The Role of Skin-Derived Somatic Cell and Tissue Cryobanks in the Conservation of Aquatic Mammals.

Anthropogenic actions, especially inadequate waste disposal, cause permanent effects on aquatic fauna, resulting in a significant loss in their population. In this scenario, in situ and ex situ conservation strategies have been developed for these species. Among these strategies is the formation of somatic cell and tissue banks derived from skin collection that act complementarily to other biotechnologies. These banks contain all the information for genomic, genetic, and proteomic analyses. They are useful in the assessment of the toxicity of pollutants on the physiology of the species and regenerative and reproductive biotechnologies. The formation of these cryobanks involves different steps, including cryopreservation, with the optimization of all steps occurring in a species-specific manner. There is a diversity of studies on aquatic mammals; however, a low quantity compared to the number of studies on land mammals, with more than 80% of species still unexplored. This is mainly due to the difficulty of execution and asepsis in collecting skin from aquatic mammals and the in vitro culture, which seems to require more particularities for it to be successful. Therefore, this review aims to address the current scenario and the steps involved in the conservation of somatic cells and tissues derived from aquatic mammal skin, as well as results that have been achieved in recent years and the prospects.

Establishment and Characterization of a Primary Fibroblast Cell Culture from the Amazonian Manatee (Trichechus inunguis).

The vulnerable status of the Amazon manatee, Trichechus inunguis, indicates the need to seek measures to guarantee its conservation. In this context, the cultivation of cells in vitro is a strategy that should at least guarantee the preservation of their genetic material. Thus, we established for the first time a primary culture of Amazonian manatee fibroblasts (TINsf) from a skin biopsy of a young male. Karyotypic analysis of the 3rd, 7th, and 12th passages confirmed the taxonomic identity of the species T. inunguis (2n = 56/NF = 92) and indicated that this culture presents genomic stability. Gene and protein expression of vimentin at the 13th passage show the predominant presence of fibroblasts in TINsf. To test the cell line's responsiveness to materials and demonstrate a possible application of this culture, it was exposed to andiroba seed oil (ASO), and its viability and proliferative capacity were evaluated. ASO demonstrated toxic effects at the highest concentrations and longest exposure times tested, reproducing results observed in human cultures, indicating the applicability of TINsf in toxicological and biotechnological studies. After cryopreservation, the TINsf line maintained its proliferative potential, indicating the establishment of a new culture available for future studies.

A chromosome-level genome assembly for the dugong (Dugong dugon).

The dugong (Dugong dugon) is a marine mammal widely distributed throughout the Indo-Pacific and the Red Sea, with a Vulnerable conservation status, and little is known about many of the more peripheral populations, some of which are thought to be close to extinction. We present a de novo high-quality genome assembly for the dugong from an individual belonging to the well-monitored Moreton Bay population in Queensland, Australia. Our assembly uses long-read PacBio HiFi sequencing and Omni-C data following the Vertebrate Genome Project pipeline to reach chromosome-level contiguity (24 chromosome-level scaffolds; 3.16 Gbp) and high completeness (97.9% complete BUSCOs). We observed relatively high genome-wide heterozygosity, which likely reflects historical population abundance before the last interglacial period, approximately 125,000 yr ago. Demographic inference suggests that dugong populations began declining as sea levels fell after the last interglacial period, likely a result of population fragmentation and habitat loss due to the exposure of seagrass meadows. We find no evidence for ongoing recent inbreeding in this individual. However, runs of homozygosity indicate some past inbreeding. Our draft genome assembly will enable range-wide assessments of genetic diversity and adaptation, facilitate effective management of dugong populations, and allow comparative genomics analyses including with other sirenians, the oldest marine mammal lineage.

Molecular Footprints on Osmoregulation-Related Genes Associated with Freshwater Colonization by Cetaceans and Sirenians.

