Bel Air South, MD, United States
Bel Air South, MD, United States

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Zhang H.,University of Connecticut | Campbell D.A.,University of California at Los Angeles | Sturm N.R.,University of California at Los Angeles | Dungan C.F.,Cooperative Oxford Laboratory | Lin S.,University of Connecticut
PLoS ONE | Year: 2011

The genus Perkinsus occupies a precarious phylogenetic position. To gain a better understanding of the relationship between perkinsids, dinoflagellates and other alveolates, we analyzed the nuclear-encoded spliced-leader (SL) RNA and mitochondrial genes, intron prevalence, and multi-protein phylogenies. In contrast to the canonical 22-nt SL found in dinoflagellates (DinoSL), P. marinus has a shorter (21-nt) and a longer (22-nt) SL with slightly different sequences than DinoSL. The major SL RNA transcripts range in size between 80-83 nt in P. marinus, and ∼83 nt in P. chesapeaki, significantly larger than the typical ≤56-nt dinoflagellate SL RNA. In most of the phylogenetic trees based on 41 predicted protein sequences, P. marinus branched at the base of the dinoflagellate clade that included the ancient taxa Oxyrrhis and Amoebophrya, sister to the clade of apicomplexans, and in some cases clustered with apicomplexans as a sister to the dinoflagellate clade. Of 104 Perkinsus spp. genes examined 69.2% had introns, a higher intron prevalence than in dinoflagellates. Examination of Perkinsus spp. mitochondrial cytochrome B and cytochrome C oxidase subunit I genes and their cDNAs revealed no mRNA editing, but these transcripts can only be translated when frameshifts are introduced at every AGG and CCC codon as if AGGY codes for glycine and CCCCU for proline. These results, along with the presence of the numerous uncharacterized 'marine alveolate group I' and Perkinsus-like lineages separating perkinsids from core dinoflagellates, expand support for the affiliation of the genus Perkinsus with an independent lineage (Perkinsozoa) positioned between the phyla of Apicomplexa and Dinoflagellata. © 2011 Zhang et al.


Dungan C.F.,Cooperative Oxford Laboratory | Bushek D.,Rutgers University
Journal of Invertebrate Pathology | Year: 2015

During the early 1950s, Sammy M. Ray discovered that his high-salt modification of fluid thioglycollate sterility test medium caused dramatic in vitro enlargement of Perkinsus marinus (=. Dermocystidium marinum) cells that coincidentally infected several experimentally cultured oyster gill tissue explants. Subsequent testing confirmed that the enlarged cells among some oyster tissues incubated in Ray's fluid thioglycollate medium (RFTM) were those of that newly described oyster pathogen. Non-proliferative in vitro enlargement, cell wall thickening, and subsequent blue-black iodine-staining of hypertrophied trophozoites (=hypnospores. =. prezoosporangia) following incubation in RFTM are unique characteristics of confirmed members of the protistan genus Perkinsus. A number of in vitro assays and manipulations with RFTM have been developed for selective detection and enumeration of Perkinsus sp. cells in tissues of infected molluscs, and in environmental samples. RFTM-enlarged Perkinsus sp. cells from tissues of infected molluscs also serve as useful inocula for initiating in vitro isolate cultures, and cells of several Perkinsus spp. from both in vitro cultures and infected mollusc tissues may be induced to zoosporulate by brief incubations in RFTM. DNAs from RFTM-enlarged Perkinsus sp. cells provide useful templates for PCR amplifications, and for sequencing and other assays to differentiate and identify the detected Perkinsus species. We review the history and components of fluid thioglycollate and RFTM media, and the characteristics of numerous RFTM-based diagnostic assays that have been developed and used worldwide since 1952 for detection and identification of Perkinsus spp. in host mollusc tissues and environmental samples. We also review applications of RFTM for in vitro manipulations and purifications of Perkinsus sp. pathogen cells. © 2015 Elsevier Inc.


Matsche M.A.,Cooperative Oxford Laboratory | Bakal R.S.,U.S. Fish and Wildlife Service | Rosemary K.M.,Cooperative Oxford Laboratory
Journal of Applied Ichthyology | Year: 2011

Sex and reproductive maturity of Atlantic and shortnose sturgeon were determined by visual examination of the gonads using laparoscopy, and were validated by histological examination of gonadal biopsies. Surgical anesthesia was induced in all fish with 250mgL-1 tricaine methanesulfonate (MS-222) and maintained throughout procedures with 85mgL-1 MS-222 on a mobile surgical cart. A pair of Ternamian EndoTip cannulae installed through the ventral body wall in each fish, allowed access for a 5-mm rigid laparoscope and biopsy forceps. Video endocamera use with the laparoscope, following air insufflation of the coelom, provided detailed, high quality imagery to aspirate the swim bladder, examine the gonad and collect biopsies without inducing iatrogenic trauma. Germinal tissue of all immature males, 25% of immature female shortnose sturgeon and 45% of immature female Atlantic sturgeon were concealed by fat preventing sex determination by visual assessment. Morphological features of gonads were used to determine sex in all remaining fish and were 100% in concordance with histological findings. Relative amount of gonadal fat; gonad size and color; presence of testicular lobes or ovarian lamellae; and color, size and density of oocytes were useful in determining reproductive stage. Gonad morphology of each reproductive stage was similar in Atlantic and shortnose sturgeon. All captive Atlantic sturgeon survived laparoscopy, gained weight at the same rate as unexamined fish and scars from incisions were no longer evident 9-12months after surgery. Laparoscopic procedures presented here offer a safe and highly reliable way to determine sex and reproductive status for Atlantic and shortnose sturgeon. © 2011 Blackwell Verlag, Berlin.


