Foraging site use and interspecific competition between bluegills and golden shiners

Synopsis Laboratory experiments examined the foraging performances of a dietary generalist, bluegill,Lepomis macrochirus, and a dietary specialist, golden shiner,Notemigonus crysoleucas, as they fed from devices simulating four foraging sites (bottom substrate, water column, submerged macrophytes, and water surface). Fishes foraged in monospecific and mixed-species groups of two and four individuals. For monospecific groups, foraging rates of bluegills did not differ among the four sites, but golden shiners had significantly higher rates on bottom and midwater sites than on plant and surface sites. The size of monospecific groups did not affect foraging rates of either species. In mixed-species trials, bluegills removed more food items than golden shiners from plant and surface sites in two- and four-fish groups and from bottom sites in two-fish groups. Bluegills' foraging performances improved with experience, golden shiners' performances did not. Experimental results are discussed with respect to interactions between bluegills and golden shiners in natural assemblages.     

The effects of planktivorous fish (golden shiners) on the ciliate community of a mesotrophic lake

A whole-lake food web manipulation suggested that planktivorousfish can play an important role in regulating the pelagic foodweb structure of mesotrophic lakes. In this study, we examinedthe impact of golden shiners (Notemigonus crysoleucas) on zooplankton,diliates, phytoplankton and nutrients. We conducted a mesocosmexperiment using treatments with and without golden shinerswith three replicates per treatment in summer. We monitoredplankton and nutrient dynamics in these mesocosms for 6 weeks.Total macrozooplankton biomass and the proportion of large crustaceansdecreased dramatically in the golden shiner treatment, whilerotifer biomass decreased only in the second half of the experiment.In the mesocosms with golden shiners, total ciliate biovolumeincreased. However, the impact of golden shiners on ciliateswas species specific. Chlorophyll a concentrations increasedand dissolved nutrients (inorganic nitrogen and phosphorus)were statistically unaffected in the golden shiner treatment.This experiment showed that golden shiners had a strong negativeimpact on macrozooplankton, a variable impact on rotifers, weakpositive impacts on ciliates and phytoplankton, and no discernibleimpact on dissolved inorganic nutrient concentrations. The resultsof this study help integrate aspects of previous research inmesotrophic lakes and provide evidence for cascading trophicinteractions from fish to protozoans in a mesotrophic lake.     

Nest association of pumpkinseed, Lepomis gibbosus, and golden shiner, Notemigonus crysoleucas

I investigated the nest association of pumpkinseeds and golden shiners in an upstate New York pond. Golden shiners spawned in about one-third of pumpkinseed nests. Field observations indicate that golden shiners preferred to spawn in nests of male pumpkinseeds that attracted conspecific females, and avoided nests of pumpkinseeds that failed to do so. Golden shiners did not spawn in any of the nests that I kept clean experimentally after abandonment by male pumpkinseeds, suggesting that a clean nest without a guarding pumpkinseed is not enough to attract shiners to spawn. In field experiments, shiner eggs that were placed away from pumpkinseed nests suffered significantly higher losses to predation than those in the nests. This indicates that golden shiners benefit from spawning in pumpkinseed nests through the protection of their young by the host pumpkinseed. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effect of suspended sediments on ninespine stickleback,Pungitius pungitius,and golden shiner,Notemigonus crysoleucas,in a current of varying velocity

Synopsis Ninespine stickleback, Pungitius pungitius, and golden shiner, Notemigonus crysoleucas, were exposed at 5 and 20°C to 0, 15, 75 and 150 JTU (Jackson Turbidity Units) of suspended sediments. Fish were tested in a trough inclined at 2.3° with an inflow rate of 27 ml sec^-1. Changes in swimming behaviour were only noted for golden shiner and at 20°C and 15, 75 and 150 JTU. Under these conditions golden shiners were more active, changing from location to location in the apparatus significantly more often at higher than at lower concentrations of suspended sediments. This behaviour is compatible with a fleeing response from a stress inducing agent.     

