Genetic characterization of salmonid alphavirus in Norway

Pancreas disease (PD), caused by salmonid alphavirus subtype 3 (SAV3), emerged in Norwegian aquaculture in the 1980s and is now endemic along the south‐western coast. In 2011, the first cases of PD caused by marine salmonid alphavirus subtype 2 (SAV2) were reported. This subtype has spread rapidly among the fish farms outside the PD‐endemic zone and is responsible for disease outbreaks at an increasing numbers of sites. To describe the geographical distribution of salmonid alphavirus (SAV), and to assess the time and site of introduction of marine SAV2 to Norway, an extensive genetic characterization including more than 200 SAV‐positive samples from 157 Norwegian marine production sites collected from May 2007 to December 2012 was executed. The first samples positive for marine SAV2 originated from Romsdal, in June 2010. Sequence analysis of the E2 gene revealed that all marine SAV2 included in this study were nearly identical, suggesting a single introduction into Norwegian aquaculture. Further, this study provides evidence of a separate geographical distribution of two subtypes in Norway. SAV3 is present in south‐western Norway, and marine SAV2 circulates in north‐western and Mid‐Norway, a geographical area which since 2010 constitutes the endemic zone for marine SAV2.     

Multilocus sequence typing (MLST) analysis of California Flavobacterium psychrophilum reveals novel genotypes and predominance of CC‐ST10 in California salmonid hatcheries

The Gram‐negative bacterium, Flavobacterium psychrophilum, is endemic to California, USA, where it is an important pathogen in salmonid aquaculture, especially in rainbow trout (Oncorhynchus mykiss). Disease outbreaks caused by F. psychrophilum in rainbow trout fingerlings can approach 90% mortality, resulting in millions of dollars of economic losses annually. The focus of this study was to investigate the genetic diversity of 49 F. psychrophilum isolates collected from disease outbreaks in 17 salmonid hatcheries in California, USA, from 2015 to 2018 using multilocus sequence typing. Results suggest California F. psychrophilum isolates are diverse, representing 11 distinct sequence types (STs), three of which were previously undescribed. Still, the majority of genotyped isolates (n = 41) belonged to a single clonal complex (CC), CC‐ST10, which is the largest CC worldwide and has been linked to disease outbreaks on several continents. Results of this study provide evidence of marked intraspecific genetic diversity of F. psychrophilum from California. The biological significance of this genetic variability is unclear but could have implications for future vaccine development and treatments. Further studies investigating the virulence, antigenic, and antimicrobial susceptibility profiles of F. psychrophilum are warranted to better understand the epizootiology of this pathogen in the Western United States.     

Factors influencing the success of salmonid egg development in river substratum

Interstitial water conditions in the hyporheic zone of the stream bed are determinants of salmonid egg hatching success. We used standardised egg exposures to develop and validate discriminant analysis and generalised linear model models linking the hatching success of brown trout (Salmo trutta) with physicochemical factors of the interstitial zone (e.g., oxygen, specific conductance, nitrate, nitrite, ammonium, pH and redox potential). Interstitial water quality was identified as a limiting factor for egg development (median of relative hatching rates = 0.23). Hatching success was unimodal in hatchery and field references incubated in free‐flowing water, but bimodal (very high or very low hatching success) in natural sediment exposures. The effects of physicochemical factors on the hatching success of Salmo trutta strongly depended on both the time and spatial scale analysed. The variables retained in the models differed between the macroscale (over all rivers), the river‐specific scale (within a river) and the microscale (at different sediment depths). Egg hatching success decreased with increased substratum depth (decrease of 26% in 150 mm compared with 50 mm). Increasingly more variable interstitial water conditions (e.g., oxygen) throughout the egg incubation period suggest progressive degradation rates in the stream substratum during the incubation period at the microscale level. Consequently, consideration of different spatial and temporal scales is necessary for the evaluation of habitat quality in salmonid conservation and catchment management plans.     

