Sussie Dalvin

The Salmon Louse Lepeophtheirus salmonis (Copepoda: Caligidae) life cycle has only two Chalimus stages.

Each year the salmon louse ( Lepeophtheirus salmonis Krøyer, 1838) causes multi-million dollar commercial losses to the salmon farming industry world-wide, and strict lice control regimes have been put in place to reduce the release of salmon louse larvae from aquaculture facilities into the environment. For half a century, the Lepeophtheirus life cycle has been regarded as the only copepod life cycle including 8 post-nauplius instars as confirmed in four different species, including L . salmonis . Here we prove that the accepted life cycle of the salmon louse is wrong. By observations of chalimus larvae molting in incubators and by morphometric cluster analysis, we show that there are only two chalimus instars: chalimus 1 (comprising the former chalimus I and II stages which are not separated by a molt) and chalimus 2 (the former chalimus III and IV stages which are not separated by a molt). Consequently the salmon louse life cycle has only six post-nauplius instars, as in other genera of caligid sea lice and copepods in general. These findings are of fundamental importance in experimental studies as well as for interpretation of salmon louse biology and for control and management of this economically important parasite.

Slit and robo: expression patterns in lung development

First described as an axonal guidance cue through its repulsive effect on neurons expressing its receptor Roundabout (Robo), the Slit ligand has effects on cell migration, axon branching and elongation. Indirect evidence implicates Slit and Robo in lung development. We now demonstrate that Slit-2 and Slit-3 are developmentally regulated in embryonic murine lung. Immunohistochemistry demonstrates Slit-2 and Slit-3 expression by the pulmonary mesenchyme and airway epithelium. Robo-1 and Robo-2 are also expressed by the developing mesenchyme and airway epithelium. As lung development progresses, Robo-1 and Robo-2 expression localizes to only the airway epithelium. We conclude Slit/Robo are expressed in temporo-spatially adjacent domains suggesting interactive roles in pulmonary bronchiolar development.

Functional characterisation of the maternal yolk-associated protein (LsYAP) utilising systemic RNA interference in the salmon louse (Lepeophtheirus salmonis) (Crustacea: Copepoda).

The salmon louse (Lepeophtheirus salmonis) is an important pathogen in salmon aquaculture and a serious threat to wild populations of salmon. Knowledge of its basic biological processes such as reproduction is crucial for the control of this parasite and can facilitate development of a vaccine. Here, a novel yolk-associated protein, LsYAP, was characterised. Quantitative PCR and in situ analysis demonstrated that transcription of LsYAP takes place in the subcuticular tissue of adult females in the reproductive phase. LsYAP protein is transported and deposited in the developing eggs in the genital segment, where further processing takes place. The sequence characteristics, histological localisation and transcript regulation suggest that LsYAP is a yolk-associated protein. In addition, the use of RNA interference is, to our knowledge, demonstrated for the first time in a copepod. Treatment of adult females with double-stranded RNA led to lethality and deformations of offspring only. This result confirms that the LsYAP protein is produced in adult females but is utilised by the offspring.

Evidence that the cAMP pathway controls emergence of both primary and appressorial germ tubes of barley powdery mildew.

Development of conidia of barley powdery mildew involves the formation of a primary germ tube (PGT), an appressorial germ tube (AGT), and an appressorium. Previously, it was found that cyclic AMP (cAMP) was involved in these developmental processes. Comparison of development on the host surface with two types of cellulose membrane revealed that frequency of PGT emergence was surface independent. On one type of cellulose, where the frequencies of both AGT and appressorial differentiation were similar to that on the host surface, cAMP levels and protein kinase A (PKA) activities had a biphasic pattern with peaks at 15 min and 4 h after inoculation (prior to PGT and AGT emergence, respectively). The effect of manipulating cAMP levels was tested on another type of cellulose membrane, which stimulated a lower degree of AGT and appressorial formation than the host surface. Cholera toxin and forskolin, activators of adenylyl cyclase, significantly increased PGT emergence, but cAMP did not. Cholera toxin, forskolin, and cAMP increased the frequency of AGT and appressorial formation, but in a time-dependent manner.

Expression of Netrin-1 and its two receptors DCC and UNC5H2 in the developing mouse lung

The ligand Netrin-1 and its receptors DCC and UNC5H2 are critical for the regulation of neuronal migration in nervous system development. Here we demonstrate expression of these molecules in lung development. The mRNA expression profiles of Netrin-1, DCC and UNC5H2 are developmentally regulated during embryonic mouse lung formation. Netrin-1 shows a bimodal expression pattern with elevated mRNA levels early followed by a second peak in late gestation. Peak expression of DCC occurs early in development whereas expression of UNC5H2 peaks late in development. We also demonstrate localization of Netrin-1, DCC and UNC5H2 during the stages of lung development. We present evidence that these proteins are modulated spatially in the mesenchyme and epithelium during lung organogenesis.

Characterisation of two vitellogenins in the salmon louse Lepeophtheirus salmonis: molecular, functional and evolutional analysis

The salmon louse Lepeophtheirus salmonis Krøyer affects a variety of wild salmonoid hosts, but is also an important pest in aquaculture, which is a globally important and rapidly growing industry. Salmon lice have large reproductive outputs, and knowledge of reproductive processes may be crucial for the control of this parasite. Here, we report on the characterisation of 2 vitellogenins (LsVit1 and LsVit2), which are the precursors of salmon-louse egg-yolk glycoprotein. The structure of LsVit1 and LsVit2 was examined and compared to that in other oviparous animals. Phylogenetic analysis of LsVit1 and LsVit2 confirmed the view that crustaceans are a polyphyletic group. Transcriptional and translational analysis demonstrated production of LsVit1 and LsVit2 in the subcuticular tissue of the adult female lice. LsVit1 and LsVit2 could also be found in maturing oocytes and developing embryos and early larval stages. LsVit2 was found to be processed into 2 smaller fragments, whereas LsVit1 was found to be full length when deposited into the oocytes. Degradation of LsVit1 and LsVit2 was characterised through embryogenesis and the early non-feeding larval stages. Finally, protein content and the level of free amino acids were analysed in embryos and larval stages and their role in nutrition and osmoregulation discussed. In conclusion, our results confirm the role of vitellogenins in reproduction as providers of embryonic and larval nutrition.

