Investigating an increase in Florida manatee mortalities using a proteomic approach.

Two large-scale Florida manatee (Trichechus manatus latirostris) mortality episodes were reported on separate coasts of Florida in 2013. The east coast mortality episode was associated with an unknown etiology in the Indian River Lagoon (IRL). The west coast mortality episode was attributed to a persistent Karenia brevis algal bloom or 'red tide' centered in Southwest Florida. Manatees from the IRL also had signs of cold stress. To investigate these two mortality episodes, two proteomic experiments were performed, using two-dimensional difference in gel electrophoresis (2D-DIGE) and isobaric tags for relative and absolute quantification (iTRAQ) LC-MS/MS. Manatees from the IRL displayed increased levels of several proteins in their serum samples compared to controls, including kininogen-1 isoform 1, alpha-1-microglobulin/bikunen precursor, histidine-rich glycoprotein, properdin, and complement C4-A isoform 1. In the red tide group, the following proteins were increased: ceruloplasmin, pyruvate kinase isozymes M1/M2 isoform 3, angiotensinogen, complement C4-A isoform 1, and complement C3. These proteins are associated with acute-phase response, amyloid formation and accumulation, copper and iron homeostasis, the complement cascade pathway, and other important cellular functions. The increased level of complement C4 protein observed in the red tide group was confirmed through the use of Western Blot.

Signatures of conserved and unique molecular features in Afrotheria.

Afrotheria is a clade of African-origin species with striking dissimilarities in appearance and habitat. In this study, we compared whole proteome sequences of six Afrotherian species to obtain a broad viewpoint of their underlying molecular make-up, to recognize potentially unique proteomic signatures. We find that 62% of the proteomes studied here, predominantly involved in metabolism, are orthologous, while the number of homologous proteins between individual species is as high as 99.5%. Further, we find that among Afrotheria, L. africana has several orphan proteins with 112 proteins showing < 30% sequence identity with their homologues. Rigorous sequence searches and complementary approaches were employed to annotate 156 uncharacterized protein sequences and 28 species-specific proteins. For 122 proteins we predicted potential functional roles, 43 of which we associated with protein- and nucleic-acid binding roles. Further, we analysed domain content and variations in their combinations within Afrotheria and identified 141 unique functional domain architectures, highlighting proteins with potential for specialized functions. Finally, we discuss the potential relevance of highly represented protein families such as MAGE-B2, olfactory receptor and ribosomal proteins in L. africana and E. edwardii, respectively. Taken together, our study reports the first comparative study of the Afrotherian proteomes and highlights salient molecular features.

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.

Papillomatous Skin Netting of Cetaceans (Cetacea: Delphinapterus leucas, Balaena mysticetus, and Eschrichtius robustus) and Sirens (Sirenia: Trichechus manatus and Dugong dugon).

The structure of the papillomatous junction between epidermis and dermis (papillomatous netting, PN) in the skin of cetaceans (white whales, bowhead, and gray whales) and sirens (American manatee, dugong) was studied and compared using histophysiological and morphogeometric methods. The relative extent of PN development proved to be similar in members of both orders, but significant differences were found in PN configuration, the volume of "free area of grille", the degree of skin vertical compression, and skin density, which influence buoyancy. The differences are discussed from the viewpoint of species biology.

Debris ingestion by the Antillean Manatee (Trichechus manatus manatus).

The Antillean manatee inhabits coastal regions of North and Northeastern Brazil and currently is considered an endangered species in the country. Aiming to gather information for the development of public policies focusing on the conservation of manatees, the National Center for Research and Conservation of Aquatic Mammals of the Chico Mendes Institute for Biodiversity has been rescuing, rehabilitating and releasing these mammals since the 1980s. Over the last 36 years, 40 manatees were released by the CMA/ICMBio and four of them were rescued again due to debris ingestion. Two of these manatees died and the other two were taken back into captivity for a new rehabilitation process. The four mammals had confirmed diagnosis of plastic debris ingestion. These findings demonstrate that the environment where the manatees live after being released had a significant amount of garbage which may hinder the success of the species conservation in Brazil.

Assessement of serum amyloid A levels in the rehabilitation setting in the Florida manatee (Trichechus manatus latirostris).

