Relative transcript quantification by Quantitative PCR: Roughly right or precisely wrong?

BACKGROUND: When estimating relative transcript abundances by quantitative real-time PCR (Q-PCR) we found that the results can vary dramatically depending on the method chosen for data analysis. RESULTS: Analyses of Q-PCR results from a salmon louse starvation experiment show that, even with apparently good raw data, different analytical approaches [1,2] may lead to opposing biological conclusions. CONCLUSION: The results emphasise the importance of being cautious when analysing Q-PCR data and indicate that uncritical routine application of an analytical method will eventually result in incorrect conclusions. We do not know the extent of, or have a universal solution to this problem. However, we strongly recommend caution when analysing Q-PCR results e.g. by using two or more analytical approaches to validate conclusions. In our view a common effort should be made to standardise methods for analysis and validation of Q-PCR results

Identification and Description of the Key Molecular Components of the Egg Strings of the Salmon Louse (Lepeophtheirus salmonis)

The salmon louse Lepeophtheirus salmonis is a parasite of Atlantic salmon and other salmonids. Every year, it causes high costs for the Norwegian aquaculture industry. While the morphology of the female genital tract has been described, knowledge of the molecular basis of reproduction is very limited. We identified nine genes which are expressed exclusively in the female cement gland, the organ responsible for cement production, which is used to hold the eggs together and keep them attached to their mother in egg strings. Six of these genes encode proteins with signal peptides and probably form the main component of the cement. Two other genes are peroxidases, which are probably important in the cement formation. The last gene is not similar to any known protein, but contains a transmembrane domain. A knockdown of all these genes leads to missing or deformed egg strings, preventing reproduction of the lice. The correct assemblage of the cement in the cement gland is essential for successful reproduction of salmon lice. Similar proteins seem to be present in other copepod species, as well.publishedVersio

Without a pinch of salt: effect of low salinity on eggs and nauplii of the salmon louse (Lepeophtheirus salmonis)

The salmon louse is an economically important parasite on Atlantic salmon and poses a major threat to aquaculture. Several treatment methods have lost their effect due to resistance development in the lice. A rather new method for combatting sea lice is freshwater treatment where the various life stages of lice are differently affected by this treatment. In this study, we analyzed the effect of freshwater on the egg strings. A 3-h treatment with freshwater had a detrimental effect on the egg strings. First, the water penetrated the string, widening it, then entering the eggs and enlarging them. Finally, the ordered structure of the egg strings collapsed, and no alive animals hatched. Shorter treatments had a lower effectivity, and treatments with brackish water also showed milder effects. The egg strings were found to have a protective effect against low salinities, as hatched nauplii died rapidly under conditions that embryos survived. We also found that embryos react to low salinity on a molecular level by changing gene expression of several genes, when incubated in brackish water. Additionally, the hatching of embryos treated with brackish water was delayed in comparison to seawater controls.publishedVersio

The transcriptomic response of adult salmon lice (Lepeophtheirus salmonis) to reduced salinity

Salmon lice (Lepeophtheirus salmonis) are marine parasitic copepods living on salmonids and are challenging for salmon aquaculture. One of several treatment methods is the application of freshwater to the fish which can lead to lice loss. However, lab experiments have shown that salmon lice, acclimated to seawater, are capable of surviving for several weeks in freshwater, when attached to a host. If not attached to a host, they die within a few hours in freshwater but can survive a longer time in brackish water. The molecular mechanisms involved in the adaptation to low salinity of the louse have not been identified yet. In this study we incubated salmon lice, being attached to a host, or detached, in seawater, brackish water and freshwater for 4 h and 1 d, sampled the animals and used RNA-Seq to identify genes involved in these mechanisms. Freshwater incubation led to a much stronger regulatory response than brackish water and a longer incubation time gave a stronger effect than a short incubation. Among the most interesting genes, upregulated in low salinity water are in addition to several transporters, several enzymes involved in amino acid metabolism and especially in the proline biosynthesis. A strong upregulation of these enzymes might lead to an accumulation of proline which is known to be used as an osmolyte in other species. While the RNA-Seq experiment was performed with female samples, qPCR showed that at least 10 genes regulated in females, were also regulated in males.publishedVersio

Atlantic Water Boundary Current Along the Southern Yermak Plateau, Arctic Ocean

The major ocean current that carries heat into the Arctic Ocean splits into three main branches of Atlantic Water (AW) and recirculations when it encounters the Yermak Plateau (YP) located north of Svalbard. While the branches that cross the plateau and recirculations have been extensively studied, there has been limited observation of the transport and variability of the Yermak branch. In this study, we present year-round observations from an array of three moorings that were deployed across the boundary current on the southern slope of the YP. The temporal-averaged sections show a surface-intensified AW core, which is strongest in winter but also persistent throughout the record within the upper 500 m. The volume transport of AW is highest in fall (1.4 ± 0.2 Sv; 1 Sv = 106 m3 s−1) and decreases to 0.8 ± 0.1 Sv in summer. Beneath a surface-intensified core, the velocity profile has a minimum at middepth, gradually increasing toward the bottom. This cold, bottom-intensified current is detectable in all seasons and reaches a maximum transport of 1.5 Sv in spring. The transport of AW is regulated by wind stress curl and coastal upwelling along the northwestern shelf of Svalbard. A positive wind stress curl increases the volume transport in the Yermak branch, thereby reducing the Svalbard branch transport. Eddy kinetic energy is surface-intensified and decreases to negligible values below 500 m. In the upper 500 m, the average baroclinic conversion in winter and summer is about 1 × 10−5 W m−3, which is 4–10 times the barotropic conversion rates.publishedVersio

