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.

The salmon louse genome: Copepod features and parasitic adaptations.

Copepods encompass numerous ecological roles including parasites, detrivores and phytoplankton grazers. Nonetheless, copepod genome assemblies remain scarce. Lepeophtheirus salmonis is an economically and ecologically important ectoparasitic copepod found on salmonid fish. We present the 695.4 Mbp L. salmonis genome assembly containing ≈60% repetitive regions and 13,081 annotated protein-coding genes. The genome comprises 14 autosomes and a ZZ-ZW sex chromosome system. Assembly assessment identified 92.4% of the expected arthropod genes. Transcriptomics supported annotation and indicated a marked shift in gene expression after host attachment, including apparent downregulation of genes related to circadian rhythm coinciding with abandoning diurnal migration. The genome shows evolutionary signatures including loss of genes needed for peroxisome biogenesis, presence of numerous FNII domains, and an incomplete heme homeostasis pathway suggesting heme proteins to be obtained from the host. Despite repeated development of resistance against chemical treatments L. salmonis exhibits low numbers of many genes involved in detoxification.

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.

Chitin synthesis and degradation in Lepeophtheirus salmonis: Molecular characterization and gene expression profile during synthesis of a new exoskeleton.

Animals with exoskeleton need to molt to grow and develop. Molting is well described in some arthropods especially insects. Chitin is a polymer of N-acetylglucosamine, and one of the major components of the exoskeleton of arthropods. Chitin is synthesized and degraded by a series of enzymes during the molting cycle. However, the presence and function of these enzymes are largely unknown in copepods such as the ectoparasite salmon louse (Lepeophtheirus salmonis) a major pest in salmonid aquaculture. Here we describe six genes found in the L. salmonis genome (LsCHS1, LsCHS2, LsGFAT, LsGNA1, LsAGM, and LsUAP) with high homology to enzymes in the chitin synthesis pathway. The transcription profiles of these enzymes together with three chitinases enzymes (LsChi1, LsChi2, and LsChi4), which have been characterized before, were examined during the synthesis of a new exoskeleton and revealed a dynamical expression concurrent with the morphological changes during the molt cycle. Further understanding of chitin metabolism and its regulation may prove useful tool to develop new pesticides.

A novel gene-family involved in spermatophore generation in the economically important salmon louse Lepeophtheirus salmonis.

The salmon louse (Lepeophtheirus salmonis) is an important parasite of Atlantic salmon (Salmo salar). It is widely spread in aquaculture facilities and leads to economic losses every year. As it has developed resistances against many common treatments, new control methods must be established. Here we characterize a novel gene family of the salmon louse, consisting of two genes, which has not been described in other species before. We analyzed temporal expression patterns of both genes, the localization of mRNA and protein. An RNAi mediated gene knockdown lead to information about the function of the protein. Overall, these two genes are expressed only in sperm ducts of male sea lice. The mucin-like proteins can additionally be found in the wall of spermatophores, which are responsible for sperm transfer to females. Knockdown showed that both genes are essential for successful fertilization of females. Overall, all results indicate that the two analyzed genes are necessary for reproduction in sea lice as they are essential for the formation of a wall surrounding the spermatophores, which is needed for fertilization. Therefore, we name them Mucin-like spermatophore wall protein 1 & 2 (MLSWP1 & MLSWP2). Analysis of sequence data from other copepod species suggests that MLSWPs are present in many copepod species and may also play a similar role in reproduction in those species.

Gene silencing reveals multiple functions of Na+/K+-ATPase in the salmon louse (Lepeophtheirus salmonis).

Na+/K+-ATPase has a key function in a variety of physiological processes including membrane excitability, osmoregulation, regulation of cell volume, and transport of nutrients. While knowledge about Na+/K+-ATPase function in osmoregulation in crustaceans is extensive, the role of this enzyme in other physiological and developmental processes is scarce. Here, we report characterization, transcriptional distribution and likely functions of the newly identified L. salmonis Na+/K+-ATPase (LsalNa+/K+-ATPase) α subunit in various developmental stages. The complete mRNA sequence was identified, with 3003 bp open reading frame encoding a putative protein of 1001 amino acids. Putative protein sequence of LsalNa+/K+-ATPase revealed all typical features of Na+/K+-ATPase and demonstrated high sequence identity to other invertebrate and vertebrate species. Quantitative RT-PCR analysis revealed higher LsalNa+/K+-ATPase transcript level in free-living stages in comparison to parasitic stages. In situ hybridization analysis of copepodids and adult lice revealed LsalNa+/K+-ATPase transcript localization in a wide variety of tissues such as nervous system, intestine, reproductive system, and subcuticular and glandular tissue. RNAi mediated knock-down of LsalNa+/K+-ATPase caused locomotion impairment, and affected reproduction and feeding. Morphological analysis of dsRNA treated animals revealed muscle degeneration in larval stages, severe changes in the oocyte formation and maturation in females and abnormalities in tegmental glands. Thus, the study represents an important foundation for further functional investigation and identification of physiological pathways in which Na+/K+-ATPase is directly or indirectly involved.

