Differences in virulence of marine and freshwater isolates of viral hemorrhagic septicemia virus in vivo correlate with in vitro ability to infect gill epithelial cells and macrophages of rainbow trout (Oncorhynchus mykiss)

Two strains of viral hemorrhagic septicemia virus (VHSV) with known different virulence characteristics in vivo were studied (by a time course approach) for their abilities to infect and translocate across a primary culture of gill epithelial cells (GEC) of rainbow trout (RBT; Oncorhynchus mykiss). The strains included one low-virulence marine VHSV (ma-VHSV) strain, ma-1p8, and a highly pathogenic freshwater VHSV (fw-VHSV) strain, fw-DK-3592B. Infectivities toward trout head kidney macrophages were also studied (by a time course method), and differences in in vivo virulence were reconfirmed, the aim being to determine any correlation between in vivo virulence and in vitro infectivity. The in vitro studies showed that the fw-VHSV isolate infected and caused a cytotoxic effect in monolayers of GEC (demonstrating virulence) at an early time point (2 h postinoculation) and that the same virus strain had translocated over a confluent, polarized GEC layer by 2 h postinoculation. The marine isolate did not infect monolayers of GEC, and delayed translocation across polarized GEC was seen by 48 h postinoculation. Primary cultures of head kidney macrophages were also infected with fw-VHSV, with a maximum of 9.5% virus-positive cells by 3 days postinfection, while for the ma-VHSV strain, only 0.5% of the macrophages were positive after 3 days of culture. In vivo studies showed that the fw-VHSV strain was highly virulent for RBT fry and caused high mortality, with classical features of viral hemorrhagic septicemia. The ma-VHSV showed a very low level of virulence (only one pool of samples from the dead fish was VHSV positive). This study has shown that the differences in virulence between marine and freshwater strains of VHSV following the in vivo infection of RBT correlate with in vitro abilities to infect primary cultures of GEC and head kidney macrophages of the same species.     

Meta-analysis Shows a Consistent and Strong Latitudinal Pattern in Fish Omnivory Across Ecosystems

Several studies have demonstrated a latitudinal gradient in the proportion of omnivorous fish species (that is, consumers of both vegetal and animal material) in marine ecosystems. To establish if this global macroecological pattern also exists in fresh and brackish waters, we compared the relative richness of omnivorous fish in freshwater, estuarine, and marine ecosystems at contrasting latitudes. Furthermore, we sought to determine the main environmental correlates of change in fish omnivory. We conducted a meta-analysis of published data focusing on change in the relative richness of omnivorous fishes in native fish communities along a broad global latitudinal gradient, ranging from 41°S to 81.5 N° including all continents except for Antarctica. Data from streams, rivers, lakes, reservoirs, estuaries, and open marine waters (ca. 90 papers covering 269 systems) were analyzed. Additionally, the relationship between the observed richness in omnivory and key factors influencing trophic structure were explored. For all ecosystems, we found a consistent increasing trend in the relative richness of omnivores with decreasing latitude. Furthermore, omnivore richness was higher in freshwaters than in marine ecosystems. Our results suggest that the observed latitudinal gradient in fish omnivory is a global ecological pattern occurring in both freshwater and marine ecosystems. We hypothesize that this macroecological pattern in fish trophic structure is, in part, explained by the higher total fish diversity at lower latitudes and by the effect of temperature on individual food intake rates; both factors ultimately increasing animal food limitation as the systems get warmer.     

Fish introduction and parasites in marine ecosystems: a need for information

Invasive species provide unique and useful systems by which to examine various ecological and evolutionary issues, both in terms of the effects on native environments and the subsequent evolutionary impacts. While biological invasions are an increasing agent of change in aquatic systems, alien species also act as vectors for new parasites and diseases. To date, colonizations by hosts and parasites have not been treated and reviewed together, although both are usually interwoven in various ways and may have unpredictable negative consequences. Fish are widely introduced worldwide and are convenient organisms to study parasites and diseases. We report a global overview of fish invasions with associated parasitological data. Data available on marine and freshwater are in sharp contrast. While parasites and diseases of inland freshwater fish, ornamental, reared and anadromous fish species are well documented, leading to the emergence of several evolutionary hypotheses in freshwater ecosystems during the last decade, the transfer of such organisms are virtually unexplored in marine ecosystems. The paucity of information available on the parasites of introduced marine fish reflects the paucity of information currently available on parasites of non-indigenous species in marine ecosystems. However, such information is crucial as it can allow estimations of the extent to which freshwater epidemiology/evolution can be directly transferred to marine systems, providing guidelines for adapting freshwater control to the marine environment.     

