RNA sequencing reveals distinct gene expression patterns during the development of parasitic larval stages of the salmon louse (Lepeophtheirus salmonis).

The salmon louse (Lepeophtheirus salmonis), an ectoparasitic copepod on salmonids, has become a major threat for the aquaculture industry. In search for new drugs and vaccines, transcriptome analysis is increasingly used to find differently regulated genes and pathways in response to treatment. However, the underlying gene expression changes going along with developmental processes could confound such analyses. The life cycle of L. salmonis consists of eight stages divided by moults. The developmental rate of salmon lice on the host is not uniform. Individual- and sex-related differences are found leading to individuals of unlike developmental status at same sampling time point after infection. In this study, we analyse L. salmonis from a time series by RNA sequencing applying a method of separating individuals of different instar age independent of sampling time point. Lice of four stages divided into up to four age groups within the stage were analysed in triplicate (total of 66 samples). Gene expression analysis shows that the method for sorting individuals was successful. Many genes show cyclic expression patterns over the moulting cycles. Overall gene expression differs more between lice of different age within the same stage than between lice of different stage but same instar age.

The role of chewing lice (Phthiraptera: Philopteridae) as intermediate hosts in the transmission of Hymenolepis microps (Cestoda: Cyclophyllidea) from the willow ptarmigan Lagopus lagopus (Aves: Tetraonidae).

The cestode Hymenolepis microps is an intestinal parasite of tetraonid birds, including the willow ptarmigan (Lagopus lagopus). This parasite is able to maintain a high prevalence and intensity throughout the year, even in a subarctic environment in bird populations with relatively low host densities, indicating effective transmission routes. Willow ptarmigan consume mainly vegetal material and active consumption of invertebrates is confined to the first two or three weeks of life. Ptarmigan are infected by different species of ectoparasites, of which two species of feather lice, Lagopoecus affinis and Goniodes lagopi, are the most abundant. In this study, we explored the hypothesis that feather lice may be suitable intermediate hosts for H. microps. We applied histological techniques and light microscopy to investigate lice for the presence of larval cestode stages (cysticercoids). We found 12 cysticercoid-like structures inside chewing lice collected on L. lagopus hosts harbouring H. microps. In addition, a polymerase chain reaction (PCR) screening of Ischnocera lice DNA, targeting the 18S rRNA gene of the cestode, showed positive results for two different short fragments of the 18S rRNA gene of H. microps which were sequenced from lice collected on birds. Both independent lines of evidence support the hypothesis that Ischnocera lice might be suitable intermediate hosts in the life cycle of H. microps in L. lagopus.

ArcTiCA: Arctic tidal constituents atlas.

Tides in the Arctic Ocean affect ocean circulation and mixing, and sea ice dynamics and thermodynamics. However, there is a limited network of available in situ tidal coefficient data for understanding tidal variability in the Arctic Ocean; e.g., the global TICON-3 database contains only 111 sites above 60°N and 21 above 70°N. At the same time, the presence of sea ice and latitude limits of satellite altimetry complicate altimetry-based retrievals of Arctic tidal coefficients. This leads to a reliance on ocean tide models whose accuracy depend on having sufficient in situ data for validation and assimilation. Here, we present a comprehensive new dataset of tidal constituents in the Arctic region, combining analyses of in situ measurements from tide gauges, ocean bottom pressure sensors and GNSS interferometric reflectometry. The new dataset contains 914 measurement sites above 60°N and 399 above 70°N, with each site being quality-assessed and expert guidance provided to help maximise the usage of the dataset. We also compare the dataset to recent tide models.

Salmon lice--impact on wild salmonids and salmon aquaculture.

