Copyright (c) 2013 University of Rhode Island All rights reserved. http://digitalcommons.uri.edu/favs_facpubs Recent documents in Fisheries, Animal and Veterinary Sciences Faculty Publications en-us Mon, 20 May 2013 09:10:39 PDT 3600 http://digitalcommons.uri.edu/favs_facpubs/15 http://digitalcommons.uri.edu/favs_facpubs/15 Fri, 08 Mar 2013 11:30:37 PST Two antimicrobial peptides (AMPs) of marine origin, tachyplesin from the Japanese horseshoe crab, Tachypleus tridentatus, and pleurocidin-amide from the winter flounder, Pseudopleuronectes americanus, were tested for their potential effectiveness in disease treatment in oyster aquaculture. Tachyplesin had a greater antimicrobial effect than pleurocidin-amideagainst a range of gram-positive and gram-negative bacteria, with minimum inhibitor concentrations in the range of 0.625 to 5ug mL^–1. Tachyplesin (50 mg mL^–1) was also more effective than pleurocidin-amide (250 mg mL^–1) against the oyster protozoan parasite Perkinsus marinus, reducing its viability to 9% versus 22% in vitro. Both peptides were unaffected by variations in pH and salinity that would be encountered in marine culture conditions. Candidate algal and yeast for expression and feed-based delivery of recombinant AMPs were largely unaffected by tachyplesin and pleurocidin at concentrations that inhibited bacterial growth but were sensitive to concentrations that reduced the viability of P. marinus. Several proteases, including those of oyster and parasitic origin, decreased AMP activity, but tachyplesin was affected to a much lesser degree than pleurocidin-amide. Coincubation of homogenates from oyster digestive tissues with tachyplesin and pleurocidin-amide was found to reduce the abundance of colony forming units in the tissue. Tachyplesin was more effective against gram-negative bacteria present in oyster tissues, whereas pleurocidin-amide was more effective against gram-positive bacteria. Tachyplesin was considered a better candidate than pleurocidin-amide for feed-based delivery applications in oyster aquaculture.

]]> Tarquin Dorrington et al. http://digitalcommons.uri.edu/favs_facpubs/14 http://digitalcommons.uri.edu/favs_facpubs/14 Fri, 08 Mar 2013 11:30:36 PST Tissue-associated bacterial community profiles generated using a nested polymerase chain reaction–denaturing gradient gel electrophoresis (DGGE) approach and culture-dependent and culture-independent isolation techniques were compared. Oyster samples were collected from 2 harvest areas along the coast of Maine, in the United States. Profiles from both isolation strategies were evaluated using Sorensen’s index of similarity and cluster analysis of gel banding patterns. Cultureindependent profiles were further evaluated using the Shannon diversity index. In general, the culture-dependent strategy resulted in a greater number of bands within a profile. BacterialDGGEprofiles were found to be highly similar within an isolation strategy, with a higher degree of unrelatedness between culture-dependent and -independent techniques. Cluster analysis identified bands present in the culture-dependent strategy and not the total DNA technique, and vice versa. Significant differences in community profiles between oyster-associated and seawater were observed, indicating a diverse group of specialist bacterial species inhabit and are able to proliferate within the oyster.

]]> Kenneth J. LA VALLEY et al. http://digitalcommons.uri.edu/favs_facpubs/13 http://digitalcommons.uri.edu/favs_facpubs/13 Fri, 08 Mar 2013 11:30:35 PST The economically important marine bivalve mollusc, Mercenaria mercenaria, (commonly called a northern quahog or hard clam), has endured considerable mortalities caused by a thraustochytrid pathogen called Quahog Parasite X (QPX). Data on the percent prevalence of QPX infections were compiled from published reports along with our data to describe the epizootiology of QPX disease.QPX infections occurred in clams collected from both cultured beds and wild populations, but a higher percentage of QPX cases (76.5%) were from cultured clam beds. In addition, samples from cultured beds had a significantly higher prevalence (29.2 ± 27.2%) of QPX infections compared with samples from wild populations (9.6 ± 9.6%). The highest prevalence of QPX infections occurred in clams from samples with an intermediate size range (shell lengths 20–55 mm). QPX infections occurred in both male and female clams, but infection prevalence does not appear to be correlated with sex or sex ratios. The geographical range of QPX-related clam mortalities was Atlantic Canada to the Eastern Shore of Virginia, USA. Only marginally significant differences were detected between the prevalence of QPX at different locations. There were no latitudinal gradients in QPX prevalence or frequencies, suggesting local factors were important in determining its distribution. Although QPX infections occurred throughout the year, no seasonal trends in the prevalence or frequencies of QPX were discernable. This summary of information available on QPX disease highlights the need for more thorough data collection regarding factors believed to be associated with its presence and severity in hard clams.