A rapid immunoperoxidase-based virus neutralization assay for salmonid alphavirus used for a serological survey in Northern Ireland

A modified virus neutralization (VN) assay was developed to replace an existing assay read on the presence or absence of virus-induced cytopathic effect (CPE). The modified assay used a monoclonal antibody to salmon pancreas disease virus as the first layer of an immunoperoxidase (IPX)-based immunostaining technique to detect viral growth. The IPX-based VN assay required only 3 days to perform, and the adoption of a 96-well microtitre format facilitated a high throughput of samples requiring small volumes of serum, cells and virus. When 352 sera from farmed salmon and 302 sera from farmed trout were tested by both the modified and the original CPE-based assays, overall correlations of 97.72 and 96.03% were, respectively, obtained (96.94% combined). When the modified assay was used to test 188 sera collected from wild salmonids in freshwater river systems in Northern Ireland, no positive results were recorded.     

The integrated development and application of controlled reproduction techniques in Pacific salmonid aquaculture

During the last several years there has been rapid progress in the development and application of controlled reproduction and associated biotechnologies to the culture of salmonids. This review focuses on the development of those techniques for Pacific salmon (Oncorhynchus species) and rainbow trout (Salmo gairdneri). These include methodologies for the induction of ovulation, storage of gametes, chromosome set manipulation, i.e., induced gynogenesis and triploidy, hormonal feminization, hormonal sterilization and hormonal masculinization of genotypic females to produce milt containing only female spermatozoa.The current and future applications of these methodologies to the commercial mariculture of salmonids and to the enhancement and management of ocean and fresh water stocks are described. The implementation of each individual methodology for the control of reproduction can in itself have a significant impact on the efficiency of culture systems. It is, however, from the combination of specific complementary methods in production systems that the greatest gains will ultimately be made.It is concluded that the utilization of single or paired controlled reproduction techniques by themselves or in combination with other biotechnologies (e.g. sterilization plus growth acceleration) offer significant advantages for both the commercial mariculture of salmonids and the rearing of salmonids by government agencies for release into the oceanic or lacustrine environment.     

Geographical distribution of salmonid alphavirus subtypes in marine farmed Atlantic salmon, Salmo salar L., in Scotland and Ireland

Sequence data from salmonid alphavirus (SAV) strains obtained from farmed marine Atlantic salmon, Salmo salar L. , over a 20-year period between 1991 and 2011 was reviewed to examine the geographical distribution of the genetically defined SAV subtypes in twelve regions across Ireland and Scotland. Of 160 different Atlantic salmon SAV strains examined, 62 belonged to subtype 1, 28 to subtype 2, 34 to subtype 4, 35 to subtype 5 and 1 to subtype 6. SAV subtypes 1, 4 and 6 were found in Ireland, while subtypes 1, 2, 4 and 5 were found in Scotland. In the majority of regions, there was a clear clustering of subtypes, with SAV subtype 1 being the dominant subtype in Ireland overall, as well as in Argyll and Bute in Scotland. SAV subtype 2 predominated in the Shetland and Orkney Islands. The emergence in Atlantic salmon of subtype 2 strains typically associated with sleeping disease in rainbow trout in Argyll and Bute, strongly suggesting transmission of infection between these species, was noted for the first time. SAV subtype 4 was the most common subtype found in the southern Western Isles, while SAV subtype 5 predominated in the northern Western Isles and north-west mainland Scotland. No single strain was dominant on sites in the western Highlands, with a number of sites in this region in particular having more than one subtype detected in different submissions. The significance of these results in relation to aspects of the epidemiology of infection, including transmission, biosecurity and wildlife reservoirs are discussed and knowledge gaps identified.     