Salmon lice, Lepeophtheirus salmonis, are naturally occurring parasites of salmon in sea water. Intensive salmon farming provides better conditions for parasite growth and transmission compared with natural conditions, creating problems for both the salmon farming industry and, under certain conditions, wild salmonids. Salmon lice originating from farms negatively impact wild stocks of salmonids, although the extent of the impact is a matter of debate. Estimates from Ireland and Norway indicate an odds ratio of 1.1:1-1.2:1 for sea lice treated Atlantic salmon smolt to survive sea migration compared to untreated smolts. This is considered to have a moderate population regulatory effect. The development of resistance against drugs most commonly used to treat salmon lice is a serious concern for both wild and farmed fish. Several large initiatives have been taken to encourage the development of new strategies, such as vaccines and novel drugs, for the treatment or removal of salmon lice from farmed fish. The newly sequenced salmon louse genome will be an important tool in this work. The use of cleaner fish has emerged as a robust method for controlling salmon lice, and aquaculture production of wrasse is important towards this aim. Salmon lice have large economic consequences for the salmon industry, both as direct costs for the prevention and treatment, but also indirectly through negative public opinion.

Life history and virulence are linked in the ectoparasitic salmon louse Lepeophtheirus salmonis.

Models of virulence evolution for horizontally transmitted parasites often assume that transmission rate (the probability that an infected host infects a susceptible host) and virulence (the increase in host mortality due to infection) are positively correlated, because higher rates of production of propagules may cause more damages to the host. However, empirical support for this assumption is scant and limited to microparasites. To fill this gap, we explored the relationships between parasite life history and virulence in the salmon louse, Lepeophtheirus salmonis, a horizontally transmitted copepod ectoparasite on Atlantic salmon Salmo salar. In the laboratory, we infected juvenile salmon hosts with equal doses of infective L. salmonis larvae and monitored parasite age at first reproduction, parasite fecundity, area of damage caused on the skin of the host, and host weight and length gain. We found that earlier onset of parasite reproduction was associated with higher parasite fecundity. Moreover, higher parasite fecundity (a proxy for transmission rate, as infection probability increases with higher numbers of parasite larvae released to the water) was associated with lower host weight gain (correlated with lower survival in juvenile salmon), supporting the presence of a virulence-transmission trade-off. Our results are relevant in the context of increasing intensive farming, where frequent anti-parasite drug use and increased host density may have selected for faster production of parasite transmission stages, via earlier reproduction and increased early fecundity. Our study highlights that salmon lice, therefore, are a good model for studying how human activity may affect the evolution of parasite virulence.

Major histocompatibility complex (MHC) variation and susceptibility to the sea louse Lepeophtheirus salmonis in Atlantic salmon Salmo salar.

The relationship between genetic variation in major histocompatibility complex (MHC) Class I and II genes and susceptibility to sea lice Lepeophtheirus salmonis (Krøyer) in Atlantic salmon Salmo salar (L.) was studied in cage-reared post smolts. Polymorphic repeat markers located in the 3' untranslated regions (3UTR) of the genes Sasa-UBA (MHC Class I) and Sasa-DAA (MHC Class II) were screened in 1004 fish sampled from 11 full-sibling families. This gave rise to a total of 7 and 5 alleles, and 17 and 13 genotypes respectively. Significant relationships between both Sasa-UBA-3UTR and Sasa-DAA-3UTR genotypes and abundance of lice were observed within the pooled material, within individual families, and within the pooled material with both markers combined. However, most of these associations were either weak, linked with variation in fish size among genotypes, or influenced by family background genome. Nevertheless, within one family, the Sasa-DAA-3UTR 248/278 genotype displayed a significantly higher (33%) abundance of lice compared with the Sasa-DAA-3UTR 208/258 genotype, and this difference was not influenced by fish size. Consequently, the results of this study indicate a link between MHC Class II and susceptibility to lice.

Does rapid intraoperative parathyroid hormone analysis predict cure in patients undergoing surgery for primary hyperparathyroidism? A prospective study.