Occurrence of reoviruses in European cyprinid fishes (Tinea tinea Lin.; Leuciscus cephalus Lin.)

Two viruses have been isolated from tench (Tinea tinea) and from chub (Leuciscus cephalus). The agents replicated in EPC-and FHM cells at 20°C forming syncytia and lysis. Electron micrographs of the virion revealed icosahedral particles with a double capsid and a size of 70–75 nm. The genome of the isolated viruses consisted of 11 segments of dsRNA. SDS-PAGE migration patterns of the RNA revealed differences between the isolates and between the reoviruses of fishes. The isolates are serologically identical and they show serological relationships to the golden shiner virus (GSV) and to a lower extent to the chum salmon virus (CSV).     

Red shiner invasion and hybridization with blacktail shiner in the upper Coosa River,USA

Human disturbance increases the invasibility of lotic ecosystems and the likelihood of hybridization between invasive and native species. We investigated whether disturbance contributed to the invasion of red shiner (Cyprinella lutrensis) and their hybridization with native blacktail shiner (C. venusta stigmatura) in the Upper Coosa River System (UCRS). Historical records indicated that red shiners and hybrids rapidly dispersed in the UCRS via large, mainstem rivers since the mid to late 1990s. We measured the occurrence and abundance of parental species and hybrids near tributary-mainstem confluences and characterized populations at these incipient contact zones by examining variation across morphological traits and molecular markers. Red shiners represented only 1.2% of total catch in tributaries yet introgression was widespread with hybrids accounting for 34% of total catch. Occurrence of red shiners and hybrids was highly correlated with occurrence of blacktail shiners, indicating that streams with native populations are preferentially colonized early in the invasion and that hybridization is a key process in the establishment of red shiners and their genome in new habitats. Tributary invasion was driven by post-F1 hybrids with proportionately greater genomic contributions from blacktail shiner. Occurrence of red shiners and hybrids and the relative abundance of hybrids significantly increased with measures of human disturbance including turbidity, catchment agricultural land use, and low dissolved oxygen concentration. Red shiners are a significant threat to Southeast Cyprinella diversity, given that 41% of these species hybridize with red shiner, that five southeastern drainages are invaded, and that these drainages are increasingly disturbed by urbanization.     

Non-pathogenic reoviruses of leafhoppers and planthoppers

Reoviruses of hoppers are reviewed with emphasis on their transmission, classification, and evolution. Plant reoviruses multiply in both plants and hopper vectors, whereas three non-pathogenic reoviruses are known; leafhopper A virus,Peregrinus maidisreovirus, andNilaparvata lugensreovirus (NLRV), that do not appear to cause disease in their insect hosts, and do not multiply in plants but share many properties with plant reoviruses. Genome analysis of NLRV would place it in the genusFijivirusin the family Reoviridae, and this assignment would be consistent with the biological and physicochemical properties of Fijiviruses, except for host range. The hypothesis that plant reoviruses originate as viruses exclusively of insects is studied.     

Spatial variation in relative abundance of a widespread, numerically dominant fish species and its effect on fish assemblage structure

Collections of fish assemblages from streams in the midwestern United States were used to examine assemblage-level effects of spatial variation in relative abundance of the red shiner, Cyprinella lutrensis, a widespread and highly abundant minnow species. This species has been widely introduced outside its native range and is suspected to have impacted local assemblages where it has become established. Given its overall dominance of midwest fish assemblages, and its suspected impact on assemblage structure, we asked if structure of the residual fish assemblages (red shiners excluded) was a function of the relative abundance of red shiners throughout the native range of C. lutrensis in the USA. Although red shiner ranked first in abundance in half of the assemblages and numerically dominated 28% of the assemblages, red shiner relative abundance in an assemblage had no detectable effect on richness, diversity, evenness, or complexity of other (residual) species in the assemblage. Relative abundance of red shiners did have a positive effect on the abundance of benthic minnows in the residual assemblage, but not on water column minnows that are ecologically most like red shiners. Environmental factors did not explain a significant amount of the variation in relative abundance of red shiners, but did explain some variation in residual assemblage structure. Although widespread and numerically dominant at many localities, red shiners do not appear to have a strong impact on local fish assemblage structure within their native range. This is in sharp contrast to the reported negative effects of red shiners on fish assemblages where they have been introduced outside their native range.     