Prevalence of piscine orthoreovirus and salmonid alphavirus in sea‐caught returning adult Atlantic salmon (Salmo salar L.) in northern Norway

Heart and skeletal muscle inflammation (HSMI) caused by piscine orthoreovirus (PRV) and pancreas disease (PD) caused by salmonid alphavirus (SAV) are among the most prevalent viral diseases of Atlantic salmon farmed in Norway. There are limited data about the impact of disease in farmed salmon on wild salmon populations. Therefore, the prevalence of PRV and SAV in returning salmon caught in six sea sites was determined using real‐time RT‐PCR analyses. Of 419 salmon tested, 15.8% tested positive for PRV, while none were positive for SAV. However, scale reading revealed that 10% of the salmon had escaped from farms. The prevalence of PRV in wild salmon (8%) was significantly lower than in farm escapees (86%), and increased with fish length (proxy for age). Sequencing of the S1 gene of PRV from 39 infected fish revealed a mix of genotypes. The observed increase in PRV prevalence with fish age and the lack of phylogeographic structure of the virus could be explained by virus transmission in the feeding areas. Our results highlight the need for studies about the prevalence of PRV and other pathogens in Atlantic salmon in its oceanic phase.     

New salmonid hosts for Tenacibaculum species: Expansion of tenacibaculosis in Chilean aquaculture

The success and sustainability of Chilean aquaculture largely depends on the control of endemic and emerging pathogens, including several species of the genus Tenacibaculum. Tenacibaculum dicentrarchi and “Tenacibaculum finnmarkense” have been detected and confirmed in Chilean Atlantic salmon (Salmo salar). However, no outbreaks of tenacibaculosis in rainbow trout (Oncorhynchus mykiss) or coho salmon (Oncorhynchus kisutch) have been reported, either in Chile or globally. The aims of this study were to determine whether the mortalities recorded for rainbow trout and coho salmon from five marine fish farms located in the Los Lagos, Aysén, and Magallanes Regions could be caused by Tenacibaculum spp. The diseased fish exhibited cutaneous haemorrhages, tail and peduncle rots, and damage on the mouth and tongue. Microbiological analysis of infected external tissues yielded 13 bacterial isolates. The isolates were identified as members of the genus Tenacibaculum through biochemical analysis (e.g. Gram-stain negative, straight rods, filamentous cells and motile by gliding), but differences existed in biochemical results, making species-level identification through biomolecular tools essential. The 16S rRNA analysis found that the majority of isolates were more closely related to “T. finnmarkense” than T. dicentrarchi, while the phylogenetic trees resulting from multilocus sequence data recovered the four main clades (clades I to IV) identified by Olsen et al. (2017, Veterinary Microbiology, 205, 39). This is the first documented occurrence of clinical tenacibaculosis in farmed rainbow trout and coho salmon globally, and it extends the known host distribution of this pathogen in Chile. Moreover, we confirm the presence of Tenacibaculum species in the Chilean Patagonia. These findings highlight the importance of establishing preventative measures to minimize the spread of this disease within the Chilean marine aquaculture industry, as well as the need for monitoring initiatives worldwide in these farmed fish species.     

Dynamics of experimental production of Thelohanellus hovorkai (Myxozoa: Myxosporea) in fish and oligochaete alternate hosts

The dynamics of development and production of Thelohanellus hovorkai (Myxozoa) were examined to investigate factors inducing haemorrhagic thelohanellosis in carp, Cyprinus carpio L. Fresh actinospores of T. hovorkai were harvested from the oligochaete alternate host, Branchiura sowerbyi, and used for infection experiments with myxosporean-free carp. Visualization of actinospores by fluorescent labelling revealed that sporoplasms penetrated the gill filaments of carp immersed in an actinospore suspension as early as 30 min post-exposure (PE). Plasmodia of T. hovorkai developed in the connective tissues of various organs and matured 3-5 weeks PE; dispersion of myxospores from degenerate plasmodia occurred 5-7 weeks PE. Challenges with a high dose of actinospores (4.5 x 10(6) spores per fish) resulted in the onset of disease, which was more easily achieved by the oral intubation of actinospores than by immersion in an actinospore suspension. Actinosporean-free B. sowerbyi were exposed to different densities of myxospores (10(4)-10(6) spores per oligochaete) and subsequently reared at different temperatures (15, 20, 25 degrees C). At 20 and 25 degrees C, actinospore releases were first detected 40-43 days PE, with multiple peaks of release (max. 7 x 10(5) actinospores day(-1)) during the next 60 days. We concluded that the developmental cycle of T. hovorkai was completed within 3-5 months at 20-25 degrees C, and that the ingestion of large numbers of actinospores orally, possibly by feeding on infected oligochaetes, resulted in a disease condition in carp.     