BACKGROUND AND AIMS: Preoperative 99mTc-sestamibi scintigraphy is used by many surgeons to identify the anatomical location of pathological parathyroid glands in patients undergoing surgical treatment for hyperparathyroidism. However, false negative results do occur. It has been suggested that intraoperative parathyroid hormone (PTH) analysis may enhance the possibility of performing successful focused, unilateral neck surgery in these patients. This study aimed to evaluate whether an adequate fall in intraoperative parathyroid hormone values predicts the removal of all hyperfunctioning parathyroid tissue and postoperative normocalcemia. MATERIAL AND METHODS: One hundred consecutive patients undergoing surgery for hyperparathyroidism had preoperative 99mTc-sestamibi scintigraphy and intraoperative parathyroid hormone (PTH) analysis. A fall in intraoperative PTH value by more than 50% of baseline value ended the procedure. This prospective study presents the clinical and biochemical results. RESULTS: The overall sensitivity of the 99mTc-sestamib scintigraphy was 88% and for single adenomas 95%. The scintigraphy failed to detect the correct pathology in all cases with multiglandular disease (7 patients). A fall in intraoperative PTH value by more than 50% of baseline value was achieved in all patients. The combination of intraoperative PTH analysis and 99mTc-sestamibi scintigraphy enabled us to limit the operation to a focused, unilateral operation in 87 of the 100 patients. All patients were normocalcemic postoperatively. CONCLUSIONS: A fall in intraoperative PTH value more than 50 % of baseline value seems to predict postoperative normocalcemia and the removal of all hyperfunctioning parathyroid tissue. Bilateral neck exploration is avoided in the majority of patients.

Three structurally and functionally conserved Hlx genes in zebrafish.

For the Hlx class, which includes homeodomains (HD) that are similar to Drosophila H2.0, few members have been identified in vertebrates. In this report, we describe three zebrafish genes, hlx1, hlx2 and hlx3, related to the murine Dbx genes. The proteins encoded by hlx1 and hlx2 have about the same sequence identity to Dbx1 (approximately 60%), suggesting that they derive from a duplication in the fish lineage. This is supported by similarities in the embryonic expression patterns and promoter sequence conservation. The zebrafish Hlx3 protein is related to murine Dbx2, but it is apparently too diverged to be orthologous. Our phylogenetic analysis of all the known HD sequences of the Hlx class also shows that it can be divided into at least two distinct families. All the Dbx-like genes have similar expression in the embryonic nervous system. However, the initial expression patterns of the zebrafish hlx genes are quite unique, suggesting that some functional divergence has occurred between fish and mammals.

Partial cloning of constitutive and inducible nitric oxide synthases and detailed neuronal expression of NOS mRNA in the cerebellum and optic tectum of adult Atlantic salmon (Salmo salar).

Studies of different species have implicated nitric oxide (NO) synthase (NOS) in various physiological and pathological events. Three major NOS isoforms are present in the brain of mammals; endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS). Little is known about the significance of the presence of these proteins in the brain. We report the first investigation into the presence of nNOS and iNOS isoforms in a teleost, adult Atlantic salmon (Salmo salar). Complementary DNA was synthesized from cerebellum and thymus mRNA using RT-PCR techniques with primers against conserved regions of NOS. Cloning and sequencing revealed a partial gene sequence of 560 bp corresponding to mammalian nNOS from cerebellum cDNA. The predicted protein sequence of identified salmon nNOS possessed 85% identity to that of mammalian nNOS. Northern blot analysis of different tissues revealed expression in brain and heart, and indicated expression of three different nNOS mRNAs in the brain. In addition, a 389 bp sequence corresponding to iNOS was identified in thymus cDNA. Salmon iNOS is almost identical to rainbow trout iNOS (95%), but shows much less amino acid identity to goldfish (65%) and mammalian (52%) iNOS. Phylogenetically, all vertebrate nNOS and iNOS homologues are clustered separately. Expression studies by means of in situ hybridization revealed abundant nNOS mRNA transcripts in distinct neuronal populations throughout the Purkinje cell layer of the corpus cerebellum and the periventricular layer of the optic tectum. Our data show that adult Atlantic salmon possess a gene encoding an nNOS isoform and putative alternatively spliced forms, which are expressed in distinct neuronal populations in the cerebellum and optic tectum, and in yet unidentified cell types in the heart. The data suggest that the arising of different vertebrate NOS isoforms is an evolutionary old event. The well conserved sequences present in salmon and mammalian nNOS may reflect their importance in protein function, whereas interspecies distributional differences in cellular expression of nNOS and sequence differences of iNOS may reflect variations and specializations in routes of NO action in the vertebrate phylogeny.

