Development of real-time PCR assays for detection of megalocytiviruses in imported ornamental fish

Megalocytiviruses have been associated globally with severe systemic disease and economic loss in farmed food fish and ornamental fish. The viruses have been spread internationally by translocation of live fish. In New Zealand, megalocytiviruses are regarded as exotic. A potential pathway for introduction has been identified, namely imported ornamental fish. In the present study, real‐time PCR assays were developed for detection of megalocytiviruses using a conserved major capsid protein gene. A SYBR green assay was developed to target all known megalocytiviruses. A second real‐time PCR assay using a molecular beacon was developed to specifically target gourami, Trichogaster trichopterus, iridovirus, a species of iridovirus previously linked to ornamental fish imports in Australia. The analytical sensitivity for the SYBR green and molecular beacon assays were 10 and 100 fg, respectively. The analytical specificity of the real‐time PCR assays determined using genomic DNA templates from three target viruses, 12 non‐target viruses and 25 aquatic bacterial species were 100%. The intra‐run and inter‐run coefficients of variation of both assays were <5%. The real‐time PCR assays developed in this study provide rapid, sensitive, and specific detection of megalocytiviruses and gourami iridovirus.     

硝化细菌对淡水水族箱水质及养殖观赏鱼影响的初步研究

本试验设立硝化细菌、水生观赏植物对照组与空白对照组进行对比试验。结果表明硝化细菌对降低淡水水族箱中COD、NH4^ -N、NO^2 -N效果显。有利于提高蓝毛足鲈等的相对生长率，对孔雀鱼、七彩神仙鱼增色有益。实验中还发现硝化细菌有明显预防细菌性肠炎病发生的作用。硝化细菌在淡水水族箱中固着种群稳定需要25—28天，在施用硝化细菌3—4周后水体COD、NH4^ -N、NO^2 -N达到稳定水平。     

Detection and molecular characterization of infectious spleen and kidney necrosis virus from major ornamental fish breeding states in Peninsular Malaysia

‘Gold standard’ OIE reference PCR assay was utilized to detect the presence of infectious spleen and kidney necrosis virus (ISKNV) in freshwater ornamental fish from Malaysia. From total of 210 ornamental fish samples representing 14 species, ISKNV was detected in 36 samples representing 5 fish species. All positive cases did not show any clinical signs of ISKNV. Three restriction enzymes analyses showed that the fish were infected by identical strains of the same virus species within Megalocytivirus genus. Major capsid protein (MCP) genes of 10 ISKNV strains were sequenced and compared with 9 other reference nucleotide sequences acquired from GenBank. Sequence analysis of MCP gene showed that all strains detected in this study were closely related to the reference ISKNV with nucleotide sequence identity that was ranging from 99.8% to 100%. In addition, phylogenetic analysis of MCP gene revealed that viruses from genus Megalocytivirus can be divided into three genotypes: genotype 1 include reference ISKNV and all other strains that were detected in this study, genotype 2 include viruses closely related to red sea bream iridovirus (RSIV), and genotype 3 include viruses closely related turbot reddish body iridovirus (TRBIV).     

The effect of photoperiod and feeding frequency on larval of the Amazonian ornamental fish Pyrrhulina brevis (Steindachner, 1876)

This study aimed to evaluate the performance of Pyrrhulina larvae, Pyrrhulina brevis, under different photoperiods and feeding frequencies. A completely randomized design was used, with four replicates in a 5 × 2 factorial, with five photoperiods (6L:18D, 12L:12D, 16L:8D, 20L:4D, 24L:0D) and two feeding frequencies (two or four times a day). Four hundred larvae with an initial length of 4.6 ± 0.09 mm were randomly distributed in 40, 1‐L containers at a density of 10 larvae/L. Feeding was performed with Artemia nauplii in the ratio of 150 Artemia nauplii/larva/feeding. After 15 days, all larvae were euthanized for measurements of length (mm), final weight (mg), survival rate (%) and larvae uniformity in weight an length (%). The data were subjected to analysis of variance and subsequently, in case of significance, a Tukey test was performed to compare means. There was no interaction between photoperiod and feeding frequency on studied variables (P > 0.05). Likewise, there was no influence of feed rate on the same variables (P > 0.05). There was only influence of photoperiod, where the larvae subjected to longest photoperiods (24L:0D and 20L:4D) showed improved weight and length (P < 0.05). Therefore, a photoperiod of 20L:4E, with feeding twice a day is recommended.     

