The battle against the introduced pathogenic monogenean Gyrodactylus salaris in Norwegian Atlantic salmon rivers and fish farms.

The introduced salmonid ectoparasite Gyrodactylus salaris has been detected on Atlantic salmon in 53 Norwegian rivers and in 39 Norwegian fish farms. In affected rivers, the mortality of Atlantic salmon juveniles is very high, estimated to a mean of 86%. G. salaris has been considered one of the biggest threats to wild Norwegian Atlantic salmon stocks. With various measures, the authorities have reduced the potential for further spread of the parasite to new rivers and fish farms, and G. salaris has been eradicated from 43 rivers and all fish farms. Furthermore, the eradication process is almost completed in five affected rivers located at the Norwegian west coast, while preparations for the eradication in the remaining five rivers in the southeastern part of Norway have begun. The goal of Norwegian management is to eradicate the introduced pathogenic G. salaris strains from all occurrences in Norway. In fish farms, the parasite has been removed by mandatory slaughter of infected fish. In rivers, G. salaris has mostly been removed by killing all the fish hosts with rotenone. The indigenous genetic Atlantic salmon stocks are re-established after eradication of the parasite. New methods are developed using chemicals that kill the parasite without killing fish in the rivers. Norwegian authorities have so far used more than NOK 1.5 billion on research, monitoring and combating G. salaris. However, the benefits are considered many times greater than the spending. Without control measures, G. salaris would likely have spread to new Atlantic salmon rivers where the same catastrophic outcome had to be expected. The Norwegian authorities seem to meet the goal in their long-term work to halt the spread of G. salaris and to eradicate the parasite in affected rivers.

Occurrence and seasonality of Gyrodactylus salaris and G. salmonis (Monogenea) on Arctic char (Salvelinus alpinus (L.)) in the Fustvatnet lake, Northern Norway.

Gyrodactylus spp. (Monogenea) were found on 16.9% (233 out of 1376) Arctic char, Salvelinus alpinus (L.), sampled from September 2010 to October 2011 in the Fustvatnet lake, Northern Norway. Two species were identified: G. salaris Malmberg, 1957, and G. salmonis Yin & Sproston, 1948. Gyrodactylus salaris was only found on Arctic char larger than 28 cm and only in samples obtained in the autumn (September and October). Gyrodactylus salmonis was found on Arctic char of all sizes (11-47 cm) and throughout the year, with a small peak in abundance in the late autumn (November). Gyrodactylus salaris was found to prefer the tail and dorsal fin. Based on the results, we recommend that surveys of Arctic char for the presence of G. salaris are based on the examination of the fins of large fish sampled during the spawning season (autumn).

Patterns of Tetracapsuloides bryosalmonae infection of three salmonid species in large, deep Norwegian lakes.

Proliferative kidney disease (PKD), caused by the myxozoan endoparasite Tetracapsuloides bryosalmonae, is of serious ecological and economical concern to wild and farmed salmonids. Wild salmonid populations have declined due to PKD, primarily in rivers, in Europe and North America. Deep lakes are also important habitats for salmonids, and this work aimed to investigate parasite presence in five deep Norwegian lakes. Kidney samples from three salmonid species from deep lakes were collected and tested using real-time PCR to detect PKD parasite presence. We present the first detection of T. bryosalmonae in European whitefish in Norway for the first time, as well as the first published documentation of the parasite in kidneys of Arctic charr, brown trout and whitefish in four lakes. The observed prevalence of the parasite was higher in populations of brown trout than of Arctic charr and whitefish. The parasite was detected in farmed, but not in wild, charr in one lake. This suggests a possible link with a depth of fish habitat and fewer T. bryosalmonae-infected and PKD-affected fish. Towards a warmer climate, cold hypolimnion in deep lakes may act as a refuge for wild salmonids, while cold deep water may be used to control PKD in farmed salmonids.

Increasing intensities of Anisakis simplex (Rudolphi, 1809 det. Krabbe, 1878) larvae with weight and sea age in returning adult Atlantic salmon, Salmo salar L., of coastal waters of Norway.

Ninety wild Atlantic salmon, Salmo salar L., (1.5-10.3 kg) were caught in the Namsen Fjord near the mouth of River Namsen, mid-Norway, and examined for the presence and distribution of Anisakis simplex (Rudolphi, 1809 det. Krabbe, 1878) larvae by digestion of the viscera and muscles in a pepsin/HCl solution. All salmon were migrating spawners after 1-4 years of feeding in the Atlantic Ocean. All 90 Atlantic salmon had A. simplex larvae in the viscera, and all, except two, had A. simplex larvae in the musculature. The number of A. simplex larvae in each fish varied between 3 and 181, and the total mean number of nematode larvae was 44.5. The intensity of A. simplex larvae was positively correlated with increasing weight and sea age of the host. However, the proportion of larvae in the muscle fillets decreased with increasing host weight and sea age. Atlantic salmon females had more A. simplex larvae than males. In all the fish examined, 70.2% of the A. simplex larvae were found in the viscera and 29.8% in the musculature. The majority (93%) of the larvae in the musculature occurred in the hypaxial sections anterior to the anus. As A. simplex larvae commonly occur in the musculature of wild Atlantic salmon, consumption of unfrozen, raw or semi-raw musculature represents a risk for humans developing anisakiasis.