Gas-containing renal stones treated with percutaneous nephrolithotomy: case report.

We present a rare case of E. coli emphysematous pyelonephritis with sepsis. The radiologic presentation consisted of multiple radiolucent gas-filled, free-floating uric acid calculi in a hydronephrotic renal pelvis. The infection was treated by intravenous fluids and antibiotics and percutaneous nephrostomy drainage. The patient was rendered stone free by percutaneous nephrolithotomy and ultrasound lithotripsy.

Ovipleistophora gen. n., a new genus for Pleistophora mirandellae-like microsporidia.

Based on ultrastructural study and molecular analysis, a new genus, Ovipleistophora, is established for Pleistophora mirandellae-like microsporidia from roach and ruff oocytes. Unlike Pleistophora, Ovipleistophora has a thick additional envelope around the meront. This envelope breaks open to release the cells into the host cell cytoplasm. The cells, becoming multinuclear sporogonic plasmodia, already have a surface coat that transforms into the sporont wall and eventually into the sporophorous vesicle wall. The surface coat and its transformation differ from those of Pleistophora, but bear some resemblance to those of Trachipleistophora. In Trachipleistophora the sporonts, however, do not form plasmodia, as they do in Ovipleistophora and Pleistophora. Small subunit ribosomal DNA analysis supports the establishment of the new genus and assignment of P. mirandellae from 2 different fish hosts to the same species. The same small subunit ribosomal DNA analysis lends support for transferring P. ovariae into the genus Ovipleistophora.

Redescription of Microsporidium takedai (Awakura, 1974) as Kabatana takedai (Awakura, 1974) comb. n.

Ultrastructural study of the microsporidian Microsporidium takedai from the muscles of masu salmon Oncorhynchus masou proved that this species can be assigned to the genus Kabatana Lom, Dyková and Tonguthai, 2000. The parasites develop within disintegrated sarcoplasm without any delimiting boundary or cyst. Cylindrical multinucleate meronts proliferate by serial constrictions into uninucleate stages which repeat the process. Eventually, the uninucleate stages transform into uninucleate sporonts, which divide once to produce sporoblasts, thus functioning as sporoblast mother cells. Spores, with a subterminally located anchoring disc and 3 to 4 turns of the polar tube coil, average 3.3 by 1.9 microm in size. The exospore is divided into small fields; the endospore frequently makes small invaginations into the spore inside. Phylogenetic analysis using SSU rDNA sequence consistently placed Kabatana takedai in a group consisting of Microgemma sp., Spraguea lophii and Glugea americanus. The K. takedai could easily be separated from the other species in the same group by 2 inserts in the SSU rDNA sequence.

Diseases, prophylaxis and treatment of the Atlantic halibut Hippoglossus hippoglossus: a review.