The genetic basis underlying adaptive physiological mechanisms has been extensively explored in mammals after colonizing the seas. However, independent lineages of aquatic mammals exhibit complex patterns of secondary colonization in freshwater environments. This change in habitat represents new osmotic challenges, and additional changes in key systems, such as the osmoregulatory system, are expected. Here, we studied the selective regime on coding and regulatory regions of 20 genes related to the osmoregulation system in strict aquatic mammals from independent evolutionary lineages, cetaceans, and sirenians, with representatives in marine and freshwater aquatic environments. We identified positive selection signals in genes encoding the protein vasopressin (AVP) in mammalian lineages with secondary colonization in the fluvial environment and in aquaporins for lineages inhabiting the marine and fluvial environments. A greater number of sites with positive selection signals were found for the dolphin species compared to the Amazonian manatee. Only the AQP5 and AVP genes showed selection signals in more than one independent lineage of these mammals. Furthermore, the vasopressin gene tree indicates greater similarity in river dolphin sequences despite the independence of their lineages based on the species tree. Patterns of distribution and enrichment of Transcription Factors in the promoter regions of target genes were analyzed and appear to be phylogenetically conserved among sister species. We found accelerated evolution signs in genes ACE, AQP1, AQP5, AQP7, AVP, NPP4, and NPR1 for the fluvial mammals. Together, these results allow a greater understanding of the molecular bases of the evolution of genes responsible for osmotic control in aquatic mammals.

From teeth to pad: tooth loss and development of keratinous structures in sirenians.

Sirenians are a well-known example of morphological adaptation to a shallow-water grazing diet characterized by a modified feeding apparatus and orofacial morphology. Such adaptations were accompanied by an anterior tooth reduction associated with the development of keratinized pads, the evolution of which remains elusive. Among sirenians, the recently extinct Steller's sea cow represents a special case for being completely toothless. Here, we used µ-CT scans of sirenian crania to understand how motor-sensor systems associated with tooth innervation responded to innovations such as keratinized pads and continuous dental replacement. In addition, we surveyed nine genes associated with dental reduction for signatures of loss of function. Our results reveal how patterns of innervation changed with modifications of the dental formula, especially continuous replacement in manatees. Both our morphological and genomic data show that dental development was not completely lost in the edentulous Steller's sea cows. By tracing the phylogenetic history of tooth innervation, we illustrate the role of development in promoting the innervation of keratinized pads, similar to the secondary use of dental canals for innervating neomorphic keratinized structures in other tetrapod groups.

Ancestral chromosomal signatures of Paenungulata (Afroteria) reveal the karyotype of Amazonian manatee (Trichechus inunguis, Sirenia: Trichechidae) as the oldest among American manatees.

BACKGROUND: Chromosomal painting in manatees has clarified questions about the rapid evolution of sirenians within the Paenungulata clade. Further cytogenetic studies in Afrotherian species may provide information about their evolutionary dynamics, revealing important insights into the ancestral karyotype in the clade representatives. The karyotype of Trichechus inunguis (TIN, Amazonian manatee) was investigated by chromosome painting, using probes from Trichechus manatus latirostris (TML, Florida manatee) to analyze the homeologies between these sirenians. RESULTS: A high similarity was found between these species, with 31 homologous segments in TIN, nineteen of which are whole autosomes, besides the X and Y sex chromosomes. Four chromosomes from TML (4, 6, 8, and 9) resulted in two hybridization signals, totaling eight acrocentrics in the TIN karyotype. This study confirmed in TIN the chromosomal associations of Homo sapiens (HSA) shared in Afrotheria, such as the 5/21 synteny, and in the Paenungulata clade with the syntenies HSA 2/3, 8/22, and 18/19, in addition to the absence of HSA 4/8 common in eutherian ancestral karyotype (EAK). CONCLUSIONS: TIN shares more conserved chromosomal signals with the Paenungulata Ancestral Karyotype (APK, 2n = 58) than Procavia capensis (Hyracoidea), Loxodonta africana (Proboscidea) and TML (Sirenia), where TML presents less conserved signals with APK, demonstrating that its karyotype is the most derived among the representatives of Paenungulata. The chromosomal changes that evolved from APK to the T. manatus and T. inunguis karyotypes (7 and 4 changes, respectively) are more substantial within the Trichechus genus compared to other paenungulates. Among these species, T. inunguis presents conserved traits of APK in the American manatee genus. Consequently, the karyotype of T. manatus is more derived than that of T. inunguis.

