Deltamethrin resistance in salmon lice: Genetic markers and molecular mechanisms

Resistance of salmon lice (Lepeophtheirus salmonis) against the pyrethroid deltamethrin used in bath treatments is common, but the mechanisms underlying resistance are unknown. In insects, deltamethrin resistance can involve mutations of voltage-gated sodium channels (Nav) considered the target-site of pyrethroids as well as metabolic detoxification through carboxylesterases (CaEs). In L. salmonis, deltamethrin resistance is mainly inherited maternally and associated with single nucleotide polymorphisms (SNPs) in the mitochondrial genome (mtDNA). In addition, a potential target-site mutation (I936V) has been identified in the L. salmonis Nav homologue LsNav1.3. This PhD thesis investigated the relative contribution of Nav target-site mutations and mtDNA mutations in deltamethrin resistance in L. salmonis. L. salmonis from farm sites were rated as deltamethrin resistant or susceptible in bioassays and genotyped for the LsNav1.3 mutation I936V and previously identified resistance-associated mitochondrial SNPs. The results provided no evidence for a role of I936V in deltamethrin resistance, while confirming its association with several mtDNA SNPs. This conclusion was further supported by the genotyping of deltamethrin resistant and susceptible L. salmonis derived from a crossing experiment. Further experiment assessed the association of mitochondrial SNPs with deltamethrin resistance. In L. salmonis from farm sites, several mitochondrial haplotypes associated with deltamethrin resistance were identified, suggesting that deltamethrin resistance evolved at least two times independently. The association of a previously unknown haplotype with deltamethrin resistance was demonstrated in toxicity and genetic studies that support the hypothesis that SNP T8600C in cytochrome c oxidase subunit 1 (Leu107Ser) is related to the resistance mechanism. Finally, potential roles of the CaE gene family in deltamethrin resistance were examined. L. salmonis CaE sequences were identified and annotated, and CaE sequences predicted to be catalytically competent were studied regarding their transcript and SNP expression in resistant and susceptible lice. Results suggested that CaEs are not major determinants of deltamethrin resistance

Genomic analysis of the carboxylesterase family in the salmon louse (Lepeophtheirus salmonis)

The pyrethroid deltamethrin and the macrocyclic lactone emamectin benzoate (EMB) are used to treat infestations of farmed salmon by parasitic salmon lice, Lepeophtheirus salmonis. While the efficacy of both compounds against Atlantic populations of the parasite has decreased as a result of the evolution of resistance, the molecular mechanisms of drug resistance in L. salmonis are currently not fully understood. The functionally diverse carboxylesterases (CaE) family includes members involved in pesticide resistance phenotypes of terrestrial arthropods. The present study had the objective to characterize the CaE family in L. salmonis and assess its role in drug resistance. L. salmonis CaE homologues were identified by homology searches in the parasite's transcriptome and genome. The transcript expression of CaEs predicted to be catalytically competent was studied using quantitative reverse-transcription PCR in drug susceptible and multi-resistant L. salmonis. The above strategy led to the identification of 21 CaEs genes/pseudogenes. Phylogenetic analyses assigned 13 CaEs to clades involved in neurodevelopmental signaling and cell adhesion, while three sequences were predicted to encode secreted enzymes. Ten CaEs were identified as being potentially catalytically competent. Transcript expression of acetylcholinesterase (ace1b) was significantly increased in multi-resistant lice compared to drug-susceptible L. salmonis, with transcript abundance further increased in preadult-II females following EMB exposure. In summary, results from the present study demonstrate that L. salmonis possesses fewer CaE gene family members than most arthropods characterized so far. Drug resistance in L. salmonis was associated with overexpression of ace1b

Key role of mitochondrial mutation Leu107Ser (COX1) in deltamethrin resistance in salmon lice (Lepeophtheirus salmonis)

