Heavy and light chain homologs of ferritin are essential for blood-feeding and egg production of the ectoparasitic copepod Lepeophtheirus salmonis

The salmon louse, Lepeophtheirus salmonis, is a hematophagous ectoparasite of salmonid fish. Due to its blood-feeding activity, the louse is exposed to great amounts of iron, which is an essential, yet potentially toxic mineral. The major known iron storage protein is ferritin, which the salmon louse encodes four genes of (LsFer1-4). Two of the ferritins are predicted to be secreted. These are one of the heavy chain homologs (LsFer1) and the light chain homolog (LsFer2). Here, we perform functional studies and characterize the two secreted ferritins. Our results show that knocking down LsFer1 and LsFer2 both negatively affect the parasite’s physiology, as it is not able to properly feed and reproduce. In a starvation experiment, the transcript levels of both LsFer1 and LsFer2 decrease during the starvation period. Combined, these results demonstrate the importance of these genes for the normal parasite biology, and they could thus potentially be targets for pest management.publishedVersio

The FTZ-F1 gene encodes two functionally distinct nuclear receptor isoforms in the ectoparasitic copepod salmon louse (Lepeophtheirus salmonis)

The salmon louse, Lepeophtheirus salmonis, is an ectoparasitic crustacean that annually inflicts substantial losses to the aquaculture industry in the northern hemisphere and poses a threat to the wild populations of salmonids. The salmon louse life cycle consists of eight developmental stages each separated by a molt. Fushi Tarazu Factor-1 (FTZ-F1) is an ecdysteroid-regulated gene that encodes a member of the NR5A family of nuclear receptors that is shown to play a crucial regulatory role in molting in insects and nematodes. Characterization of an FTZ-F1 orthologue in the salmon louse gave two isoforms named αFTZ-F1 and βFTZ-F1, which are identical except for the presence of a unique N-terminal domain (A/B domain). A comparison suggest conservation of the FTZ-F1 gene structure among ecdysozoans, with the exception of nematodes, to produce isoforms with unique N-terminal domains through alternative transcription start and splicing. The two isoforms of the salmon louse FTZ-F1 were expressed in different amounts in the same tissues and showed a distinct cyclical expression pattern through the molting cycle with βFTZ-F1 being the highest expressed isoform. While RNA interference knockdown of βFTZ-F1 in nauplius larvae and in pre-adult males lead to molting arrest, knockdown of βFTZ-F1 in pre-adult II female lice caused disruption of oocyte maturation at the vitellogenic stage. No apparent phenotype could be observed in αFTZ-F1 knockdown larvae, or in their development to adults, and no genes were found to be differentially expressed in the nauplii larvae following αFTZ-F1 knockdown. βFTZ-F1 knockdown in nauplii larvae caused both down and upregulation of genes associated with proteolysis and chitin binding and affected a large number of genes which are in normal salmon louse development expressed in a cyclical pattern. This is the first description of FTZ-F1 gene function in copepod crustaceans and provides a foundation to expand the understanding of the molecular mechanisms of molting in the salmon louse and other copepods.publishedVersio

Mining Lepeophtheirus salmonis RNA-Seq data for qPCR reference genes and their application in Caligus elongatus

Lepeophtheirus salmonis and Caligus elongatus are two parasitic copepod species posing a significant threat to salmonid aquaculture. Consequently, several gene expression studies are executed each year to gain new knowledge and treatment strategies. Though, to enable accurate gene expression measurements by quantitative real time PCR, stable reference genes are needed. Previous studies have mainly focused on a few genes selected based on their function as housekeeping genes, as these are often stably expressed in various cells and tissues. In the present study, however, RNA-sequencing data from 127 L. salmonis samples from different life stages and diverse environmental conditions were used to identify new candidate reference genes displaying low variation. From this, six genes were selected, and the stability validated by qPCR on samples from different life stages. Since neither a genome nor comprehensive RNA sequencing data are available for C. elongatus, homologous genes to those identified for L. salmonis were identified within a C. elongatus transcriptome assembly and validated by qPCR in different life stages. Overall, the genes eukaryotic translation initiation factor 1A (EIF1A) and serine/threonine-protein phosphatase 1 (PP1) displayed the highest stability in L. salmonis, while the combination of PP1 and ribosomal protein S13 (RPS13) was found to have the highest stability in C. elongatus. These genes are well-suited reference genes for qPCR applications which allow for accurate normalization of target genes.publishedVersio