]]> M. MAILLE LYONS et al. http://digitalcommons.uri.edu/favs_facpubs/12 http://digitalcommons.uri.edu/favs_facpubs/12 Fri, 08 Mar 2013 11:30:33 PST Perkinsus marinus is responsible for disease and mortality of the American oyster, Crassostrea virginica. To inves-tigate the interactions between P. marinus and oyster hemocytes, protease activity was measured in plasma of oysters collected 4 hr, 24 hr, 4 days, and 2 mo after experimental infection with P. marinus. A significant increase in protease activity was observed in oyster plasma 4 hr after injection with P. marinus, followed by a sharp decrease within 24 hr. Gelatin-impregnated gel electrophoresis showed the presence of 2 major bands (60 and 112 kDa) and 3 less prevalent bands (35, 92, and 200 kDa) with metalloproteinaselike activity in the plasma of noninfected oysters. Additional bands in the 40- to 60-kDa range, corresponding to P. marinus serine proteases, were observed in oyster plasma at early time points after infection. A transient, but significant, decrease in the activity of oyster metalloproteinases was observed at early time points after infection. Coincubation of oyster plasma with P. marinus extracellular products resulted in a decrease in oyster metalloproteinases and several P. marinus proteases. This study provides insights into the role of proteases in the pathogenesis of Dermo disease.

]]> P. Munoz et al. http://digitalcommons.uri.edu/favs_facpubs/11 http://digitalcommons.uri.edu/favs_facpubs/11 Fri, 08 Mar 2013 11:30:32 PST Species of Perkinsus are responsible for high mortalities of bivalve molluscs world-wide. Techniques to accurately estimate parasites in tissues are required to improve understanding of perkinsosis. This study quantifies the number and tissue distribution of Perkinsus marinus in Crassostrea virginica by modern stereology and immunohistochemistry. Mean total number of trophozoites were (mean ± SE) 11.80 ± 3.91 million and 11.55 ± 3.88 million for the optical disector and optical fractionator methods, respectively. The mean empirical error between both stereological approaches was 3.8 ± 1.0%. Trophozoites were detected intracellularly in the following tissues: intestine (30.1%), Leydig tissue (21.3%), hemocytes (14.9%), digestive gland (11.4%), gills (6.1%), connective tissues (5.7%), gonads (4.1%), palps (2.2%), muscle (1.9%), mantle connective (0.8%), peri-cardium (0.7%), mantle epithelium (0.1%), and heart (0.1%). The remaining 0.6% were found extracellularly. Percentages of trophozoite stages were (mean ± SE): large, log-phase trophonts, i.e., signet rings, 97.0 ± 1.2%; meronts, 2.0 ± 0.9%; clusters of small, log-phase trophonts, i.e., merozoites, 1.0 ± 0.5%. Levels of infection in hemocytes and Leydig tissue were representative of total parasite intensity. These techniques are a powerful tool to follow parasite distribution and invasion, and to further explore mechanisms of Perkinsus spp. pathogenesis in bivalves.