Herpesvirus and adenovirus surveillance in threatened wild West Indian (Trichechus manatus) and Amazonian manatees (Trichechus inunguis), Brazil.

THE FAMILY TRICHECHIDAE (ORDER SIRENIA) COMPRISES THREE SPECIES: African (Trichechus senegalenses), West Indian (T. manatus), WIM)], and the Amazonian manatees (T. inunguis, AMM). Whereas WIM inhabits both riverine and coastal systems in the western Atlantic, AMM is the only exclusively freshwater sirenian, endemic to the Amazon River Basin. The study of infectious agents is essential to species conservation, especially considering that both species are classified as Vulnerable by the IUCN Red List and as Endangered by the Brazilian Red List. The current knowledge about viral agents in sirenians is scarce. Herpesviruses and adenovirus are DNA viruses able to infect and cause disease in a wide range of hosts. Herein, we used panPCR protocols to survey herpesvirus and adenovirus in blood samples of wild WIM (n = 23) and AMM (n = 26) under human care in Brazil. Herpesvirus DNA was detected in one juvenile female WIM (1/23; 4.3%; 95% CI -4.7 - 13.3) from Ceará state and in four AMM (two juvenile females, a juvenile male, and an adult female; 4/26; 15.4%; 95% CI 0.5 - 30.3) from Amazonas state. The two different gammaherpesvirus DNA polymerase sequence types identified (one per species, a sequence type in a WIM and another one in three AMM) were highly similar (99% nucleotide identity) to Trichechid herpesvirus 1, reported in West Indian manatees of Florida (USA), and 100% identical when translated into amino acids. A herpesviral glycoprotein B sequence was identified in two AMM. None of the samples was positive to adenovirus. To the authors' knowledge, this is the first herpesvirus detection in manatees from South America, expanding the herpesvirus geographical range, and the first in WIM and AMM worldwide. Our findings suggest (i) that West Indian and Amazonian manatees are possibly the natural hosts of the detected herpesvirus, and (ii) coevolution of that gammaherpesvirus with Trichechus. Future studies are necessary to characterize the obtained virus and elucidate potential pathological effects (if any) in these 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.

Polycyclic aromatic hydrocarbons in marine mammals: A review and synthesis.

Most marine mammal species and populations are listed as endangered, threatened, or depleted under the Endangered Species Act and the Marine Mammal Protection Act. Organic contaminants such as polycyclic aromatic hydrocarbons from anthropogenic activities are part of the threat to marine mammals. The evaluation of the potential bioaccumulation of these compounds by marine mammals is a tool for adoption of policies to reduce polycyclic aromatic hydrocarbons discharges to the marine environment, where important players such as the oil and gas industries, maritime transport and sewage companies operate. This review seeks to present a bibliographic survey covering all published peer reviewed works of the contents of polycyclic aromatic hydrocarbons in biological tissues of marine mammals. It intended to compare the sampling protocols, procedures for preservation of the tissues, and the analytical method applied to quantify the polycyclic aromatic hydrocarbons, no to criticize any of them but to review the data and discuss how they can be compared.