A plasmid-based reverse genetics system for animal double-stranded RNA viruses

Mammalian orthoreoviruses (reoviruses) are highly tractable experimental models for studies of double-stranded (ds) RNA virus replication and pathogenesis. Reoviruses infect respiratory and intestinal epithelium and disseminate systemically in newborn animals. Until now, a strategy to rescue infectious virus from cloned cDNA has not been available for any member of the Reoviridae family of dsRNA viruses. We report the generation of viable reovirus following plasmid transfection of murine L929 (L) cells using a strategy free of helper virus and independent of selection. We used the reovirus reverse genetics system to introduce mutations into viral capsid proteins sigma1 and sigma3 and to rescue a virus that expresses a green fluorescent protein (GFP) transgene, thus demonstrating the tractability of this technology. The plasmid-based reverse genetics approach described here can be exploited for studies of reovirus replication and pathogenesis and used to develop reovirus as a vaccine vector.     

Validation of a quantitative PCR diagnostic method for detection of the microsporidian Ovipleistophora ovariae in the cyprinid fish Notemigonus crysoleucas

Microsporidian parasites are easily detected by light microscopy when infections are heavy and spores are present. However, early infections without spores, or light infections with low numbers of spores, are easily missed. This limitation has made it difficult to conduct investigations into microsporidian prevalence and transmission. In this study, we developed a quantitative TaqMan polymerase chain reaction assay to assess the presence of Ovipleistophora ovariae in the tissues of the cyprinid fish Notemigonus crysoleucas (golden shiner). The efficiency of the primer set was 100.8%, with a correlation coefficient of threshold position to copy number of 0.997 over 9 logs using a plasmid containing the cloned reaction product. No product was produced from other closely related microsporidian species (Nucleospora salmonis, Pseudoloma neurophila, Glugea stephani, Heterosporis sp., and O. mirandella). The coefficient of variation for replicate assays done on different days was 12.4%. The assay detects O. ovariae reliably at less than 10 genomic copies and 0.14 spores per reaction, but maximum sensitivity is only achieved when sonication is included as part of the DNA purification step. Using the assay, we found 4.44 x 10(1) to 7.91 x 10(6) copies microg(-1) host DNA in female golden shiners, with the spore density increasing during the spawning season. The parasite was also detected for the first time in the testes of male golden shiners at 2.60 x 10(1) to 8.62 x 102 copies microg(-1) host DNA.     

The reovirus sigma1s protein is a determinant of hematogenous but not neural virus dissemination in mice

Nonstructural protein σ1s is a critical determinant of hematogenous dissemination by type 1 reoviruses, which reach the central nervous system (CNS) by a strictly blood-borne route. However, it is not known whether σ1s contributes to neuropathogenesis of type 3 reoviruses, which disseminate by both vascular and neural pathways. Using isogenic type 3 viruses that vary only in σ1s expression, we observed that mice survived at a higher frequency following hind-limb inoculation with σ1s-null virus than when inoculated with wild-type virus. This finding suggests that σ1s is essential for reovirus virulence when inoculated at a site that requires systemic spread to cause disease. Wild-type and σ1s-null viruses produced comparable titers in the spinal cord, suggesting that σ1s is dispensable for invasion of the CNS. Although the two viruses ultimately achieved similar peak titers in the brain, loads of wild-type virus were substantially greater than those of the σ1s-null mutant at early times after inoculation. In contrast, wild-type virus produced substantially higher titers than the σ1s-null virus in peripheral organs to which reovirus spreads via the blood, including the heart, intestine, liver, and spleen. Concordantly, viral titers in the blood were higher following infection with wild-type virus than following infection with the σ1s-null mutant. These results suggest that differences in viral brain titers at early time points postinfection are due to limited virus delivery to the brain by hematogenous pathways. Transection of the sciatic nerve prior to hind-limb inoculation diminished viral spread to the spinal cord. However, wild-type virus retained the capacity to disseminate to the brain following sciatic nerve transection, indicating that wild-type reovirus can spread to the brain by the blood. Together, these results indicate that σ1s is not required for reovirus spread by neural mechanisms. Instead, σ1s mediates hematogenous dissemination within the infected host, which is required for full reovirus neurovirulence.     