Variation in the hyphal growth rate and the virulence of two genotypes of the crayfish plague organism Aphanomyces astaci

Crayfish plague, a devastating disease of freshwater crayfish, is caused by an oomycete organism, Aphanomyces astaci. Currently five genotypes of A. astaci are known, but variable features between the strains or genotypes have not been studied extensively. This study analysed 28 isolates of the As genotype and 25 isolates of the Ps1 genotype and reveals that the radial growth rate is significantly (P < 0.001) different between these two genotypes, although highly variable inside the genotype As. Two Ps1 genotype isolates and two As genotype isolates with different radial growth rates were tested in an infection trial. Clear differences were detected in the development of mortality in the test groups. The representatives of the Ps1 genotype caused total mortality within a short time span. The As genotype isolates were much less virulent. The slow‐growing As isolate showed higher virulence than the As isolate with a high growth capacity. Although slow growth could be one survival strategy of the pathogen, several other mechanisms are involved in the pathogenicity and warrant further studies.     

Pathogenicity of Saprolegnia species associated with outbreaks of salmonid saprolegniosis in Japan

The present study was conducted to evaluate the pathogenicity and pathology of Saprolegnia salmonis NJM 9851 and Saprolegnia parasitica NJM 9868, isolated from outbreaks of saprolegniosis, against the immature stages of five species of salmonids. The cumulative mortalities of the tested fish groups that were exposed to 2 × 10 ⁵  spore/L concentrations of S. salmonis NJM 9851 were 90% for brown trout, 93.3% for sockeye salmon and 100% for rainbow tout, masu salmon, and Japanese char. In contrast, all salmonid species exposed to 2 × 10 ⁵  spore/L concentrations of S. parasitica NJM 9868 experienced cumulative mortalities of 100%. The histopathological changes of the saprolegniosis lesions found in all sites of infection were loss of the epidermis, edema of the hypodermis and different degrees of degenerative changes in the underlying musculature. It is clear from our results that S. salmonis NJM 9851 and S. parasitica NJM 9868 are highly pathogenic species to five species of salmonid fishes     

Variations in growth in haemoglobin genotypes of Atlantic cod

In the present paper are described the growth properties of three different haemoglobin genotypes of juvenile Atlantic cod (Gadus morhua) reared at 7, 10, 13 and 16 °C. In addition one group was reared under temperature steps i.e. moved successively from 16 to 13 and 10 °C. The genotype Hb-I(2/2)displayed the overall highest growth rate in the temperature range 13–16 °C, whereas the Hb- I(1/1)genotype showed the highest overall growth at the lowest temperature (7 °C). Accordingly, we found a significant interaction between genotype and temperature. The differences in growth were largest when cod were reared under the temperature step regime where the Hb-I(2/2)genotype displayed 17 and 24% higher growth than Hb-I(1/1)and Hb-I(1/2),respectively. Optimal temperature for growth (T_opt.G) varied between the genotypes with the genotype Hb-I(1/2)displaying the highest (mean ± SE) T_opt.G (14.5 ± 1:0.8 °C) and Hb-I(1/1)the lowest (12.5 ± 0.2 °C). The biological significance of this link between biochemical genetic variation and physiological properties might be the influences on growth pattern, ultimate size and age at first maturity.     