Salmonellosis in a young Amazonian manatee (Trichechus inunguis) / Salmonelose em um filhote de peixe-boi-da-amazônia (Trichechus inunguis)

ABSTRACT: The objective of this study was to describe a case of salmonellosis in an Amazonian manatee (Trichechus inunguis). The animal was rescued from a tributary of the Capim River, in the municipality of Goianésia do Pará, Pará, Brazil, kept in a pool, and died approximately five months after being rescued. The main changes observed at necropsy were that the cecum and colon had serosal hyperemia, wall edema, thickened mucosa with ulcerated areas and covered by a thin layer of fibrin, and a yellowish liquid content. Histologically, there was moderate multifocal fibrinonecrotic typhlocolitis associated with intense bacterial colonization. Salmonella enterica subsp. enterica (rough) was isolated in cecum and colon fragments. This is the first report of salmonellosis in an Amazonian manatee.

RESUMO: O objetivo deste trabalho é descrever um caso de salmonelose em um peixe-boi-da-amazônia (Trichechus inunguis). O animal foi resgatado em um afluente do rio Capim, no município de Goianésia do Pará, Pará, Brasil, sendo mantido em uma piscina e morrendo aproximadamente cinco meses após o resgate. As principais alterações observadas na necropsia foram ceco e colón com hiperemia da serosa; edema da parede; mucosa espessada, com áreas ulceradas e revestida por uma fina camada de fibrina; e conteúdo líquido amarelado. Histologicamente havia tiflocolite fibrinonecrótica, multifocal, moderada, associada a intensa colonização bacteriana. Em fragmentos de ceco e cólon foi isolada Salmonella enterica subsp. enterica (rugosa). Este é o primeiro relato de salmonelose em peixe-boi-da-amazônia.

Molecular confirmation of twinning in the West Indian Manatee (Trichechus manatus) / Confirmação molecular de gemelaridade em peixes-boi marinho (Trichechus manatus)

Abstract: Few twinning events have been recorded in the West Indian manatee (Trichechus manatus, Sirenia: Trichechidae) and no previous published study has provided confirmation of this phenomenon based in molecular tools. Here we investigate a possible case of twinning in an endangered Brazilian population of T. manatus using molecular tools. We analyzed two male neonates found stranded in Ceará State, on the northeastern coast of Brazil. The DNA of both individuals was isolated, and 10 microsatellite loci were amplified and genotyped. Following the identification of the alleles, the probabilities of identity by descent (∆7 and ∆8) and relatedness (rxy) were calculated using estimators that evaluate inbreeding. The two individuals shared most of the alleles, with differences in the genotypes being identified in only two loci. All the estimators identified a level of relatedness compatible with that found between siblings (selfed or outbred), indicating they were dizygotic twins. This is the first confirmed case of fraternal twins in free-ranging West Indian manatees in South America. The recognition of this type of twinning provides elements to improve actions for the rehabilitation of stranded animals and their subsequent release to the environment.

Resumo: Poucos eventos de gemelaridade foram registrados para o peixe-boi marinho (Trichechus manatus, Sirenia: Trichechidae) e nenhum estudo previamente publicado confirmou esse fenômeno com base em ferramentas moleculares. Aqui investigamos um possível caso de gemelaridade em uma ameaçada população brasileira de T. manatus utilizando ferramentas moleculares. Foram analisados dois neonatos machos encontrados encalhados no Ceará, costa nordeste do Brasil. O DNA dos indivíduos foi isolado e 10 loci microsatélites foram amplificados e genotipados. Após a identificação dos alelos, as probabilidades de identidade por descendência (∆7 e ∆8) e relação (rxy) foram calculadas usando estimadores que avaliam endogamia. Os dois indivíduos partilharam a maioria dos alelos, com diferenças nos genótipos sendo identificadas em apenas dois loci. Todos os estimadores identificaram um nível de parentesco compatível com o encontrado entre irmãos (com e sem endogamia), o que aponta para o fato de serem gêmeos dizigóticos. Este é o primeiro caso confirmado de gêmeos fraternais em peixes-boi marinho de vida livre na América do Sul. O reconhecimento deste tipo de gemelaridade fornece elementos para aprimorar ações que visem a reabilitação de animais encalhados e sua posterior soltura ao ambiente.