The acute phase protein serum amyloid A (SAA) has been previously shown to have value as a biomarker of inflammation and infection in many species, including manatees (Trichechus manatus latirostris). In the current study, results from an automated assay for SAA were used in a rehabilitation setting. Reference intervals were established from clinically normal manatees using the robust method: 0-46 mg/L. More than 30-fold higher mean SAA levels were observed in manatees suffering from cold stress and boat-related trauma. Poor correlations were observed between SAA and total white blood count, percentage of neutrophils, albumin, and albumin/globulin ratio. A moderate correlation was observed between SAA and the presence of nucleated red blood cells. The sensitivity of SAA testing was 93% and the specificity was 98%, representing the highest combined values of all the analytes. The results indicate that the automated method for SAA quantitation can provide important clinical data for manatees in a rehabilitation setting.

Fatty acid profiles as a potential lipidomic biomarker of exposure to brevetoxin for endangered Florida manatees (Trichechus manatus latirostris).

Fatty acid signature analysis (FASA) is an important tool by which marine mammal scientists gain insight into foraging ecology. Fatty acid profiles (resulting from FASA) represent a potential biomarker to assess exposure to natural and anthropogenic stressors. Florida manatees are well studied, and an excellent necropsy program provides a basis against which to assess this budding tool. Results using samples from 54 manatees assigned to four cause-of-death categories indicated that those animals exposed to or that died due to brevetoxin exposure (red tide, or RT samples) demonstrate a distinctive hepatic fatty acid profile. Discriminant function analysis indicated that hepatic fatty acids could be used to classify RT versus non-RT liver samples with reasonable certainty. A discriminant function was derived based on 8 fatty acids which correctly classified 100% of samples from a training dataset (10 RT and 25 non-RT) and 85% of samples in a cross-validation dataset (5 RT and 13 non-RT). Of the latter dataset, all RT samples were correctly classified, but two of thirteen non-RT samples were incorrectly classified. However, the "incorrect" samples came from manatees that died due to other causes during documented red tide outbreaks; thus although the proximal cause of death was due to watercraft collisions, exposure to brevetoxin may have affected these individuals in ways that increased their vulnerability. This use of FASA could: a) provide an additional forensic tool to help scientists and managers to understand cause of death or debilitation due to exposure to red tide in manatees; b) serve as a model that could be applied to studies to improve assessments of cause of death in other marine mammals; and c) be used, as in humans, to help diagnose metabolic disorders or disease states in manatees and other species.

Carbon and nitrogen stable isotope turnover rates and diet-tissue discrimination in Florida manatees (Trichechus manatus latirostris).

The Florida manatee (Trichechus manatus latirostris) is a herbivorous marine mammal that occupies freshwater, estuarine and marine habitats. Despite being considered endangered, relatively little is known about its feeding ecology. The present study expands on previous work on manatee feeding ecology by providing critical baseline parameters for accurate isotopic data interpretation. Stable carbon and nitrogen isotope ratios were examined over a period of more than 1 year in the epidermis of rescued Florida manatees that were transitioning from a diet of aquatic forage to terrestrial forage (lettuce). The mean half-life for (13)C turnover was 53 and 59 days for skin from manatees rescued from coastal and riverine regions, respectively. The mean half-life for (15)N turnover was 27 and 58 days, respectively. Because of these slow turnover rates, carbon and nitrogen stable isotope analysis in manatee epidermis is useful in summarizing average dietary intake over a long period of time rather than assessing recent diet. In addition to turnover rate, a diet-tissue discrimination value of 2.8 per thousand for (13)C was calculated for long-term captive manatees on a lettuce diet. Determining both turnover rate and diet-tissue discrimination is essential in order to accurately interpret stable isotope data.

Detection and identification of plasma progesterone metabolites in the female Florida manatee (Trichechus manatus latirostris) using GC/MS/MS.

Florida manatees (Trichechus manatus latirostris) have relatively low peripheral concentrations of progesterone (P4). The objective of this study was to determine if these relatively low P4 concentrations are associated with a high ratio of progestin metabolites and to document metabolite concentrations from individual blood samples obtained from manatees during diestrus or pregnancy. Metabolites known to exist in elephants-terrestrial manatee relatives-were targeted. These included 5alpha-reduced progestins (5alpha-pregnane-3,20-dione [5alpha-DHP] and 3alpha-hydroxy-5alpha-pregnan-20-one [5alpha-P3-OH]) and 17alpha-hydroxyprogesterone (17alpha-OHP), which occurs in Asian elephants. An additional, inactive metabolite, 20alpha-hydroxyprogesterone (20alpha-OHP), indicative of P4 overproduction, was also targeted. Progesterone itself was the predominant progestin detected in pregnant and nonpregnant manatee plasma (n = 10) using gas chromatography-mass spectrometry with tandem quadrupole detectors (GC/MS/MS). Progesterone concentrations in pregnant females varied from early (moderate to high) through mid and late (low) pregnancy. Progesterone concentrations ranged from low to high in nonpregnant, nonlactating females. The most commonly detected metabolite was 5alpha-P3-OH (n = 7), which occurred in pregnant (lower limit of detection [LLOD] to high) and nonpregnant (trace to high) females. The 5alpha-DHP metabolite was also detected in pregnant (LLOD to moderate) and nonpregnant (low) females. The 17alpha-OHP metabolite was not detected in any tested female. The 20alpha-OHP metabolite was detected in one nonpregnant, nonlactating, captive female (LLOD). Metabolites were most prevalent during early pregnancy, concurrent with maximum P4 concentrations. Based on their concentrations in peripheral circulation, we inferred that these metabolites may have, opposite to elephants, a limited physiologic role during luteal, pregnant, and nonpregnant phases in the manatee.

