Comparative Molecular Characterization of Novel and Known Piscine Toti-Like Viruses.

Totiviridae is a virus family well known to infect uni-cellular organisms like fungi and protozoa. In more recent years, viruses characterized as toti-like viruses, have been found in primarily arthropods, but also a couple in planarians and piscine species. These toti-like viruses share phylogenetic similarities to totiviruses; however, their genomes also includes additional coding sequences in either 5' or 3' ends expected to relate to more advanced infection mechanisms in more advanced hosts. Here, we applied next generation sequencing (NGS) technologies and discovered three new toti-like viruses, one in wild common carp and one in bluegill from the USA and one in farmed lumpsucker from Norway. These are named common carp toti-like virus 1 (CCTLV-1), bluegill toti-like virus 1 (BGTLV-1), and Cyclopterus lumpus toti-like virus (CLuTLV), respectively. The genomes of these viruses have been characterized and compared to the three previously known piscine toti-like viruses, piscine myocarditis virus (PMCV) found in Atlantic salmon and the two from golden shiner, now named golden shiner toti-like virus 1 and 2 (GSTLV-1 and -2), and also to totiviruses and other toti-like viruses. We found that four piscine toti-like viruses had additional gene(s) in the 3' end of the genome, and also clustered phylogenetically based on both capsid and RdRp-genes. This cluster constituted a distant branch in the Totiviridae, and we suggest this should be defined as a separate genus named Pistolvirus, to reflect this major cluster of piscine toti-like viruses. The remaining two piscine toti-like viruses differentiated from these by lacking any additional 3' end genes and also by phylogenetical relation, but were both clustering with arthropod viruses in two different clusters.

Pasteurella spp. Infections in Atlantic salmon and lumpsucker.

The use of cleaner fish as a delousing method in Norwegian salmonid aquaculture has increased tremendously over the last few years. This has led to the emergence of a new large industry of farming lumpsuckers (Cyclopterus lumpus L.). The use of lumpsuckers as cleaner fish has, however, not been problem-free. Bacterial diseases cause high mortalities with pasteurellosis as one of the major emerging diseases. During the past few years, outbreaks of pasteurellosis in farmed Atlantic salmon (Salmo salar L.) have become more frequent. This has led to an increasing concern that this disease will become common in salmon farming as well. The purpose of this study was to investigate the susceptibility of Atlantic salmon to Pasteurella spp. infection and the possibility of lumpsuckers transmitting pasteurellosis to Atlantic salmon. Atlantic salmon were experimentally challenged, either by bath or by cohabitation with challenged lumpsuckers, using two different strains of Pasteurella spp. (originating from lumpsucker and Atlantic salmon, respectively). No clinical signs of pasteurellosis were observed on any of the Atlantic salmon. The lumpsuckers were, however, equally susceptible to both isolates. In addition, clear differences in histopathological changes were observed between individuals challenged with the two isolates.

Molecular characterization and functional analysis of components of the TOR pathway of the salmon louse, Lepeophtheirus salmonis (Krøyer, 1838).

The salmon louse Lepeophtheirus salmonis (Copepods, Caligida) is a marine ectoparasite infecting salmonid fishes in the northern hemisphere. At present, salmon lice infections are the most severe disease problem in the salmon farming industry causing significant economic losses. Due to development of resistance towards available chemotherapeutants, it is clear that new chemotherapeutants or non-chemical control methods are essential to manage the parasite in the future. The TOR signaling pathway is present in all metazoans and is a major regulator of cellular activity according to nutrient availability. In this study, we identified the TOR pathway genes in salmon louse; LsTSC1, LsTSC2, LsRheb, LsTOR, LsRaptor and LsRictor. RNA interference mediated gene silencing was performed to elucidate the functional role of each member of the pathway. Our results show that interference of the TOR signaling pathway either directly or indirectly inhibits many biological processes including egg maturation. In addition, the effect of gene knock-down results in more comprehensive physiological defects when targeting TORC1 and the upstream regulator Rheb. This is the first report on the TOR pathway in the salmon louse and that our research contributes to the basic knowledge of the parasite that could lead to development of novel treatment methods.