An understated danger: Antimicrobial resistance in aquaculture and pet fish in Switzerland,a retrospective study from 2000 to 2017

Aquaculture is a rapidly growing field of food production. However, morbidity and mortality are higher in aquaculture species than in domestic animals. Bacterial diseases are a leading cause of farmed fish morbidity and are often treated with antimicrobials. Since most Swiss fish farms release effluents directly into surface water without treatment and since aquaculture fish are consumed by humans, antimicrobial resistance (AMR) and multi-resistance in aquaculture fish are important for environmental and public health. In this study, AMR tests for 14 antimicrobials were performed on 1,448 isolates from 1,134 diagnostic laboratory submissions from farmed and ornamental fish submissions for the period from 2000 to 2017. Amoxicillin, gentamycin and norfloxacin had the lowest proportion of resistant samples. However, AMR was highly variable over time. Resistance proportions were higher in: (a) ornamental fish compared with farmed fish, (b) fish from recirculation systems compared with those from other farming systems and (c) isolates originating from skin compared with those originating from inner organs. Multiple resistances were common. The results of this study provide useful data for Swiss fish veterinarians and some interesting hypotheses about risk factors for AMR in aquaculture and pet fish in Switzerland. However, further research is needed to define risk factors.     

Population growth and mass mortality of an estuarine fish,Acanthopagrus butcheri,unlawfully introduced into an inland lake

• 1. In 2006, two periods of hypoxia resulted in the death of approximately 35 tonnes of black bream (Acanthopagrus butcheri) in Lake Indoon, a small inland lake in Western Australia.
• 2. Acanthopagrus butcheri was the first fish species to be recorded in this lake, along with the mosquitofish (Gambusia holbrooki) which was also observed during sampling in 2006. Acanthopagrus butcheri appears to have been introduced to Lake Indoon between 1998 and 2003 and formed a self‐sustaining population. It is believed to have been deliberately introduced for the purpose of creating a recreational fishery, despite the existence of substantial penalties for illegal translocation of fish in Western Australia.
• 3. Recent human‐induced environmental changes, including rising groundwater and salinization, have probably aided the establishment of both species in Lake Indoon. The importance of salinity to recruitment success by A. butcheri was indicated by the presence of only two age classes in 2006, with estimated recruitment dates coinciding with the years of highest recorded salinity in the lake.
• 4. The ‘fish kills’ provided an opportunity to examine aspects of A. butcheri biology in a relatively low salinity environment which is atypical for this estuarine species. In particular, the recruitment period in Lake Indoon was delayed until autumn/winter, rather than spring/summer as seen in other populations. Biological responses in Lake Indoon have implications for natural populations living in estuaries with modified salinity regimes.
• 5. The ecological, social and economic impacts potentially arising from the introduction of fish to Lake Indoon, which is an important migratory bird habitat and a recreational amenity for local residents and tourists, illustrate the complexities of fish translocation and the need for rigorous assessment before stocking to identify potential costs and benefits.

Copyright © 2008 John Wiley & Sons, Ltd.     

Isolation and characterization of a rhabdovirus from co-infection of two viruses in mandarin fish