Blazer V.S.,U.S. Geological Survey | Pinkney A.E.,U.S. Fish and Wildlife Service | Jenkins J.A.,U.S. Geological Survey | Iwanowicz L.R.,U.S. Geological Survey | And 3 more authors.
Science of the Total Environment | Year: 2013

Reduced recruitment of yellow perch has been noted for a number of years in certain urbanized watersheds (South and Severn Rivers) of the Chesapeake Bay. Other rapidly developing watersheds such as Mattawoman Creek are more recently showing evidence of reduced recruitment of anadromous fishes. In this study, we used a battery of biomarkers to better document the reproductive health of adult yellow perch collected during spring spawning in 2007-2009. Perch were collected in the South and Severn Rivers, Mattawoman Creek and the less developed Choptank and Allen's Fresh watersheds for comparison. Gonadosomatic indices, plasma reproductive hormone concentrations, plasma vitellogenin concentrations and gonad histology were evaluated in mature perch of both sexes. In addition, sperm quantity (cell counts) and quality (total and progressive motility, spermatogenic stage and DNA integrity), were measured in male perch. Many of these biomarkers varied annually and spatially, with some interesting statistical results and trends. Male perch from the Choptank and Allen's Fresh had generally higher sperm counts. In 2008 counts were significantly lower in the perch from the Severn when compared to other sites. The major microscopic gonadal abnormality in males was the proliferation of putative Leydig cells, observed in testes from Severn and less commonly, Mattawoman Creek perch. Observations that could significantly impact egg viability were an apparent lack of final maturation, abnormal yolk and thin, irregular zona pellucida. These were observed primarily in ovaries from Severn, South and less commonly Mattawoman Creek perch. The potential association of these observations with urbanization, impervious surface and chemical contaminants is discussed. © 2012.


Bronson E.,Maryland Zoo in Baltimore | Spiker H.,Wildlife and Heritage Service | Driscoll C.P.,Cooperative Oxford Laboratory
Journal of Wildlife Diseases | Year: 2014

American black bears (Ursus americanus) in Maryland, USA, live in forested areas in close proximity to humans and their domestic pets. From 1999 to 2011, we collected 84 serum samples from 63 black bears (18 males; 45 females) in five Maryland counties and tested them for exposure to infectious, including zoonotic, pathogens. A large portion of the bears had antibody to canine distemper virus and Toxoplasma gondii, many at high titers. Prevalences of antibodies to zoonotic agents such as rabies virus and to infectious agents of carnivores including canine adenovirus and canine parvovirus were lower. Bears also had antibodies to vector-borne pathogens common to bears and humans such as West Nile virus, Borrelia burgdorferi, Rickettsia rickettsii, and Anaplasma phagocytophilum. Antibodies were detected to Leptospira interrogans serovars Pomona, Icterohaemorrhagiae, Canicola, Grippotyphosa, and Bratislava. We did not detect antibodies to Brucella canis or Ehrlichia canis. Although this population of Maryland black bears demonstrated exposure to multiple pathogens of concern for humans and domesticated animals, the low levels of clinical disease in this and other free-ranging black bear populations indicate the black bear is likely a spillover host for the majority of pathogens studied. Nevertheless, bear populations living at the human–domestic-wildlife interface with increasing human and domestic animal exposure should continue to be monitored because this population likely serves as a useful sentinel of ecosystem health. © Wildlife Disease Association 2014.


Matsche M.A.,Cooperative Oxford Laboratory | Gibbons J.,South Carolina Department of Natural Resources
Fish Physiology and Biochemistry | Year: 2012

Shortnose sturgeon (Acipenser brevirostrum) spawning migrations on the Cooper River are impeded by Pinopolis Dam, Lake Moultrie, South Carolina. Sturgeon and other species aggregate below the dam in late winter/early spring and are subjected to a variety of stressors stemming from crowding, poor habitat quality, and injuries that appear to be caused by boat propeller or turbine strikes. Spawning has been documented in the tailrace, but reproductive success has not been verified as no juveniles have been captured. Fish within the dam tailrace were captured by gill net during winter, 2005 and 2007-2011, and physiological condition was assessed using a panel of hematologic and biochemical indices. Plasma phosphorus and calcium were significantly higher in females, while PCV and aspartate aminotransferase were significantly higher in males, indicating sex-specific physiological changes triggered during maturity. A marked leucopenia, accompanied by lymphopenia and neutrophilia, was evident in both sexes and was consistent across years, indicating that these fish were under chronic stress. Testosterone and estradiol levels and hematologic and biochemical reference intervals are provided for comparative purposes. © 2012 Springer Science+Business Media B.V.