Identification and characterisation of the ecdysone biosynthetic genes neverland, disembodied and shade in the salmon louse Lepeophtheirus salmonis (Copepoda, Caligidae).

The salmon louse is a marine ectoparasitic copepod on salmonid fishes. Its lifecycle consists of eight developmental stages, each separated by a molt. In crustaceans and insects, molting and reproduction is controlled by circulating steroid hormones such as 20-hydroxyecdysone. Steroid hormones are synthesized from cholesterol through catalytic reactions involving a 7,8-dehydrogenase Neverland and several cytochrome P450 genes collectively called the Halloween genes. In this study, we have isolated and identified orthologs of neverland, disembodied and shade in the salmon louse (Lepeophtheirus salmonis) genome. Tissue-specific expression analysis show that the genes are expressed in intestine and reproductive tissue. In addition, levels of the steroid hormones ecdysone, 20-hydroxyecdysone and ponasterone A were measured during the reproductive stage of adult females and in early life stages.

The ecdysone receptor (EcR) is a major regulator of tissue development and growth in the marine salmonid ectoparasite, Lepeophtheirus salmonis (Copepoda, Caligidae).

The function of the ecdysone receptor (EcR) during development and molting has been thoroughly investigated in some arthropods such as insects but rarely in crustacean copepods such as the salmon louse Lepeophtheirus salmonis (L. salmonis) (Copepoda, Caligidae). The salmon louse is an ectoparasite on Atlantic salmon that has major economical impact in aquaculture due to the cost of medical treatment methods to remove lice from the fish. Handling of salmon louse infestations is further complicated by development of resistance towards available medicines. Understanding of basic molecular biological processes in the salmon louse is essential to enable development of new tools to control the parasite. In this study, we found L. salmonis EcR (LsEcR) transcript to be present in the neuronal somata of the brain, nuclei of muscle fibres and the immature intestine of the salmon louse. Furthermore, we explored the function of LsEcR during development using RNA interference mediated knock-down and through infection trials. Our results show that knock-down of LsEcR in the salmon louse is associated with hypotrophy of several tissues, delayed development and mortality. In addition, combined knock-down of LsEcR/LsRXR resulted in molting arrest during early larval stages.

Molecular characterisation of the salmon louse, Lepeophtheirus salmonis salmonis (Krøyer, 1837), ecdysone receptor with emphasis on functional studies of female reproduction.

The salmon louse Lepeophtheirus salmonis (Copepoda, Caligidae) is an important parasite in the salmon farming industry in the Northern Hemisphere causing annual losses of hundreds of millions of dollars (US) worldwide. To facilitate development of a vaccine or other novel measures to gain control of the parasite, knowledge about molecular biological functions of L. salmonis is vital. In arthropods, a nuclear receptor complex consisting of the ecdysone receptor and the retinoid X receptor, ultraspiracle, are well known to be involved in a variety of both developmental and reproductive processes. To investigate the role of the ecdysone receptor in the salmon louse, we isolated and characterised cDNA with the 5'untranslated region of the predicted L. salmonis EcR (LsEcR). The LsEcR cDNA was 1608 bp encoding a 536 amino acid sequence that demonstrated high sequence similarities to other arthropod ecdysone receptors including Tribolium castaneum and Locusta migratoria. Moreover, in situ analysis of adult female lice revealed that the LsEcR transcript is localised in a wide variety of tissues such as ovaries, sub-cuticula and oocytes. Knock-down studies of LsEcR using RNA interference terminated egg production, indicating that the LsEcR plays important roles in reproduction and oocyte maturation. We believe this is the first report on the ecdysone receptor in the economically important parasite L. salmonis.