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Koi herpesvirus (KHV) causes a highly infectious disease afflicting common carp and koi, Cyprinus carpio L. Various molecular and antibody-based detection methods have been used to elucidate the rapid attachment and dissemination of the virus throughout carp tissues, facilitating ongoing development of effective diagnostic approaches. In situ hybridization (ISH) was used here to determine the target tissues of KHV during very early infection, after infecting carp with a highly virulent KHV isolate. Analysis of paraffin-embedded tissues (i.e. gills, skin, spleen, kidney, gut, liver and brain) during the first 8 h and following 10 days post-infection (hpi; dpi) revealed positive signals in skin mucus, gills and gut sections after only 1 hpi. Respiratory epithelial cells were positive as early as 2 hpi. Viral DNA was also detected within blood vessels of various tissues early in the infection. Notable increases in signal abundance were observed in the gills and kidney between 5 and 10 dpi, and viral DNA was detected in all tissues except brain. This study suggests that the gills and gut play an important role in the early pathogenesis of this Alloherpesvirus, in addition to skin, and demonstrates ISH as a useful diagnostic tool for confirmation of acutely infected carp.

Hygienic measures such as disinfection are important tools for the maintenance of fish health in aquaculture. While little information is available on the disinfection of water intended for fish containment, Huwa-San®, a disinfectant used in food and water industries, was used for daily treatment at concentrations of approximately 60 ppm over a total period of 3 months (experiment 1) with a 3-week treatment-free interval after 2 months (experiment 2). During this period, koi herpesvirus (KHV) was added to the water of two aquaria, one used as a normal contact control, the other one receiving daily water disinfectant treatments that prevented KHV infection of carp. In the second experiment, Huwa-San® treatment was interrupted and KHV infection was prevalent. However, when naive fish were introduced to the same aquarium after re-application of disinfectant, KHV could not be detected in those naive fish. Whilst KHV could not be detected in samples where disinfectant had been applied, it was present in samples of naive fish cohabiting with infection contact control animals which had undergone no disinfectant treatment over experiments 1 and 2. The results presented here show that water treatment with a disinfectant may prevent transmission of infectious KHV to naive carp cohabited with infected carp.

Background. The study is a contribution to Project CELFISH which involves genetic identification of populations of fish species presenting a particular economic importance or having a potential to be used in the so-called commercial substitutions. The EU fish trade has been showing a distinct trend of more and more fish species previously unknown to consumers being placed on the market. Molecular assays have become the only way with which to verify the reliability of exporters. This paper is aimed at pinpointing genetic markers with which to label and differentiate between two populations of splendid alfonsino, Beryx splendens Lowe, 1834, a species highly attractive to consumers in Asia and Oceania due to the meat taste and low fat content. Materials and methods. DNA was isolated from fragments of fins collected at local markets in Japan (MJ) (n = 10) and New Zealand (MNZ) (n = 18). The rhodopsin gene (RH1) fragment and 16 microsatellite DNA fragments (SSR) were analysed in all the individuals. The sequences obtained were processed using the BioEdit and BLAST software, whereas SSR data were processed with the GeAlEX analysis package. Results. The BioEdit software-aided comparison of MJ and MNZ nucleotide sequences of the rhodopsin gene fragments were identical and showed 100% agreement with the alfonsino sequence deposited under access number DQ197832. The preliminary analysis of SSR markers showed all the loci analysed in both populations to be polymorphic, and when randomly selected specimens were assigned to the original populations. The affinity test correctly identified the provenance of all those specimens. Conclusion. The results obtained constitute a tool for molecular differentiation between alfonsino populations collected in the FAO 81 (New Zealand) and FAO 71 (Japan) areas for the purpose of catch quota control and for checking the agreement between the label declaration and the actual product.

Hybrids of koi, Cyprinus carpio x crucian carp, Carassius carassius and koi x goldfish, Carassius auratus, proved to be susceptible to koi herpesvirus (KHV, syn. CyHV-3) and developed KHV disease (KHVD). While hybrids of koi x goldfish were partly resistant to mortality following infection by immersion, most koi x crucian carp hybrids died after bath infection. KHV DNA was detected in dead fish but also in all surviving animals by different polymerase chain reactions (PCRs). According to these results, hybrid crossbreeding does not seem to prevent severe losses associated with KHV in terms of inducing KHVD. The present study showed severe losses after a waterborne KHV infection of between 35% and 100% in koi x goldfish and koi x crucian carp hybrids as well as in SPF carp.