]]> Antonio Remacha-Trivino et al. http://digitalcommons.uri.edu/favs_facpubs/10 http://digitalcommons.uri.edu/favs_facpubs/10 Mon, 25 Feb 2013 11:35:24 PST We compared 2 methods of direct gene delivery into live rainbow trout Oncorhynchus mykiss tissue, with the final goal of developing DNA-based vaccines for bacterial diseases in salmonids. The introduction of plasmid constructs containing the luciferase and β-galactosidase reporter genes was achieved either by direct injection or by particle bombardment with DNA-coated gold microparticles. Luciferase expression was observed in homogenates of trout flank muscle and skin 2 d after injection of 10 to 100 pg of DNA per fish or bombardment of 1 pm gold particles coated with 5 to 25 pg DNA per fish at helium pressures ranging from 2750 to 12 400 kPa. Expression levels increased over 10 d and persisted for at least 60 d after injection. For particle bombardment, the most reproducible levels of luciferase expression were obtained with the eye as a target (83% of fish positive versus 59% of fish positive when the flank was the target). The levels of luciferase expression observed after particle bombardment were significantly lower (t-test, p < 0.02) than the levels measured after direct injection. Immunohistochemical analysis indicated β-galactosidase gene expression in muscle cells at the site of injection and in the dermis, epidermis and muscle after bombardment. These 2 methods may prove valuable for the development of a new generation of DNA-based vaccines for fish.

]]> Marta Gomez-Chiarri et al. http://digitalcommons.uri.edu/favs_facpubs/9 http://digitalcommons.uri.edu/favs_facpubs/9 Fri, 22 Feb 2013 08:05:19 PST To determine if population density in areas closed to fishing in Narragansett Bay is causing differences in the reproductive potential of the organisms, this study used two approaches to determine the reproductive condition of the animals. The first approach consisted in employing a gravimetric condition index (CI) to evaluate the general condition of quahogs from nine different sites, 3 sites open conditionally for fishing (conditional areas) and 6 sites closed to fishing. The second approach was a determination of gonadal index (GI) of a subset of the sample sites, by histological observation of gonadal tissue sections. Initial sampling included determination of CI only, and lasted from March 25 to Sep. 22, 2005. The subset of six of the sites was sampled the next year (2006), every three weeks from April 15 until Sep. 28 to determine CI and the gonadal index (GI). Results show that there is a significant difference between the CI of northern quahogs from conditional areas and quahogs from closed areas (P

]]> DORA C. MARROQUIN-MORA et al. http://digitalcommons.uri.edu/favs_facpubs/8 http://digitalcommons.uri.edu/favs_facpubs/8 Fri, 22 Feb 2013 08:05:16 PST The relationship between the somatic growth rate (G) and feeding level (unfed, intermediate, and maximum rations) of age-0 juvenile cod Gadus morhua and haddock Melanogrammus aeglefinus was quantified at different temperatures. Laboratory trials were conducted using 2 sizeclasses of cod (3.6 to 5.6 cm standard length [SL], and 8.1 to 12.4 cm SL) at 5, 8, 12, and 15°C, and 1 size-class of haddock (6.0 to 9.6 cm SL) at 8 and 12°C. The shape of the growth-feeding relationship was well described by a 3-parameter asymptotic function for cod and by a linear function for haddock (R² range = 0.837 to 0.966). The growth rate and scope for growth were maximum at 12°C, whereas growth efficiency was greatest (26.0 to 32.2%) at temperatures between 5 and 8°C. Juvenile cod held at 15°C exhibited reduced rates and efficiencies of somatic growth compared to fish at other temperatures. Biochemical-based growth indicators for age-0 juveniles were calibrated from measurements of the amounts of RNA, DNA, and protein in white muscle samples. A multiple linear regression using RNA:DNA and temperature as independent variables explained a significant portion of the variability observed in G of juvenile cod (R² = 0.716) and haddock (R² = 0.637). This relationship may be useful in estimating recent growth of age-0 juvenile cod and haddock in the field.

