Mechanism behind Resistance against the Organophosphate Azamethiphos in Salmon Lice (Lepeophtheirus salmonis)

Acetylcholinesterase (AChE) is the primary target for organophosphates (OP). Several mutations have been reported in AChE to be associated with the reduced sensitivity against OP in various arthropods. However, to the best of our knowledge, no such reports are available for Lepeophtheirus salmonis. Hence, in the present study, we aimed to determine the association of AChE(s) gene(s) with resistance against OP. We screened the AChE genes (L. salmonis ace1a and ace1b) in two salmon lice populations: one sensitive (n=5) and the other resistant (n=5) for azamethiphos, a commonly used OP in salmon farming. The screening led to the identification of a missense mutation Phe362Tyr in L. salmonis ace1a, (corresponding to Phe331 in Torpedo californica AChE) in all the samples of the resistant population. We confirmed the potential role of the mutation, with reduced sensitivity against azamethiphos in L. salmonis, by screening for Phe362Tyr in 2 sensitive and 5 resistant strains. The significantly higher frequency of the mutant allele (362Tyr) in the resistant strains clearly indicated the possible association of Phe362Tyr mutation in L. salmonis ace1a with resistance towards azamethiphos. The 3D modelling, short term survival experiments and enzymatic assays further supported the imperative role of Phe362Tyr in reduced sensitivity of L. salmonis for azamethiphos. Based on all these observations, the present study, for the first time, presents the mechanism of resistance in L. salmonis against azamethiphos. In addition, we developed a rapid diagnostic tool for the high throughput screening of Phe362Tyr mutation using High Resolution Melt analysis.乙酰胆碱酯酶（Acetylcholinesterase, AChE）是有机磷酸酯类（organophosphates, OP）的主要作用靶点。已有研究报道，多种节肢动物体内的AChE发生突变后，会降低其对有机磷酸酯类的敏感性。但据我们所知，目前尚无关于鲑疮痂鱼虱（Lepeophtheirus salmonis）此类突变的相关报道。因此，本研究旨在探究AChE基因与鲑疮痂鱼虱对有机磷酸酯类抗药性的关联。 我们对两个鲑鱼虱种群的AChE基因（鲑疮痂鱼虱ace1a与ace1b）进行了筛查：其中一个种群对甲基吡啶磷（azamethiphos，鲑鱼养殖中常用的有机磷酸酯类杀虫剂）敏感（样本量n=5），另一个种群则对其具有抗药性（样本量n=5）。 筛查结果显示，在抗药性种群的所有样本中，均存在鲑疮痂鱼虱ace1a基因上的错义突变Phe362Tyr（该位点对应加州电鳐（Torpedo californica）AChE的Phe331位点）。 我们通过对2个敏感株与5个抗药性株开展Phe362Tyr位点筛查，验证了该突变可降低鲑疮痂鱼虱对甲基吡啶磷的敏感性。抗药性菌株中突变等位基因（362Tyr）的频率显著升高，这表明鲑疮痂鱼虱ace1a基因的Phe362Tyr突变与其对甲基吡啶磷的抗药性存在潜在关联。 三维结构建模、短期存活实验与酶活测定进一步证实了Phe362Tyr突变在降低鲑疮痂鱼虱对甲基吡啶磷敏感性过程中的关键作用。基于上述结果，本研究首次阐明了鲑疮痂鱼虱对甲基吡啶磷产生抗药性的分子机制。 此外，本研究还建立了一种基于高分辨率熔解曲线分析（High Resolution Melt analysis）的快速检测方法，可用于Phe362Tyr突变的高通量筛查。