After substantial investments in research, the Atlantic halibut Hippoglossus hippoglossus is now being cultivated commercially in Norway, Iceland, Scotland and Canada. As with other domesticated species, disease problems have been experienced. This review summarizes the current state of knowledge of diseases of the Atlantic halibut, and their diagnosis, prophylaxis and treatment. In economic terms, the most important losses have been suffered at the larval and juvenile stages. The most important infections are caused by nodaviruses, causative agents of Viral Encephalopathy and Retinopathy (VER), which are the major reason why Norway's production of halibut fry has been level since 1995. An aquatic birnavirus, Infectious Pancreatic Necrosis Virus, is also an important agent of mortality. Vibrio anguillarum, Flexibacter ovolyticus and atypical Aeromonas salmonicida are the major bacterial pathogens. The protozoan parasites recorded include Ichthyobodo sp., the microsporidium Enterocytozoon sp., Trichodina hippoglossi, and the metazoan pathogens include myxozoans, helminths, Entobdella hippoglossi, Lepeophtheirus hippoglossi and other parasitic copepods. Experimental vaccines have been tested against V anguillarum and atypical A. salmonicida, with good results. A recombinant vaccine against nodaviruses is under development. A few trials have been carried out on non-specific immunostimulants, but no such treatment is currently available. A number of efficacy and pharmacokinetic trials with various antibacterial agents have also been published.

Phylogenetic analysis of infectious salmon anaemia virus isolates from Norway, Canada and Scotland.

The sequences of gene segments 2 and 8 from 10 different isolates of infectious salmon anaemia virus (ISAV) sampled in Norway, Canada and Scotland between 1987 and 1999 were determined and compared. Pairwise comparisons revealed a high degree of homology between the European isolates, with identities of 98 to 100% for both genes examined. The Canadian isolate showed identities of 84 and 87 to 88% with the European isolates for the nucleotide sequence of segments 2 and 8, respectively. Phylogenetic analyses were performed to establish the interrelationship between the European virus isolates. The evolutionary rate based on 4 Norwegian isolates clustered together in the analysis of segment 2 was calculated to be 0.96 x 10(-3) nucleotides site(-1) yr(-1). On the basis of this mutation rate it was estimated that the Norwegian Glesvaer 90 and Canadian Bay of Fundy 97 isolates diverged around 1900, which coincides with transportation of salmonids between Europe and North America starting in the late nineteenth century.

Strain variation, based on the hemagglutinin gene, in Norwegian ISA virus isolates collected from 1987 to 2001: indications of recombination.

Infectious salmon anemia (ISA) is caused by a virus that probably belongs to the Orthomyxoviridae and was first recorded in Norway in 1984. The disease has since spread along the Norwegian coast and has later been found in Canada, Scotland, the Faroe Islands, Chile, and the USA. This study presents sequence variation of the hemagglutinin gene from 37 ISA virus isolates, viz. one isolate from Scotland, one from Canada and 35 from Norway. The hemagglutinin gene contains a highly polymorphic region (HPR), which together with the rest of the gene sequence provides a good tool for studies of epizootics. The gene shows temporal and geographical sequence variation, where certain areas are dominated by distinct groups of isolates. Evidence of transmission of ISA virus isolates within and between regions is given. It is suggested that the hemagglutinin gene from different isolates may recombine. Possible recombination sites are found within the HPR and in the 5'-end flanking region close to the HPR.

Small subunit ribosomal DNA phylogeny of microsporidia with particular reference to genera that infect fish.

Molecular data have proved useful in the study of microsporidia phylogeny. Previous studies have shown that there are several important differences between phylogenies based on rRNA and morphological data. In the present study, small subunit (SSU) rDNA sequences were obtained from 7 different fish-infecting microsporidia from 4 different genera (Glugea Thélohan, 1891, Loma Morrison and Sprague, 1981, Pleistophora Gurley, 1893, and Spraguea Weissenberg, 1976). The lengths of the SSU rDNA genes in these species were between 1,332 and 1,343 base pairs. Phylogenetic analysis was performed using parsimony, maximum likelihood, and Kimura 2-parameter with neighbor joining. The analyses revealed that the microsporidia could be divided into 3 major groups. With the exception of Nucleospora salmonis Hedrick, Groff, and Baxa, 1991, all the microsporidia infecting fishes occurred in the same group. The analysis showed that Pleistophora mirandellae Vaney and Conte, 1901 and Pleistophora aguillarum Hoshina, 1951 are not species of Pleistophora. Furthermore, the analysis showed that Loma is not a member of Glugeidae Thélohan, 1892.

