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

Kiranpreet Kaur,Tor Einar Horsberg,Marit Jørgensen Bakke,Kari Olli Helgesen,Israel Silman

doi:10.1371/journal.pone.0124220

Abstract

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.

Highlights

Acetylcholinesterase (AChE), encoded by ace genes, is a serine hydrolase that plays a critical role in neurotransmission at cholinergic synapses and neuromuscular junctions
We have recently identified and characterized the two genes coding for AChE in L. salmonis [8]
To characterize the salmon lice strains with regard to their sensitivity to azamethiphos, small scale treatments of fish infested with salmon lice were performed

Summary

Introduction

Acetylcholinesterase (AChE), encoded by ace genes, is a serine hydrolase that plays a critical role in neurotransmission at cholinergic synapses and neuromuscular junctions. AChE is a target for two main classes of anti-cholinergic agents, organophosphates (OP) and carbamates (CB). OP and CB bind to the active site of AChE, and inactivate the enzyme by phosphorylating or carbamylating a serine residue in the enzyme’s catalytic center [1]. Azamethiphos Resistance in Salmon Lice the cleavage of the transmitter, acetyl choline (ACh), and results in elevated levels of ACh in the synaptic cleft thereby causing excitation, paralysis and death [2]. OPs have been used for treatment against salmon lice (Lepeophtheirus salmonis), a marine ectoparasitic copepod on salmonid species, in Norwegian salmonid aquaculture since the late 1970s. When the use of azamethiphos was terminated during 1999, the problem of reduced sensitivity in salmon lice against azamethiphos was wide-spread. The cause of resistance was not determined

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