Co-infection of two viruses has been observed in mandarin fish (Siniperca chuatsi), but the two viruses have not been characterized. In this study, a rhabdovirus has been isolated from the co-infected two viruses extracted from the diseased mandarin fish, and its morphological structure and partial biochemical and biophysical characteristics have been observed and analyzed. The isolated rhabdovirus has a typical bullet shape, and is therefore called S. chuatsi rhabdovirus (SCRV). And, the isolated rhabdovirus produced a higher titer (10^8.5 TCID₅₀ ml^− 1) than did the co-infecting viruses (10^6.5 TCID₅₀ ml^− 1). Subsequently, the viral genome RNA was extracted, and used as template to clone the complete nucleoprotein (N) gene by RT-PCR amplification. Cloning and sequencing of the SCRV N protein revealed 42%-31% amino acid identities to that of trout rhabdovirus 903/87 and the rhabdoviruses in genus Vesiculovirus. SDS-PAGE separation of the isolated SCRV and other two rhabdoviruses also revealed obvious polypeptide profile difference. Moreover, the anti-SCRV N protein antibody was prepared, and the anti-SCRV N protein antibody only could recognize the SCRV N protein, whereas no antigenicity was detected in other two rhabdoviruses. The data suggested that the SCRV should be a rhabdovirus member related to the genus Vesiculovirus in the Rhabdoviridae.     

A comparison of electric fishing and gillnetting to examine the effects of anthropogenic disturbance on riverine fish communities

Abstract Quantitative electric fishing and multiple mesh-size gillnetting of fish communities in the Hawkesbury-Nepean River system, near Sydney, were carried out upstream of, within and well below each of three major points of treated sewage effluent discharge. Approximately half of the fish sampled were caught by electric fishing, comprising 515 individuals in 3 h of electric fishing (172 fish per hour). The remaining 439 fish were caught in 72 h of gillnetting (six fish per hour). In total, 16 fish species were collected by electric fishing and only seven species by the gill nets. Differences were found in the total abundances and numbers of species collected by each method in relation to points of treated sewage effluent discharge, but neither community variable showed consistent differences between the areas above, within and well below points of treated sewage effluent discharge in all three river regions. However, classification and ordination of electric fishing catch data produced distinct fish community groups in relation to sewage discharge, while similar analyses of the gillnetting data failed to reveal such differences in community structure. It was suggested that the increased sampling efficiency of electric fishing, both in terms of the number of fish sampled per unit effort and number of species collected, allowed for significant increases in the power of univariate and multivariate analysis methods in detecting such environmental disturbances in large rivers.     

Entrainment of larval fish assemblages from the inner shelf into the East Australian Current and into the western Tasman Front

Entrainment and transport of larval fish assemblages by the East Australian Current (EAC) were examined from the coastal waters of northern New South Wales (NSW) to the western Tasman Front, via the separation of the EAC from the coast, during the austral spring of 2004. Shore‐normal transects from the coast to the EAC off northern NSW revealed an inner shelf assemblage of near‐shore families (Clupeidae, Engraulidae, Platycephalidae and Triglidae), an EAC assemblage dominated by Myctophidae and Gonostomatidae, and a broadly distributed assemblage over the continental shelf dominated by Scombridae and Carangidae. Further south and after the EAC had separated from the coast, we observed a western Tasman Front assemblage of inner shelf and shelf families (Clupeidae, Engraulidae, Serranidae, Scombridae, Carangidae, Bothidae and Macroramphosidae). The abundance of these families declined with distance from the coast. Surprisingly, there was no distinctive or abundant larval fish assemblage in the chlorophyll‐ and zooplankton‐enriched waters of the Tasman Sea. Water type properties (temperature‐salinity, T‐S), the larval fish assemblages and family‐specific T‐S signatures revealed the western Tasman Front to be an entrained mix of EAC and coastal water types. We found an abundance of commercially important species including larval sardine (Sardinops sagax, Clupeidae), blue mackerel (Scomber australasicus, Scombridae) and anchovy (Engraulis australis, Engraulidae). The entrainment and transport of larval fish from the northern inner shelf to the western Tasman Front by the EAC reflects similar processes with the Gulf Stream Front and the Kuroshio Extension.     

