Genetic Analysis of Betanodaviruses in Subclinically Infected Aquarium Fish and Invertebrates

Betanodaviruses causing viral nervous necrosis (VNN) have been detected and isolated from several species of cultured marine fish worldwide. In Korea, VNN was identified in several species of cultured marine fish. This study presents data on the amplified nested PCR product (420 bp) of 11 nodavirus strains from different species of apparently healthy aquarium fish and invertebrates collected in one private commercial aquarium in Korea. Phylogenetic analyses based on the partial nucleotide sequence (177 bases) of the RNA2 coat protein gene were identical to the redspotted grouper nervous necrosis virus (RGNNV) genotype (96%–100%). The presence of the RGNNV type of betanodaviruses in these subclinically infected aquarium fish and invertebrates imported from different countries probably indicates that the samples were contaminated inside the aquarium and represents a serious challenge for its management of viral nervous necrosis. These positive samples can be an inoculum source of betanodavirus infection to other susceptible fish species inside the aquarium.     

Metagenomic analysis of the marine coastal invertebrates of South Korea as assessed by Ilumina MiSeq

Metagenomic analysis was carried out for the first time on the marine coastal invertebrates of South Korea. Samples collected from coastal areas of Korea were filtered through a 63 µm mesh, then their 18S rDNA V4 regions were amplified. High-throughput sequencing of PCR amplicons using Illumina MiSeq and BLAST against the SILVA database showed that a total of 319 eukaryotic Operational Taxonomic Units were identified at the species level. Annelida, Arthropoda, Mollusca, Nematoda, and Platyhelminthes and for 92.23% of the total 103 metazoan species belonging to 101 genera, 75 families, and 10 phyla. Of these, several taxa previously unreported to exist in Korea were detected at the family level compared with the integrated database from three main Korean biodiversity DBs (MABIK, KOMBIS, and MRBR). Analysis of beta diversity of the community structure of invertebrates indicated that the composition of marine invertebrates is likely to be affected by habitat type rather than geographical distance. The present study showed that metagenomic high-throughput technology can be used to unravel species diversity and for various studies regarding marine invertebrate community structure.     

Phylogeographical structure in Zelkova serrata in Japan and phylogeny in the genus Zelkova using the polymorphisms of chloroplast DNA

The genetic differentiation inherent in Zelkova serrata in Japan and the southern portion of the Korean Peninsula was examined by comparing a chloroplast DNA (cpDNA) sequence over a 16?k baselength in 40 individual samples collected from an area covering the natural distribution range of Z. serrata in Japan and the southern portion of the Korean Peninsula. We detected over 50 single-nucleotide polymorphisms in the protein-coding and intergenic regions, and over 30 insertions/deletions in the intergenic region. From the polymorphisms detected in the cpDNA, 14 haplotypes were identified. These 14 haplotypes had cluster-like structures and genetic differentiation between the clusters was large. Closely related haplotypes existed in adjacent regions. One haplotype existed in both Japan and the Korean Peninsula. By comparison with other Zelkova species, Z. serrata is apparently distinct from European and East Asian Zelkova species and Z. serrata is closest to the Ulmus species in the genus Zelkova. The effects of the analyzed length of the cpDNA sequence on the detection of polymorphisms were analyzed by re-sampling simulation.     

Geographical variation in species diversity: A comparison of marine polychaetes and nematodes

The aim of this study was to determine if marine species diversity was influenced by geographical location and whether it was higher at lower latitudes. Artificial collectors (made of nylon pan scourers) were employed as a standard substratum for the colonisation of marine invertebrates inhabiting subtidal (12 to 15 m) hard, rocky bottom substrata. These artificial substrate units (ASUs) were deployed at different latitudes including northern and southern temperate (South West England, UK and New Zealand), tropical (Trinidad and Tobago, West Indies) and polar (Signy Island, Antarctica) areas. The polychaetes, representative of the macrofauna and the nematodes, representative of the meiofauna fractions of the total invertebrate fauna collected were analysed.Neither polychaete nor nematode species diversity showed a trend based on latitude and each taxon showed a different pattern of diversity variation in relation to location. Polychaete diversity varied from area to area with highest species diversity occurring in the southern temperate (New Zealand). Nematode species diversity however was similar for the northern and southern temperate (UK and New Zealand) and the tropical area (Trinidad and Tobago). Thus, although the number of locations studied was limited, these data do not conform to a gradient in species diversity with latitude as has been previously supposed. The success of ASUs to compare species diversities in standardised habitat units augurs well for their future use in other ecological areas such as biogeographical or pollution studies.     