Host gill attachment causes blood-feeding by the salmon louse (Lepeophtheirus salmonis) chalimus larvae and alters parasite development and transcriptome

Background: Blood-feeding is a common strategy among parasitizing arthropods, including the ectoparasitic salmon louse (Lepeophtheirus salmonis), feeding off its salmon host’s skin and blood. Blood is rich in nutrients, among these iron and heme. These are essential molecules for the louse, yet their oxidative properties render them toxic to cells if not handled appropriately. Blood-feeding might therefore alter parasite gene expression. Methods: We infected Atlantic salmon with salmon louse copepodids and sampled the lice in two different experiments at day 10 and 18 post-infestation. Parasite development and presence of host blood in their intestines were determined. Lice of similar instar age sampled from body parts with differential access to blood, namely from gills versus lice from skin epidermis, were analysed for gene expression by RNA-sequencing in samples taken at day 10 for both experiments and at day 18 for one of the experiments. Results: We found that lice started feeding on blood when becoming mobile preadults if sitting on the fish body; however, they may initiate blood-feeding at the chalimus I stage if attached to gills. Lice attached to gills develop at a slower rate. By differential expression analysis, we found 355 transcripts elevated in lice sampled from gills and 202 transcripts elevated in lice sampled from skin consistent in all samplings. Genes annotated with “peptidase activity” were among the ones elevated in lice sampled from gills, while in the other group genes annotated with “phosphorylation” and “phosphatase” were pervasive. Transcripts elevated in lice sampled from gills were often genes relatively highly expressed in the louse intestine compared with other tissues, while this was not the case for transcripts elevated in lice sampled from skin. In both groups, more than half of the transcripts were from genes more highly expressed after attachment. Conclusions: Gill settlement results in an alteration in gene expression and a premature onset of blood-feeding likely causes the parasite to develop at a slower pace.publishedVersio

Roles of three putative salmon louse (Lepeophtheirus salmonis) prostaglandin E2synthases in physiology and host–parasite interactions

The salmon louse (Lepeophtheirus salmonis) is a parasite of salmonid fish. Atlantic salmon (Salmo salar) exhibit only a limited and ineffective immune response when infested with this parasite. Prostaglandins (PGs) have many biological functions in both invertebrates and vertebrates, one of which is the regulation of immune responses. This has led to the suggestion that prostaglandin E2 (PGE2) is important in the salmon louse host–parasite interaction, although studies of a salmon louse prostaglandin E2 synthase (PGES) 2 gene have not enabled conformation of this hypothesis. The aim of the present study was, therefore, to characterize two additional PGES-like genes.publishedVersio

Dark neutrino interactions make gravitational waves blue

New interactions of neutrinos can stop them from free streaming in the early Universe even after the weak decoupling epoch. This results in the enhancement of the primordial gravitational wave amplitude on small scales compared to the standard $\Lambda$CDM prediction. In this paper we calculate the effect of dark matter neutrino interactions in CMB tensor $B$-modes spectrum. We show that the effect of new neutrino interactions generates a scale or $\ell$ dependent imprint in the CMB $B$-modes power spectrum at $\ell \gtrsim 100$. In the event that primordial $B$-modes are detected by future experiments, a departure from scale invariance, with a blue spectrum, may not necessarily mean failure of simple inflationary models but instead may be a sign of non-standard interactions of relativistic particles. New interactions of neutrinos also induce a phase shift in the CMB B-mode power spectrum which cannot be mimicked by simple modifications of the primordial tensor power spectrum. There is rich information hidden in the CMB $B$-modes spectrum beyond just the tensor to scalar ratio.Comment: 31 pages, 10 figures. Version published in Phys. Rev.

A novel approach to co-expression network analysis identifies modules and genes relevant for moulting and development in the Atlantic salmon louse (Lepeophtheirus salmonis)