Role of Dendritic Cells in Sjögren's Syndrome

Sjögrens syndrome (SS) is a chronic inflammatory and lymphoproliferative autoimmune disease of unknown aetiology. It is characterised by progressive mononuclear cell infiltration of the salivary and lacrimal glands and a decreased glandular secretion, resulting in dryness of the mouth and eyes (xerostomia and keratoconjunctivitis sicca, respectively). Dendritic cells (DC) are considered to be the most potent antigen‐presenting cells. Because of their central role in initiating an immune response while maintaining tolerance, impaired function of these cells might lead to the break of peripheral tolerance and initiation of immune responses to self‐antigens. This review will focus on the possible role of DC in SS.

A method for stable gene knock-down by RNA interference in larvae of the salmon louse (Lepeophtheirus salmonis)

The salmon louse (Lepeophtheirus salmonis), an ectoparasitic copepod of salmonid fish, is a major threat to aquaculture in Norway, Ireland, Scotland and Canada. Due to rise in resistance against existing pesticides, development of novel drugs or vaccines is necessary. Posttranscriptional gene silencing by RNA interference (RNAi), when established in a high throughput system is a potential method for evaluation of molecular targets for new medical compounds or vaccine antigens. Successful use of RNAi has been reported in several stages of salmon lice. However, when we employed a previously described protocol for planktonic stages, no reproducible down-regulation of target genes was gained. In the present study, we describe a robust method for RNAi, where nauplius larvae are soaked in seawater added double stranded RNA (dsRNA). In order to test for when dsRNA may be introduced, and for the efficacy and duration of RNAi, we performed a series of experiments on accurately age determined larvae, ranging from the hatching egg to the copepodid with a salmon louse coatomer and a putative prostaglandin E synthase gene. Presumptive knock-down was monitored by real time PCR. Significant gene silencing was obtained only when nauplius I larvae were exposed to dsRNA during the period in which they molted to nauplius II. A knock down effect could be detected 2days after soaking, and it remained stable until the last measurement, on day 12. Soaking nauplius I larvae, knock-down was verified for six additional genes with a putative role in molting. For one chitinase, a loss-of-function phenotype with abnormal swimming was obtained. Hence, RNAi, induced in the nauplius, may facilitate studies of the molecular biology of the louse, such as the function of specific genes in developmental processes and physiology, host recognition, host-parasite interaction, and, in extension, the engineering of novel medicines.

RNA interference mediated knockdown of the KDEL receptor and COPB2 inhibits digestion and reproduction in the parasitic copepod Lepeophtheirus salmonis

Retrograde transport of proteins from the endoplasmic reticulum to the Golgi is an essential part of the secretory pathway that all newly synthesised secreted and membrane proteins in eukaryotic cells undergo. The aim of this study was to characterise two components of the retrograde transport pathway in the parasitic copepod Lepeophtheirus salmonis (salmon louse) on a molecular and functional level. LsKDELR and LsCOPB2 were confirmed to be the salmon louse homologues of the chosen target proteins by sequence similarity. Ontogenetic analysis by qRT-PCR revealed the highest expression levels of both genes in adult females and the earliest larval stage. LsKDELR and LsCOPB2 localisation in adult females was detected by immunofluorescence and in situ hybridisation, respectively. Both LsKDELR and LsCOPB2 were found in the ovaries, the oocytes and the gut. LsKDELR and LsCOPB2 were knocked down by RNA interference in preadult females, which was confirmed by qRT-PCR. LsCOPB2 knockdown lice had a significantly higher mortality and failed to develop normally, while both LsCOPB2 and LsKDELR knockdown caused disturbed digestion and the absence of egg strings. This shows the potential of LsKDELR and LsCOPB2 as suitable target candidates for new pest control methods.

A trypsin-like protease with apparent dual function in early Lepeophtheirus salmonis (Krøyer) development

BackgroundTrypsin-like serine proteases are involved in a large number of processes including digestive degradation, regulation of developmental processes, yolk degradation and yolk degradome activation. Trypsin like peptidases considered to be involved in digestion have been characterized in Lepeophtheirus salmonis. During these studies a trypsin-like peptidase which differed in a number of traits were identified.ResultsAn intronless trypsin-like serine peptidase (LsTryp10) from L., salmonis was identified and characterized. LsTryp10 mRNA is evenly distributed in the ovaries and oocytes, but is located along the ova periphery. LsTryp10 protein is deposited in the oocytes and all embryonic cells. LsTryp10 mRNA translation and concurrent degradation after fertilization was found in the embryos demonstrating that LsTryp10 protein is produced both by the embryo and maternally. The results furthermore indicate that LsTryp10 protein of maternal origin has a distribution pattern different to that of embryonic origin.ConclusionBased on present data and previous studies of peptidases in oocytes and embryos, we hypothesize that maternally deposited LsTryp10 protein is involved in regulation of the yolk degradome. The function of LsTryp10 produced by the embryonic cells remains unknown. To our knowledge a similar expression pattern has not previously been reported for any protease.