Type X Toxoplasma gondii in a wild mussel and terrestrial carnivores from coastal California: new linkages between terrestrial mammals, runoff and toxoplasmosis of sea otters

Sea otters in California are commonly infected with Toxoplasma gondii. A unique Type X strain is responsible for 72% of otter infections, but its prevalence in terrestrial animals and marine invertebrates inhabiting the same area was unknown. Between 2000 and 2005, 45 terrestrial carnivores (lions, bobcats, domestic cats and foxes) and 1396 invertebrates (mussels, clams and worms) were screened for T. gondii using PCR and DNA sequencing to determine the phylogeographic distribution of T. gondii archetypal I, II, III and Type X genotypes. Marine bivalves have been shown to concentrate T. gondii oocysts in the laboratory, but a comprehensive survey of wild invertebrates has not been reported. A California mussel from an estuary draining into Monterey Bay was confirmed positive for Type X T. gondii by multilocus PCR and DNA sequencing at the B1 and SAG1 loci. This mussel was collected from nearshore marine waters just after the first significant rainfall event in the fall of 2002. Of 45 carnivores tested at the B1, SAG1, and GRA6 typing loci, 15 had PCR-confirmed T. gondii infection; 11 possessed alleles consistent with infection by archetypal Type I, II or III strains and 4 possessed alleles consistent with Type X T. gondii infection. No non-canonical alleles were identified. The four T. gondii strains with Type X alleles were identified from two mountain lions, a bobcat and a fox residing in coastal watersheds adjacent to sea otter habitat near Monterey Bay and Estero Bay. Confirmation of Type X T. gondii in coastal-dwelling felids, canids, a marine bivalve and nearshore-dwelling sea otters supports the hypotheses that feline faecal contamination is flowing from land to sea through surface runoff, and that otters can be infected with T. gondii via consumption of filter-feeding marine invertebrates.     

Occurrence of viral haemorrhagic septicaemia virus (VHSV) in wild marine fish species in the coastal regions of Norway

This study was aimed at determining the occurrence of viral haemorrhagic septicaemia virus (VHSV) in selected stocks of wild fish species in the coastal regions of Norway. Six cruises were undertaken covering areas which included the coastal regions of northern Norway, the Norwegian Sea, the Barents Sea, and the Skagerrak area down to the Danish border. Collected, pooled samples of internal organs (kidney, spleen and heart) from 8395 fish were examined for the presence of VHSV by inoculation on BF-2 cells. Identification of virus was performed by a standard ELISA procedure with monoclonal antibody IP5B11, which is specific for the VHSV nucleocapsid protein (N-protein). VHSV was isolated from blue whiting and Norway pout in Skagerrak. No positive samples were detected in the northern coastal regions of Norway, the Norwegian Sea, or the Barents Sea. The findings indicate a very low occurrence of VHSV in the coastal regions of Norway. Geographically, the only positive samples were obtained from fish collected in areas where VHSV has previously been found in different species of fish.     

Effect of lipopolysaccharides from Vibrio alginolyticus on the Mx gene expression and virus recovery from gilthead sea bream (Sparus aurata L.) experimentally infected with Nodavirus

Infections with nodavirus affect a wild and farmed fish species throughout the world, mostly from the marine environment. The aim of this work was to determine the immune status of gilthead sea bream that comes as a result of a Nodavirus infection, induced by activation of the interferon response pathway by lipopolysaccharides from Vibrio alginolyticus and the expression of interferoninduced Mx protein in liver samples. The enhancement of Mx protein gene expression was detected in liver samples of experimentally nodavirus infected fish and, furthermore, the immunostimulant LPS of V. alginolyticus decreased almost three times the virus titration with respect to no-immunized or infected with nodavirus group of fish.     

Detection of hepatopancreatic parvovirus (HPV) in wild shrimp from India by nested polymerase chain reaction (PCR)

The prevalence of hepatopancreatic parvovirus (HPV) in wild penaeid shrimp samples from India was studied by nested polymerase chain reaction (PCR) using primers designed in our laboratory. The virus could be detected in 9 out of 119 samples by non-nested PCR. However, by nested PCR 69 out of 119 samples were positive. The PCR results were confirmed by hybridization with digoxigenin-labelled DNA probe. Shrimp species positive by non-nested PCR included Penaeus monodon, Penaeus indicus and Penaeus semisulcatus and by nested PCR Parapenaeopsis stylifera, Penaeus japonicus, Metapenaeus monoceros, M. affinis, M. elegans, M. dobsoni, M. ensis and Solenocera choprai. This is the first report on the prevalence of HPV in captured wild shrimp from India.     