Genetic population structure of Gyrodactylus thymalli (Monogenea) in a large Norwegian river system.

The extent of geographic genetic variation is the result of several processes such as mutation, gene flow, selection and drift. Processes that structure the populations of parasite species are often directly linked to the processes that influence the host. Here, we investigate the genetic population structure of the ectoparasite Gyrodactylus thymalli Zitnan, 1960 (Monogenea) collected from grayling (Thymallus thymallus L.) throughout the river Glomma, the largest watercourse in Norway. Parts of the mitochondrial dehydrogenase subunit 5 (NADH 5) and cytochrome oxidase I (COI) genes from 309 G. thymalli were analysed to study the genetic variation and investigated the geographical distribution of parasite haplotypes. Three main clusters of haplotypes dominated the three distinct geographic parts of the river system; one cluster dominated in the western main stem of the river, one in the eastern and one in the lower part. There was a positive correlation between pairwise genetic distance and hydrographic distance. The results indicate restricted gene flow between sub-populations of G. thymalli, most likely due to barriers that limit upstream migration of infected grayling. More than 80% of the populations had private haplotypes, also indicating long-time isolation of sub-populations. According to a molecular clock calibration, much of the haplotype diversity of G. thymalli in the river Glomma has developed after the last glaciation.

Severe mortality in wild Atlantic salmon Salmo salar due to proliferative kidney disease (PKD) caused by Tetracapsuloides bryosalmonae (myxozoa).

Extensive mortality in Atlantic salmon fry was reported in the River Aelva from 2002 to 2004. Dead fish were collected in late summer 2006, and live fish were sampled by electrofishing in September the same year. At autopsy and in histological sections, the fish kidneys were found to be pale and considerably enlarged. Proliferative lesions with characteristic PKX cells were seen in a majority of the fish. DNA from kidney samples of diseased fish was subjected to PCR and sequencing, and the amplified sequences matched those of Tetracapsuloides bryosalmonae. We concluded that this myxozoan transmitted from bryozoans was the main cause of the observed mortality in salmon fry in 2006. Results from quantitative electrofishing in 2005 and 2006, combined with the observed fry mortality from 2002 to 2004, show that the smolt production in the river is severely reduced and that T. bryosalmonae is the most likely explanation for this decline. The present study is the first to report a considerable negative population effect in wild Atlantic salmon due to proliferative kidney disease (PKD). It also represents the northernmost PKD outbreak in wild fish. The river is regulated for hydroelectric power purposes, causing reduced water flow and elevated summer temperatures, and the present PKD outbreak may serve as an example of increased disease vulnerability of northern fish populations in a warmer climate.

Identification of genetic markers associated with Gyrodactylus salaris resistance in Atlantic salmon Salmo salar.

Gyrodactylus salaris Malmberg, 1957 is a freshwater monogenean ectoparasite of salmonids, first recorded in Norway in 1975 and responsible for extensive epizootics in wild Atlantic salmon Salmo salar L. The susceptibility of different populations of Atlantic salmon to G. salaris infection differs markedly, with fish from the Baltic being characterised as relatively resistant whereas those from Norway or Scotland are known to be (extremely) susceptible. Resistance to Gyrodactylus infection in salmonids has been found to be heritable and a polygenic mechanism of control has been hypothesised. The current study utilises a 'Quantitative trait loci' (QTL) screening approach in order to identify molecular markers linked to QTL influencing G. salaris resistance in B1 backcrosses of Baltic and Scottish salmon. Infection patterns in these fish exhibited 3 distinct types; susceptible (exponential parasite growth), responding (parasite load builds before dropping) and resistant (parasite load never increases). B1 backcross fish were screened at 39 microsatellite markers and single marker-trait associations were examined using general linear modelling. We identified 10 genomic regions associated with heterogeneity in both innate and acquired resistance, explaining up to 27.3% of the total variation in parasite loads. We found that both innate and acquired parasite resistance in Atlantic salmon are under polygenic control, and that salmon would be well suited to a selection programme designed to quickly increase resistance to G. salaris in wild or farmed stocks.

Infections with Gyrodactylus spp. (Monogenea) in Romanian fish farms: Gyrodactylus salaris Malmberg, 1957 extends its range.