长江上游珍稀特有鱼类国家级自然保护区干流段鱼类组成及其多样性

2010年9-11月、2011年5-7月和9-11月、2012年5-7月在长江上游珍稀特有鱼类国家级自然保护区干流宜宾、合江及江津江段进行了4次鱼类资源调查,共采集鱼类样本27 836尾,隶属于7目17科111种(亚种),其中长江上游特有鱼类27种,产漂流性卵鱼类45种,外来鱼类6种。宜宾、合江及江津江段鱼类物种数分别为85、91及91种,宜宾与合江江段鱼类相似度指数为69.23%,合江与江津江段为71.70%,宜宾与江津江段为77.78%。铜鱼(Coreius heterodon)、南方鲇(Silurus meridionalis)、长鳍吻(Rhinogobio ventralis)及圆口铜鱼(C.guichenoti)为保护区干流主要经济鱼类,产漂流性卵鱼类占渔获物总重量的66.35%。鱼类Shannon-Wiener多样性指数在4次调查中江津江段均最高,宜宾次之,合江最低,但三江段之间多样性指数均无显著性差异(P>0.05)。与历史资料相比,该江段鱼类资源明显减少,急需加强保护措施。     

鳜脑组织细胞系的建立及其病毒敏感性研究

鱼类细胞是开展鱼类病毒分离鉴定、功能基因分析以及生物制品制备等研究的重要物质基础。鳜(Siniperca chuatsi)是深受养殖者和消费者欢迎的养殖品种。随着鳜鱼养殖产量的逐年增加,其病害问题尤其是病毒病问题也日趋严重,但是,可用于鳜鱼病毒分离和基因功能分析的鳜细胞系缺乏。本研究采用组织块消化法,对来源鳜脑组织的细胞进行原代培养,建立了鳜脑组织细胞系,命名为MFB。MFB细胞在28℃含10%胎牛血清的L-15中已稳定传代超过70次,第25代鳜脑组织细胞的染色体众数为56。采用免疫荧光细胞化学技术(β-tubulin和Neu-N)鉴定MFB细胞的神经元纯度,结果显示,培养的MFB细胞为神经元类细胞。病毒敏感性实验结果显示,鳜蛙虹彩病毒(MFRaIV)、大口黑鲈蛙虹彩病毒(LMBRaIV)和大鲵虹彩病毒(GSIV)均可在MFB细胞中产生典型细胞病变效应,病毒滴度分别为108.68±0.12、108.36±0.15、1010.15±1.85 TCID50/mL。使用脂质体Lipofectamine®2000将pEGFP-N1转入MFB细胞,转染效率可达20%。本研究建立的鳜脑组织细胞系不仅对多种蛙虹彩病毒敏感,而且转染质粒效率较高,为鳜病毒性病原的分离及基因功能研究奠定了前期基础。     

Growth of Amazon ornamental fish Nannostomus beckfordi larvae (Steindachner, 1876) submitted to different stocking densities and feeding management in captivity conditions

The study evaluated productive performance of larvae Nannostomus beckfordi submitted to feeding management (Experiment one) and different stock density (Experiment two) in captivity conditions. The first experiment evaluated feeding rate (100 and 200 nauplii of artemia larvae^?1 day^?1) and feeding frequency (2 and 4× per day). Second experiment evaluated different stock density (1, 5, 10, 20, 40 larvae per litre). At the end of 15 days, for both experiment, the survival and productive performance such as total length (TL), final weight (FW), specific development rate (SDR), specific growth rate (SGR), uniformity for weight (UW) uniformity for length (UL) and relative condition factor (Kr) was determined. For the main results, there is no significant difference to the rate and feeding frequency. The stock density has no significant difference for UW, Kr and survival. Nonetheless, a reduction in the TL, FW, SDR, SGR and FW with the increase in density (20 for 40 larvae per litre) was observed. Thus, for this species Nannostomus beckfordi during to initial stage using exogenous feeding, it is recommended 20 larvae per litre fed with 100 nauplii per larvae having two meals per day.     