The pyrethroid deltamethrin (DTM) is used to treat Atlantic salmon (Salmo salar) against salmon louse (Lepeophtheirus salmonis) infestations. However, DTM resistance has evolved in L. salmonis and is currently common in the North Atlantic. This study aimed to re-assess the association between DTM resistance and mitochondrial (mtDNA) mutations demonstrated in previous reports. Among 218 L. salmonis collected in Scotland in 2018–2019, 89.4% showed DTM resistance in bioassays, while 93.6% expressed at least one of four mtDNA single nucleotide polymorphisms (SNPs) previously shown to be resistance associated. Genotyping at further 14 SNP loci allowed to define three resistance-associated mtDNA haplotypes, named 2, 3 and 4, occurring in 72.0%, 14.2% and 7.3% of samples, respectively. L. salmonis strains IoA-02 (haplotype 2) and IoA-10 (haplotype 3) both showed high levels (~ 100-fold) of DTM resistance, which was inherited maternally in crossing experiments. MtDNA haplotypes 2 and 3 differed in genotype for 17 of 18 studied SNPs, but shared one mutation that causes an amino acid change (Leu107Ser) in the cytochrome c oxidase subunit 1 (COX1) and was present in all DTM resistant while lacking in all susceptible parasites. We conclude that Leu107Ser (COX1) is a main genetic determinant of DTM resistance in L. salmonis

Mutations in voltage-gated sodium channels from pyrethroid resistant salmon lice (Lepeophtheirus salmonis)

Background Parasitic salmon lice (Lepeophtheirus salmonis) cause high economic losses in Atlantic salmon farming. Pyrethroids, which block arthropod voltage‐gated sodium channels (Nav1), are used for salmon delousing. However, pyrethroid resistance is common in L. salmonis. The present study characterised Nav1 homologues in L. salmonis in order to identify channel mutations associated to resistance, called kdr (knockdown) mutations. Results Genome scans identified three L. salmonis Nav1 homologues, LsNav1.1, LsNav1.2 and LsNav1.3. Arthropod kdr mutations map to specific Nav1 regions within domains DI‐III, namely segments S5 and S6 and the linker helix connecting S4 and S5. The above channel regions were amplified by RT‐PCR and sequenced in deltamethrin‐susceptible and deltamethrin‐resistant L. salmonis. While LsNav1.1 and LsNav1.2 lacked nucleotide polymorphisms showing association to resistance, LsNav1.3 showed a non‐synonymous mutation in S5 of DII occurring in deltamethrin‐resistant parasites. The mutation is homologous to a previously described kdr mutation (I936V, numbering according to Musca domestica Vssc1) and was present in two pyrethroid‐resistant L. salmonis strains (allele frequencies of 0.800 and 0.357), but absent in two pyrethroid‐susceptible strains. Conclusions The present study indicates that a kdr‐mutation in LsNaV 1.3 may contribute to deltamethrin resistance in L. salmonis

Investigation of deltamethrin resistance in salmon lice (Lepeophtheirus salmonis) provides no evidence for roles of mutations in voltage-gated sodium channels

BACKGROUND The pyrethroid deltamethrin is used to treat infestations of farmed salmon by parasitic salmon lice, Lepeophtheirus salmonis (Krøyer). However, the efficacy of deltamethrin for salmon delousing is threatened by resistance development. In terrestrial arthropods, knockdown resistance (kdr) mutations of the voltage‐gated sodium channel (Nav), the molecular target for pyrethroids, can cause deltamethrin resistance. A putative kdr mutation of an L. salmonis sodium channel homologue (LsNav1.3 I936V) has previously been identified. At the same time, deltamethrin resistance of L. salmonis has been shown to be inherited maternally and to be associated with mitochondrial DNA (mtDNA) mutations. The present study assessed potential roles of the above putative kdr mutation as a determinant of deltamethrin resistance in laboratory strains and field populations of L. salmonis. RESULTS The deltamethrin resistant L. salmonis strain IoA‐02 expresses the LsNav1.3 I936V mutation but was susceptible to the non‐ester pyrethroid etofenprox, a compound against which pyrethroid resistant arthropods are usually cross‐resistant if resistance is caused by Nav mutations. In a family derived from a cross between an IoA‐02 male and a drug‐susceptible female lacking the kdr mutation, deltamethrin resistance was not associated with the genotype at the LsNav1.3 locus (p>0.05). Similarly, in Scottish field populations of L. salmonis, LsNav1.3 I936V showed no association with deltamethrin resistance. In contrast, genotypes at the mtDNA loci A14013G and A9030G were significantly associated with deltamethrin resistance (P less than 0.001). CONCLUSION In the studied L. salmonis isolates, deltamethrin resistance was unrelated to the LsNav1.3 I936V mutation, but showed close association with mtDNA mutations