Interactions between body mass and water temperature cause energetic bottlenecks in juvenile steelhead

Across taxa, it is generally accepted that there are fitness advantages to rapid growth early in life. For stream‐dwelling salmonids, however, high temperatures and associated energetic costs during the summer growing season might offset or even prevent the competitive advantage of large body size. Our overall objective was to understand the relative importance of factors that can cause variation in growth rates in an age‐0 cohort of wild steelhead (Oncorhynchus mykiss) in Idaho, USA, where temperatures approach, and temporarily exceed, their tolerance level. For individually tagged fish inhabiting the same stream reach, we found that growth rates were negatively related to fish mass (slopes of the two best approximating models were both ?0.024). Comparing growth rates from 16 different stream reaches throughout the watershed, we found that temperature‐induced metabolic cost was the single best approximating model (AIC w_i = 1.0) of the variation in individual growth rates. The bioenergetic model showed that mass‐specific metabolic costs decreased with mass, but the absolute energetic demands increased over the same size range. Because temperature had a multiplicative effect on metabolic cost, our results suggest that the effect of food limitation increased with fish size. We conclude that high water temperatures pose energetic bottlenecks and can be a potentially strong mechanism limiting growth in juvenile salmonids in summer, particularly as streams in the region experience warming trends.     

草鱼生长相关优势基因型的聚合效果分析

为研究草鱼(Ctenopharyngodon idella)生长性状相关分子标记聚合效果,本研究选择利用候选基因关联分析方法获得10个与生长性状相关SNP标记,分别位于草鱼载脂蛋白A-I-1基因(apoA-I-1)、丙酮酸激酶1型(PKL)、羧肽酶A1(CPA1)、柠檬酸合酶(CS)、醛缩酶B(Aldo-B)、生长催乳...     

大口黑鲈生长性状相关标记的聚合效果分析

选择已经获得的8个与大口黑鲈(Micropterus salmoides)生长性状相关的分子标记, 其中4个单核苷酸多态性标记分别位于IGF-I、POU1F1、PSSIII和MSTN基因上, 4个微卫星位点分别是JZL60、JZL67、MisaTpw76和MisaTpw117。从养殖群体中筛选出具有4个以上优势基因型数量的大口黑鲈亲鱼20尾进行群体繁殖, 其中雌鱼9尾, 雄鱼11尾。所产子代进行同塘饲养, 9月龄时随机挑选288尾进行体质量测量与STR基因分型。结果显示:子代体质量为103~401 g, 含有生长优势基因型的数量在1~6之间, 含有1~6个生长优势基因型的个体其平均体质量依次为227.83、239.56、258.81、273.02、302.50和305.60 g, 生长优势基因型的数量与大口黑鲈生长速度呈正相关。进一步分析表明子代中优势基因型的平均数量为2.99, 相比亲本群体的优势基因型平均数量(2.36)得到提高。研究结果说明, 利用有限的与生长相关的优势基因型进行聚合可以获得具有优良生长性状的大口黑鲈, 也为下一步大口黑鲈分子标记辅助育种提供理论依据。

     

Utilization of tissue habitats by Myxobolus wulii Landsberg & Lom, 1991 in different carp hosts and disease resistance in allogynogenetic gibel carp: redescription of M. wulii from China and Japan

Myxobolus wulii (= Myxosoma magna ) was first described from the gills of goldfish, Carassius auratus auratus, in China. Subsequently, a myxosporean infecting the hepatopancreas of allogynogenetic gibel carp, C. auratus gibelio , was designated as a different species, Myxobolus guanqiaoensis , although the morphological features were almost identical to those of M. wulii . In Japan, an unidentified Myxobolus sp. was found in the gills and hepatopancreas of goldfish. Morphological and molecular analyses in the present study identified these myxosporeans as M. wulii , which was thus shown to use different habitats in the host fish. Phylogenetic analyses of small subunit ribosomal RNA gene sequences showed that M. wulii is closely related to two gill-infecting Myxobolus species, M. ampullicapsulatus and M. longisporus . Fish infected with M. wulii in the hepatopancreas exhibit swollen abdomens and chronic mortality. Hepatopancreas tissues are virtually destroyed and replaced with plasmodia of M. wulii . A remarkable difference in susceptibility to M. wulii between two clones of allogynogenetic gibel carp was observed, suggesting that resistance to the myxosporean infection was established in a clone of fish bred by allogynogenesis.