals. Although federal authorities have ruled out water chemical pollution, the cause of these deaths is still awaiting conclusive laboratory results. Present work seeks to summarize information currently available on non-essential metals and those of great toxicological relevance in Sirenia (dugongs and manatees), highlighting its body distribution, presence in blood, and its relationship with their geographical distribution, gender and age, whenever possible. This paper focuses on the five elements: As, Cr, Hg, Pb and Cd, which are commonly considered as threats for marine mammals and reported in Sirenia. Some of these metals (Cr and Cd) were thought to be related to the recent deaths in Tabasco. All five elements are accumulated by Sirenia at different levels. Metal presence is associated to their diet but does not necessarily imply adverse effects for dugongs and manatees. Toxicological aspects and the human consumption risk in case of any illegal or traditional consumption in some cultures are discussed. Important toxicological research areas that need to be addressed are highlighted.

Thromboelastography in wild Florida manatees (Trichechus manatus latirostris).

BACKGROUND: Thromboelastography (TEG) provides a comprehensive evaluation of blood clot formation. This test can be used to identify abnormalities in coagulation by assessing multiple aspects of the clotting cascade, including the speed of clot initiation and formation, clot strength, and ultimately fibrinolysis. Thromboembolic disease has been hypothesized to play a role in the pathophysiology of cold stress syndrome (CSS), an important cause of mortality in the threatened Florida manatee (Trichechus manatus latirostris). OBJECTIVES: The objective of this study was to establish thromboelastography RIs using the TEG 5000 with citrated whole blood samples and kaolin activation in wild, healthy manatees. METHODS: In December 2014 and January 2015, 29 wild manatees (17 male and 12 female) were blood sampled as part of the annual wild manatee health assessments organized by United States Geological Survey (USGS). TEG was performed using citrated kaolin-activated samples. RESULTS: The samples were obtained from manatees caught in Crystal River, Citrus County, and used to identify the mean ± SD of normal TEG parameters: R = reaction time 2.1 minutes (0.8), K = clot formation time 0.8 min (0), α angle = 83.1° (2), MA = maximum amplitude 75 mm (7.6), and LY30 = clot lysis 0.41% (0.68). No significant differences were found between manatee size, sex, or time between sampling and running the test. CONCLUSIONS: Manatee TEG parameters demonstrate a relatively hypercoagulable condition when compared to other mammals. This information will facilitate detection of changes in hemostasis during injury and disease and provide a valuable reference range.