Background The salmon louse (Lepeophtheirus salmonis) is an obligate ectoparasitic copepod living on Atlantic salmon and other salmonids in the marine environment. Salmon lice cause a number of environmental problems and lead to large economical losses in aquaculture every year. In order to develop novel parasite control strategies, a better understanding of the mechanisms of moulting and development of the salmon louse at the transcriptional level is required. Methods Three weighted gene co-expression networks were constructed based on the pairwise correlations of salmon louse gene expression profiles at different life stages. Network-based approaches and gene annotation information were applied to identify genes that might be important for the moulting and development of the salmon louse. RNA interference was performed for validation. Regulatory impact factors were calculated for all the transcription factor genes by examining the changes in co-expression patterns between transcription factor genes and deferentially expressed genes in middle stages and moulting stages. Results Eight gene modules were predicted as important, and 10 genes from six of the eight modules have been found to show observable phenotypes in RNA interference experiments. We knocked down five hub genes from three modules and observed phenotypic consequences in all experiments. In the infection trial, no copepodids with a RAB1A-like gene knocked down were found on fish, while control samples developed to chalimus-1 larvae. Also, a FOXO-like transcription factor obtained highest scores in the regulatory impact factor calculation. Conclusions We propose a gene co-expression network-based approach to identify genes playing an important role in the moulting and development of salmon louse. The RNA interference experiments confirm the effectiveness of our approach and demonstrated the indispensable role of a RAB1A-like gene in the development of the salmon louse. We propose that our approach could be generalized to identify important genes associated with a phenotype of interest in other organisms.publishedVersio

Salmon louse (Lepeophtheirus salmonis) transcriptomes during post molting maturation and egg production, revealed using EST-sequencing and microarray analysis

<p>Abstract</p> <p>Background</p> <p><it>Lepeophtheirus salmonis </it>is an ectoparasitic copepod feeding on skin, mucus and blood from salmonid hosts. Initial analysis of EST sequences from pre adult and adult stages of <it>L. salmonis </it>revealed a large proportion of novel transcripts. In order to link unknown transcripts to biological functions we have combined EST sequencing and microarray analysis to characterize female salmon louse transcriptomes during post molting maturation and egg production.</p> <p>Results</p> <p>EST sequence analysis shows that 43% of the ESTs have no significant hits in GenBank. Sequenced ESTs assembled into 556 contigs and 1614 singletons and whenever homologous genes were identified no clear correlation with homologous genes from any specific animal group was evident. Sequence comparison of 27 <it>L. salmonis </it>proteins with homologous proteins in humans, zebrafish, insects and crustaceans revealed an almost identical sequence identity with all species.</p> <p>Microarray analysis of maturing female adult salmon lice revealed two major transcription patterns; up-regulation during the final molting followed by down regulation and female specific up regulation during post molting growth and egg production. For a third minor group of ESTs transcription decreased during molting from pre-adult II to immature adults. Genes regulated during molting typically gave hits with cuticula proteins whilst transcripts up regulated during post molting growth were female specific, including two vitellogenins.</p> <p>Conclusion</p> <p>The copepod <it>L.salmonis </it>contains high a level of novel genes. Among analyzed <it>L.salmonis </it>proteins, sequence identities with homologous proteins in crustaceans are no higher than to homologous proteins in humans. Three distinct processes, molting, post molting growth and egg production correlate with transcriptional regulation of three groups of transcripts; two including genes related to growth, one including genes related to egg production. The function of the regulated transcripts is discussed in relation to post molting morphological changes in adult female salmon louse. There is clear evidence that transcription of the major yolk proteins is not induced before some of the post molting growth of abdomen and the genital segment has occurred. A hallmark for the observed growth is transcription of many putative cuticula proteins prior to the size increase.</p

Transcriptomic and targeted immune transcript analyses confirm localized skin immune responses in Atlantic salmon towards the salmon louse

Atlantic salmon (Salmo salar) are highly susceptible to infestations with the ectoparasite Lepeophtheirus salmonis, the salmon louse. Infestations elicit an immune response in the fish, but the response does not lead to parasite clearance, nor does it protect against subsequent infestations. It is, however, not known why the immune response is not adequate, possibly because the local response directly underneath the louse has been poorly evaluated. The present study describes the transcriptomic response by RNA sequencing of skin at the site of copepodid attachment. Analysing differentially expressed genes, 2864 were higher and 1357 were lower expressed at the louse attachment site compared to uninfested sites in the louse infested fish, while gene expression at uninfested sites were similar to uninfested control fish. The transcriptional patterns of selected immune genes were further detailed in three skin compartments/types: Whole skin, scales only and fin tissue. The elevation of pro-inflammatory cytokines and immune cell marker transcripts observed in whole skin and scale samples were not induced in fin, and a higher cytokine transcript level in scale samples suggest it can be used as a nonlethal sampling method to enhance selective breeding trials. Furthermore, the immune response was followed in both skin and anterior kidney as the infestation developed. Here, newly moulted preadult 1 stage lice induced a higher immune response than chalimi and adult lice. Overall, infestation with salmon louse induce a modest but early immune response with an elevation of mainly innate immune transcripts, with the response primarily localized to the site of attachment.publishedVersio

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.publishedVersio