PERMANENT GENETIC RESOURCES: Eighteen new polymorphic microsatellite markers for the endangered Florida manatee, Trichechus manatus latirostris

Here we describe 18 polymorphic microsatellite loci for Trichechus manatus latirostris (Florida manatee), isolated using a polymerase chain reaction‐based technique. The number of alleles at each locus ranged from two to four (mean = 2.5) in specimens from southwest (n = 58) and northeast (n = 58) Florida. Expected and observed heterozygosities ranged from 0.11 to 0.67 (mean = 0.35) and from 0.02 to 0.78 (mean = 0.34), respectively. Departures from Hardy–Weinberg equilibrium occurred at two loci. There was no evidence of genotypic disequilibrium for any pair of loci. For individual identification, mean random‐mating and θ‐corrected match probabilities were 9.36 × 10^?7 and 1.95 × 10^?6, respectively.     

Detection of hydrodynamic stimuli by the Florida manatee (Trichechus manatus latirostris)

Florida manatees inhabit the coastal and inland waters of the peninsular state. They have little difficulty navigating the turbid waterways, which often contain obstacles that they must circumnavigate. Anatomical and behavioral research suggests that the vibrissae and associated follicle–sinus complexes that manatees possess over their entire body form a sensory array system for detecting hydrodynamic stimuli analogous to the lateral line system of fish. This is consistent with data highlighting that manatees are tactile specialists, evidenced by their specialized facial morphology and use of their vibrissae during feeding and active investigation/manipulation of objects. Two Florida manatees were tested in a go/no-go procedure using a staircase method to assess their ability to detect low-frequency water movement. Hydrodynamic vibrations were created by a sinusoidally oscillating sphere that generated a dipole field at frequencies from 5 to 150 Hz, which are below the apparent functional hearing limit of the manatee. The manatees detected particle displacement of less than 1?μm for frequencies of 15–150 Hz and of less than a nanometer at 150 Hz. Restricting the facial vibrissae with various size mesh openings indicated that the specialized sensory hairs played an important role in the manatee’s exquisite tactile sensitivity.     

Characterization of kidney epithelial cells from the Florida manatee, Trichechus manatus latirostris

Summary The West-Indian manatee,Trichechus manatus latirostris, is a herbivorous marine mammal found in the coastal waters of Florida. Because of their endangered status, animal experimentation is not allowed. Therefore, a cell line was developed and characterized from tissue collected during necropsies of the manatees. A primary cell culture was established by isolating single cells from kidney tissue using both enzymatic and mechanical techniques. Primary manatee kidney (MK) cells were subcultured for characterization. These cells were morphologically similar to the cell lines of epithelial origin. An immunocytochemistry assay was used to localize the cytokeratin filaments common to cells of epithelial origin. At second passage, epithelial-like cells had an average population-doubling time of 48 h, had an optimum seeding density of 5×10³ cells/cm², and readily attached to plastic culture plates with a high level of seeding efficiency. Although the epithelial-like cells had a rapid growth rate during the first three passages, the cloning potential was low. These cells did not form colonies in agar medium, were serum dependent, had a limited life span of approximately nine passages, and possessed cell-contact inhibition. These data suggest that the cells were finite (noncontinuous growth), did not possess transformed properties, and were of epithelial origin. These cells are now referred to as MK epithelial cells.     

Description of the Oocysts of Two New Species of Eimeria (Apicomplexa: Eimerüdae) from the Florida Manatee, Trichechus manatus (Sirenia: Trichechidae)