黄海生态区保护空缺分析

黄海生态区是世界自然基金会筛选出来的全球最优先保护的43个海洋生态区之一。目前, 我国已在黄海生态区内建立多处海洋保护地, 有效保护了其海洋生态系统及生物多样性。然而我国海洋保护地管理和建设中仍有许多问题, 存在较多保护空缺区域。因此, 完善黄海生态区内的海洋保护地建设是非常关键的。本文利用空缺分析法研究了黄海生态区内的重要滨海湿地、关键物种以及重点海域, 以识别保护空缺理论图斑, 从而提出新建保护地的提议。根据空缺分析结果, 黄海生态区内的主要保护空缺为辽河口湿地、曹妃甸海草床、天津塘沽和黄骅湿地、胶东半岛湿地、舟山群岛等。建议优化整合黄河口海洋保护地, 扩大或新建斑海豹(Phoca largha)、勺嘴鹬(Eurynorhynchus pygmeus)保护地, 并加强对东亚江豚(Neophocaena asiaeorientalis sunameri)以及小须鲸(Balaenoptera acutorostrata)的调查研究, 以形成更有效的海洋保护地网络。     

东北亚地区野猪种群mtDNA遗传结构及系统地理发生

研究测定了中国东北、华北及四川西部72个野猪(Susscrofa)个体线粒体控制区全序列,并结合GenBank报道的日本野猪(S.s.leucomystax)、琉球野猪(S.s.riukiuanus)72个同源区序列,分析了东北亚地区野猪线粒体DNA的变异及系统地理格局。在东北亚地区野猪的线粒体控制区共发现42个变异位点,均为转换,共定义了34个单元型。单元型之间的系统发生分析表明,东北亚地区野猪来自同一祖先。东北亚地区野猪现生种群具有显著的种群遗传结构,其中日本野猪与分布于中国东北地区的东北野猪之间亲缘关系较近;而琉球野猪则与华北野猪间亲缘关系较近,与日本野猪和东北野猪间的关系相对较远。嵌套进化枝系统地理分析(Nestedcladephylogeographicalanalysis,NCPA)表明:东北亚地区野猪由同一祖先经过长距离的迁徙而形成现生各种群(或亚种);琉球野猪应起源于大陆野猪,其种群演化可能经历了片断化事件;华北野猪呈现南部种群遗传多样性高的特点,其种群内部曾经历了一次分布区由南向北的扩张     

Conservation of the insect assemblages of the Cape Peninsula biodiversity hotspot

The Cape Peninsula is an area of outstanding biological importance, not only for to its high levels of floristic diversity and endemism, but also for its number of localised endemic invertebrates. Little is known of the spatial distribution of invertebrates across the Peninsula, or how best to conserve them. Sampling by visual searches assisted by aerial and aquatic hand-nets was undertaken throughout the Peninsula. The most important areas for insect diversity on the Peninsula, and associated environmental variables, were determined. The ‘Peninsula effect’ was also investigated. Nine Red Listed species and five new species for the Peninsula were recorded. This high number of Red Listed species (for those few groups that have been assessed) emphasises the biological importance of the Cape Peninsula. Table Mountain had the most Red Listed species, while Cape Point had many species not found in the other areas. Noordhoek Wetland is very important for aquatic Coleoptera. Small hills on the Peninsula are important for overall insect diversity. Elevation, slope, aspect, distance to water and vegetation structure were the most important environmental variables in determining the insect assemblages. The Peninsula effect appears to have no influence on these particular insect assemblages of the Cape Peninsula. The high number of new Peninsula records for well-known taxonomic groups indicates that still little is known of the insect assemblages across the Peninsula. Nevertheless, areas of conservation priority identified in this study are Table Mountain (for Red Listed species), Noordhoek (for aquatic Coleoptera) and Cape Point and the small hills across the Peninsula (for their unique invertebrate assemblages). Conservation of a variety of elevations, including steep and flat areas, all aspects of mountains, as well as both the wet and dry areas, overall will contribute to the conservation of the insects.     