The Relevance of Ecological Transitions to Intelligence in Marine Mammals.

Macphail's comparative approach to intelligence focused on associative processes, an orientation inconsistent with more multifaceted lay and scientific understandings of the term. His ultimate emphasis on associative processes indicated few differences in intelligence among vertebrates. We explore options more attuned to common definitions by considering intelligence in terms of richness of representations of the world, the interconnectivity of those representations, the ability to flexibly change those connections, and knowledge. We focus on marine mammals, represented by the amphibious pinnipeds and the aquatic cetaceans and sirenians, as animals that transitioned from a terrestrial existence to an aquatic one, experiencing major changes in ecological pressures. They adapted with morphological transformations related to streamlining the body, physiological changes in respiration and thermoregulation, and sensory/perceptual changes, including echolocation capabilities and diminished olfaction in many cetaceans, both in-air and underwater visual focus, and enhanced senses of touch in pinnipeds and sirenians. Having a terrestrial foundation on which aquatic capacities were overlaid likely affected their cognitive abilities, especially as a new reliance on sound and touch, and the need to surface to breath changed their interactions with the world. Vocal and behavioral observational learning capabilities in the wild and in laboratory experiments suggest versatility in group coordination. Empirical reports on aspects of intelligent behavior like problem-solving, spatial learning, and concept learning by various species of cetaceans and pinnipeds suggest rich cognitive abilities. The high energy demands of the brain suggest that brain-intelligence relationships might be fruitful areas for study when specific hypotheses are considered, e.g., brain mapping indicates hypertrophy of specific sensory areas in marine mammals. Modern neuroimaging techniques provide ways to study neural connectivity, and the patterns of connections between sensory, motor, and other cortical regions provide a biological framework for exploring how animals represent and flexibly use information in navigating and learning about their environment. At this stage of marine mammal research, it would still be prudent to follow Macphail's caution that it is premature to make strong comparative statements without more empirical evidence, but an approach that includes learning more about how animals flexibly link information across multiple representations could be a productive way of comparing species by allowing them to use their specific strengths within comparative tasks.

Angioarchitecture of collateral arteries of the aortic arch of Antillean manatee (Trichechus manatus manatus Linnaeus, 1758).

The aortic arrangement is an important structure associated with the maintenance of homeostasis. Based on this information, this study was conducted to describe the collateral arteries of the aortic arch of Antillean manatee and define the standard model for the species. Three specimens, an adult male, adult female and a male neonate, all strandings on the coast of the state of Rio Grande do Norte, Brazil, were used. The study was performed in the Laboratory of Morphophysiology of Vertebrates of the Federal University of Rio Grande do Norte, where in situ photographs were taken to demonstrate their topography. Subsequently, their hearts were removed and fixed in 10% formaldehyde and after 72 hr were dissected and analysed, obtaining schematic drawings and photographs of the vascular arrangement. The aortic arch was represented by three collateral arteries identified as the brachiocephalic trunk, left common carotid artery and left subclavian artery. This arrangement was similar to that found for other sirenians, and yet, for other mammals like hooded seal, murine, margarita island capuchin, black-handed tamarin, Mongolian gerbil and human. The morphological similarity presented in this study with different species of mammals, including humans, may contribute valuable information from an evolutionary point of view.

Reproductive Science Methods for Wild, Fully-Marine Mammals: Current Approaches and Future Applications.

