Michael K. Stoskopf

M6P/IGF2R Imprinting Evolution in Mammals

Imprinted gene identification in animals has been limited to eutherian mammals, suggesting a significant role for intrauterine fetal development in the evolution of imprinting. We report herein that M6P/IGF2R is not imprinted in monotremes and does not encode for a receptor that binds IGF2. In contrast, M6P/IGF2R is imprinted in a didelphid marsupial, the opossum, but it strikingly lacks the differentially methylated CpG island in intron 2 postulated to be involved in imprint control. Thus, invasive placentation and gestational fetal growth are not required for imprinted genes to evolve. Unless there was convergent evolution of M6P/ IGF2R imprinting and receptor IGF2 binding in marsupials and eutherians, our results also demonstrate that these two functions evolved in a mammalian clade exclusive of monotremes.

The Kinetics of Oxytetracycline Degradation in Deionized Water under Varying Temperature, pH, Light, Substrate, and Organic Matter

Abstract The kinetics of oxytetracycline (OTC) degradation in deionized water was studied under the influence of various environmental factors. The experiment was conducted with a solution of 10 μg aqueous OTC /mL in 600-mL glass beakers under controlled laboratory conditions. The aqueous concentration of OTC was determined by high-performance liquid chromatography. Low temperatures (4°C) favored high drug stability, and high temperatures (43°C) speeded OTC degradation, resulting in a very short half-life of 0.26 ± 0.11 d. Light exposure caused photodecomposition, reflecting degradation rates threefold higher than those under dark conditions. Acidic conditions (pH 3.0) favored drug stability (half-life = 46.36 ± 4.92 d), and alkaline conditions (pH 10.0) increased the degradation rate (half-life = 9.08 ± 4.22 d). The presence of a substrate (bentonite clay) resulted in an approximate 17% decrease in OTC concentration within 5 min of contact. Addition of organic matter (fish feed) along with the substrate ...

PLASMODIUM RELICTUM AS A CAUSE OF AVIAN MALARIA IN WILD-CAUGHT MAGELLANIC PENGUINS (SPHENISCUS MAGELLANICUS

Avian malaria (Plasmodium relictum) caused significant mortality in wild-caught Magellanic penguins (Spheniscus magellanicus) in 1986 at the Blank Park Zoo in Des Moines, Iowa (USA). In early winter, wild birds were captured off the southern coast of Chile and flown to Detroit, Michigan for a 38 day quarantine. After quarantine, 18 birds were dispersed to Lansing, Michigan, six to a facility in Maine, and 46 to Des Moines, Iowa. Upon arrival in Des Moines, several penguins became weak and inactive, had to be force-fed, and died after 2 days. Gross lesions at postmortem included splenomegaly, hepatomegaly, and pulmonary edema. Histopathological examination revealed numerous intraendothelial schizonts in spleen, lung, liver, heart and kidney. Schizonts were generally 16 to 28 μm by 11 to 16 μm and contained merozoites of two distinct sizes (macromerozoites, nuclei 1.0 μm micromerozoites, nuclei 0.5 μm). Based on the morphology of the abundant exoerythrocytic forms, a tentative diagnosis of avian malaria (Plasmodium sp.) was made. Subsequent transmission electron microscopic examination of schizonts in formalized tissue revealed merozoites with tear-shaped rhoptries. Antimalarial therapy was initiated early but deaths continued for 5 mo. Mortality, which eventually totaled 83%, occurred in three distinct waves, each separated by a hiatus of approximately 1 mo. Despite examinations of repeated blood smears, intraerythrocytic Plasmodium relictum was not detected until late in the outbreak. Diagnosis was based on morphologic characteristics including schizonts with eight to 12 merozoites/segmenter and round gametocytes that displaced and turned the infected erythrocyte nucleus. In addition to malaria, penguins showed evidence of aspergillosis, bacterial enteritis (Escherichia coli; Proteus sp.; and Edwardsiella sp.), and helminthiasis (Contracaecum sp. and Tetrabothrius sp). Based on gross and histological lesions, disease prevalence in this group of penguins was malaria 58%, aspergillosis 61%, enteritis 60%, helminthiasis 26%. Epidemiologic investigation including group transport history, disease prevalence in co-quarantined birds not sent to Des Moines and climatological data implicated Des Moines as the likely site for initial exposure, although information is not conclusive. Stress and concurrent disease certainly contributed to the severe mortality in this group of penguins infected with P. relictum.