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.

Microsomal triglyceride transfer protein in the ectoparasitic crustacean salmon louse (Lepeophtheirus salmonis).

The salmon louse, Lepeophtheirus salmonis, is an endemic ectoparasite on salmonid fish that is challenging for the salmon farming industry and wild fish. Salmon lice produce high numbers of offspring, necessitating sequestration of large amounts of lipids into growing oocytes as a major energy source for larvae, most probably mediated by lipoproteins. The microsomal triglyceride transfer protein (MTP) is essential for the assembly of lipoproteins. Salmon lice have three L. salmonis MTP (LsMTP) transcript variants encoding two different protein isoforms, which are predicted to contain three ß-sheets (N, C, and A) and a central helical domain, similar to MTPs from other species. In adult females, the LsMTPs are differently transcribed in the sub-cuticular tissues, the intestine, the ovary, and in the mature eggs. RNA interference-mediated knockdown of LsMTP in mature females gave offspring with significantly fewer neutral lipids in their yolk and only 10-30% survival. The present study suggests the importance of LsMTP in reproduction and lipid metabolism in adult female L. salmonis, a possible metabolic bottleneck that could be exploited for the development of new anti-parasitic treatment methods.

The genome sequence of Atlantic cod reveals a unique immune system.

Atlantic cod (Gadus morhua) is a large, cold-adapted teleost that sustains long-standing commercial fisheries and incipient aquaculture. Here we present the genome sequence of Atlantic cod, showing evidence for complex thermal adaptations in its haemoglobin gene cluster and an unusual immune architecture compared to other sequenced vertebrates. The genome assembly was obtained exclusively by 454 sequencing of shotgun and paired-end libraries, and automated annotation identified 22,154 genes. The major histocompatibility complex (MHC) II is a conserved feature of the adaptive immune system of jawed vertebrates, but we show that Atlantic cod has lost the genes for MHC II, CD4 and invariant chain (Ii) that are essential for the function of this pathway. Nevertheless, Atlantic cod is not exceptionally susceptible to disease under natural conditions. We find a highly expanded number of MHC I genes and a unique composition of its Toll-like receptor (TLR) families. This indicates how the Atlantic cod immune system has evolved compensatory mechanisms in both adaptive and innate immunity in the absence of MHC II. These observations affect fundamental assumptions about the evolution of the adaptive immune system and its components in vertebrates.

Molecular characterization and functional analysis of a salmon louse (Lepeophtheirus salmonis, Krøyer 1838) heme peroxidase with a potential role in extracellular matrixes.

Heme peroxidases are the most abundant type of peroxidase catalyzing a H2O2-dependent oxidation of a wide variety of substrates. They are involved in numerous processes like the innate immune response, hormone and prostaglandin synthesis and crosslinking of proteins within extracellular matrixes (ECM) as well as molecules within the cuticle and chorion of arthropods and nematodes. In the present study, a Lepeophtheirus salmonis heme peroxidase (LsHPX) 1 was characterized. Amino acids in the active site of heme peroxidases were conserved, and the predicted protein sequence showed the highest similarity to genes annotated as chorion peroxidases and genes suggested to be involved in cuticle hardening or adhesion. LsHPX1 exhibited a dynamic expression during ontogenesis and during the nauplius molting cycle. Transcripts were localized to muscle cells near the muscle-tendon junction, in nerve tissue especially at neuromuscular junctions, subcuticular epithelium, subepithelial cells facing the hemolymph, exocrine glands within the subepithelial tissue and in isolated cells within the testis. Knock-down of LsHPX1 in nauplius larvae decreased the swimming activity of emerging copepodids. Histological analysis of knock-down animals revealed increased spacing between myofibers and changes in subepithelial and exocrine gland tissue. Considering these results, the potential role of LsHPX1 in crosslinking molecules of salmon louse ECMs is discussed.

Characterization of a novel RXR receptor in the salmon louse (Lepeophtheirus salmonis, Copepoda) regulating growth and female reproduction.