A study of the salt lakes and salt springs of Eyre Peninsula, South Australia

An 18-month-study of 40 saline wetlands, ranging from 6 to 336 g l⁻¹, on the west and southern coasts of Eyre Peninsula yielded 88 species of invertebrates, some aquatic plants and a fish. The invertebrates are taxonomically diverse and include 38 crustaceans, 28 insects, 12 molluscs and significantly an aquatic spider, a nemertean, two polychaetes, two sea anemones, a sponge and a bryzoan. Most were tolerant of wide fluctuations in salinity, there being 51 halobionts, 21 halophils and only 16 salt-tolerant freshwater species. Many invertebrates are restricted to the thalassic springs where marine molluscs dominated. Athalassic wetlands were dominated by crustaceans and were of two basic types—coastal and continental. There is evidence of the former evolving biologically into the later, and for some lakes to be still in transition. There is also evidence of increasing salinity in recent decades and already two lakes exhibit severe secondary salinity. Like other salt lakes in Australia the fauna is regionally distinctive. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Guest Editors: J. John & B. Timms Salt Lake Research: Biodiversity and Conservation—Selected papers from the 9th Conference of the International Society for Salt Lake Research     

Development of in situ hybridization and RT-PCR assay for the detection of a nodavirus (PvNV) that causes muscle necrosis in Penaeus vannamei

A nodavirus (tentatively named PvNV, Penaeus vannamei nodavirus) that causes muscle necrosis in P. vannamei was found in Belize in 2004. From 2004 to 2006, shrimp samples collected from Belize exhibited clinical signs, white, opaque lesions in the tails and histopathology similar to those of shrimps infected by infectious myonecrosis virus (IMNV). Histological examination revealed multifocal necrosis and hemocytic fibrosis in the skeletal muscle. In addition, basophilic, cytoplasmic inclusions were found in striated muscle, lymphoid organ and connective tissues. However, IMNV was not detected in these shrimps by either RT-PCR or in situ hybridization, suggesting that these lesions may be caused by another RNA virus. Thus, a cDNA library was constructed from total RNA extracted from hemolymph collected from infected shrimp. One clone (designated PvNV-4) with a 928 bp insert was sequenced and found to be similar (69% similarity when comparing the translated amino acid sequences) to the capsid protein gene of MrNV (Macrobrachium rosenbergii nodavirus). The insert of PvNV-4 was labeled with digoxigenin-11-deoxyuridine triphosphate (dUTP) and hybridized to tissue sections of P. vannamei with muscle necrosis collected in Belize and from laboratory bioassays. The samples were positive for PvNV infection. Positively reacting tissues included skeletal muscle, connective tissues, the lymphoid organ, and hemocytes in the heart and gills. In addition, we experimentally infected both P. vannamei and P. monodon with PvNV prepared from Belize samples. A nested RT-PCR assay developed from the PvNV-4 cloned sequence showed that both species are susceptible to PvNV infection.     

The Distribution of Francisella-like Bacteria Associated with Coastal Waters in Norway

We report the diversity and distribution of Francisella species in Norwegian coastal and fresh waters following a nationwide survey in which water and sediment samples were collected from locations spanning almost the entire Norwegian coastline. In total, samples were obtained from 149 and 64 seawater and freshwater sites, respectively. DNA extracts from these environmental samples were initially screened by polymerase chain reaction (PCR) using Francisella genus-specific 16S rDNA primers. Positive samples were then amplified with genus-specific primers targeting Francisella succinate dehydrogenase A gene and Francisella philomiragia group-specific sequences for the SAICAR synthetase/phosphoribosylamine-glycine ligase gene. Francisella-related bacteria were identified in approximately 30% of seawater sampled sites, mainly in southern Norway, although a single positive sample was identified in the far north of the country. No PCR positives were identified from the freshwater sources. Sequences related to recognised species, both pathogenic and environmental, were identified, with the majority closely associated with F. philomiragia. However, a number of identified sequences probably represent previously undescribed species. Our data provide evidence of a significant background of Francisella spp. in geographical areas associated with outbreaks of fish francisellosis in Norway.     

Barcoding Antarctic Biodiversity: current status and the CAML initiative,a case study of marine invertebrates

The Census of Antarctic Marine Life (CAML) aims to collate DNA barcode data for Antarctic marine species. DNA barcoding is a technique that uses a short gene sequence from a standardised region of the genome as a diagnostic ‘biomarker’ for species. This study aimed to quantify genetic data currently available in GenBank in order to establish whether a representative cross-section of Antarctic marine taxa and bio-geographic areas has been sequenced and to propose priorities for barcoding, with a particular emphasis on marine invertebrate species. It was found that, amongst marine invertebrate fauna, sequence information covers a limited range of taxa and areas—mainly Crustacea, Annelida and Mollusca from the Weddell Sea and the Antarctic Peninsula. Only 15% of genes sequenced in Antarctic marine invertebrates were the standard barcode gene cytochrome c oxidase subunit 1 (CO1), the majority were other nuclear and mitochondrial genes. There is an urgent need for more in-depth genetic barcoding and species identification studies in Antarctic science, from a range of taxa and areas, given the rate of climate-driven habitat changes that might lead to extinctions in the region. CAML hopes to redress the balance, by collecting and sequencing over the circum-Antarctic area, using material from voyages that occurred during 2008 and 2009, within the framework of the International Polar Year (IPY).     