Determining reproductive rates of marine mammal populations can give insight into their persistence and resilience in changing environments. As our marine environments continue to degrade along developed coastal fringes and as mankind's influences extend across even our widest oceans, there is a concern that the reproductive functioning of marine mammals may be affected adversely. Since many marine mammal species and populations are still in the recovery phase post-commercial harvest, and yet others are endangered or threatened by ongoing pressures, further environmental changes may represent direct or indirect threats to their reproductive potential. In this chapter, we review the current methods employed to investigate various aspects of reproductive science in fully-marine mammals, including direct observation of reproductive behavior and output, endocrinology to determine reproductive state, and assisted reproductive technologies to enhance reproductive outcomes. In particular, we focus on the most recent developments and innovations to reproductively sample marine mammals. Two case studies are presented to illustrate the challenges thrown up to researchers studying free-ranging marine mammals, and to highlight diversity in research approach. The North Atlantic right whale is on the brink of extinction through historic overharvest and present-day entanglement and ship strike. Environmental disruption to their migration routes and declining population health has resulted in reduced reproductive rates. In contrast, the main current threats to the reproductive success and survival of the vulnerable dugong are extreme weather events that affect availability of its seagrass diet. Climate disruption with increasingly severe coastal storms and flooding threaten the health of coastal seagrass beds, and consequently reproductive success and survival of this species. It is anticipated that climate change may have diverse and often serious effects on marine mammal reproduction in populations around the globe.

Affinity of Staphylococcal a and Streptococcal G Proteins to West Indian Manatee ( Trichechus manatus manatus) Immunoglobulins.

The West Indian manatee ( Trichechus manatus manatus), a subspecies that inhabits coastal areas of Central and South America, has been listed as a vulnerable species because of the rapid decline in its population. Commercially available immunologic reagents specific for sirenians are lacking, limiting the development of sensitive immunodiagnostic assays. We observed the affinity of the microbial proteins A and G to T. m. manatus immunoglobulins. Manatee serum pools were analyzed using enzyme-linked immunosorbent assay (ELISA) to determine the affinity intensity followed by western blotting to confirm the specific binding of proteins A and G to immunoglobulins. The ELISA demonstrated maximum affinity of both proteins until the serum dilution of 1:12,800, with a similar affinity for both proteins. Because both A and G proteins exhibited affinity to manatee immunoglobulins, they can be used to develop sensitive immunodiagnostic assays for this species, contributing to manatee conversation procedures.

Selection on different genes with equivalent functions: the convergence story told by Hox genes along the evolution of aquatic mammalian lineages.

BACKGROUND: Convergent evolution has been a challenging topic for decades, being cetaceans, pinnipeds and sirenians textbook examples of three independent origins of equivalent phenotypes. These mammalian lineages acquired similar anatomical features correlated to an aquatic life, and remarkably differ from their terrestrial counterparts. Whether their molecular evolutionary history also involved similar genetic mechanisms underlying such morphological convergence nevertheless remained unknown. To test for the existence of convergent molecular signatures, we studied the molecular evolution of Hox genes in these three aquatic mammalian lineages, comparing their patterns to terrestrial mammals. Hox genes are transcription factors that play a pivotal role in specifying embryonic regional identity of nearly any bilateral animal, and are recognized major agents for diversification of body plans. RESULTS: We detected few signatures of positive selection on Hox genes across the three aquatic mammalian lineages and verified that purifying selection prevails in these sequences, as expected for pleiotropic genes. Genes found as being positively selected differ across the aquatic mammalian lineages, but we identified a substantial overlap of their developmental functions. Such pattern likely resides on the duplication history of Hox genes, which probably provided different possible evolutionary routes for achieving the same phenotypic solution. CONCLUSIONS: Our results indicate that convergence occurred at a functional level of Hox genes along three independent origins of aquatic mammals. This conclusion reinforces the idea that different changes in developmental genes may lead to similar phenotypes, probably due to the redundancy provided by the participation of Hox paralogous genes in several developmental functions.

Antifungal Resistance and Virulence Among Candida spp. from Captive Amazonian manatees and West Indian Manatees: Potential Impacts on Animal and Environmental Health.