BACKGROUND: Nuclear receptors have crucial roles in all metazoan animals as regulators of gene transcription. A wide range of studies have elucidated molecular and biological significance of nuclear receptors but there are still a large number of animals where the knowledge is very limited. In the present study we have identified an RXR type of nuclear receptor in the salmon louse (Lepeophtheirus salmonis) (i.e. LsRXR). RXR is one of the two partners of the Ecdysteroid receptor in arthropods, the receptor for the main molting hormone 20-hydroxyecdysone (E20) with a wide array of effects in arthropods. RESULTS: Five different LsRXR transcripts were identified by RACE showing large differences in domain structure. The largest isoforms contained complete DNA binding domain (DBD) and ligand binding domain (LBD), whereas some variants had incomplete or no DBD. LsRXR is transcribed in several tissues in the salmon louse including ovary, subcuticular tissue, intestine and glands. By using Q-PCR it is evident that the LsRXR mRNA levels vary throughout the L. salmonis life cycle. We also show that the truncated LsRXR transcript comprise about 50% in all examined samples. We used RNAi to knock-down the transcription in adult reproducing female lice. This resulted in close to zero viable offspring. We also assessed the LsRXR RNAi effects using a L. salmonis microarray and saw significant effects on transcription in the female lice. Transcription of the major yolk proteins was strongly reduced by knock-down of LsRXR. Genes involved in lipid metabolism and transport were also down regulated. Furthermore, different types of growth processes were up regulated and many cuticle proteins were present in this group. CONCLUSIONS: The present study demonstrates the significance of LsRXR in adult female L. salmonis and discusses the functional aspects in relation to other arthropods. LsRXR has a unique structure that should be elucidated in the future.

Phylogenomic and functional analyses of salmon lice aquaporins uncover the molecular diversity of the superfamily in Arthropoda.

BACKGROUND: An emerging field in biomedical research is focusing on the roles of aquaporin water channels in parasites that cause debilitating or lethal diseases to their vertebrate hosts. The primary vectorial agents are hematophagous arthropods, including mosquitoes, flies, ticks and lice, however very little is known concerning the functional diversity of aquaporins in non-insect members of the Arthropoda. Here we conducted phylogenomic and functional analyses of aquaporins in the salmon louse, a marine ectoparasitic copepod that feeds on the skin and body fluids of salmonids, and used the primary structures of the isolated channels to uncover the genomic repertoires in Arthropoda. RESULTS: Genomic screening identified 7 aquaporin paralogs in the louse in contrast to 42 in its host the Atlantic salmon. Phylogenetic inference of the louse nucleotides and proteins in relation to orthologs identified in Chelicerata, Myriapoda, Crustacea and Hexapoda revealed that the arthropod aquaporin superfamily can be classified into three major grades (1) classical aquaporins including Big brain (Bib) and Prip-like (PripL) channels (2) aquaglyceroporins (Glp) and (3) unorthodox aquaporins (Aqp12-like). In Hexapoda, two additional subfamilies exist as Drip and a recently classified entomoglyceroporin (Eglp) group. Cloning and remapping the louse cDNAs to the genomic DNA revealed that they are encoded by 1-7 exons, with two of the Glps being expressed as N-terminal splice variants (Glp1_v1, -1_v2, -3_v1, -3_v2). Heterologous expression of the cRNAs in amphibian oocytes demonstrated that PripL transports water and urea, while Bib does not. Glp1_v1, -2, -3_v1 and -3_v2 each transport water, glycerol and urea, while Glp1_v2 and the Aqp12-like channels were retained intracellularly. Transcript abundance analyses revealed expression of each louse paralog at all developmental stages, except for glp1_v1, which is specific to preadult and adult males. CONCLUSIONS: Our data suggest that the aquaporin repertoires of extant arthropods have expanded independently in the different lineages, but can be phylogenetically classified into three major grades as opposed to four present in deuterostome animals. While the aquaporin repertoire of Atlantic salmon represents a 6-fold redundancy compared to the louse, the functional assays reveal that the permeation properties of the different crustacean grades of aquaporin are largely conserved to the vertebrate counterparts.

Molecular characterization and knock-down of salmon louse (Lepeophtheirus salmonis) prostaglandin E synthase.