Background. Sturgeons have long been extinct in Polish inland waters. A substantial effort has recently been put into their restitution, covering the drainage areas of two major Polish rivers, the Oder and the Vistula. The stocked fishes are clinically healthy, but very little is known about their potential to transmit viral diseases including koi herpes virus (KHV) to healthy fishes of other species, which may pose a threat to the disease-free zones. This study was intended to determine if sturgeons could be asymptomatic carriers of KHV. Materials and methods. A total of 29 sturgeons (two species; length 8–37 cm) originating from fish farms in northern Poland with a known KHV history in common carp or koi in the area were examined: 15 Russian sturgeons, Acipenser gueldenstaedtii, with clinical signs of a disease and 14 asymptomatic Atlantic sturgeons, A. oxyrinchus. The former were sent to the laboratory alive while the latter were sent fixed in ethanol. As it is required for detection of a latent KHV infection in acipenserids, two independent procedures were applied. The preliminary results were obtained using PCR. Those findings were subsequently confirmation by nested PCR. The latter procedure consists of sequence analysis of PCR products and direct detection of KHV infected cells in tissue materials by in-situ hybridization on nucleic acid level or indirect immunofluorescence on KHV protein level. Results. KHV genome parts were found in nine Russian sturgeons and four Atlantic sturgeons. Comparison of PCR results obtained from three primer pairs used for KHV diagnostic in sturgeon showed that those designed by Bercovier et al. were most sensitive and robust for this purpose. In order to confirm the presence of viral particles the most useful method was in-situ hybridization (ISH), allowing the detection of KHV in gill samples obtained from live sturgeons. Conclusion. This preliminary study shows that sturgeons can be carriers of KHV. Therefore a viral diagnostics is highly recommended not only for sturgeons obtained from the environment but also for fertilized eggs, fry, and fish intended for re-stocking measurements of inland waters.

Background. Monitoring the genetic diversity of fish populations can provide information necessary to determine fishing quotas for the commercially caught species. One of the species subjected to intensive exploitation is the yellowstripe scad, Selaroides leptolepis (Cuvier, 1833), commonly occurring in the coastal waters of Vietnam, Cambodia, Malaysia, and Indonesia. This paper is the third part of a larger project: ”Development of a genetic-based system for identification of food products from fisheries and aquaculture introduced to the European Union customs area”. Materials and methods. Fin fragments of the yellowstripe scad, Selaroides leptolepis, were obtained in 2012 and 2013 from local markets in Vietnam (SVN), Cambodia (SKH), Malaysia (MMY), and Indonesia (MID). The analyses focused on the amplification and sequencing of a fragment of the nuclear rhodopsin gene which utilized as identification label. Subsequently, in the obtained SVN, SKH, MMY, and MID samples, 11 microsatellite regions (SRR) were analysed. Sequence analyses were performed using the BioEdit and BLAST software, while the analysis of the obtained SRR data was conducted using the GenAlEx software. Results. The analysis of the obtained loci variants divided the investigated populations into two genetically homogeneous groups: the Vietnamese–Indonesian group and the Malaysian–Cambodian group. The analysis of genetic diversity revealed statistically significant deviations from the Hardy–Weinberg equilibrium in the investigated populations and indicated the Vietnamese population as the most stable, while the Malaysian population as the least stable. Conclusion. The study provides information on the genetic diversity of the investigated populations and allows assignment of the captured fish to the geographical regions specified. Moreover, indicate that among analysed populations the safest populations were those caught in the local fisheries (continental shelf) of Vietnam and Cambodia due to their high mean genetic diversity.

Parasitic copepods of the genus Achtheres commonly infect perch, Perca fluviatilis, and zander, Sander lucioperca, in Europe. The aim of this study was to verify the specific identity of these copepods. The parasites were examined morphologically, biometrically and genetically. Statistical processing of the biometrical data was based on both empirical measurements and transformed data related to total length and genital trunk width. Principal component analysis was applied to both sets of data. DNA of both parasite 'forms' was amplified using two sets of primers (EU5.8S+EU18S and 18SF1+28SR) and the products were subjected to restriction fragment length polymorphism (RFLP). Morphological differences were found in the overall shape of the copepod bodies as well as in the details of the armament of some appendages. The morphometric study emphasized the importance of second maxillae and genital process as the variables most distinctly distinguishing the two 'forms'. The two 'forms' of Achtheres differed in the DNA sequence amplified by one set of primers. RFLP revealed even more extensive differences between these two copepods. We concluded that the copepods parasitizing perch should be referred to as Achtheres percarum von Nordmann, 1832, whereas a long-forgotten name, A. sandrae Gadd, 1901, should be applied to the copepods from zander.

