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.     

Digesta passage rates in the Florida manatee (Trichechus manatus latirostris)

The Florida manatee, Trichechus manatus latirostris (Sirenia: Trichechidae), is an herbivorous marine mammal found within coastal areas throughout the state of Florida, which feeds on both fresh and salt water sea grasses. Manatees, like other Sirenians, are a tropical species with little tolerance for water temperatures below 20°C, rely on a relatively poor nutritional food source, and have a low metabolic rate. Although manatees are hindgut fermenting herbivores, they are very efficient at extracting nutrients from the plants on which they feed. Slow passage rates of digesta have been suggested to be a factor in this increased efficiency. Two studies monitored the digesta passage times and mixing of particulate digesta within the manatee digestive tract using MicroGrits colored corncob grit as a fecal marker. Fecal samples were collected subsequently from four manatees in Study 1 and 3 manatees in Study 2, grit pieces removed, counted, and measured. The digesta passage times ranged from 6 and 10 days in Study 1, and 4.3 and 8.3 days in Study 2, supporting data presented in previous studies. When two different colored markers were administered on sequential days, minimal to no mixing was seen in recovered feces, suggesting that the digesta from a given day traveled through the tract as a bolus. Less than 1% of the marker fed was recovered and we hypothesize that perpendicular folds of the large intestine may be the major contributing factor, with pieces being retained and eventually digested. Zoo Biol 26:503–515, 2007. © 2007 Wiley‐Liss, Inc.     

Tactile discrimination of textures by Florida manatees (Trichechus manatus latirostris)

Two male Florida manatees (Trichechus manatus latirostris) demonstrated sensitive tactile discrimination in a two‐alternative forced choice task, using a modified staircase method. Stimuli were acrylic plates with vertical gratings of ridges and grooves. The standard stimulus, present on every trial, had 2 mm gratings and the comparison stimuli had wider gratings. The blindfolded subjects were trained to demonstrate discrimination by pressing the target with wider gratings. Discrimination thresholds (75% correct) for the subjects were 2.05 mm and 2.15 mm, corresponding to Weber fractions of 0.025 and 0.075, respectively. These results indicate thresholds on similar stimuli comparable to humans (index finger tasks) and better than harbor seals, Phoca vitulina, and the closely related Antillean manatee, Trichechus manatus manatus. Memory for the tactile task was quite stable for both subjects, over 2 yr in the case of one of the subjects. Video analysis of responses indicated that bristle‐like hairs, perioral bristles, and skin on the oral disk were involved in the discrimination response.     

Evaluation of adrenocortical function in Florida manatees (Trichechus manatus latirostris)

The study objectives were to determine the predominant manatee glucocorticoid; validate assays to measure this glucocorticoid and adrenocorticotropic hormone (ACTH); determine diagnostic thresholds to distinguish physiological vs. pathological concentrations; identify differences associated with sex, age class, female reproductive status, capture time, and lactate; and determine the best methods for manatee biologists and clinicians to diagnose stress. Cortisol is the predominant manatee glucocorticoid. IMMULITE 1000 assays for cortisol and ACTH were validated. Precision yielded intra‐ and inter‐assay coefficients of variation for serum cortisol: ≤23.5 and ≤16.7%; and ACTH: ≤6.9 and ≤8.5%. Accuracy resulted in a mean adjusted R²≥0.87 for serum cortisol and ≥0.96 for ACTH. Assay analytical sensitivities for cortisol (0.1 µg/dl) and ACTH (10.0 pg/ml) were verified. Methods were highly correlated with another IMMULITE 1000 for serum cortisol (r=0.97) and ACTH (r=0.98). There was no significant variation in cortisol or ACTH with sex or age class and no correlation with female progesterone concentrations. Cortisol concentrations were highest in unhealthy manatees, chronically stressed by disease or injury. ACTH was greatest in healthy free‐ranging or short‐term rehabilitating individuals, peracutely stressed by capture and handling. Cortisol concentrations ≥1.0 µg/dl were diagnostic of chronic stress; ACTH concentrations ≥87.5 pg/ml were diagnostic of peracute stress. In healthy long‐term captive manatees, cortisol (0.4±0.2 µg/dl) and ACTH (47.7±15.9 pg/ml) concentrations were lower than healthy free‐ranging, short‐term rehabilitated or unhealthy manatees. Capture time was not significantly correlated with cortisol; ACTH correlation was borderline significant. Cortisol and ACTH were positively correlated with lactate. Zoo Biol 30:17–31, 2011. © 2010 Wiley‐Liss, Inc.     

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.     

Polymorphic microsatellite DNA markers for the Florida manatee (Trichechus manatus latirostris)

Florida manatees (Trichechus manatus latirostris) are marine mammals that inhabit the coastal waters and rivers of the southeastern USA, primarily Florida. Previous studies have shown that Florida manatees have low mitochondrial DNA variability, suggesting that nuclear DNA loci are necessary for discriminatory analyses. Here we report 10 polymorphic microsatellite loci with an average of 4.2 alleles per locus, and average heterozygosity of 50.1%. These loci have been developed for use in population studies, parentage assignment, and individual identification.     