Marine biodiversity: patterns, threats and conservation needs

Marine biodiversity is higher in benthic rather than pelagic systems, and in coasts rather than the open ocean since there is a greater range of habitats near the coast. The highest species diversity occurs in the Indonesian archipelago and decreases radially from there. The terrestrial pattern of increasing diversity from poles to tropics occurs from the Arctic to the tropics but does not seem to occur in the southern hemisphere where diversity is high at high latitides. Losses of marine diversity are highest in coastal areas largely as a result of conflicting uses of coastal habitats. The best way to conserve marine diversity is to conserve habitat and landscape diversity in the coastal area. Marine protected areas are only a part of the conservation strategy needed. It is suggested that a framework for coastal conservation is integrated coastal area management where one of the primary goals is sustainable use of coastal biodiversity.     

Acquisition of fishes and aquatic invertebrates for zoological collections. Is there a future?

The majority of the freshwater fishes in the ornamental trade now originate from captive‐bred sources, as do a large proportion of the freshwater species exhibited in public aquariums. In contrast, commercial operators who also supply marine specimens to the ornamental trade remove directly from the wild approximately 98% of the marine fishes and invertebrates exhibited in public aquariums. The common perception prevails that captive propagation is inherently a better alternative to obtaining animals from the wild. Although captive propagation has been shown to have many benefits for terrestrial species, there are a number of features unique to marine species that challenge the idea that every species should be bred in captivity. Some of the key issues relating to the development of widespread conservation‐oriented captive propagation programs include: 1) the high taxonomic diversity in marine animals; 2) the resultant variety in their reproductive methods; 3) their ecological, behavioral, physiological, and nutritional needs; and 4) our general lack of knowledge on their husbandry and medical care. There are several characteristics of marine fish and invertebrate populations that make them suitable candidates for sustainable harvest. For instance, marine teleosts are “r‐selected,” meaning that they have an extremely high fecundity, and most marine teleosts have a wide distribution and the ability to disperse over long distances. In locations considered for fish collection, appropriate management techniques should be employed to ensure that fishes and invertebrates are collected with as little impact on the ecosystem as possible. The collection of marine fishes and invertebrates for public aquariums and the hobby trade should be managed like a fishery to ensure long‐term sustainability. The public aquarium community should support marine organism certification initiatives, such as the Marine Aquarium Council (MAC). Marine organism certification will create market incentives that encourage and support quality and sustainable practices by creating consumer demand and confidence for certified organisms, practices, and industry participants. The creation of refuges that supply propagules to harvested areas, the rotation of areas fished, species‐specific size limits and seasons, and standardization of collecting, handling, and transportation techniques should be used to manage these fisheries and harvest areas. Zoo Biol 22:519–527, 2003. © 2003 Wiley‐Liss, Inc.     

Viral encephalopathy and retinopathy (VER) in Atlantic salmon Salmo salar after intraperitoneal challenge with a nodavirus from Atlantic halibut Hippoglossus hippoglossus

Homogenate of tissue from juveniles of Atlantic halibut Hippoglossus hippoglossus suffering from viral encephalopathy and retinopathy (VER) was used to challenge smolt of Atlantic salmon Salmo salar with an initial average weight of 110 g. The nodavirus was administered in the form of an intraperitoneal injection, and the fish were kept for 134 d post challenge. Genotype characterisation of the nodavirus was performed by sequencing the RNA1 and RNA2 segments, and a quantitative real-time PCR (Q-PCR) assay was developed. Tissues from different organs were stained by immunohistochemistry (IHC). Samples were collected at random on Days 7, 25, 45, 69, 125 and 134 after challenge. Mortality, clinical signs and pathology of VER were observed only in the challenged group. The Q-PCR detected positive fish only in the challenged group, all of which were positive on all days of sampling. An increase in relative virus concentrations was observed from Day 7 to Day 25 post challenge. The increased level of virus concentration was maintained in the medulla oblongata throughout the experiment, suggesting persistence or slow elimination of the virus over time. The IHC detected positive cells on Days 34, 70 and 74. These results suggest that the nodavirus is transported to the medulla oblongata from the intraperitoneal injection site and is able to replicate in salmon. When injected, this nodavirus isolate caused mortality and established a persistent infection in the challenged salmon throughout the experiment. This susceptibility suggests that co-location of salmon and marine species should be avoided until further studies of possible transmission have been carried out.