Tetrahymena sp. infection in guppies, Poecilia reticulata Peters: parasite characterization and pathology of infected fish

Tetrahymena sp. infection was diagnosed in guppies imported from Singapore. The parasite was isolated (Tet-NI) and optimally cultured in vitro in RM-9 medium. Cytological analyses [silver-staining and scanning electron microscopy (SEM)] revealed a pyriform-shaped, 64 × 41-μm holotrich ciliate without caudal cilium, containing a macro-nucleus (18.25 × 16.83 μm) and micro-nucleus (5.73 × 5.40 μm). Wet-mount examination and histological analyses of fish exposed to the parasite by co-habitation, immersion and infection by i.p. (intra-peritoneal) and i.m. (intra-muscular) injection revealed numerous ciliates on the skin, and in the gill and caudal fin blood vessels. Ciliates surrounded internal organs, the peri-orbital region of the eye, and were observed inside developing guppy embryos. Some muscle necrosis was associated with infection, but little or no inflammatory response. Immersion, co-habitation and i.m. injection caused relatively high infection rates and levels in the skin and tail, and lower infection in the gill blood vessels and internal organs; i.p. injection caused higher infection in the gill blood vessels and internal organs. Co-habited fish had relatively high infection levels in the hind-gut sub-mucosa. This is the first report of controlled systemic infection by Tetrahymena sp.     

Development of an aquatic pathogen database (AquaPathogen X) and its utilization in tracking emerging fish virus pathogens in North America

The AquaPathogen X database is a template for recording information on individual isolates of aquatic pathogens and is freely available for download (http://wfrc.usgs.gov). This database can accommodate the nucleotide sequence data generated in molecular epidemiological studies along with the myriad of abiotic and biotic traits associated with isolates of various pathogens (e.g. viruses, parasites and bacteria) from multiple aquatic animal host species (e.g. fish, shellfish and shrimp). The cataloguing of isolates from different aquatic pathogens simultaneously is a unique feature to the AquaPathogen X database, which can be used in surveillance of emerging aquatic animal diseases and elucidation of key risk factors associated with pathogen incursions into new water systems. An application of the template database that stores the epidemiological profiles of fish virus isolates, called Fish ViroTrak, was also developed. Exported records for two aquatic rhabdovirus species emerging in North America were used in the implementation of two separate web-accessible databases: the Molecular Epidemiology of Aquatic Pathogens infectious haematopoietic necrosis virus (MEAP-IHNV) database (http://gis.nacse.org/ihnv/) released in 2006 and the MEAP- viral haemorrhagic septicaemia virus (http://gis.nacse.org/vhsv/) database released in 2010.     

Non-native fish in the fresh waters of Portugal, Azores and Madeira Islands: a growing threat to aquatic biodiversity

Abstract  Portugal, the Azores, and Madeira had a combined total of twenty six non-native fish species introductions, with the mainland having the highest number (twenty three), followed by the Azores with ten and Madeira with two records. Fifteen of these species established in Portugal, six in the Azores and one in Madeira. The oldest known introduction (1792) was goldfish, Carassius auratus (L.), while the newest (2005) was tinfoil barb, Barbonymus schwanenfeldii (Bleeker). Ten species had been introduced in Portugal by the beginning of 20th century, but the rate of introduction continues to increase, with four species introduced since 2000. In mainland Portugal, the Douro, the Tejo and the Guadiana drainages had the highest number of introduction records, while the Lima and the Mira drainages had the fewest. Management measures are offered to reduce current rate of fish introductions in Portuguese watersheds.     

Connectivity,habitat, and flow regime influence fish assemblage structure: Implications for environmental water management in a perennial river of the wet–dry tropics of northern Australia

1. Environmental water management seeks to balance competing demands between the water needed to sustain human populations and their economic activities and that required to sustain functioning freshwater ecosystems and the species they support. It must be predicated on an understanding of the environmental, hydrological, and biological factors that determine the distribution and abundance of aquatic species.
2. The Daly River of the wet–dry tropics of northern Australia consists of a perennially flowing main stem and large tributaries, as well as many small to large naturally intermittent tributaries, and associated off‐channel wetlands. Increased groundwater abstraction to support irrigated agriculture during the dry season threatens to reduce dry‐season flows that maintain perenniality and persistence of freshwater fishes.
3. Fish assemblages were surveyed at 55 locations during the dry season over a 2‐year period with the goal of establishing the key landscape‐scale and local‐scale (i.e. habitat) drivers of fish species distribution.
4. Longitudinal (upstream/downstream) and lateral (river/floodplain) gradients in assemblage structure were observed with the latter dependent on the position in the river landscape. Underlying these gradients, stream flow intermittency influenced assemblage composition, species richness, and body size distributions. Natural constraints to dispersal were identified and their influence on assemblage structure was also dependent on position within the catchment.
5. Eight distinct assemblage types were identified, defined by differences in the abundance of species within five groups differing in functional traits describing body size, spawning requirements, and dispersal capacity. These functional groups largely comprised species widely distributed in northern Australia.
6. The results of the study are discussed with reference to the environmental flow needs of the Daly River and other rivers of northern Australia. The findings may also be applied to environmental flow management in savannah rivers elsewhere.