BACKGROUND: The salmon parasite Gyrodactylus salaris Malmberg, 1957 has caused high mortalities in many Atlantic salmon, Salmo salar, populations, mainly in Norway. The parasite is also present in several countries across mainland Europe, principally on rainbow trout, Oncorhynchus mykiss, where infections do not seem to result in mortalities. There are still European countries where there are potential salmonid hosts for G. salaris but where the occurrence of G. salaris is unknown, mainly due to lack of investigations and surveillance. Gyrodactylus salaris is frequently present on rainbow trout in low numbers and pose a risk of infection to local salmonid populations if these fish are subsequently translocated to new localities. METHODS: Farmed rainbow trout Oncorhynchus mykiss (n = 340), brook trout, Salvelinus fontinalis (n = 186), and brown trout, Salmo trutta (n = 7), and wild brown trout (n = 10) from one river in Romania were sampled in 2008 and examined for the presence of Gyrodactylus spp. Alltogether 187 specimens of Gyrodactylus spp. were recovered from the fish. A subsample of 76 specimens representing the different fish species and localities were subjected to species identification and genetic characterization through sequencing of the ribosomal internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1). RESULTS: Two species of Gyrodactylus were found, G. salaris and G. truttae Gläser, 1974. This is the first time G. salaris is diagnosed in Romania. Gyrodactylus salaris was found to infect rainbow trout, brown trout and brook trout in eight of the 12 farms examined. The prevalence and intensity of infections were generally low in all farms. Gyrodactylus truttae was present on brook trout in one farm and on wild brown trout in the river studied. This also represents the first record of this parasite in Romania. Analyses of sequences of the cox1 gene of G. salaris from Romania revealed four haplotypes, all previously undescribed. While it is not unlikely that the infections in Romanian fish farms originate directly from imported rainbow trout, the current data is not sufficient to conclude on this and does not exclude that the infections can originate from hosts in the local water systems. The study shows that there are still unknown populations and variants (haplotypes) of G. salaris present in European rainbow trout aquaculture, all or many of them with unknown biological characteristics such as host specificity and virulence. As some strains might be pathogenic to Atlantic salmon, the importance of carrying out surveillance and keeping a high focus on control with import and export of live fish for aquaculture purposes is important. CONCLUSIONS: Gyrodactylus salaris and G. truttae are for the first time found on salmonids in Romania. All mitochondrial haplotypes recovered were previously undescribed and this indicates that there is still an unknown diversity of this parasite present in localities not previously examined. The virulence of the haplotypes found in Romania is unknown and requires establishing.

Gyrodactylus spp. diversity in native and introduced minnow (Phoxinus phoxinus) populations: no support for "the enemy release" hypothesis.

BACKGROUND: Translocation of native species and introduction of non-native species are potentially harmful to the existing biota by introducing e.g. diseases, parasites and organisms that may negatively affect the native species. The enemy release hypothesis states that parasite species will be lost from host populations when the host is introduced into new environments. METHODS: We tested the enemy release hypothesis by comparing 14 native and 29 introduced minnow (Phoxinus phoxinus) populations in Norway with regard to the ectoparasitic Gyrodactylus species community and load (on caudal fin). Here, we used a nominal logistic regression on presence/absence of Gyrodactylus spp. and a generalized linear model on the summed number of Gyrodactylus spp. on infected populations, with individual minnow heterozygosity (based on 11 microsatellites) as a covariate. In addition, a sample-based rarefaction analysis was used to test if the Gyrodactylus-species specific load differed between native and introduced minnow populations. An analysis of molecular variance was performed to test for hierarchical population structure between the two groups and to test for signals of population bottlenecks the two-phase model in the Wilcoxon signed-rank test was used. To test for demographic population expansion events in the introduced minnow population, we used the kg-test under a stepwise mutation model. RESULTS: The native and introduced minnow populations had similar species compositions of Gyrodactylus, lending no support to the enemy release hypothesis. The two minnow groups did not differ in the likelihood of being infected with Gyrodactylus spp. Considering only infected minnow populations it was evident that native populations had a significantly higher mean abundance of Gyrodactylus spp. than introduced populations. The results showed that homozygotic minnows had a higher Gyrodactylus spp. infection than more heterozygotic hosts. Using only infected individuals, the two minnow groups did not differ in their mean number of Gyrodactylus spp. However, a similar negative association between heterozygosity and abundance was observed in the native and introduced group. There was no evidence for demographic bottlenecks in the minnow populations, implying that introduced populations retained a high degree of genetic variation, indicating that the number of introduced minnows may have been large or that introductions have been happening repeatedly. This could partly explain the similar species composition of Gyrodactylus in the native and introduced minnow populations. CONCLUSIONS: In this study it was observed that native and introduced minnow populations did not differ in their species community of Gyrodactylus spp., lending no support to the enemy release hypothesis. A negative association between individual minnow host heterozygosity and the number of Gyrodactylus spp. was detected. Our results suggest that the enemy release hypothesis does not necessarily limit fish parasite dispersal, further emphasizing the importance of invasive fish species dispersal control.

Isolation of a novel fish thymidylate kinase gene, upregulated in Atlantic salmon (Salmo salar L.) following infection with the monogenean parasite Gyrodactylus salaris.

Analysis of differential gene expression in salmon (Salmo salar) blood following infection with the monogenean parasite Gyrodactylus salaris, resulted in the isolation of a thymidylate kinase gene not previously described from fish and which showed similarity to an LPS-inducible thymidylate kinase gene isolated from mouse macrophages. This salmon TYKi-like gene may play a role in an innate generalised response to pathogen infection as it was upregulated in salmon following infection with the parasite, and also in response to injection with the immunostimulants LPS and Poly I:C, used to emulate bacterial and viral infections, respectively. The possible role of this gene in the biosynthesis of mitochondrial DNA in activated macrophages, in response to G. salaris infection is discussed.