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

Kaur K, Helgesen KO, Bakke MJ, Horsberg TE - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: NMBU School of Veterinary Science, Sea Lice Research Centre, PO Box 8146 Dep., NO-0033 Oslo, Norway.

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.

No MeSH data available.


Related in: MedlinePlus

Survival analysis plot.Kaplan-Meier survival plot of all three genotypes: homozygote sensitive (SS), heterozygote (RS) and homozygote resistant (RR). Three salmon infested with Ls F were treated for 30 minutes with 0.1 mgL-1 azamethiphos in a bath treatment. All detached salmon lice were removed during the exposure and the following 2.5 hours. Detached parasites were also removed 24 hours later. Two parasites were excluded from the analysis as they attached to the tank wall after detaching from the fish. The rest of the parasites were picked off the fish 8 days post treatment. These salmon lice were regarded as alive, while the detached were regarded as dead. All salmon lice were genotyped by PatoGen AS in Ålesund, Norway using a TaqMan assay. The upper dotted line is the RR group, the solid line is the RS group, while the lower broken line is the SS group. One of the RS parasites died between 200 minutes and 24 hours after start of exposure, but the exact time is unknown. In this plot the time of death is set to 250 minutes. The cut-off limit is set to 300 minutes.
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pone.0124220.g006: Survival analysis plot.Kaplan-Meier survival plot of all three genotypes: homozygote sensitive (SS), heterozygote (RS) and homozygote resistant (RR). Three salmon infested with Ls F were treated for 30 minutes with 0.1 mgL-1 azamethiphos in a bath treatment. All detached salmon lice were removed during the exposure and the following 2.5 hours. Detached parasites were also removed 24 hours later. Two parasites were excluded from the analysis as they attached to the tank wall after detaching from the fish. The rest of the parasites were picked off the fish 8 days post treatment. These salmon lice were regarded as alive, while the detached were regarded as dead. All salmon lice were genotyped by PatoGen AS in Ålesund, Norway using a TaqMan assay. The upper dotted line is the RR group, the solid line is the RS group, while the lower broken line is the SS group. One of the RS parasites died between 200 minutes and 24 hours after start of exposure, but the exact time is unknown. In this plot the time of death is set to 250 minutes. The cut-off limit is set to 300 minutes.

Mentions: The Kaplan-Meier survival analysis of the SS genotype demonstrated that the median survival time was 25 minutes (95% CI: 15–30 min). A Wilcoxon test showed highly significant differences between the groups (χ2 = 64.7, DF = 2, p< 0.0001). The survival plot is displayed in Fig 6.


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

Kaur K, Helgesen KO, Bakke MJ, Horsberg TE - PLoS ONE (2015)

Survival analysis plot.Kaplan-Meier survival plot of all three genotypes: homozygote sensitive (SS), heterozygote (RS) and homozygote resistant (RR). Three salmon infested with Ls F were treated for 30 minutes with 0.1 mgL-1 azamethiphos in a bath treatment. All detached salmon lice were removed during the exposure and the following 2.5 hours. Detached parasites were also removed 24 hours later. Two parasites were excluded from the analysis as they attached to the tank wall after detaching from the fish. The rest of the parasites were picked off the fish 8 days post treatment. These salmon lice were regarded as alive, while the detached were regarded as dead. All salmon lice were genotyped by PatoGen AS in Ålesund, Norway using a TaqMan assay. The upper dotted line is the RR group, the solid line is the RS group, while the lower broken line is the SS group. One of the RS parasites died between 200 minutes and 24 hours after start of exposure, but the exact time is unknown. In this plot the time of death is set to 250 minutes. The cut-off limit is set to 300 minutes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403986&req=5

pone.0124220.g006: Survival analysis plot.Kaplan-Meier survival plot of all three genotypes: homozygote sensitive (SS), heterozygote (RS) and homozygote resistant (RR). Three salmon infested with Ls F were treated for 30 minutes with 0.1 mgL-1 azamethiphos in a bath treatment. All detached salmon lice were removed during the exposure and the following 2.5 hours. Detached parasites were also removed 24 hours later. Two parasites were excluded from the analysis as they attached to the tank wall after detaching from the fish. The rest of the parasites were picked off the fish 8 days post treatment. These salmon lice were regarded as alive, while the detached were regarded as dead. All salmon lice were genotyped by PatoGen AS in Ålesund, Norway using a TaqMan assay. The upper dotted line is the RR group, the solid line is the RS group, while the lower broken line is the SS group. One of the RS parasites died between 200 minutes and 24 hours after start of exposure, but the exact time is unknown. In this plot the time of death is set to 250 minutes. The cut-off limit is set to 300 minutes.
Mentions: The Kaplan-Meier survival analysis of the SS genotype demonstrated that the median survival time was 25 minutes (95% CI: 15–30 min). A Wilcoxon test showed highly significant differences between the groups (χ2 = 64.7, DF = 2, p< 0.0001). The survival plot is displayed in Fig 6.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: NMBU School of Veterinary Science, Sea Lice Research Centre, PO Box 8146 Dep., NO-0033 Oslo, Norway.

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.

No MeSH data available.


Related in: MedlinePlus