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

Abstract

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.

Keywords: Carboxylesterase; Deltamethrin; Emamectin benzoate; Resistance; Salmon lice.

Graphical abstract

Fig. 1

Phylogenetic relationship of carboxylesterases (CaEs) in Lepeophtheirus salmonis, Drosophila melanogaster, and Apis mellifera. The alignment was constructed using Multiple Sequence Comparison by Log-Expectation (MUSCLE) and phylogenetic relationship was conducted by Maximum likelihood (ML) analysis using RaxML. ML bootstrap support values (BS) (percentage of 1000 BS) are provided next to the nodes. L. salmonis (LS) CaEs are highlighted in red. DM D. melanogaster. AM: A. mellifera. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Conserved motifs in L. salmonis carboxylesterase (CaE) sequences. L. salmonis CaE sequences were aligned against the reference Drosophila melanogaster acetylcholine esterase (DmAChE) sequence. Amino acid residues were numbered according to DmAChE. Conserved catalytic triad residues (Ser238, Glu/Asp367, and His480) are shown in green. Additional conserved amino acid residues within the active site (oxyanion hole G149 and G150, putative catalytic tetrad residue Ser264 (Oakeshott et al., 2005)) are shown in blue. Conserved disulphide bridges (Cys66, Cys98 and Cys292, Cys307) are shown in yellow. “-” indicates a gap in the alignment. ¹NCBI accession number. ²RT-PCR followed by Sanger sequencing was used to confirm cDNA sequences, which were deposited in the European Nucleotide Archive (see Table S5 for accession numbers). ³EnsemblMetazoa accession number. ⁴GXSXG: Nucleophilic elbow. ⁵Family affiliation according to Pfam (PF) and InterPro (IPR) entries. The carboxylesterase family type B belongs to the superfamily α/β hydrolase fold (PF00561, IPR029058). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Effect of deltamethrin exposure on carboxylesterase (CaE) transcript expression in L. salmonis. Preadult-II females and adult males of the drug susceptible strain IoA-00 and the multi-resistant strain IoA-02 were exposed to deltamethrin (0.05 μg L⁻¹; 2.0 μg L⁻¹) for 30 min and allowed to recover for 24 h in seawater before esterase transcript expression was determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Gene expression was expressed as relative units (RUs) calculated from the mean normalised ratios (n = 6 ± SE) between the estimated relative copy numbers of target genes and the estimated relative copy numbers of the reference genes. Bars bearing stars are significantly different (Dunn's test post-hoc comparisons to the control group).

Fig. 4

Effect of emamectin benzoate exposure on carboxylesterase (CaE) transcript expression in L. salmonis. Preadult-II females and adult males of the drug susceptible strain IoA-00 and the multi-resistant strain IoA-02 were exposed to deltamethrin (25 μg L⁻¹; 150 μg L⁻¹) for 30 min and allowed to recover for 24 h in seawater before esterase transcript expression was determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Gene expression was expressed as relative units (RUs) calculated from the mean normalised ratios (n = 6 ± SE) between the estimated relative copy numbers of target genes and the estimated relative copy numbers of the reference genes. Bars bearing stars are significantly different (Dunn's test post-hoc comparisons to the control group); *significant at p < 0.05, **significant at p < 0.01.