Non‐native species modify the isotopic structure of freshwater fish communities across the globe

Multiple anthropogenic pressures including the widespread introductions of non‐native species threaten biodiversity and ecosystem functioning notably by modifying the trophic structure of communities. Here, we provided a global evaluation of the impacts of non‐native species on the isotopic structure (δ¹³C and δ¹⁵N) of freshwater fish communities. We gathered the stable isotope values (n = 4030) of fish species in 496 fish communities in lentic (lakes, backwaters, reservoirs) and lotic (running waters such as streams, rivers) ecosystems throughout the world and quantified the isotopic structure of communities. Overall, we found that communities containing non‐native species had a different isotopic structure than communities without non‐native species. However, these differences varied between ecosystem types and the trophic positions of non‐native species. In lotic ecosystems, communities containing non‐native species had a larger total isotopic niche than communities without non‐native species. This was primarily driven by the addition of non‐native predators at the top of the food chain that increased δ¹⁵N range without modifying the isotopic niche size of native species. In lentic ecosystems, non‐native primary consumers increased δ¹⁵N range and this was likely driven by an increase of resource availability for species at higher trophic levels, increasing food chain length. The introduction of non‐native secondary consumers at the centre of the isotopic niche of recipient communities decreased the core isotopic niche size, the δ¹³C range of recipient communities and the total isotopic niche of coexisting native species. These results suggested a modified contribution of the basal resources consumed (e.g. multi‐chain omnivory) and an increase level of competition with native species. Our results notably imply that, by affecting the isotopic structure of freshwater fish communities at a global scale, non‐native species represent an important source of perturbations that should be accounted for when investigating macro‐ecological patterns of community structure and biotic interactions.     

Climate effects on fish body size–trophic position relationship depend on ecosystem type

The energetic demand of consumers increases with body size and temperature. This implies that energetic constraints may limit the trophic position of larger consumers, which is expected to be lower in tropical than in temperate regions to compensate for energy limitation. Using a global dataset of 3635 marine and freshwater ray‐finned fish species, we addressed if and how climate affects the fish body size–trophic position relationship in both freshwater and marine ecosystems, while controlling for the effects of taxonomic affiliation. We observed significant fish body size–trophic position relationships for different ecosystems. However, only in freshwater systems larger tropical fish presented a significantly lower trophic position than their temperate counterparts. Climate did not affect the fish body size–trophic position relationship in marine systems. Our results suggest that larger tropical freshwater fish may compensate for higher energetic constraints feeding at lower trophic positions, compared to their temperate counterparts of similar body size. The lower latitudinal temperature range in marine ecosystems and/or their larger ecosystem size may attenuate and/or compensate for the energy limitation of larger marine fish. Based on our results, temperature may determine macroecological patterns of aquatic food webs, but its effect is contingent on ecosystem type. We suggest that freshwater ecosystems may be more sensitive to warming‐induced alterations in food web topology and food chain length than marine ecosystems.     