On the occurrence and morphology of Gyrodactylus pterygialis from saithe Pollachius virens in a Norwegian fjord.

A total of 308 Pollachius virens from a fjord near Bergen, western Norway, were examined for Gyrodactylus pterygialis over a 14-mo period; G. pterygialis showed the highest prevalence and density in winter/spring (November, January, March, and April pooled) but was absent in some months (August, September, and November). The morphology of the opisthaptoral hard parts of G. pterygialis were studied throughout the survey, and their variation is discussed with respect to host species, host locality, and different temperatures. On the basis of the present and previous descriptions, it is suggested that G. pterygialis is a parasite infecting gadine fishes of the genera Pollachius and Gadus, and that records from Pacific Eleginus and White Sea herring are distinct from G. pterygialis sensu Bychowsky and Poljansky, 1953.

Experimental infection of Atlantic salmon (Salmo salar) with marine Eubothrium sp. (Cestoda: Pseudophyllidea): observations on the life cycle, aspects of development and growth of the parasite.

The life cycle of marine Eubothrium sp. (Cestoda: Pseudophyllidea), from Atlantic salmon (Salmo salar L.) was experimentally completed in one year and included only one intermediate host (Acartia tonsa Dana) (Copepoda: Calanoida). Adult cestodes were collected from farmed salmon, and ripe eggs released by the cestodes were fed to Acartia tonsa. Ingested eggs hatched in the gut and the larvae developed in the haemocoel of the copepod for 15 days at 16 degrees C. A total of 170 seawater-reared salmon were exposed to infected copepods and the total prevalence of Eubothrium sp. in the salmon after infection was 95.3%, with a mean intensity of 15.0 (range 1-87). The infected salmon were kept in the laboratory where the growth of the cestodes was studied for eleven months. Mean length of the cestodes increased with time, but a large variation among the cestodes was observed. Growth and maturation of the cestodes were dependent on host size and the number of worms present in the intestine. No evidence of mortality of Eubothrium sp. was observed during the experimental period.

Small subunit rDNA phylogeny of Bacillidium sp. (Microspora, Mrazekiidae) infecting oligochaets.

Small subunit (SSU) rDNA has been sequenced from a microsporidium, identified as a member of the genus Bacillidium obtained from an oligochaete. The length of the amplified PCR product was 1386 bp which is currently the longest microsporidium SSU sequence known. Phylogenetic analysis using 28 microsporidia SSU sequences, using 3 different tree-building methods indicated that Bacillidium sp. may be one of the earliest branches on the microsporidia tree. However, bootstrapping failed to give a high score (more than 50%) for the position of Bacillidium sp. The branch leading to Bacillidium sp. was long, indicating that this species is not closely related to any of the other microsporidia so far studied by means of rDNA.

Observations on an intranuclear microsporidian in lymphoblasts from farmed Atlantic halibut larvae (Hippoglossus hippoglossus L.).

An intranuclear microsporidian was observed in lymphoblasts from the kidney of farmed Atlantic halibut larvae (Hippoglossus hippoglossus). In addition to spores, intranuclear pre-spore stages were observed. Uninucleated stages with a slight thickening of the cell membrane were the most simple developmental stages observed. Multinucleated developmental stages contain electron-lucent vesicles and electron-dense discs. The spores are ovoid and measure 2.9 x 1.2 micron (mean). Sporophorous vesicles, diplokarya and mitochondria were not observed in any of the developmental stages. The developmental stages observed are similar to those of the family Enterocytozoonidae, genus Enterocytozoon. However, there are several differences between the present species and E. bieneusi from enterocytes in man. The relationship to the other species in the genus, E. salmonis can only be determined when all the developmental stages of the species from Atlantic halibut are identified.