Resistance towards deltamethrin (DMT) in the crustacean ectoparasite Lepeophtheirus salmonis (Caligidae) is a problem on fish farms lining the North Atlantic Ocean. Two Norwegian strains with different susceptibility towards DMT were crossed in the parental generation (P0), females from a sensitive strain were crossed with males from a resistant strain and vice versa. Individual susceptibility towards DMT was assessed in the second filial generation (F2). DMT resistance was only found in F2 descendants when the P0 females were from the resistant strain, pointing to maternal inheritance. Since maternal inheritance might be linked to the mitochondrial (mt) genome, the nucleotide sequences and the gene expressions of mt-genes were analysed. Twenty non-synonymous single nucleotide polymorphisms (SNPs) were identified in mt-transcripts from resistant F2 parasites, including SNPs in two cytochrome C oxidase subunits (COX1 and COX3) and two subunits of the NADH dehydrogenase complex (ND1 and ND5) previously linked to DMT resistance in the salmon louse. Differential expression analysis between the sensitive and resistant strain revealed strain effect in seven out of twelve mt-genes. The current study also show that DNA fragmentation (indicating apoptosis) was affected by DMT exposure in skeletal muscle tissue and that resistant parasites undergo less apoptosis than sensitive parasites.

Salmon lice (Lepeophtheirus salmonis) constitute a major challenge during the production of farmed Atlantic salmon in Norway. Preventive measures are considered to have a higher impact on sustainable control than lice treatment. Therefore, the studies presented here aimed to document the preventive effects of probiotic Aliivibrio spp. on lice infestation in experimental challenges. A reduction in salmon lice attachment success (58–65%) was observed in two separate aquarium trials, where Atlantic salmon were exposed to different compositions of Aliivibrio species 91 and 155 days prior to lice challenge. In a third trial, no difference in attachment was observed in groups exposed to probiotics 58 days prior to lice challenge compared to controls. However, a relative reduction in lice counts was seen on movable stages later in the trial. High levels of probiotic bacteria had no impact on lice viability in an in vitro bioassay on the preadult life stage; thus, the mechanism behind the preventative effect remains unknown. In conclusion, probiotic Aliivibrio bacteria can likely be used as a preventive tool to reduce salmon louse infestations in the salmon industry. The mechanism is still unknown, and this novel approach to lice control warrants further investigation to understand its optimal use and potential.

Background: Hydrogen peroxide (H₂O₂) is one of the delousing agents used to control sea lice infestations in salmonid aquaculture. However, some Lepeophtheirus salmonis populations have developed resistance towards H₂O₂. An increased gene expression and activity of catalase, an enzyme that breaks down H₂O₂, have been detected in resistant lice, being therefore introduced as a resistance marker in the salmon industry. In the present study the aim was to validate the use of catalase expression as a marker and to identify new candidate genes as additional markers to catalase, related to H₂O₂ resistance in L. salmonis. Methods: A sensitive and an H₂O₂ resistant laboratory strain (P0 generation, not exposed to H₂O₂ for several years) were batch crossed to generate a cohort with a wide range of H₂O₂ sensitivities (F2 generation). F2 adult females were then exposed to H₂O₂ to separate sensitive and resistant individuals. Those F2 lice, the P0 lice and field-collected resistant lice (exposed to H₂O₂ in the field) were used in an RNA sequencing study. Results: Catalase was upregulated in resistant lice exposed to H₂O₂ compared to sensitive lice. This was, however, not the case for unexposed resistant P0 lice. Several other genes were found differentially expressed between sensitive and resistant lice, but most of them seemed to be related to H₂O₂ exposure. However, five genes were consistently up- or downregulated in the resistant lice independent of exposure history. The upregulated genes were: one gene in the DNA polymerase family, one gene encoding a Nesprin-like protein and an unannotated gene encoding a small protein. The downregulated genes encoded endoplasmic reticulum resident protein 29 and an aquaporin (Glp1_v2). Conclusions: Catalase expression seems to be induced by H₂O₂ exposure, since it was not upregulated in unexposed resistant lice. This may pose a challenge for its use as a resistance marker. The five new genes associated with resistance are put forward as complementary candidate genes. The most promising was Glp1_v2, an aquaglyceroporin that may serve as a passing channel for H₂O₂. Lower channel number can reduce the influx or distribution of H₂O₂ in the salmon louse, being directly involved in the resistance mechanism.

Polymerase chain reaction (PCR) is a widely used molecular biology technique for amplifying specific DNA sequences. However, amplifying templates with a high GC content (>60 %) poses challenges owing to strong hydrogen bonds and secondary structure formation, hindering DNA polymerase activity and primer annealing. Given these challenges, our study focuses on refining PCR protocols for the nicotinic acetylcholine receptor subunits, pivotal for understanding signal transduction in various organisms and potential important drug targets. We optimized the PCR protocol to efficiently amplify the beta1 and alpha1 subunits of the nicotinic acetylcholine receptor from Ixodes ricinus (Ir-nAChRb1) and Apis mellifera (Ame-nAChRa1). Ir-nAChRb1 and Ame-nAChRa1 have open reading frames of 1743 and 1884 bp, respectively, with overall GC contents of 65 % and 58 %. Various DNA polymerases and organic additives have been evaluated at different annealing temperatures. The tailored protocol incorporated organic additives, such as DMSO and betaine, increased enzyme concentration, and adjusted annealing temperatures. This study demonstrates the importance of a multipronged approach involving various organic molecules, DNA polymerases, PCR conditions, and primer adjustments to overcome the challenges of amplifying GC-rich sequences.

The development of resistance in salmon lice (Lepeophtheirus salmonis) to antiparasitic agents presents a challenge for parasite management in aquaculture. Existing bioassay protocols for monitoring drug sensitivity primarily rely on preadult and adult lice, requiring significant resources and manual labor. In this study, we present a novel high-throughput bioassay protocol utilizing the copepodite life stage, established in 96-well microtiter plates. The protocol was evaluated for two of the ten substances tested, deltamethrin and imidacloprid, by direct comparisons between copepodite and preadult bioassays in a fully sensitive strain (LsGulen). In addition, comparison of bioassay performance with deltamethrin between the two life stages was carried out on a pyrethroid resistant strain (LsBjugn). Our results demonstrate that the copepodite bioassay reliably estimates compound efficacy, clearly discriminating between the deltamethrin sensitive and resistant strain. For both deltamethrin and imidacloprid, the bioassays on copepodids yielded higher EC50-values compared to bioassays on preadults. The copepodite assay enables screening of multiple compounds with minimal resource requirements. It could also serve as method for sensitivity testing in field, if good representation of the larger sea lice population is assured by sampling egg strings from multiple females. Furthermore, the copepodid bioassay allows for direct association between copepodite phenotypes and maternal genotypes, facilitating studies of inheritance and resistance mechanisms. While not a replacement for established preadult assays, the copepodite protocol offers complementary advantages in efficiency, throughput, and the ability to resolve intra-population sensitivity differences. These findings represent the first reported comparison of deltamethrin susceptibility between copepodid and preadult life stages in L. salmonis.