Metagenomic Analysis of RNA Viruses in a Fresh Water Lake

Freshwater lakes and ponds present an ecological interface between humans and a variety of host organisms. They are a habitat for the larval stage of many insects and may serve as a medium for intraspecies and interspecies transmission of viruses such as avian influenza A virus. Furthermore, freshwater bodies are already known repositories for disease-causing viruses such as Norwalk Virus, Coxsackievirus, Echovirus, and Adenovirus. While RNA virus populations have been studied in marine environments, to this date there has been very limited analysis of the viral community in freshwater. Here we present a survey of RNA viruses in Lake Needwood, a freshwater lake in Maryland, USA. Our results indicate that just as in studies of other aquatic environments, the majority of nucleic acid sequences recovered did not show any significant similarity to known sequences. The remaining sequences are mainly from viral types with significant similarity to approximately 30 viral families. We speculate that these novel viruses may infect a variety of hosts including plants, insects, fish, domestic animals and humans. Among these viruses we have discovered a previously unknown dsRNA virus closely related to Banna Virus which is responsible for a febrile illness and is endemic to Southeast Asia. Moreover we found multiple viral sequences distantly related to Israeli Acute Paralysis virus which has been implicated in honeybee colony collapse disorder. Our data suggests that due to their direct contact with humans, domestic and wild animals, freshwater ecosystems might serve as repositories of a wide range of viruses (both pathogenic and non-pathogenic) and possibly be involved in the spread of emerging and pandemic diseases.     

Host and geographic range extensions of the North American strain of viral hemorrhagic septicemia virus

Viral hemorrhagic septicemia virus (VHSV) was isolated from populations of Pacific sardine Sardinops sagax from the coastal waters of Vancouver Island, British Columbia, Canada, and central and southern California, USA. The virus was also isolated from Pacific mackerel Scomber japonicus in southern California, from eulachon or smelt Thaleichthys pacificus, and surf smelt Hypomesus pretiosus pretiosus from Oregon, USA. Mortality and skin lesions typical of viral hemorrhagic septicemia in other marine fish species were observed among sardine in Canada and in a few surf smelt from Oregon, but the remaining isolates of VHSV were obtained from healthy appearing fish. The prevalence of VHSV among groups of apparently healthy sardine, mackerel and smelt ranged from 4 to 8%, in California and Oregon. A greater prevalence of infection (58%) occurred in groups of sardine sampled in Canada that sustained a naturally occurring epidemic during 1998-99. A captive group of surf smelt in Oregon exhibited an 81% prevalence of infection with clinical signs in only a few fish. The new isolates were confirmed as North American VHSV and were closely related based on comparisons of the partial nucleotide sequence of the glycoprotein (G) gene. The VHSV isolates from sardine in Canada and California were the most closely related, differing from isolates obtained from other marine fish species and salmonids in British Columbia, Canada, Alaska and Washington, USA. These new virus isolations extend both the known hosts (sardine, mackerel and 2 species of smelt) and geographic range (Oregon and California, USA) of VHSV.     

Assessing the diversity and specificity of two freshwater viral communities through metagenomics

Transitions between saline and fresh waters have been shown to be infrequent for microorganisms. Based on host-specific interactions, the presence of specific clades among hosts suggests the existence of freshwater-specific viral clades. Yet, little is known about the composition and diversity of the temperate freshwater viral communities, and even if freshwater lakes and marine waters harbor distinct clades for particular viral sub-families, this distinction remains to be demonstrated on a community scale.To help identify the characteristics and potential specificities of freshwater viral communities, such communities from two lakes differing by their ecological parameters were studied through metagenomics. Both the cluster richness and the species richness of the Lake Bourget virome were significantly higher that those of the Lake Pavin, highlighting a trend similar to the one observed for microorganisms (i.e. the specie richness observed in mesotrophic lakes is greater than the one observed in oligotrophic lakes). Using 29 previously published viromes, the cluster richness was shown to vary between different environment types and appeared significantly higher in marine ecosystems than in other biomes. Furthermore, significant genetic similarity between viral communities of related environments was highlighted as freshwater, marine and hypersaline environments were separated from each other despite the vast geographical distances between sample locations within each of these biomes. An automated phylogeny procedure was then applied to marker genes of the major families of single-stranded (Microviridae, Circoviridae, Nanoviridae) and double-stranded (Caudovirales) DNA viruses. These phylogenetic analyses all spotlighted a very broad diversity and previously unknown clades undetectable by PCR analysis, clades that gathered sequences from the two lakes. Thus, the two freshwater viromes appear closely related, despite the significant ecological differences between the two lakes. Furthermore, freshwater viral communities appear genetically distinct from other aquatic ecosystems, demonstrating the specificity of freshwater viruses at a community scale for the first time.     