Background. Genetic traceability of seafood as well as population identification using molecular methods provide useful information about the fish origin and are important for protection of overfished populations, as well as for monitoring illegal, unreported, and unregulated (IUU) fisheries. The presently reported study focused on Indian mackerel, Rastrelliger kanagurta (Cuvier, 1816)—a pelagic species with a wide range of distribution—especially important for many tropical countries, such as India, Philippines, and Thailand. This paper is the first part of a larger project: ”Development of a genetic-based system for identification of food products from fisheries and aquaculture introduced to the European Union customs area”. Materials and methods. Samples consisting of fin fragments of Indian mackerel were obtained from local markets in Thailand (MTH), Vietnam (SVN), Cambodia (SKH), and Madagascar (SMG) within 2012–2013. Two genes were analysed: nuclear rhodopsin gene (RH1) and mitochondrial D-loop (D-loop) region through RFLP analysis simulation and sequencing. Additionally, the samples from Cambodia and Madagascar were analysed with eight microsatellite loci (SSR). The data processing was aided by GenAlEx 6.5 and GeneClass2 software. Results. A comparison of the RH1 gene section revealed a total homology among the studied samples. A comparative analysis of D-loop sequences in the studied groups revealed intrapopulational diversity for MTH-, SKH-, SMG-, and SVN samples, at the level of 1, 1, 0.5, and 0.6 percentage points, respectively. Furthermore, the D-loop sequences identified a characteristic restriction site for SMG population. Based on the allele frequencies, we randomly assigned selected individuals to their original populations. GeneClass2 software correctly assigned only 16 out of 21 individuals to either the Cambodian or the Madagascar population, which jointly constituted 76% of all samples. We demonstrated, using AMOVA and GenAlEx 6.5, that the highest level of variability occurred among individuals within the respective populations, while the lowest interpopulation diversity was between the SMG and SKH populations. Conclusion. Our results may help the relevant authorities in the countries of the European Union to identify Indian mackerel and especially its products and trace them to the respective locality. Our findings may also be used for species-specific conservation measures hopefully undertaken by fisheries authorities of the countries where we took our samples. Results on other fish species, prepared in the frames of the same project, will be presented in other papers that will follow soon.

A new monogenean species Neoheterobothrium hippoglossini sp. n. from the gill cavity of Hippoglossina stomata Steindachner, 1876 is described and illustrated. The new species differs from its seven congeners in a number of morphological features and in having a different host. The validity of the genus Neoheterobothrium is discussed. Neoheterobothrium mcdonaldi Payne, 1987 is relegated from the genus and from the subfamily Choricotylinae and placed in one of the remaining subfamilies of the family Diclidophoridae.

The genetic variability of whitefish, Coregonus lavaretus (L.), was studied based on 114 individuals from nine populations inhabiting Polish lakes, including the Szczecin Lagoon, and from one population each from lakes in Austria and Switzerland. Differences within and among populations were assessed with mitochondrial ND-1 gene sequences that were PCR amplified and digested with ten restriction enzymes. The ten composite haplotypes obtained were sequenced and analyzed with MEGA4 software. The highest intrapopulation variability was noted in the whitefish populations inhabiting lakes Insko, Miedwie, Marianowo, Wisola, Œremskie, Morzycko, the Szczecin Lagoon, and Lake Lucerne, which possessed from two to five composite haplotypes. In contrast, populations inhabiting lakes Woœwin, Czarne, and Traunsee were fixed for the most common haplotype H2.

Characterization of asymptomatic KHV carriers may help understand virus transmission and storage. Such information allows farmers to minimize KHV on farms where this virus is present, also in common carp (Cyprinus carpio) monocultures. As asymptomatic KHV carriers, freshwater mollusks from the Unionidae family (swan mussels, Anodonta cygnea) and crustaceans from the Gammaridae family (scud, Gammarus pulex) were studied because of their unique method of feeding by accumulating bacterial and viral particles. The KHV genome was detected by nested PCR and confirmed by PCR recognizing the KHV glycoprotein gene in ORF 56 (KHV-U). Both PCR and nested PCR, which recognize the KHV thymidine kinase gene, always produced negative results in the swan mussels and scud samples.