]]> Myron Arms Peck et al. http://digitalcommons.uri.edu/favs_facpubs/7 http://digitalcommons.uri.edu/favs_facpubs/7 Fri, 15 Feb 2013 09:25:22 PST The advent of an aquaculture industry for summer flounder Paralichthys dentatus requires that optimal methods be identified for hatchery production. Two experiments were conducted to test strategies for larval rearing and for weaning newly metamorphosed juveniles from live to artificial diets. Rearing of larvae in ‘‘green water’’ (with algae added) resulted in better survival (76.1 6 6.5%) from days 5–42 after hatching than did rearing in ‘‘clear water’’ (no algae added; 27.8 6 13.6%), although no differences in growth were apparent. When fish were weaned from live feed beginning at day 45 versus day 57 by either a ‘‘gradual’’ method (7-d weaning period) or an ‘‘immediate’’ method (no weaning period), better survival and growth were obtained with fish weaned at the later age. For both agegroups, fish weaned by the gradual method exhibited better growth, but not better survival, than those weaned by the immediate method. With these data as examples, commercial hatcheries can conduct cost : benefit analyses of the different rearing methods.

]]> David A. Bengtson et al. http://digitalcommons.uri.edu/favs_facpubs/6 http://digitalcommons.uri.edu/favs_facpubs/6 Fri, 15 Feb 2013 09:25:21 PST
Significant differences in growth and prespawning body morphology were detected among three stocks of Atlantic salmon reared in a common marine environment. Smolts originating from river-specific broodstock of the Machias, East Machias, and Dennys populations were reared at two marine net-pen facilities for 25 months. Significant differences in stock-specific growth were observed among two stocks at both sites, suggesting a genetic basis for the observed phenotypic variation. There was a significant stock effect to the total measured phenotypic variation based on collected truss network analyses. Linear discriminant function analysis of a truss network of morphometric distances allowed for 73% accuracy of stock classification. A thin-plate spline procedure characterized the Machias body form as having a shortened narrow caudal peduncle region, a compressed body with an elongated trunk, and a deeper head region relative to the other two stocks. Phenotypic variation may be associated with hydrological characteristics of the Machias watershed.

]]> Timothy F. Sheehan et al. http://digitalcommons.uri.edu/favs_facpubs/5 http://digitalcommons.uri.edu/favs_facpubs/5 Fri, 15 Feb 2013 09:25:18 PST Larval summer flounder Paralichthys dentatus were hatched and reared through metamorphosis in the laboratory. At several points in the rearing cycle, larvae were removed from their rearing chambers and placed in small bowls, where they were fed known quantities of the rotifer Brachionus plicatilis (flounder larvae age 6, 10, or 13 d posthatch) or nauplii of brine shrimp Artemia sp. (flounder larvae age 23, 28, 34, or 47 d posthatch). Counts of prey organisms remaining in the bowls after 24 h allowed us to calculate numbers of prey consumed. Consumption rates increased from 62 rotifers/24 h (6-d larvae) to 301 rotifers/24 h (13-d larvae) and from 59 brine shrimp/24 h (23-d larvae) to 394 brine shrimp/24 h (47-d larvae). These rates can be used to calculate the approximate rotifer and brine shrimp production necessary for rearing summer flounder through metamorphosis in aquaculture hatcheries.

]]> David A. Bengtson et al. http://digitalcommons.uri.edu/favs_facpubs/4 http://digitalcommons.uri.edu/favs_facpubs/4 Tue, 31 Jul 2012 12:01:41 PDT The elemental composition of otoliths may provide valuable information for establishing connectivity between fish nursery grounds and adult fish populations. Concentrations of Rb, Mg, Ca, Mn, Sr, Na, K, Sr, Pb, and Ba were determined by using solution-based inductively coupled plasma mass spectrometry in otoliths of young-of-the year tautog (Tautoga onitis) captured in nursery areas along the Rhode Island coast during two consecutive years. Stable oxygen (δ¹⁸O) and carbon (δ¹³C) isotopic ratios in young-of-the year otoliths were also analyzed with isotope ratio mass spectrometry. Chemical sig natures dif fered sig nif icantly among the distinct nurseries within Narragansett Bay and the coastal ponds across years. Significant differences were also observed within nurseries from year to year. Classification accuracy to each of the five tautog nursery areas ranged from 85% to 92% across years. Because accurate classification of juvenile tautog nursery sites was achieved, otolith chemistry can potentially be used as a natural habitat tag.