淡水鱼类功能生态学研究进展

在全球变化和人类活动的影响下,生物多样性正以前所未有的速度丧失,全球生物正经受第六次生物多样性危机。淡水生态系统是最脆弱的生态系统之一。淡水鱼类作为淡水生态系统的重要组成部分,承受着日趋严重的气候变化、栖息地退化、生物入侵和过度捕捞等压力,面临巨大的威胁。在此背景下,如何准确评估鱼类种群和群落对环境变化的响应,以及鱼类群落结构和功能的变化对生态系统功能的影响是淡水鱼类多样性和淡水生态系统保护的关键问题。近年来,淡水鱼类功能生态学的快速发展为解答这一问题提供了一个框架。系统地介绍了淡水鱼类功能生态学主要研究内容、方法、进展及其应用,并着重介绍了淡水鱼类功能特征及其与环境的关系、环境变化下的功能生态学响应研究。据此提出了淡水鱼类功能生态学未来的重点研究方向,指出了其在鱼类多样性保护和资源利用等领域的应用前景。     

一种新型草鱼呼肠孤病毒人工感染方法

研究从患病草鱼中新分离到一株草鱼呼肠孤病毒（Grass carp reovirus,GCRV）,对其进行了病毒纯化与电镜观察、基因组RT-PCR分型以及病毒量定量分析等,并在此基础上探索了一种新的病毒人工感染方法。取病鱼肌肉组织进行病毒纯化与电镜观察,观察到大量病毒粒子,直径在70-80 nm。病毒基因组RT-PCR扩增结果表明,该草鱼呼肠孤病毒新分离株属基因型Ⅱ型GCRV;通过绝对定量的方法,对病毒悬液的浓度进行了测定,为2.97×103 copy/μL。通过灌胃法,对3个组别的实验鱼分别感染不同浓度的病毒液,同时设置灌胃PBS的组别作为对照组。结果显示,3个实验组死亡率均在80%左右,而对照组仅出现一例死亡个体。实验组死亡个体体表发黑,腹部、鳍条基部以及鳃盖处均有明显的出血症状,为草鱼出血病的典型症状。随机选取死亡的个体进行RT-PCR检测,均能检测出Ⅱ型GCRV的条带。以上结果说明,灌胃法可以作为一种新的方法,用于草鱼的GCRV人工感染实验。     

Evolution and biogeography of an emerging quasispecies: diversity patterns of the fish Viral Hemorrhagic Septicemia virus (VHSv)

Viral Hemorrhagic Septicemia virus (VHSv) is an RNA rhabdovirus that causes one of the most important finfish diseases, affecting over 70 marine and freshwater species. It was discovered in European cultured fish in 1938 and since has been described across the Northern Hemisphere. Four strains and several substrains have been hypothesized, whose phylogenetic relationships and evolutionary radiation are evaluated here in light of a quasispecies model, including an in-depth analysis of the novel and especially virulent new substrain (IVb) that first appeared in the North American Laurentian Great Lakes in 2003. We analyze the evolutionary patterns, genetic diversity, and biogeography of VHSv using all available RNA sequences from the glycoprotein (G), nucleoprotein (N), and non-virion (Nv) genes, with Maximum Likelihood and bayesian approaches. Results indicate that the G gene evolves at an estimated rate of μ=2.58×10(-4) nucleotide substitutions per site per year, the N gene at μ=4.26×10(-4), and Nv fastest at μ=1.25×10(-3). Phylogenetic trees from the three genes largely are congruent, distinguishing strains I-IV as reciprocally monophyletic with high bootstrap and posterior probability support. VHSv appears to have originated from a marine ancestor in the North Atlantic Ocean, diverging into two primary clades: strain IV in North America (the Northwestern Atlantic Ocean), and strains I-III in the Northeastern Atlantic region (Europe). Strain II may comprise the basal group of the latter clade and diverged in Baltic Sea estuarine waters; strains I and III appear to be sister groups (according to the G and Nv genes), with the former mostly in European freshwaters and the latter in North Sea marine/estuarine waters. Strain IV is differentiated into three monophyletic substrains, with IVa infecting Northeastern Pacific salmonids and many marine fishes (with 44 unique G gene haplotypes), IVb endemic to the freshwater Great Lakes (11 haplotypes), and a newly-designated IVc in marine/estuarine North Atlantic waters (five haplotypes). Two separate substrains independently appeared in the Northwestern Pacific region (Asia) in 1996, with Ib originating from the west and IVa from the east. Our results depict an evolutionary history of relatively rapid population diversifications in star-like patterns, following a quasispecies model. This study provides a baseline for future tracking of VHSv spread and interpreting its evolutionary diversification pathways.     