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.     

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.     

Health Assessment and Seroepidemiologic Survey of Potential Pathogens in Wild Antillean Manatees (Trichechus manatus manatus)

The Antillean manatee (Trichechus manatus manatus), a subspecies of the West Indian manatee, inhabits fresh, brackish, and warm coastal waters distributed along the eastern border of Central America, the northern coast of South America, and throughout the Wider Caribbean Region. Threatened primarily by human encroachment, poaching, and habitat degradation, Antillean manatees are listed as endangered by the International Union for the Conservation of Nature. The impact of disease on population viability remains unknown in spite of concerns surrounding the species’ ability to rebound from a population crash should an epizootic occur. To gain insight on the baseline health of this subspecies, a total of 191 blood samples were collected opportunistically from wild Antillean manatees in Belize between 1997 and 2009. Hematologic and biochemical reference intervals were established, and antibody prevalence to eight pathogens with zoonotic potential was determined. Age was found to be a significant factor of variation in mean blood values, whereas sex, capture site, and season contributed less to overall differences in parameter values. Negative antibody titers were reported for all pathogens surveyed except for Leptospira bratislava, L. canicola, and L. icterohemorrhagiae, Toxoplasma gondii, and morbillivirus. As part of comprehensive health assessment in manatees from Belize, this study will serve as a benchmark aiding in early disease detection and in the discernment of important epidemiologic patterns in the manatees of this region. Additionally, it will provide some of the initial tools to explore the broader application of manatees as sentinel species of nearshore ecosystem health.     

Systematics of the Gyrosigma balticum complex (Bacillariophyta), including three new species

Three new species in the genus Gyrosigma Hassall (G. caliG. Reid sp. nov., G. gibbyiG. Reid sp. nov. and G. murphyi G. Reid sp. nov) are described. A cladistic analysis on the basis of 10 morphological characters, was undertaken to investigate the interrelationships of these new species within Gyrosigma. As well as ‘standard’ cladistic analysis, which apportions equal weight to all characters, a posteriori differential weighting was also investigated. Pleurosigma subtilis Brébisson is transferred to Gyrosigma, as G. subtile (Brébisson) G. Reid comb. nov., because it is more closely related to other species of Gyrosigma than to Pleurosigma Smith. G. balticum var. californicum Grunow in Cleve and Möller is elevated to specific status [as G. californicum (Grunow in Cleve and Möller) G. Reid stat. nov.], because it does not form a monophyletic group with G. balticum (Ehrenberg) Rabenhorst.     

New species of the diatom genus Fryxelliella (Bacillariophyta), Fryxelliella pacifica sp. nov., from the tropical Mexican Pacific

During phytoplankton monitoring of coasts off Salina Cruz, Oaxaca, in the tropical Mexican Pacific, a new species, Fryxelliella pacifica sp. nov., was found and is described in this paper. The species is solitary, with cells of medium size, discoid with three relatively large ocelli on the valve face, located close to the margins (3–5 areolae from the margins) and placed symmetrically. Significantly, it possesses the morphological characters that distinguish the genus Fryxelliella from related genera: the presence of the 'circumferential marginal tube' (siphon marginalis), the external subcircular or subtriangular apertures at the valve margins, and the 'juxtaposed rectangular plates' in the valve mantle. The species that appears to be the most closely related is Fryxelliella floridana Prasad, an extant species and the type of the genus. However Fryxelliella pacifica differs from it (i) the size and shape of the cell; (ii) the size, location and structure of the ocelli (which additionally are not elevated); (iii) the shape and density of the subcircular to subtriangular marginal apertures; (iv) the external morphology of the rimoportulae (short process, two concentric tubes with the outer tube tip as a crown); and (v) it is marine rather than brackish. Externally the rimoportulae have a rather complex structure of two concentric tubes: the exterior tube has a tip divided like a crown. In spite of the fact this species was found in plankton samples, it is considered to inhabit sandy sediments (epipsammic) or as tychoplanktonic.     

Characterization of a new species in the genus Didymosphenia and of Cymbella janischii (Bacillariophyta) from Connecticut,USA

Two non-native stalk-forming diatoms that were recently observed in the West Branch of the Farmington River, a tributary of the Connecticut River in Connecticut (USA), are characterized morphologically and barcode marker sequences were obtained for each of them. Cymbella janischii, the dominant stalk-forming species during the summer of 2012, previously had not been found in the northeastern USA. Samples of C. janischii were examined microscopically and used to obtain four sequences of the barcode marker, the V4 region of the 18S rDNA gene. Phylogenetic analysis indicated that the four independent sequences of C. janischii were distinct from, but most closely related to, published sequences of C. janischii from Idaho and C. mexicana from Texas, USA. A second non-native stalk-producing diatom, resembling Didymosphenia geminata, was found in November 2012 – June 2013 and first reported as Didymosphenia sp. Over this period, the observed cells had a compressed morphology and were consistently small compared with D. geminata. Sequences of the V4 region, obtained from three independent direct polymerase chain reactions (PCR) of single cells isolated from the Connecticut samples, indicated a close relationship to three published sequences of D. geminata from Italy, New Zealand and the USA, and to D. siberica and D. dentata from Russia. Frustules of the cells used in the PCR reactions were recovered and examined using scanning electron microscopy, providing a direct link between the observed morphology and sequence data. The morphology of the novel Connecticut Didymosphenia taxon was compared with that of other Didymosphenia taxa, being most similar to D. pumila, D. laticollis, D. grunowii and smaller cells of D. geminata. Didymosphenia sp. had a triundulate morphology with a consistent length of 40–60 µm. Given the unique morphological features of this diatom, it is proposed as a new species, Didymosphenia hullii Khan-Bureau, sp. nov.     