The salmon louse (Lepeophtheirus salmonis) is a major parasite of salmonid fish in the marine environment. The interaction between the parasite and the host upon infection is not completely understood. However, it is clear that the parasite influences the host and its immune system. Prostaglandins produced by parasites such as flatworms, roundworms and ticks are documented or assumed to play a role in immunomodulation of the host. In the salmon louse, the effect of prostaglandins on the host is assumed, but remains to be documented. In this study, a salmon louse prostaglandin E2 synthase (LsPGES2) is characterized. Ontogenetic analysis showed that LsPGES2 is relatively stable expressed during development. The highest level of expression was seen in the free living stages, although elevated levels of LsPGES2 were also found in adult females. In copepodids, LsPGES2 is found around muscle cells, while it is observed in the reproductive organs of adult female lice. LsPGES2 expression was knocked-down by RNA interference in nauplii, but emerging copepodids did not display any changes in morphology nor ability to infect and develop to adult stages on fish. Additional knock-down of LsPGES2 in adult female lice did not produce any characteristic changes in phenotype nor reproductive output. It is concluded that under these experimental conditions, knock-down of LsPGES2 did not affect any essential functions of the salmon louse, neither in the free-living nor the parasitic stages.

Molecular characterisation and functional analysis of LsChi2, a chitinase found in the salmon louse (Lepeophtheirus salmonis salmonis, Krøyer 1838).

The salmon louse (Lepeophtheirus salmonis spp.) is an economically important parasite on Atlantic salmon reared in aquaculture globally. Production and degradation of chitin, a major component of the exoskeleton, is the target of some pesticides (Di/Teflubenzuron) used in management of lice on farmed fish. These chemicals inhibit molting of the salmon louse leading to the death of the parasite. We found three chitinases (LsChi1, LsChi2 and LsChi4) in the salmon louse genome. Sequence analysis and phylogeny showed that they belong to the GH18 type of chitinase group and show high sequence similarity to chitinases found in other crustaceans and in insects. Expression patterns were different for all three chitinases suggesting different functions during louse development. Furthermore, the function of LsChi2 was further explored through the use of RNA interference and infection trials. Copepodids with knock down of LsChi2 transcripts were deformed and showed a highly reduced infection success.

Characterisation of iron regulatory protein 1A and 1B in the blood-feeding copepod Lepeophtheirus salmonis.

During its parasitic life stages, the marine ectoparasitic copepod Lepeophtheirus salmonis ingests large amounts of host blood, which contains high amounts of iron. Iron is an essential micronutrient, but also toxic in high dosages, and blood-feeding parasites like the salmon louse must thus possess an efficient system to handle the excess iron. Iron regulatory protein 1 and 2 (IRP1 and IRP2) are known to play crucial roles in this process, by regulating several proteins involved in iron transport and storage, depending on the cellular iron concentration. To gain knowledge about the regulation of the iron metabolism in salmon lice, two IRP homologues (LsIRP1A and LsIRP1B) were identified by sequence and predicted structure similarity to known IRPs in other species. In situ hybridisation revealed that LsIRP1A and LsIRP1B mRNAs were expressed in the ovaries, oviducts and vitellogenic oocytes of adult females. Transcription levels of LsIRP1A and LsIRP1B mRNAs did not differ significantly between the different developmental stages of the salmon louse. Adults in the absence of blood as a feed source had decreased levels of LsIRP1A, but not LsIRP1B mRNA. RNA binding experiments indicated the presence of functioning IRP in salmon lice. In order to explore the biological functions of LsIRP1A and LsIRP1B, the mRNAs of both proteins were knocked down by RNA interference (RNAi) in preadult females. The knockdown was confirmed by qRT-PCR. LsIRP1B knockdown lice produced less offspring than control lice due to slightly shorter egg strings and had decreased levels of transcripts involved in egg development. Knockdown of both LsIRP1A and LsIRP1B caused increased expression of a salmon louse Ferritin (LsFer). These results confirm that salmon lice have two IRP1 homologues, LsIRP1A and LsIRP1B, and might suggest a function in cellular iron regulation in the reproductive organs and eggs.

Evaluation of emamectin benzoate and substance EX against salmon lice in sea-ranched Atlantic salmon smolts.

Experimental releases of Atlantic salmon smolts treated with emamectin benzoate (EB) against salmon lice have previously been used to estimate the significance of salmon lice on the survival of migrating smolts. In recent years, the salmon louse has developed reduced sensitivity to EB, which may influence the results of such release experiments. We therefore tested the use of 2 anti-lice drugs: EB was administered to salmon smolts in high doses by intra-peritoneal injection and the prophylactic substance EX (SubEX) was administered by bathing. A third, untreated control group was also established. Salmon were challenged with copepodids of 2 strains of salmon lice (1 EB-sensitive strain and 1 with reduced EB-sensitivity) in mixed-group experimental tanks. At 31 d post-challenge, the numbers of pre-adult lice on treated fish were around 20% compared with the control fish, with minor or no differences between the 2 treatments and lice strains. Both treatments therefore appeared to give the smolts a high degree of protection against infestation of copepodids of salmon lice. However, significantly lower growth of the EB-treatment group indicates that bathing the fish in SubEX is less stressful for smolts than intra-peritoneal injection of EB.