Copy number variation of a fatty acid desaturase gene Fads2 associated with ecological divergence in freshwater stickleback populations

Fitness of aquatic animals can be limited by the scarcity of nutrients such as long-chain polyunsaturated fatty acids, especially docosahexaenoic acid (DHA). DHA availability from diet varies among aquatic habitats, imposing different selective pressures on resident animals to optimize DHA acquisition and synthesis. For example, DHA is generally poor in freshwater ecosystems compared to marine ecosystems. Our previous work revealed that, relative to marine fishes, several freshwater fishes evolved higher copy numbers of the fatty acid desaturase2 (Fads2) gene, which encodes essential enzymes for DHA biosynthesis, likely compensating for the limited availability of DHA in freshwater. Here, we demonstrate that Fads2 copy number also varies between freshwater sticklebacks inhabiting lakes and streams with stream fish having higher Fads2 copy number. Additionally, populations with benthic-like morphology possessed higher Fads2 copy number than those with planktivore-like morphology. This may be because benthic-like fish mainly feed on DHA-deficient prey such as macroinvertebrates whereas planktivore-like fish forage more regularly on DHA-rich prey, like copepods. Our results suggest that Fads2 copy number variation arises from ecological divergence not only between organisms exploiting marine and freshwater habitats but also between freshwater organisms exploiting divergent resources.     

Virus susceptibility of the fish cell line SAF-1 derived from gilt-head seabream

The recently reported SAF-1 cell line from fins of gilt-head seabream was evaluated for susceptibility to lymphocystis disease virus (LDV) and to several salmonid fish viruses, such as infectious haematopoietic necrosis virus (IHNV), viral haemorrhagic septicemia virus (VHSV) and several strains of infectious pancreatic necrosis virus (IPNV). LDV, VHSV and IHNV replicated well in the cultured fin cells as demonstrated by cell lysis and increases in viral titer. The potential use of this cell line to detect viruses from fish marine species is discussed.     

The functional role of producer diversity in ecosystems

Over the past several decades, a rapidly expanding field of research known as biodiversity and ecosystem functioning has begun to quantify how the world's biological diversity can, as an independent variable, control ecological processes that are both essential for, and fundamental to, the functioning of ecosystems. Research in this area has often been justified on grounds that (1) loss of biological diversity ranks among the most pronounced changes to the global environment and that (2) reductions in diversity, and corresponding changes in species composition, could alter important services that ecosystems provide to humanity (e.g., food production, pest/disease control, water purification). Here we review over two decades of experiments that have examined how species richness of primary producers influences the suite of ecological processes that are controlled by plants and algae in terrestrial, marine, and freshwater ecosystems. Using formal meta-analyses, we assess the balance of evidence for eight fundamental questions and corresponding hypotheses about the functional role of producer diversity in ecosystems. These include questions about how primary producer diversity influences the efficiency of resource use and biomass production in ecosystems, how primary producer diversity influences the transfer and recycling of biomass to other trophic groups in a food web, and the number of species and spatial /temporal scales at which diversity effects are most apparent. After summarizing the balance of evidence and stating our own confidence in the conclusions, we outline several new questions that must now be addressed if this field is going to evolve into a predictive science that can help conserve and manage ecological processes in ecosystems.     

Immunoprophylaxis in fish by injection of mouse antibody genes

Antibodies are a crucial part of the body's specific defense against infectious diseases and have considerable potential as therapeutic and prophylactic agents in humans and animals. The development of recombinant single-chain antibodies allows a genetic application strategy for prevention of infectious diseases. To test this in a fish model, a gene construct encoding a neutralizing single-chain antibody to the fish-pathogenic rhabdovirus VHSV (viral hemorrhagic septicemia virus) was administered to rainbow trout by intramuscular injection of plasmid DNA. Circulating recombinant antibodies could later be detected in the fish, and protective immunity to the viral disease was established.     