Kimmel D.G.,University of Cambridge | Boicourt W.C.,University of Cambridge | Pierson J.J.,University of Cambridge | Roman M.R.,University of Cambridge | Zhang X.,Cooperative Oxford Laboratory
Journal of Plankton Research | Year: 2010

The biological response of mesozooplankton (250-2000 m size range) to hypoxia in the northern Gulf of Mexico was investigated using an optical plankton counter (OPC) and a high-capacity pump. We sampled the water column in an area with the most persistent occurrence of hypoxia every 4 h for a 24-h period in both years. The amount of hypoxic bottom water differed between 2006 and 2007, with 2006 having more widespread bottom hypoxia than 2007. Large-sized mesozooplankton (>1000 m) were more abundant in 2006 and were found in greater abundance and biomass in hypoxic water. Small- (250-500 m) and mid-sized (500-1000 m) mesozooplankton showed diel variability, but did not appear to respond to hypoxia. The opposite pattern was observed in 2007, where smaller-sized mesozooplankton were dominant and diel variability in this size class was not detected, whereas large- and mid-sized mesozooplankton did show evidence of diel variability in 2007. Using a vital staining technique (neutral red), we found a significantly higher proportion of stained mesozooplankton in oxic, surface waters compared with deep, hypoxic waters. These findings show that mesozooplankton vertical and diel distributions differed between a year with widespread, bottom hypoxia and a year with a thin layer of hypoxic water. It remains unclear as to what is driving these size differences, the direct impact of hypoxia on mesozooplankton individual or egg mortality, differential predation in the water column or other factors such as more nutrient input related to increases in zooplankton production. © The Author 2010.


Matsche M.A.,Cooperative Oxford Laboratory
Journal of Applied Ichthyology | Year: 2011

Dose-response experiments were conducted at 14 and 24°C to evaluate the efficacy and physiological effects of tricaine methanesulfonate (MS-222) to induce and maintain surgical anesthesia in juvenile Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus). Anesthetic induction time, duration of hyperactivity, recovery time and total handling time of fish were inversely related to MS-222 concentration and water temperature. Minimum effective concentration of MS-222 to maintain anesthesia with fewest signs of stress was 85mgL-1. Sensitivity to stimuli and body movements progressively increased when fish were exposed to a lower maintenance concentration (70mgL-1) of MS-222, resulting in reduced biopsy success rates and traumatic injury to internal organs during laparoscopy as fish regained consciousness. Anesthesia with MS-222 resulted in bradychardia, near medullary collapse, elevated signs of stress (plasma cortisol and reddening of the skin) and a generalized hemo-concentration consisting of erythrocyte swelling and increased protein and monovalent ion concentrations. Magnitude of hematologic changes and stress indicators increased with decreasing MS-222 concentration and increased water temperature while plasma chemistry changes increased in magnitude with decreasing MS-222 concentration. This study demonstrates that rapid induction of surgical anesthesia with a relatively high concentration of MS-222 results in reduced signs of physiological stress, and that empirical evaluation of maintenance dosage is important to achieve the best balance between safety, efficacy and stressful side effects for invasive surgical procedures. © 2011 Blackwell Verlag, Berlin.


Two methods for colorimetric in situ DNA probe hybridization (CISH) assays on paraffin-embedded tissue sections were compared. The heated method used heat (90-100°C) to denature DNA in the sample prior to probe hybridization, while the unheated method used a standard hybridization temperature of 42°C. Both procedures were tested on tissue samples that harbored the mollusk protozoan pathogens Perkinsus marinus, P. chesapeaki, or Haplosporidium nelsoni, the protozoan and bacterial fish pathogens Myxobolus cerebralis (myxosporidean) or Renibacterium salmoninarum (bacterial), or the crab viral pathogen Callinectes sapidus reovirus. Samples were fixed in either formalin or Davidson's fixative and embedded in paraffin for histological examination. The heated method is labor intensive and highly prone to human error, while the unheated method is less labor intensive and can be completed in a shorter period of time. Both methods yielded similar hybridization results. The use of complex and expensive prehybridization buffers did not improve the performances of the tested CISH assays. Prehybridization heat denaturation of DNA in assayed samples increased both assay duration and loss of samples but did not improve hybridization signals.


A portable electro-immobilization and laparoscopy system is described that is suitable for sex determination, gonadal biopsy and immediate release of largemouth bass Micropterus salmoides. Continuous direct current at a power density of 52·2μWcm-1 for 2min was sufficient to immobilize fish for surgery, but induced a mild, transient hypokalaemia and hyperglycaemia. Insertion of a 4·8mm laparoscopic instrument set through the urogenital pore provided access to the gonads for examination and biopsy with mild tissue trauma. © 2013 The Fisheries Society of the British Isles.

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