Two new species of Eimeria (Apicomplexa: Eimerüdae) are described from the feces of the Florida manatee, Trichechus manatus latirostrts (Sirenia: Trichechidae). Oocysts of Eimeria manatus n. sp. are spherical to subsphencal, 11.8 × 10.7 (10.5–13.5 × 9.0–13.5) μ m , with a smooth, thin, bilayered wall; shape index (length/width) 1.1 (1.0–1.3). Micropyle and oocyst residuum absent; polar granule(s) usually present. Sporocysts are ovoid, 8.6 × 5.1 (8.0–9.5 × 5.0–5.5) μm, with thin, membrane–like walls and a knoblike Stieda body; shape index 1.7 (1.4–1.8). Sporozoites elongate, each with a large posterior refractile body. The sporocyst residuum consists of a small cluster or row of few to many small granules. Oocysts of Eimeria nodulosa n. sp. are spherical to subspherical, 15.6 × 14.7 (14.5–17.5 × 13.0–16.0) μm, with a distinctly bilayered wall; shape index 1.1 (1.0–1.2). Unsporulated and freshly sporulated oocysts often possess large, knob–like structures on the external surface of the oocyst wall that support a thin membrane or filament. Micropyle, oocyst residuum and polar granule absent. Sporocysts are ovoid, 10.6 × 5.9 (9.5–12.0 × 5.0–6.5) μm, with a smooth, thin wall and knob–like Stieda body; shape index 1.8 (1.5–2.1). Sporozoites granular and elongate, each with a large posterior refractile body. The sporocyst residuum consists of a loose aggregate or scattered mass of moderately sized granules.     

Composition of the milks of the bottlenose dolphin (Tursiops trucatus) and the Florida manatee (Trichechus manatus latirostris)

Milk samples from four individual bottlenose (Tursiops truncatus) and two Florida manatees (Trichechus manatus latirostris) of known lactation stages were analyzed for protein, carbohydrate and lipid composition, as well as for activity levels of alpha-lactalbumin, the regulatory protein of lactose synthase. The milk from both species had relatively high protein and lipid levels, as reported previously for other marine mammals. The major proportion of the lipid was in the form of triglycerides. Dolphin milk contained an average of 2.2% neutral sugars, which was essentially all in the form of lactose, as determined by several criteria. Manatee milk samples contained 0.6% of neutral sugars, and a larger proportion (about 2%) of amino sugars. Lactose was not detected by enzymatic assay or paper chromatography, but HPLC analysis indicated the presence of low levels of lactose together with two components that were tentatively identified as oligosaccharides. alpha-Lactalbumin activity, determined by assay with bovine galactosyltransferase, was found in both dolphin and manatee milk. The level in dolphin milk was comparable with that found in bovine and other milk, but the level in the manatee was less than 10% of that in the dolphin.     

A PCR assay for gender assignment in dugong (Dugong dugon) and West Indian manatee (Trichechus manatus)

Gender assignment for some aquatic mammals in the field is difficult. Molecular sexing from tissue biopsies is possible as males are heterogametic. Here we describe a multiplex PCR assay that amplifies the male specific SRY gene and differentiates ZFX and ZFY gametologues in two sirenian species, dugong (Dugong dugon) and West Indian manatee (Trichechus manatus). The assay was validated with animals of known gender and proved accurate and robust to experimental failure.     

Helminths of the Florida manatee, Trichechus manatus latirostris, with a discussion and summary of the parasites of sirenians

We examined 215 Florida manatees (Trichechus manatus latirostris) at necropsy to determine the helminth fauna. Six species were identified: Heterocheilus tunicatus (Nematoda: Ascaridoidea); Anoplocephala sp. (Cestoda: Cyclophyllidea); and 4 species of trematodes, Cochleotrema cochleotrema (Digenea: Opisthotrematidae), Chiorchis fabaceus (Digenea: Paramphistomatidae), Nudacotyle undicola (Digenea: Nudacotylidae), and Moniligerum blairi (Digenea: Opisthotrematidae). Seventy-three percent of the manatees examined were infected with at least 1 species of helminth. The mean number of species of helminths per infected manatee was 1.9 with a range of 1-4. Fifty-nine manatees were helminth-free; 30 of these were calves. No associations were found between the intensity of helminth infections and host sex, age class, season, and geographic location of recovery, or cause of death. Differences in parasite prevalence between age classes were highly significant for Chiorchis, Cochleotrema, and Heterocheilus, due to a low number of infected calves. A higher prevalence of Cochleotrema was found in manatees recovered from eastern Florida, and Heterocheilus was evident in significantly more manatees from western and souther Florida. Comparisons in the parasite fauna are made among Florida manatees and other sirenian populations, and a brief review of sirenian parasites is included.     