Morphological and molecular characteristics of selected species of Minidiscus (Bacillariophyta,Thalassiosiraceae)

We report on taxonomically distinguishing morphological features of six diatom taxa from the marine, nanoplanktonic genus, Minidiscus, found in Eastern Canada and neighbouring waters. Four of the six were also investigated using genetic approaches. We found that the culture deposited in the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP) as CCMP 496 was morphologically closest to the published type image of M. trioculatus, whereas other isolates were morphologically and/or genetically divergent. Consequently, we propose three new taxa. One monoclonal line isolated from the Bay of Fundy is proposed as a new variety, M. trioculatus var. monoculatus. The well-studied nanoplanktonic diatom strain, CCMP 495, currently considered to be ‘M. trioculatus’ was morphologically and genetically divergent from CCMP 496 and we propose specific status for it, M. variabilis. We isolated a new strain from the Gulf of St Lawrence that was nearly genetically identical in 18S and ITS regions to CCMP 495, but showed distinct morphological differences and we propose a new form for this strain. Culture-based data were supplemented by those from natural seawater whenever possible. Two species (M. comicus, M. chilensis) known from our region, but unavailable in culture, are presented for comparison.     

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.     

Characterization of highly informative cross-species microsatellite panels for the Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) including five novel primers

The Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) are threatened species of aquatic mammals in the order Sirenia. Sirenian conservation and management actions would benefit from a more complete understanding of genetic diversity and population structure. Generally, species-specific microsatellite markers are employed in conservation genetic studies; however, robust markers can be difficult and costly to isolate. To increase the number of available markers, dugong and manatee microsatellite primers were evaluated for cross-species amplification. Furthermore, one manatee and four dugong novel primers are reported. After polymerase chain reaction optimization, 23 (92%) manatee primers successfully amplified dugong DNA, of which 11 (48%) were polymorphic. Of the 32 dugong primers tested, 27 (84%) yielded product in the manatee, of which 17 (63%) were polymorphic. Dugong and manatee primers were compared and the most informative markers were selected to create robust and informative marker-panels for each species. These cross-species microsatellite marker-panels can be employed to assess other sirenian populations and can provide beneficial information for the protection and management of these unique mammals.     

Four new species of Pleurosigma (Bacillariophyta) from Alexandria, Egypt

Four new species of Pleurosigma are described from the region of Alexandria in Egypt: P. williamsii sp. nov. , P. mariutensis sp. nov. , P. ramlehensis sp. nov. and P. rushdyensis sp. nov. The taxa had been previously misidentified as P. rigidum W. Sm., P. elongatum W. Sm., P. speciosum var. sumatricum H. Perag. and P. formosum W. Sm., respectively. Due to the close resemblance of P. williamsii, P. mariutensis , P. ramlehensis and P. rushdyensis to P. rigidum , P. elongatum , P. speciosum var. sumatricum and P. formosum and the lack of good illustrations of these previously described species, their valvar features are shown and discussed in relation to the new taxa. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 140 , 77–92.     

The systematics of Seminavis (Bacillariophyta) : the lost identities of Amphora angusta,A. ventricosa and A. macilenta

Amphora angusta Gregory, Amphora ventricosa Gregory and Amphora macilenta Gregory have been misunderstood for the last 140 years. Gregory described these species from sublittoral sediments off the west coast of Scotland. Although the illustrations were excellent for their time (1857), it is impossible to be confident of identification using Gregory's paper, and subsequent authors have created such confusion that records of these taxa must be regarded as untrustworthy unless backed by photographic evidence. In this paper, the first to describe in detail the recently established genus Seminavis, we show that A. angusta is in fact a species of Amphora Ehrenberg, whereas A. ventricosa sensu Gregory represents two independent species of the genus Seminavis D. G. Mann, namely Seminavis ventricosa (Gregory) Garcia-Baptista (non S. ventricosa sensu Garcia-Baptista 1993) and Seminavis arranensis Danielidis & D. G. Mann, sp. nov. The form usually known as A. ventricosa is neither of these and requires a new name, Seminavis robusta Danielidis & D. G. Mann, sp. nov. The long forgotten and misclassified species A. macilenta Gregory is shown to be yet another Seminavis species, requiring a new combination as S. macilenta (Gregory) Danielidis & D. G. Mann, comb. nov.; contrary to previous claims, it is separate from A. ergadensis Gregory, which is a true Amphora. Valve and girdle ultrastructure, plastid arrangement and auxosporulation are described for Seminavis and resemble those in the Naviculaceae sensu stricto.