Effect of Cecropin B and a Synthetic Analogue on Propagation of Fish Viruses In Vitro

Abstract Cecropins and other natural antimicrobial peptides are widely distributed in animals from insects to mammals. These proteins have been shown to be major constituents of the innate immune systems of animals for nonspecific defense of the host against various bacteria and parasites. Therefore, exploitation of this natural innate defense system may lead to the development of effective methods for protecting fish from invasion by microbial pathogens. Recently, we have demonstrated that the introduction of cecropin transgenes into Japanese medaka (Oryzias latipes) conferred resistance to infection by fish bacterial pathogens. Aside from a few reports documenting the antiviral effect of antimicrobial peptides including cecropins against mammalian viruses, there is no evidence for the effect of these peptides against fish viruses. In this article we present results of in vitro characterization of native cecropin B and a synthetic analogue, CF17, against several important fish viral pathogens—namely, infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), snakehead rhabdovirus (SHRV), and infectious pancreatic necrosis virus (IPNV). Upon coincubation of these peptides and viruses, the viral titers yielded in fish cells were reduced from several fold to 104-fold. Direct disruption of the viral envelope and disintegration of the viral capsids may be involved in the inhibition of viral replication by the peptides. Results of our studies demonstrate the potential of manipulating antimicrobial peptide genes by transgenesis to combat viral infection in fish.     

Relative Invasion Risk for Plankton across Marine and Freshwater Systems: Examining Efficacy of Proposed International Ballast Water Discharge Standards

Understanding the implications of different management strategies is necessary to identify best conservation trajectories for ecosystems exposed to anthropogenic stressors. For example, science-based risk assessments at large scales are needed to understand efficacy of different vector management approaches aimed at preventing biological invasions associated with commercial shipping. We conducted a landscape-scale analysis to examine the relative invasion risk of ballast water discharges among different shipping pathways (e.g., Transoceanic, Coastal or Domestic), ecosystems (e.g., freshwater, brackish and marine), and timescales (annual and per discharge event) under current and future management regimes. The arrival and survival potential of nonindigenous species (NIS) was estimated based on directional shipping networks and their associated propagule pressure, environmental similarity between donor-recipient ecosystems (based on salinity and temperature), and effects of current and future management strategies (i.e., ballast water exchange and treatment to meet proposed international biological discharge standards). Our findings show that current requirements for ballast water exchange effectively reduce invasion risk to freshwater ecosystems but are less protective of marine ecosystems because of greater environmental mismatch between source (oceanic) and recipient (freshwater) ecoregions. Future requirements for ballast water treatment are expected to reduce risk of zooplankton NIS introductions across ecosystem types but are expected to be less effective in reducing risk of phytoplankton NIS. This large-scale risk assessment across heterogeneous ecosystems represents a major step towards understanding the likelihood of invasion in relation to shipping networks, the relative efficacy of different invasion management regimes and seizing opportunities to reduce the ecological and economic implications of biological invasions.     

A Spatial Modeling Approach to Predicting the Secondary Spread of Invasive Species Due to Ballast Water Discharge

Ballast water in ships is an important contributor to the secondary spread of invasive species in the Laurentian Great Lakes. Here, we use a model previously created to determine the role ballast water management has played in the secondary spread of viral hemorrhagic septicemia virus (VHSV) to identify the future spread of one current and two potential invasive species in the Great Lakes, the Eurasian Ruffe (Gymnocephalus cernuus), killer shrimp (Dikerogammarus villosus), and golden mussel (Limnoperna fortunei), respectively. Model predictions for Eurasian Ruffe have been used to direct surveillance efforts within the Great Lakes and DNA evidence of ruffe presence was recently reported from one of three high risk port localities identified by our model. Predictions made for killer shrimp and golden mussel suggest that these two species have the potential to become rapidly widespread if introduced to the Great Lakes, reinforcing the need for proactive ballast water management. The model used here is flexible enough to be applied to any species capable of being spread by ballast water in marine or freshwater ecosystems.