Seroepidemiology of TmPV1 infection in captive and wild Florida manatees (Trichechus manatus latirostris)

In 1997, cutaneous papillomatosis caused by Florida manatee (Trichechus manatus latirostris [Tm]) papillomavirus 1 (TmPV1) was detected in seven captive manatees at the Homosassa Springs Wildlife State Park, Florida, USA, and, subsequently, in two wild manatees from the adjacent Homosassa River. Since then, papillomatosis has been reported in captive manatees housed in other locations, but not in wild animals. To determine TmPV1 antibody prevalence in captive and wild manatees sampled at various locations throughout Florida coastal regions, virus-like particles, composed of the L1 capsid protein of TmPV1, were generated with a baculovirus expression system and used to measure anti-TmPV1 antibodies in an enzyme-linked immunosorbent assay. Serologic analysis of 156 manatees revealed a TmPV1 antibody prevalence of 26.3%, with no significant difference between captive (n=39) and wild (n=117) manatees (28.2% and 25.6%, respectively). No antibody-positive wild animal showed PV-induced cutaneous lesions, whereas papillomatosis was observed in 72.7% of antibody-positive captive manatees. Our data indicate that Florida manatees living in the wild are naturally infected by TmPV1 but rarely show TmPV1-induced papillomatosis. Hence, it appears that the wild population would not be harmed in a case of contact with captive animals without visible lesions and productive infections, which could be thus released into the wild.     

Development of 51 genomic microsatellite DNA markers of guppy (Poecilia reticulata) and their application in closely related species

Fifty‐one microsatellite DNA markers of guppy (Poecilia reticulata) were developed. All of the markers detected moderate allelic variation. The number of alleles for each locus ranged from two to 10. Observed and expected heterozygosities varied from 0.10 to 0.63 and from 0.23 to 0.77, respectively. Three additional fish species of family Poeciliidae were used for the cross‐species amplification of these markers. It was found that only four to 16 markers detected polymorphism in these three fish species.     

Parentage test in broad-snouted caimans (Caiman latirostris, Crocodylidae) using microsatellite DNA

In this study, microsatellite markers, developed for Alligator mississipiensis and Caiman latirostris, were used to assess parentage among individuals from the captive colony of Caiman latirostris at the University of São Paulo, in Piracicaba, São Paulo, Brazil. Many of the females in the colony were full siblings, which made maternal identification difficult due to genotypic similarity. Even so, the most likely mother could be identified unambiguously among offspring in most of the clutches studied. Two non-parental females displayed maternal behavior which would have misled managers in assigning maternity based on behavior alone. This set of variable loci demonstrates the utility of parentage testing in captive propagation programs.     

Purification and characterization of the major whey proteins from the milks of the bottlenose dolphin (Tursiops truncatus), the Florida manatee (Trichechus manatus latirostris), and the beagle (Canis familiaris)

The major whey proteins of the milks of the dolphin, manatee, and beagle were purified by gel filtration and ion exchange chromatography and characterized and identified by molecular weight determination, amino acid analysis, N-terminal sequencing, and activity measurements. The major whey protein components from all three species were found to be monomeric beta-lactoglobulins. These proteins were all active in binding retinol. Dolphin milk contained two beta-lactoglobulins (designated 1 and 2) which showed a slight difference in molecular weight and considerably divergent N-terminal sequences, whereas the other milks only contained a single form of beta-lactoglobulin. alpha-Lactalbumins were purified from dolphin and dog milks and were active in promoting lactose synthesis by bovine galactosyltransferase. The dolphin protein had an N-terminal sequence more similar to ruminant alpha-lactalbumins than to those known from other species. Although alpha-lactalbumin activity has been detected in manatee milk at low levels, the corresponding protein was not isolated. In addition, dog milk was found to contain high levels of lysozyme (greater than 1.0 mg/ml), which were identified by activity and sequencing. The functional and evolutionary implications of these results are discussed.     

Isolation and characterization of microsatellite markers in the invasive shrub Mahonia aquifolium (Berberidaceae) and their applicability in related species

Microsatellite loci were isolated from a Mahonia aquifolium cultivar. We describe the variability of 10 loci in invasive European and native North American M. aquifolium and their transspecies amplification in native Mahonia repens and Mahonia pinnata from North America and one species of the related genus Berberis (Berberis vulgaris), native to Europe. The markers should be useful to reveal the genetic origin of invasive Mahonia populations and differences in the genetic make up between invasive and native populations.     