Protamine precipitation of two reovirus particle types from polluted waters.

Two forms of virus particle are released from reovirus-infected cell cultures, infectious reovirus and potentially infectious reovirus (PIV). PIV particle forms have a complete outer coat and are not infectious until the outer coat is altered or removed. The PIV concentration in polluted waters, however, has not been determined. Protamine sulfate precipitation, using 0.25% fetal bovine serum and 0.005% protamine sulfate for the first precipitation of the sample and 0.0025% for the second, was employed to concentrate infectious reovirus and PIV from water and sewage. Infectious reovirus and PIV particles were concentrated over 500-fold from river water inoculated with virus, and virus recoveries of between 80 and 100% were achieved. Virus precipitates stored at -20 degrees C as a protamine-virus concentrate showed a 5% loss of PIV after 14 days. Virus preparations were assayed, before and after treatment, with 200 micrograms of chymotrypsin per ml, using a fluorescent-antibody procedure. Protamine sulfate precipitation and fluorescent-antibody detection are effective ways to recover and assay reoviruses present in raw sewage.     

Inhibition of reovirus type 3 binding to host cells by sialylated glycoproteins is mediated through the viral attachment protein.

The interaction of mammalian reoviruses with sialylated glycoproteins was studied and found to be highly serotype specific in that attachment of type 3 Dearing reovirus to murine L cell receptors could be strongly inhibited by bovine submaxillary mucin (BSM), fetuin, and alpha 1 acid glycoprotein, albeit at different efficiencies, whereas attachment of type 1 Lang reovirus was inhibited only by fetuin. We subsequently demonstrated, by using reassortants between type 3 and 1 reoviruses, that inhibition of reovirus attachment to cell receptors was specified by the viral attachment protein gene S1. Using a solid-phase binding assay, we further demonstrated that the ability of reovirus type 3 or reassortant 1HA3 and the inability of reovirus type 1 or reassortant 3HA1 to bind avidly to BSM was a property of the viral S1 genome segment and required the presence of sialic acid residues on BSM oligosaccharides. Taken together, these results demonstrated that there is a serotype-specific difference in the ability of the reovirus attachment protein, sigma 1, to interact with sialylated oligosaccharides of glycoproteins. Interaction of reovirus type 3 with sialylated oligosaccharides of BSM is dramatically affected by the degree of O-acetylation of their sialic acid residues, as indicated by the findings that chemical removal of O-acetyl groups stimulated reovirus type 3 attachment to BSM, whereas preferential removal of residues lacking or possessing reduced amounts of O-acetyl groups per sialic acid molecule with Vibrio cholerae sialidase abolished binding. We also demonstrated that BSM was 10 times more potent in inhibiting attachment of infectious reovirus to L cells than was V. cholerae-treated BSM. The results are consistent with the hypothesis that sialylated oligosaccharides on host cells or erythrocytes may act as binding sites or components of binding sites for type 3 reovirus through a specific interaction with the virus attachment protein.     