Relationship between barnacle epibiotic load and hematologic parameters in loggerhead sea turtles (Caretta caretta), a comparison between migratory and residential animals in Pamlico Sound, North Carolina.

Abstract Health status of a total of 57 loggerhead sea turtles (Caretta caretta; 42 migratory and 15 residential turtles) was analyzed using body condition and hematologic parameters. A subset of 18 juvenile migratory loggerhead sea turtles in the fall of 1997 and 15 residential turtles in the summer of 2000 were analyzed for barnacle epibiota. The migratory group had significantly higher red blood cell counts and percent heterophils and significantly lower percent lymphocyte and absolute eosinophil counts, as well as significantly lower plasma concentrations of calcium, sodium, chloride, potassium, glucose, alkaline phosphatase, and anion gap. Many of these variations may be because of physiology of migration. A positive association between turtle weight and hematocrit was detected and may be because of larger turtles diving for longer periods of time. There were no significant differences of epibiota load, health of the turtles, or condition index between turtles captured during the two events.

Metabolomic Investigations of American Oysters Using 1H-NMR Spectroscopy

The Eastern oyster (Crassostrea virginica) is a useful, robust model marine organism for tissue metabolism studies. Its relatively few organs are easily delineated and there is sufficient understanding of their functions based on classical assays to support interpretation of advanced spectroscopic approaches. Here we apply high-resolution proton nuclear magnetic resonance (1H NMR)-based metabolomic analysis to C. virginica to investigate the differences in the metabolic profile of different organ groups, and magnetic resonance imaging (MRI) to non-invasively identify the well separated organs. Metabolites were identified in perchloric acid extracts of three portions of the oyster containing: (1) adductor muscle, (2) stomach and digestive gland, and (3) mantle and gills. Osmolytes dominated the metabolome in all three organ blocks with decreasing concentration as follows: betaine > taurine > proline > glycine > ß-alanine > hypotaurine. Mitochondrial metabolism appeared most pronounced in the adductor muscle with elevated levels of carnitine facilitating ß-oxidation, and ATP, and phosphoarginine synthesis, while glycogen was elevated in the mantle/gills and stomach/digestive gland. A biochemical schematic is presented that relates metabolites to biochemical pathways correlated with physiological organ functions. This study identifies metabolites and corresponding 1H NMR peak assignments for future NMR-based metabolomic studies in oysters.

ISOLATION OF A RETROVIRUS AND A HERPESVIRUS FROM A CAPTIVE CALIFORNIA SEA LION

A non-oncogenic retrovirus was isolated from an explanted skin biopsy from a captive California sea lion (Zalophus californianus) with a history of recurring skin lesions. The morphology of the viral particles in electron photomicrographs was characteristic of a foamy virus, a retrovirus in the subfamily Spumavirinae. Viral cytopathic effects consistent with foamy virus infection were observed in subsequent explants of skin and lymph nodes and co-cultivated peripheral blood leukocytes. The sea lion with the persistent foamy virus infection later died from pericarditis caused by Pasteurella multocida. A herpesvirus was isolated from explants of lung.

Implementing recovery of the red wolf-integrating research scientists and managers

Abstract The United States Fish and Wildlife Service (USFWS) developed guidelines for the composition and role of endangered species recovery implementation teams, but few teams have been established and their success has not been evaluated. Using the recovery program of the red wolf (Canis rufus) as a model, we describe the genesis, function, and success of the Red Wolf Recovery Implementation Team (RWRIT) in helping guide the establishment of a viable red wolf population in eastern North Carolina. In operation since 1999, the RWRIT meets bi-annually to review USFWS progress and provide recommendations aimed at maximizing success of species recovery. The team is comprised of 8 research scientists from disciplines including population genetics, canid ecology, population ecology, veterinary medicine, and captive management. Representation from each of these disciplines is deemed necessary for proper evaluation of recovery progress and assessment of future needs. Meeting attendance by the USFWS field management team ensures both proper reporting of past progress and future implementation of management recommendations. Over time, RWRIT members have assumed specific assignments or data analyses, further contributing to the recovery effort. Through the combined efforts of the USFWS field team and the RWRIT, the threat of introgression of coyote (Canis latrans) genes into the red wolf population has been substantially curtailed within the recovery area, and red wolf numbers and range have increased. The RWRIT serves as an example of a recovery implementation team that is successfully incorporating the principles of adaptive management and whose template could be adapted to other endangered species.