]]> Ivan Mateo et al. http://digitalcommons.uri.edu/favs_facpubs/3 http://digitalcommons.uri.edu/favs_facpubs/3 Tue, 31 Jul 2012 11:56:42 PDT Seventeen bacterial strains previously identified as Vibrio harveyi (Baumann et al. 1981) or V. carchariae (Grimes et al. 1984) and the type strains of V. harveyi, V. carchariae and V. campbellii were analyzed by 16S ribosomal DNA (rDNA) sequencing. Four clusters were identified in a phylogenetic analysis performed by comparing a 746 base pair fragment of the 16S rDNA and previously published sequences of other closely related Vibrio species. The type strains of V. harveyi and V. carchariae and about half of the strains identified as V. harveyi or V. carchariae formed a single, wellsupported cluster designed as ‘bona fide’ V. harveyi/carchariae. A second more heterogeneous cluster included most other strains and the V. campbellii type strain. Two remaining strains are shown to be more closely related to V. rumoiensis and V. mediterranei. 16S rDNA sequencing has confirmed the homogeneity and synonymy of V. harveyi and V. carchariae. Analysis of API20E biochemical profiles revealed that they are insufficient by themselves to differentiate V. harveyi and V. campbellii strains. 16S rDNA sequencing, however, can be used in conjunction with biochemical techniques to provide a reliable method of distinguishing V. harveyi from other closely related species.

]]> Eric J. Gauger et al. http://digitalcommons.uri.edu/favs_facpubs/2 http://digitalcommons.uri.edu/favs_facpubs/2 Tue, 31 Jul 2012 11:51:20 PDT An epizootic causing mortality among cultured summer flounder Paralichthys dentatus occurred in summer of 1998 at a land-based facility on Narragansett Bay, Rhode Island, USA. The disease, flounder infectious necrotizing enteritis (FINE), was characterized by reddening around the anal area, distended abdomens filled with opaque serosanguineous fluid, enteritis and necrosis of the posterior intestine. In extreme cases of the disease, the posterior intestine was detached from the anus and was observed coming out the vent. The intestine of individuals that recovered from the dsease ended in a blind-sac; the abdomens of these fish were distended, due to food and water inside the intestinal blind-sac. A bacterium was isolated from ascites fluid and kidney of moribund flounder and identified as the causative agent in challenge experiments. The pathogen was identified as Vibno carchariae by morphological and biochemical characteristics and sequence of the 16S rRNA. The LD₅₀ estimate was 5 x 10⁵ colony-forming units injected intraperitoneally into 100 to 200 g summer flounder.

]]> Bruno Soffientino et al. http://digitalcommons.uri.edu/favs_facpubs/1 http://digitalcommons.uri.edu/favs_facpubs/1 Tue, 31 Jul 2012 11:46:18 PDT Shellfish production is often affected by bacterial pathogens that cause high losses in hatcheries and nurseries. We evaluated the relative survival of larvae and juveniles of 3 Crassostrea virginica oyster lines: (1) GHP, a Rhode Island line; (2) NEHY, a line resistant to dermo and multinucleated sphere X diseases; and (3) FLOWERS, a line resistant to Roseovarius oyster disease, experimental challenge with Vibrio spp. isolates RE22 and RE101, causative agents of bacillary necrosis in Pacific oyster larvae, and the type strain of Roseovarius crassostreae, causative agent of Roseovarius oyster disease. All of the isolates were able to induce significant mortalities in oyster larvae and juveniles. Susceptibility to bacterial challenge in larvae was significantly higher at 25°C than at 20°C. Susceptibility decreased with oyster age; mean survival time ranged from 24 h in oyster larvae to more than 6 wk in juveniles. Significant differences in susceptibility to bacterial challenge were observed between oyster lines; NEHY was the most resistant line overall. Extracellular products (ECPs) from Vibrio sp. RE22 and R. crassostreae, as well as viable bacteria, were toxic to hemocytes from the 3 oyster lines, suggesting that ECPs are involved in pathogenesis and that external and mucosal barriers to infection are major contributors to resistance to bacterial challenge. These protocols will be useful in the elucidation of mechanisms of bacterial pathogenesis and resistance to infection in oysters.

]]> Javier Gomez-Leon et al.