     

Long-term effects of human influence on fish community structure and fisheries in Berlin waters: an urban water system

Commercial catch and fisheries monitoring data were compared with hydrological and water course characteristics conditions to identify the cause of changes in fisheries production in Berlin water courses since the 1950s. The urban parts of Berlin are densely populated, and the waters are under pressure from shipping, hydraulic engineering, pollution and recreation use. Most Berlin waters are polytrophic or hypertrophic, with an annual nutrient input of 595 t of total phosphorous and 8640 t of total nitrogen. A total of 34 fish species were recorded, eight of which were introduced. According to historical records, seven native fish species are extinct or missing. At present, the fish community is dominated by a few eurytopic species, i.e. roach, Rutilus rutilus (L.), common bream, Abramis brama (L.), silver bream, Blicca bjoerkna (L.), and perch, Perca fluviatilis L., which contribute 85% of all individuals caught. Two 'faunal breaks' were identified. The first was the changing of river character from a barbel to bream zone caused by damming and river regulation. This started in the thirteenth century and ended at the beginning of the twentieth century with the extinction of anadromous fishes. The second break was during the 1960s and 1970s, with the eutrophication of Berlin waters and the near total loss of submerged macrophytes. All phytophilic fish species declined greatly and a mass development of eurytopic species started. The catch of pike, Esox lucius L., common carp, Cyprinus carpio L., and tench, Tinca tinca (L.), became negligible, and at present, eel, Anguilla anguilla (L.), and pikeperch, Stizostedion lucioperca (L.), are the main commercial fish species. Concomitantly, the profitability of the fisheries declined and was linked to a decrease in the number of people employed in fisheries by more than 75% compared with 1950.     

微生物促进传统鱼酱油发酵和品质改善的研究进展

鱼酱油是一种在东亚和东南亚地区被广泛使用的海鲜调味品,一般是以新鲜海鱼或虾为原料,并拌入大量的食盐(一般20%~30%),在开放环境下经过长期缓慢发酵而制成,具有味道鲜美、营养丰富等特点。传统的鱼酱油生产存在发酵周期过长、生物胺积累、产品品质不一致等问题,利用特定的微生物发酵剂有望解决上述问题。文章在概述传统鱼酱油生产工艺的基础上,着重介绍了传统鱼酱油中的产酶微生物种类,以及利用微生物促进鱼酱油发酵和改善鱼酱油产品品质的研究和应用情况。     

Utilization of the earthworm,Eisenia fetida (Savigny, 1826) as an alternative protein source in fish feeds processing: A review

Use of fish meal in aquaculture is gradually becoming unsustainable due to competition, cost and ecological challenges hence the need to expand the alternative protein sources. The earthworm, Eisenia fetida is among the non‐conventional protein sources, which have been tested with relatively promising results, thanks to its high protein levels, proper amino acid profile, high reproduction rate, low mortalities, fast growth and ease of production. The feasibility of using E. fetida for commercial fish feed production depends on the fundamental knowledge of its growth and reproductive biology, as well as the production methods. On the other hand, the nutritional suitability of E. fetida is determined by its amino acid composition and meal processing methods. Therefore, this study reviews the biological, biochemical composition as well as production and processing methods, as critical aspects for sustainable production and utilization of the earthworm in commercial fish feed production. Further, the study provides some recommendations and options to provide nutritionally complete and economically viable fish feed for efficient and sustainable aquaculture production systems.