A gene expression signature of confinement in peripheral blood of red wolves (Canis rufus)

The stresses that animals experience as a result of modification of their ecological circumstances induce physiological changes that leave a signature in profiles of gene expression. We illustrate this concept in a comparison of free range and confined North American red wolves (Canis rufus). Transcription profiling of peripheral blood samples from 13 red wolf individuals in the Alligator River region of North Carolina revealed a strong signal of differentiation. Four hundred eighty‐two out of 2980 transcripts detected on Illumina HumanRef8 oligonucleotide bead arrays were found to differentiate free range and confined wolves at a false discovery rate of 12.8% and P < 0.05. Over‐representation of genes in focal adhesion, insulin signalling, proteasomal, and tryptophan metabolism pathways suggests the activation of pro‐inflammatory and stress responses in confined animals. Consequently, characterization of differential transcript abundance in an accessible tissue such as peripheral blood identifies biomarkers that could be useful in animal management practices and for evaluating the impact of habitat changes on population health, particularly as attention turns to the impact of climate change on physiology and in turn species distributions.

Percutaneous malathion absorption by anuran skin in flow-through diffusion cells.

There is increased concern about the sublethal effects of organophosphorous (OP) compounds on human and animal health, including the potential role of OP compounds in the global decline of amphibian populations. Malathion is one of the most widely used OP pesticides with numerous agricultural and therapeutic applications, and exposure to environmentally applied malathion can lead to adverse systemic effects in anurans. Cutaneous absorption is considered a potentially important route of environmental exposure to OP compounds for amphibians, especially in aquatic environments. One in vitro system commonly used to determine the absorption kinetics of xenobiotics across the skin is the two-compartment Teflon flow-through diffusion cell system. To establish cutaneous absorption kinetics of malathion, six full thickness skin samples taken from both the dorsal and ventral surfaces of each of three bullfrogs (Rana catesbeiana) and three marine toads (Bufo marinus) were placed into two-compartment Teflon flow-through diffusion cells perfused with modified amphibian Ringers solution. A 26μg/cm(2) dose of malathion-2,3-(14)C diluted in 100% ethanol was applied to each sample (0.44-0.45μCi). Perfusate was collected at intervals over a 6h period and analyzed for (14)C in a scintillation counter. At the end of 6h, surface swabs, tape strips, biopsy punches of the dosed area of skin, and peripheral samples were oxidized and analyzed for residue effects. Malathion absorption was greater across the ventral skin compared to dorsal skin in both bullfrogs and marine toads.

A technique for dorsal subcutaneous implantation of heart rate biotelemetry transmitters in black ducks : Application in an aircraft noise response study

A technique for heart rate biotelemetry transmitter implantation was developed to monitor heart rate fluctuations of Black Ducks (Anas rubripes) in response to simulated aircraft noise in a large outdoor enclosure. A dorsal subcutaneous approach, with subcutaneous tunneling of lead wires, was employed for placement of the 32 g transmitters. A base-apex lead configuration, with leads anchored at the dorsal cervico-thoracic junction and the caudal keel, yielded the maximal ECG wave-form deflection for triggering the transmitter. Heart rates of six Black Ducks (three in each of two separate trials) were monitored for 3 days pre-noise to establish a baseline, and then for 4 days of simulated aircraft noise. The noise stimulus replicated an FB-111 military jet, and was played 48 times per day at a peak volume of 110 db. Daily mean heart rates, used as indicators of metabolic rates, did not increase in response to noise. Recognizable acute heart rate increases corresponding with a noise event occured with increased frequency during the first day of noise presentation, but on subsequent days the responses did not differ significantly from baseline. Acute heart rate responses to aircraft noise diminished rapidly, indicating the ability of Black Ducks to habituate to the auditory component of low altitude aircraft overflights.