A single mutation in the carboxy terminus of reovirus outer-capsid protein sigma 3 confers enhanced kinetics of sigma 3 proteolysis,resistance to inhibitors of viral disassembly,and alterations in sigma 3 structure

Mammalian reoviruses undergo acid-dependent proteolytic disassembly within endosomes, resulting in formation of infectious subvirion particles (ISVPs). ISVPs are obligate intermediates in reovirus disassembly that mediate viral penetration into the cytoplasm. The initial biochemical event in the reovirus disassembly pathway is the proteolysis of viral outer-capsid protein sigma 3. Mutant reoviruses selected during persistent infection of murine L929 cells (PI viruses) demonstrate enhanced kinetics of viral disassembly and resistance to inhibitors of endocytic acidification and proteolysis. To identify sequences in sigma 3 that modulate acid-dependent and protease-dependent steps in reovirus disassembly, the sigma 3 proteins of wild-type strain type 3 Dearing; PI viruses L/C, PI 2A1, and PI 3-1; and four novel mutant sigma 3 proteins were expressed in insect cells and used to recoat ISVPs. Treatment of recoated ISVPs (rISVPs) with either of the endocytic proteases cathepsin L or cathepsin D demonstrated that an isolated tyrosine-to-histidine mutation at amino acid 354 (Y354H) enhanced sigma 3 proteolysis during viral disassembly. Yields of rISVPs containing Y354H in sigma3 were substantially greater than those of rISVPs lacking this mutation after growth in cells treated with either acidification inhibitor ammonium chloride or cysteine protease inhibitor E64. Image reconstructions of electron micrographs of virus particles containing wild-type or mutant sigma 3 proteins revealed structural alterations in sigma 3 that correlate with the Y354H mutation. These results indicate that a single mutation in sigma 3 protein alters its susceptibility to proteolysis and provide a structural framework to understand mechanisms of sigma 3 cleavage during reovirus disassembly.     

Reovirus variants with mutations in genome segments S1 and L2 exhibit enhanced virion infectivity and superior oncolysis

Reovirus preferentially replicates in transformed cells and is being explored as a cancer therapy. Immunological and physical barriers to virotherapy inspired a quest for reovirus variants with enhanced oncolytic potency. Using a classical genetics approach, we isolated two reovirus variants (T3v1 and T3v2) with superior replication relative to wild-type reovirus serotype 3 Dearing (T3wt) on various human and mouse tumorigenic cell lines. Unique mutations in reovirus λ2 vertex protein and σ1 cell attachment protein were associated with the large plaque-forming phenotype of T3v1 and T3v2, respectively. Both T3v1 and T3v2 exhibited higher infectivity (i.e., a higher PFU-to-particle ratio) than T3wt. A detailed analysis of virus replication revealed that virus cell binding and uncoating were equivalent for variant and wild-type reoviruses. However, T3v1 and T3v2 were significantly more efficient than T3wt in initiating productive infection. Thus, when cells were infected with equivalent input virus particles, T3v1 and T3v2 produced significantly higher levels of early viral RNAs relative to T3wt. Subsequent steps of virus replication (viral RNA and protein synthesis, virus assembly, and cell death) were equivalent for all three viruses. In a syngeneic mouse model of melanoma, both T3v1 and T3v2 prolonged mouse survival compared to wild-type reovirus. Our studies reveal that oncolytic potency of reovirus can be improved through distinct mutations that increase the infectivity of reovirus particles.     

Lymphocytes protect against and are not required for reovirus-induced myocarditis.