Endringsprosess i Tromsø kommune En studie av endringsstrategi i offentlig virksomhet

Denne oppgaven studerer hvilken påvirkning valg av endringsstrategi har på endringsprosessen og resultatet av endringen i en offentlig virksomhet. Den forsøker også å gi en forklaring på årsaken til at endringsstrategien påvirker prosess og resultat. Oppgaven er bygget på en case studie av en endringsprosess i Tromsø kommune, og er basert på intervju av informanter i organisasjonen. Endringen består i hovedsak av omorganisering fra fagorganisert til prosessorganisert struktur, og informantene ble i ulik grad berørt av endringen. Bakgrunnen for problemstillingen er de stadige endringer som gjennomføres i kommuneadministrasjoner, samt at de aller fleste endringsprosesser anses som mislykkede. Oppgaven studerer casen sett i lys av de to mest typiske endringsstrategiene, E og O, og de ulike dimensjonene disse bygger på. Informantene gir uttrykk for hvordan de har opplevd disse ulike dimensjonene i endringsprosessen, og hvordan dette har påvirket dem og resultatet av endringen. Hovedinntrykket fra informantene er at endringen opplevdes top-down styrt i henhold til strategi E, men siden ansattes rettigheter vedrørende endringer på egen arbeidsplass står sterkt i offentlig sektor, ble det lagt opp til en medvirkningsprosess. Denne medvirkningen ble ikke opplevd som bred og dyp nok, og skapte derav noen negative effekter blant de ansatte både i prosessen og resultatet. Dog ble utfallet av omorganiseringen akseptert og betraktet som en god løsning blant informantene

Wind-Driven Variability in the Spitsbergen Polar Current and the Svalbard Branch Across the Yermak Plateau

The Yermak Plateau (YP) acts as a guidance or barrier for the West Spitsbergen Current (WSC), which either crosses the plateau or flows around it to enter the Arctic Ocean. Closer to the West Spitsbergen coast, the Spitsbergen Polar Current (SPC) also flows over the YP in a narrow passage between the Svalbard Branch (SB) and the coast. A 2-year ocean observing program combined with altimetry and re-analysis wind data has given new knowledge on the variability and dynamics of the SPC and SB. The variability in the SPC and SB is controlled by the sea surface pressure gradient driven by the wind stress along the West Spitsbergen coast and locally on the YP. A peak-to-peak volume transport variability of 0.8 Sv and a positive heat transport anomaly of 3 TW were found in the SPC. The variability in the SB is mainly controlled by the upstream wind stress curl field along the West Spitsbergen Shelf where the negative wind stress curl field force the barotropic WSC branch directly into the SB. The peak-to-peak variability in the SB can exceed 4 Sv and in January 2016, an episodic heat flux was estimated to be 10 TW. Hence, an increasing number of winter cyclones affecting Svalbard will increase the volume transport variability and pulses of warm water to the shelf areas north of Svalbard.publishedVersio

Sex differences in the early life stages of the salmon louse Lepeophtheirus salmonis (Copepoda: Caligidae)

Salmon lice are ectoparasites on salmonids and feed on blood, mucus, and skin from their hosts. This causes high annual costs for treatment and control for the aquaculture industry. Salmon lice have a life cycle consisting of eight life stages. Sex determination by eye is only possible from the sixth stage onwards. A molecular sex determination has not been carried out so far, even though few individual sex-linked SNPs have been reported. In the present study, we used known sex-specific SNPs as a basis to sequence the complete sex-specific gene variants and used the sequence information to develop a sex determination assay. This assay could be used to determine the developmental speed of the two sexes already in the earliest life stages. Additionally, we sampled salmon lice in the nauplius II stage, determined the sex of each individual, pooled their RNA according to their sex, and used RNA sequencing to search for differences in gene expression and further sex-specific SNPs. We succeeded in developing a sex-determination assay that works on DNA or RNA from even the earliest larval stages of the salmon louse after hatching. At these early developmental stages, male salmon lice develop slightly quicker than females. We detected several previously unknown, sex-specific SNPs in our RNA-data seq, but only very few genes showed a differential expression between the sexes. Potential connections between SNPs, gene expression, and development are discussed.publishedVersio

Chitin synthases are critical for reproduction, molting, and digestion in the salmon louse (Lepeophtheirus salmonis)

Chitin synthase (CHS) is a large transmembrane enzyme that polymerizes Uridine diphosphate N-acetylglucosamine into chitin. The genomes of insects often encode two chitin synthases, CHS1 and CHS2. Their functional roles have been investigated in several insects: CHS1 is mainly responsible for synthesizing chitin in the cuticle and CHS2 in the midgut. Lepeophtheirus salmonis is an ectoparasitic copepod on salmonid fish, which causes significant economic losses in aquaculture. In the present study, the tissue-specific localization, expression, and functional role of L. salmonis chitin synthases, LsCHS1 and LsCHS2, were investigated. The expressions of LsCHS1 and LsCHS2 were found in oocytes, ovaries, intestine, and integument. Wheat germ agglutinin (WGA) chitin staining signals were detected in ovaries, oocytes, intestine, cuticle, and intestine in adult female L. salmonis. The functional roles of the LsCHSs were investigated using RNA interference (RNAi) to silence the expression of LsCHS1 and LsCHS2. Knockdown of LsCHS1 in pre-adult I lice resulted in lethal phenotypes with cuticle deformation and deformation of ovaries and oocytes in adult lice. RNAi knockdown of LsCHS2 in adult female L. salmonis affected digestion, damaged the gut microvilli, reduced muscular tissues around the gut, and affected offspring. The results demonstrate that both LsCHS1 and LsCHS2 are important for the survival and reproduction in L. salmonis.publishedVersio