Production of nitric oxide by peripheral blood mononuclear cells from the Florida manatee, Trichechus manatus latirostris.

Florida manatees (Trichechus manatus latirostris) are exposed to many conditions in their habitat that may adversely impact health and impair immune function in this endangered species. In an effort to increase the current knowledge base regarding the manatee immune system, the production of an important reactive nitrogen intermediate, nitric oxide (NO), by manatee peripheral blood mononuclear cells (PBMC) was investigated. PBMC from healthy captive manatees were stimulated with LPS, IFN-gamma, or TNF-alpha, either alone or in various combinations, with NO production assessed after 24, 48, 72, and 96 h of culture. NO production in response to LPS stimulation was significantly greater after 48, 72, or 96 h of culture compared to NO production after 24h of culture. A specific inhibitor of inducible nitric oxide synthase (iNOS), L-NIL (L-N(6)-(1-iminoethyl)lysine), significantly decreased NO production by LPS-stimulated manatee PBMC. Manatee specific oligonucleotide primers for iNOS were designed to measure expression of relative amounts of mRNA in LPS-stimulated manatee PBMC from captive manatees. NO production by PBMC from manatees exposed to red tide toxins was analyzed, with significantly greater NO production by both unstimulated and LPS stimulated PBMC from red tide exposed compared with healthy captive or cold-stress manatees. Free-ranging manatees produced significantly lower amounts of nitric oxide compared to either captive or red tide rescued manatees. Results presented in this paper contribute to the current understanding of manatee immune function and represent the first report of nitric oxide production in the immune system of a marine mammal.

The visual pigments of the West Indian manatee (Trichechus manatus).

Manatees are unique among the fully aquatic marine mammals in that they are herbivorous creatures, with hunting strategies restricted to grazing on sea-grasses. Since the other groups of (carnivorous) marine mammals have been found to possess various visual system adaptations to their unique visual environments, it was of interest to investigate the visual capability of the manatee. Previous work, both behavioral (Griebel & Schmid, 1996), and ultrastructural (Cohen, Tucker, & Odell, 1982; unpublished work cited by Griebel & Peichl, 2003), has suggested that manatees have the dichromatic color vision typical of diurnal mammals. This study uses molecular techniques to investigate the cone visual pigments of the manatee. The aim was to clone and sequence cone opsins from the retina, and, if possible, express and reconstitute functional visual pigments to perform spectral analysis. Both LWS and SWS cone opsins were cloned and sequenced from manatee retinae, which, upon expression and spectral analysis, had lambda(max) values of 555 and 410 nm, respectively. The expression of both the LWS and SWS cone opsin in the manatee retina is unique as both pinnipeds and cetaceans only express a cone LWS opsin.

Spectral-tuning mechanisms of marine mammal rhodopsins and correlations with foraging depth.

It has been observed that deep-foraging marine mammals have visual pigments that are blue shifted in terms of their wavelength of maximal absorbance (lambda(max)) when compared to analogous pigments from terrestrial mammals. The mechanisms underlying the spectral tuning of two of these blue-shifted pigments have recently been elucidated and depend on three amino acid substitutions (83Asn, 292Ser, and 299Ser) in dolphin rhodopsin, but only one amino acid substitution (308Ser ) in the dolphin long-wavelength-sensitive pigment. The objective of this study was to investigate the molecular basis for changes in the spectral sensitivity of rod visual pigments from seven distantly related marine mammals. The results show a relationship between blue-shifted rhodopsins (lambda(max) < or = 490 nm), deep-diving foraging behavior, and the substitutions 83Asn and 292Ser. Species that forage primarily near the surface in coastal habitats have a rhodopsin with a lambda(max) similar to that of terrestrial mammals (500 nm) and possess the substitutions 83Asp and 292Ala, identical to rhodopsins from terrestrial mammals.