A common-garden experiment to quantify evolutionary processes in copepods: the case of emamectin benzoate resistance in the parasitic sea louse Lepeophtheirus salmonis.

BACKGROUND: The development of pesticide resistance represents a global challenge to food production. Specifically for the Atlantic salmon aquaculture industry, parasitic sea lice and their developing resistance to delousing chemicals is challenging production. In this study, seventeen full sibling families, established from three strains of Lepeophtheirus salmonis displaying differing backgrounds in emamectin benzoate (EB) tolerance were produced and quantitatively compared under a common-garden experimental design. Lice surviving to the preadult stage were then exposed to EB and finally identified through the application of DNA parentage testing. RESULTS: With the exception of two families (19 and 29%), survival from the infectious copepod to preadult stage was very similar among families (40-50%). In contrast, very large differences in survival following EB exposure were observed among the families (7.9-74%). Family survival post EB exposure was consistent with the EB tolerance characteristics of the strains from which they were established and no negative effect on infection success were detected in association with increased EB tolerance. Two of the lice families that displayed reduced sensitivity to EB were established from a commercial farm that had previously used this chemical. This demonstrates that resistant alleles were present on this farm even though the farm had not reported treatment failure. CONCLUSIONS: To our knowledge, this represents the first study where families of any multi-cellular parasite have been established and compared in performance under communal rearing conditions in a common-garden experiment. The system performed in a predictable manner and permitted, for the first time, elucidation of quantitative traits among sea lice families. While this experiment concentrated on, and provided a unique insight into EB sensitivity among lice families, the experimental design represents a novel methodology to experimentally address both resistance development and other evolutionary questions in parasitic copepods.

Human-induced evolution caught in action: SNP-array reveals rapid amphi-atlantic spread of pesticide resistance in the salmon ecotoparasite Lepeophtheirus salmonis.

BACKGROUND: The salmon louse, Lepeophtheirus salmonis, is an ectoparasite of salmonids that causes huge economic losses in salmon farming, and has also been causatively linked with declines of wild salmonid populations. Lice control on farms is reliant upon a few groups of pesticides that have all shown time-limited efficiency due to resistance development. However, to date, this example of human-induced evolution is poorly documented at the population level due to the lack of molecular tools. As such, important evolutionary and management questions, linked to the development and dispersal of pesticide resistance in this parasite, remain unanswered. Here, we introduce the first Single Nucleotide Polymorphism (SNP) array for the salmon louse, which includes 6000 markers, and present a population genomic scan using this array on 576 lice from twelve farms distributed across the North Atlantic. RESULTS: Our results support the hypothesis of a single panmictic population of lice in the Atlantic, and importantly, revealed very strong selective sweeps on linkage groups 1 and 5. These sweeps included candidate genes potentially connected to pesticide resistance. After genotyping a further 576 lice from 12 full sibling families, a genome-wide association analysis established a highly significant association between the major sweep on linkage group 5 and resistance to emamectin benzoate, the most widely used pesticide in salmonid aquaculture for more than a decade. CONCLUSIONS: The analysis of conserved haplotypes across samples from the Atlantic strongly suggests that emamectin benzoate resistance developed at a single source, and rapidly spread across the Atlantic within the period 1999 when the chemical was first introduced, to 2010 when samples for the present study were obtained. These results provide unique insights into the development and spread of pesticide resistance in the marine environment, and identify a small genomic region strongly linked to emamectin benzoate resistance. Finally, these results have highly significant implications for the way pesticide resistance is considered and managed within the aquaculture industry.

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.

New insights into the molecular phylogeny of Balantidium (Ciliophora, Vetibuliferida) based on the analysis of new sequences of species from fish hosts.

We obtained sequences of the small subunit ribosomal RNA (rRNA) for two new isolates of Balantidium from fishes, Balantidium polyvacuolum and Balantidium ctenopharingodoni. This is the first introduction of molecular data of Balantidium species from fish hosts in the phylogenetic analyses of the ciliate subclass Trichostomatia. Despite the fact that these species share morphological characteristics common to other species of Balantidium, the phylogenetic analysis of their sequences has shown that they are to be placed in a different branch closely related to the so-called Australian clade. Thus, our results indicate that the genus Balantidium is polyphyletic and possibly should be represented by two different genera; however, the analysis of more species from other poikilothermic hosts (amphibians, reptiles) should be made before a revised taxonomical proposal could be made.