This work aimed at evaluating the antifungal susceptibility and production of virulence factors by Candida spp. isolated from sirenians in Brazil. The isolates (n = 105) were recovered from the natural cavities of Amazonian and West Indian manatees and were tested for the susceptibility to amphotericin B, itraconazole, and fluconazole and for the production of phospholipases, proteases, and biofilm. The minimum inhibitory concentrations (MICs) for amphotericin B ranged from 0.03 to 1 µg/mL, and no resistant isolates were detected. Itraconazole and fluconazole MICs ranged from 0.03 to 16 µg/mL and from 0.125 to 64 µg/mL, respectively, and 35.2% (37/105) of the isolates were resistant to at least one of these azole drugs. Concerning the production of virulence factors, phospholipase activity was observed in 67.6% (71/105) of the isolates, while protease activity and biofilm production were detected in 50.5% (53/105) and 32.4% (34/105) of the isolates, respectively. Since the natural cavities of manatees are colonized by resistant and virulent strains of Candida spp., these animals can act as sources of resistance and virulence genes for the environment, conspecifics and other animal species, demonstrating the potential environmental impacts associated with their release back into their natural habitat.

Yeast microbiota of natural cavities of manatees (Trichechus inunguis and Trichechus manatus) in Brazil and its relevance for animal health and management in captivity.

The aim of this study was to characterize the yeast microbiota of natural cavities of manatees kept in captivity in Brazil. Sterile swabs from the oral cavity, nostrils, genital opening, and rectum of 50 Trichechus inunguis and 26 Trichechus manatus were collected. The samples were plated on Sabouraud agar with chloramphenicol and incubated at 25 °C for 5 days. The yeasts isolated were phenotypically identified by biochemical and micromorphological tests. Overall, 141 strains were isolated, of which 112 were from T. inunguis (Candida albicans, Candida parapsilosis sensu stricto, Candida orthopsilosis, Candida metapsilosis, Candida guilliermondii, Candida pelliculosa, Candida tropicalis, Candida glabrata, Candida famata, Candida krusei, Candida norvegensis, Candida ciferri, Trichosporon sp., Rhodotorula sp., Cryptococcus laurentii) and 29 were from T. manatus (C. albicans, C. tropicalis, C. famata, C. guilliermondii, C. krusei, Rhodotorula sp., Rhodotorula mucilaginosa, Rhodotorula minuta, Trichosporon sp.). This was the first systematic study to investigate the importance of yeasts as components of the microbiota of sirenians, demonstrating the presence of potentially pathogenic species, which highlights the importance of maintaining adequate artificial conditions for the health of captive manatees.

Growth curve of free-ranging Trichechus inunguis / Curva de crescimento Trichechus inunguis de vida livre

The objective of this work was to describe growth curves for free-ranging Amazonian manatees in Brazil. Data analyzed included age estimates and biometrics from 60 Amazonian manatees (33 males and 27 females) captured between 1993 and 2006 by local residents of the mid-Solimões and Pirativa Rivers, in Amazonia, and collected by the Instituto de Desenvolvimento Sustentável Mamirauá and Centro Nacional de Conservação e Manejo de Mamíferos Aquáticos/ICMBio. The non-linear mathematical model used to analyze the data was the von Bertalanffy's equation; two growth curves were calculated, one for males L(t)male = 299.4[1-e-0.0897507(t+6.55696)], and one for females L(t)female = 256.1[1-e-0.23731(t+ 3.01921)]. Based on these equations, length at birth for the Amazonian manatee is estimated at 133.2 cm (average = 113.0 cm; SD = 34.4 cm) for males, and 131.0 cm (average = 124.7 cm; SD = 22.0 cm) for females. Among the males, the annual growth rate was 0.09 cm (95 percent CI: ± 0.002), with a maximum length of 299.4 cm (95 percent CI: ± 2.039), while females presented a growth rate of 0.24 cm per year (95 percent CI: ± 0.004), reaching up to 256.1 cm (95 percent CI: ± 0.4832) of total length. Even though von Bertalanffy's equation is not commonly used for sirenians, and considering that when previously applied it did not obtain satisfactory results, in this study it proved adequate for the establishment of the growth curve for free-ranging Amazonian manatees.

