QTL ANALYSIS OF EMAMECTIN BENZOATE SUSCEPTIBILITY IN THE SALMON LOUSE Lepeophtheirus salmonis

Transcription

1 QTL ANALYSIS OF EMAMECTIN BENZOATE SUSCEPTIBILITY IN THE SALMON LOUSE Lepeophtheirus salmonis Dr Greta Carmona-Antoñanzas 1 mm

2 Pest Control Globally, the control of invertebrate disease vectors, crop-infesting pests, common urban pests and parasites of livestock significantly relies on the use of pesticides. Alternative strategies to control Lepeophtheirus salmonis include: * Biological control (i.e. cleaner fish) * Selective breeding * Sea cage design (i.e. snorkel) * Vaccination etc. Under development The main groups of chemical salmon delousing agents include organophosphates, pyrethroids, avermectins, benzoylureas and disinfectants L. salmonis pesticides in Scotland hydrogen peroxide organophosphates avermectins pyrethroids Azamethiphos Azamethiphos Emamectin Increased tolerance to 1979 Benzoate (EMB) EMB Dichlorvos 1997 Cypermethrin Deltamethrin

3 Resistance to Pesticides Continued use of the same delousing agent can lead to the development of drug resistance Genetic adaptation (heritable) that allow a PEST to survive lethal doses of a PESTICIDE or CHEMOTHERAPEUTANT (DRUG) Emergence of resistance is understood as the HERITABLE decrease in a population s susceptibility to a chemical Continued drug selection Today the major emphasis in resistance research lies on unravelling the underlying molecular mechanisms, in an attempt to use this knowledge to control the development and spread of resistant populations, provide insights into new drug development. Knowledge on the mechanisms involved has not kept pace with resistance in invertebrate pests

4 Molecular Mechanisms of Resistance to EMB AVERMECTINS Gamma amino butyrate gated chloride channels (GABA Cl) Glutamate gated chloride channels (Glu Cl) Telford et al insects and nematodes Chloride receptor Major mechanisms of resistance GABA / Glutamate Chloride ion In Lepeophtheirus salmonis GABA and Glu chloride channels have been cloned (Tribble et al., 2007) P-glycoprotein (ABCB transporters) has been cloned (Heumann et al., 2012) P-glycoprotein gene expression has been investigated (Heumann et al., 2012)

5 Aim of the Project Limitations of studying candidate genes, 1. Different target genes in crustaceans 2. Detoxifying enzymes contain numerous members Our approach? QTL analysis to identify GENETIC MARKERS associated with resistance would support: Development of diagnostic tests for susceptibility assessment More specific & informative than traditional bioassays Hints for efficient pest management and control strategies Rotation between drugs Mixtures of drugs Reduce environmental impact

6 L. salmonis strains & husbandry Reciprocal crosses of laboratory maintained salmon lice families P0 PARASITE MOTILITY ATTACHMENT BEHAVIOUR Susceptible M Resistant F Resistant M Susceptible F March 2015 March 2015 * About 50% loss per generation due to mortality Gravid female F1 Copepodid: Infective stage Flamarique et al., 2016 F1 copepodid challenge

7 Toxic Response (%) Experimental Design F1 sibling crosses M F 1 May F2 lice (phenotype) 60 2 Time-to-Response Bioassay 50 IoA-00 Susceptible M 40 IoA-02 Resistant M 30 Fam C2 M 20 Fam C6 M 10 Fam C7 M Emamectin benzoate exposure time (h)

8 Genotyping ddrad alleles 846,655,574 raw reads FastQC Reads of low quality score (< 20), ambiguous barcode or RAD tags were excluded 687,240,845 filtered reads RAD-markers common RAD-markers bi-allelic loci 140,077 4,796 4,574 Total RAD-markers identified in 190 individuals Loci retrieved in 140 individuals Loci with 2 alleles only

9 Linkage Map Lep-Map2 software Linkage map parameters were LOD = 4.5 and minimum SizeLimit = 3 The map generated contained Informative SNP markers = 3,630 Linkage groups = 11 Map length = centimorgans Unique loci = 1,525 Full Family C7 P0s and F2 lice LG Number of Markers Unique loci Length (cm) Total 3,630 1,525 1, Substantial gaps in the LG01 (not shown): - Total length: cm - gaps over 50 cm

10 P-value P-value Association Analysis Surviving trait LG markers 1.E-16 1.E-15 1.E-14 1.E-13 1.E-12 1.E-11 1.E-10 1.E-09 1.E-08 1.E-07 1.E-06 1.E-05 1.E-04 1.E-03 1.E-02 1.E-01 1.E Genetic markers LG07 2 markers Bonferroni s correction Linkage group 1 Linkage group 2 Linkage group 3 Linkage group 4 Linkage group 5 Linkage group 6 Linkage group 7 Linkage group 8 Linkage group 9 Linkage group 10 Linkage group 11

11 SYBR KASP Genotyping Assays Marker ID Trait Sequences submited to LGC Genomics Total length Surviving TAAATTCTATTTAAATTAAGCAATATTGAAAAATATATATCTTTCAAATA[A/C]AAGGAATTTTAAAAACTTTATTCGTAATGGTAATATTTCAAAGTATCATT Surviving TCCCCTTTTTGATTTAAAGTCCAGTGAAACACGACAGAATTGATTTAAAA[C/A]CTACTGACCTCATTCATTTTGTTTCAGGTCTTATCCAATAGCTTCGCCTA Surviving GTTTACCTTAAAAATATGTAGAAGTGAAAACGGATTTTATAAGTTTCTAA[C/T]TTTGAGGAACTCACCCTGTAGTGAAAATAAAGAGTACGTCCTTCGATATG Surviving TATACGTGGTTTCAAGTTAATTCATCCCCCTACTCCCTTATTTTTTTATA[C/A]CTGCCACAATTATTGACACGAGATATAACTAATAACTATTATAAAAATAT Surviving GCCTTTTTTTAACATAGCGAAATTCTTTAATATTTACAACTCTAGGTGTT[A/C]GAGATGGTCCTTCTGAGAGGGAAAGAAAGACTCTTTATAGATATTTCAGC Surviving GTATGAATGAGTATTAATATGTTCTATTTGTATGGATTATTATAAAATAT[A/G]TATTAGAAGTAAACAGTCCCTGTTGATTCAGAAAAAAAGACTGGCTATAA 105 Surviving LG03 Marker HEX HEX A/A A/A A/B A/A A/B NTC A/B NTC NTC

12 Conclusions High-density genetic map available for the salmon louse Time-to-response -associated markers are located in two LGs Different genes are likely to be involved in resistance One marker associated to sex-determination was found This study provide the basis for QTL analysis in salmon louse Next Sequencing more F2 families in the analysis: Improve the quality of the genetic map Identify new markers * * LG 03 LG linkage groups 3,630 informative SNP markers Development of molecular assays includes testing these markers: In parental strains Unrelated strains Field populations Basis for unravelling resistance mechanisms: Selection of genomic regions for gene prediction

13 Acknowledgements Thank you! Institute of Aquaculture, University of Stirling Project PI: Dr Armin Sturm ddrad library construction: Dr John Taggart Bioinformatics: Dr Michaël Bekaert, MSc David Guidi Sea louse biology: Prof James Bron Sea lice husbandry (MERL): Dr William Roy, Sally Boyd University of Edinburgh Project PI: Dr Karim Gharbi, Edinburgh Genomics Dr Ross Houston, Roslin Institute