Isolation and characterization of 10 microsatellite DNA markers in the oriental honey buzzard (Pernis ptilorhyncus)

We designed 10 new microsatellite markers for the oriental honey buzzard (Pernis ptilorhyncus) and tested them on 44 individuals. The number of alleles ranged from two to 12 with an average of five, while the observed heterozygosity ranged from 0.273 to 1.0. Linkage disequilibrium was not found. These markers are being used in a study of the ecology of this species in Taiwan. We also tested their utility on eight other raptor species.     

Isolation and characterization of microsatellite markers in the gecko Gekko swinhonis and cross‐species amplification in other gekkonid species

Gekko swinhonis is a gekkonid lizard endemic to China, inhabiting Loess Plateau, Huabei Plain, Huanghuai Plain and areas north of the Yangtze River. We characterized 21 dinucleotide microsatellite loci from an AC/AG‐enriched genomic library of G. swinhonis. The number of alleles per locus ranged from eight to 24 and the observed and expected heterozygosities ranged from 0.160 to 0.834 and from 0.584 to 0.917, respectively. We also tested the utility of these loci in other Gekko and Hemidactylus species; many of these loci can be cross‐amplified.     

Isolation and characterization of microsatellite markers in Acca sellowiana (Berg) Burret

Acca sellowiana has commercial potential because of the quality and the unique flavor of its fruit. Conservation of natural populations and management of breeding programmes would benefit from the availability of molecular markers that could be used to characterize levels and distribution of genetic variability. Thus, 13 microsatellite markers were developed from an enriched genomic library of A. sellowiana. They were characterized using 40 samples. The expected and observed heterozygosities ranged from 0.513 to 0.913 and from 0.200 to 0.889, respectively. These are the first microsatellite loci characterized from A. sellowiana that will contribute to improve researches on the genetic conservation, characterization and breeding.     

Isolation and characterization of microsatellite markers in Acca sellowiana (Berg) Burret

Acca sellowiana has commercial potential due to the quality and the unique flavor of its fruit. Conservation of natural populations and management of breeding programmes would benefit from the availability of molecular markers that could be used to characterize levels and distribution of genetic variability. Thus, 13 microsatellite markers were developed from an enriched genomic library of A. sellowiana. They were characterized using 40 samples. The expected and observed heterozygosities ranged from 0.513 to 0.913 and from 0.200 to 0.889, respectively. These are the first microsatellite loci characterized from A. sellowiana that will contribute to improve researches on its genetic conservation, characterization and breeding.     

Isolation and characterization of polymorphic microsatellite markers in Galapagos prickly pear (Opuntia) cactus species

The Opuntia (prickly pear) genus contains over 200 species. Six of them are endemic to the Galapagos archipelago. Although these cacti are ‘keystone’ species of the Galapagos’ semi‐arid ecosystem, they have never been studied in detail. Because of their current threatened status and their important role in the ecosystem, we developed 16 microsatellite markers to study the population genetic structure of some of these species. These markers display a high level of polymorphism with numbers of alleles per locus ranging from six to 53. Results also revealed possible polyploidy in these cacti.     

Erythropoiesis and granulopoiesis in the West Indian manatee, Trichechus manatus (Mammalia: Sirenia)

The West Indian manatee, Trichechus manatus, has amedullary, pachyostotic long bones. Since marrow cavities of the long bones typically are the locations of hemopoiesis in adult mammals, the manatee has evolved an alternative primary site of hemopoiesis. Histological examinations of spleens, livers, kidneys, and vertebral bodies indicate that the last-named location is the main site of erythropoiesis and granulopoiesis in T. manatus. This conclusion is based on the presence in the vertebrae of bone-marrow-containing diagnostic cell types including erythroblasts, megakaryocytes, and myelocytes, which are the precursor cells of erythrocytes, platelets, and granular leukocytes, respectively. No developing lymphocytes were found, so that the location of lymphopoiesis remains unknown.