O objetivo deste trabalho foi descrever curvas de crescimento para peixes-boi-da-Amazônia de vida livre no Brasil. Foram utilizados dados etários e biométricos de 60 peixes-boi-da-Amazônia, 33 machos e 27 fêmeas, capturados entre 1993 e 2006 por pessoas ribeirinhas do médio Solimões e do Rio Pirativa, na região Amazônica, oriundos do Instituto de Desenvolvimento Sustentável Mamirauá e do Centro Nacional de Conservação e Manejo de Mamíferos Aquáticos/ICMBio. O modelo matemático não-linear utilizado para análise desses dados foi a equação derivada de von Bertalanffy, sendo calculadas duas equações de crescimento, uma para os machos L(t)macho = 299,4[1-e-0,0897507(t+6,55696)], e outra para as fêmeas L(t)fêmea = 256,1[1-e-0,23731(t+ 3,01921)]. Com o desenvolvimento dessas equações, o comprimento do peixe-boi-da-Amazônia ao nascer foi de 133,2 cm (média = 113,0 cm; SD = 34,4 cm) quando macho, e 131,0 cm (média = 124,7 cm; SD = 22,0 cm) quando fêmeas. Nos machos a taxa de crescimento anual obtida foi de 0,09 cm (IC 95 por cento: ± 0,002), atingindo uma envergadura máxima de 299,4 cm (IC 95 por cento: ± 2,039), enquanto as fêmeas apresentaram uma taxa de crescimento de 0,24 cm ao ano (IC 95 por cento: ± 0,004), podendo alcançar até 256,1 cm (IC 95 por cento: ± 0,4832) de envergadura. Apesar da equação de von Bertalanffy não ser comumente utilizada para sirênios, e quando aplicada anteriormente não ter obtido resultados satisfatórios, neste estudo apresentou-se adequada para a elaboração da curva de crescimento para peixe-boi-da-Amazônia de vida livre.

Ocorrência de infecção Cryptosporidium spp. em peixe-boi marinho (Trichechus manatus) / Occurrence of Cryptosporidium spp. infection in antillean manatee (Trichechus manatus)

A criptosporidiose constitui-se como uma zoonose que pode afetar o homem e uma ampla variedade de animais domésticos e silvestres, principalmente indivíduos imunodeficientes. O objetivo desse trabalho foi registrar a ocorrência de infecção por Cryptosporidium em peixe-boi marinho. Após ser constatada a mudança de comportamento de um peixe-boi marinho mantido nos oceanários do Centro Mamíferos Aquáticos, ICMBio - FMA, animal foi submetido à exame clínico e, posteriormente, à coleta de amostra fecal. As amostras fecais foram analisadas pela técnica de Kinyoun, teste de imunofluorescência direta e pelo corante 4'.6'-Diamidino-2-Phenilindole (DAPI). No exame clínico, o animal apresentou sinais de desconforto abdominal. Os resultados obtidos nas análises de microscopia de luz e fluorescente revelaram a presença de oocistos de Cryptosporidium nas fezes desse peixe-boi.

Cryptosporidiosis is a zoonosis which can affect man and a wide range of domestic and wild animals, mainly immunodeficient individuals. The objective of this paper was reported the occurrence of a Cryptosporidium infection in Antillean manatee. After an unusual behavior of an Antillean manatee kept in captivity at the Centro Mamíferos Aquáticos, ICMBio - FMA, clinical examination and posterior fecal sampling was performed. Fecal samples were examined by the Kinyoun technique, Direct Immunofluorescence Test and also examined by 4'.6'-Diamidino-2-Phenylindole (DAPI) staining. At the clinical examination, the animal showed signs of abdominal pain. The results obtained by light and fluorescence microscopy analysis showed the presence of Cryptosporidium spp. oocyst in feces of this manatee.