Many studies suggest that host lymphocytes are damaging, rather than protective, in virally induced myocarditis. We have investigated the role of lymphocyte-based immunity in murine myocarditis by using a myocarditic reovirus (reovirus serotype 3 8B), nonmyocarditic reoviruses, adoptive transfer experiments, and mice with severe combined immunodeficiency (SCID mice). Prior to infection, passive transfer of monoclonal antibodies specific for 8B capsid proteins protected neonatal mice against 8B-induced myocarditis, indicating that humoral immunity can protect against myocarditis. Some monoclonal antibodies acted by blocking viral spread to and/or replication in the heart. Passive transfer of reovirus-immune, but not naive, spleen cells prior to infection protected neonatal mice from 8B-induced myocarditis. Depletion of either CD4 or CD8 T cells resulted in increased viral titer in the heart but did not abrogate immune cell-mediated protection against myocardial injury. This shows that both CD4 and CD8 T cells can act independently to protect myocardial tissue from reovirus infection. In addition, reovirus 8B caused extensive myocarditis in SCID mice. This confirms a prior report (B. Sherry, F. J. Schoen, E. Wenske, and B. N. Fields, J. Virol. 63:4840-4849, 1989) that T cells are not required for reovirus-induced myocarditis and demonstrates for the first time that B cells are not required for reovirus-induced myocarditis. We used SCID mice and a panel of reoviruses to assess (i) the relationship between growth in the heart and myocardial damage and (ii) the possibility that nonmyocarditic reoviruses exhibit a myocarditic phenotype in the absence of functional lymphocytes. Growth in the heart was not the sole determinant of myocarditic potential in SCID mice. Although 8B induced myocarditis in SCID mice, no or minimal myocarditis was found in SCID mice infected with four reovirus strains previously shown (B. Sherry and B. N. Fields, J. Virol. 63:4850-4856, 1989) to be nonmyocarditic or poorly myocarditic in normal neonatal mice. We conclude that (i) humoral immunity and cellular immunity are protective against, and not required for, reovirus-induced myocarditis and (ii) the potential to induce cardiac damage is a property of the virus independent of lymphocyte-based immunity.     

Preliminary characterization of a reovirus isolated from golden ide Leuciscus idus melanotus

Some characteristics of a reovirus recently isolated from golden ide Leuciscus idus melanotus and tentatively designated as golden ide reovirus (GIRV) were determined. Spherical non-enveloped particles with an outer capsid of about 70 nm and an inner capsid of about 50 nm were observed by electron microscopy. The density of the virus determined in CsCl gradients was 1.36 g ml-1. The genome contained 11 segments of dsRNA. GIRV differed from other aquareoviruses by a slight reduction of infectivity after treatment with chloroform and by the absence of forming syncytia in cell monolayers.     

Reovirus Induction of and Sensitivity to Beta Interferon in Cardiac Myocyte Cultures Correlate with Induction of Myocarditis and Are Determined by Viral Core Proteins

Reovirus-induced acute myocarditis in mice serves as a model to investigate non-immune-mediated mechanisms of viral myocarditis. We have used primary cardiac myocyte cultures infected with a large panel of myocarditic and nonmyocarditic reassortant reoviruses to identify determinants of viral myocarditic potential. Here, we report that while both myocarditic and nonmyocarditic reoviruses kill cardiac myocytes, viral myocarditic potential correlates with viral spread through cardiac myocyte cultures and with cumulative cell death. To address the role of secreted interferon (IFN), we added anti-IFN-α/β antibody to infected cardiac myocyte cultures. Antibody benefited nonmyocarditic more than myocarditic virus spread (P < 0.001), and this benefit was associated with the reovirus M1 and L2 genes. There was no benefit for a differentiated skeletal muscle cell line culture (C2C12 cells), suggesting cell type specificity. IFN-β induction in reovirus-infected cardiac myocyte cultures correlated with viral myocarditic potential (P = 0.006) and was associated with the reovirus M1, S2, and L2 genes. Sensitivity to the antiviral effects of IFN-α/β added to cardiac myocyte cultures also correlated with viral myocarditic potential (P = 0.004) and was associated with the same reovirus genes. Several reoviruses induced IFN-β levels discordant with their myocarditic phenotypes, and for those tested, sensitivity to IFN-α/β compensated for the anomalous induction levels. Thus, the combination of induction of and sensitivity to IFN-α/β is a determinant of reovirus myocarditic potential. Finally, a nonmyocarditic reovirus induced cardiac lesions in mice depleted of IFN-α/β, demonstrating that IFN-α/β is a determinant of reovirus-induced myocarditis. This provides the first identification of reovirus genes associated with IFN induction and sensitivity and provides the first evidence that IFN-β can be a determinant of viral myocarditis and reovirus disease.