Mis-spliced transcripts of nicotinic acetylcholine receptor alpha6 are associated with field evolved spinosad resistance in Plutella xylostella (L.)

Abstract

The evolution of insecticide resistance is a global constraint to agricultural production. Spinosad is a new, low-environmental-risk insecticide that primarily targets nicotinic acetylcholine receptors (nAChR) and is effective against a wide range of pest species. However, after only a few years of application, field evolved resistance emerged in the diamondback moth, Plutella xylostella, an important pest of brassica crops worldwide. Spinosad resistance in a Hawaiian population results from a single incompletely recessive and autosomal gene, and here we use AFLP linkage mapping to identify the chromosome controlling resistance in a backcross family. Recombinational mapping with more than 700 backcross progeny positioned a putative spinosad target, nAChR alpha 6 (Pxalpha6), at the resistance locus, PxSpinR. A mutation within the ninth intron splice junction of Pxalpha6 results in mis-splicing of transcripts, which produce a predicted protein truncated between the third and fourth transmembrane domains. Additional resistance-associated Pxalpha6 transcripts that excluded the mutation containing exon were detected, and these were also predicted to produce truncated proteins. Identification of the locus of resistance in this important crop pest will facilitate field monitoring of the spread of resistance and offer insights into the genetic basis of spinosad resistance in other species.

Figure 1. Spinosad resistance is associated with a single linkage group in Plutella xylostella.

146 AFLP genotypes were generated from a female informative backcross and assigned into 30 linkage groups. χ² values for each linkage group were calculated by comparing genotypes inherited by backcross spinosad bioassay survivors with untreated controls. A directional bias towards spinosad susceptible or resistant grandparental origin is shown. Linkage group 1 (LG01) was significantly associated with spinosad resistance after Bonferroni correction for multiple comparisons (LG01, χ² = 15.53, P>0.0001). The remaining 29 linkage groups identified here were not associated with resistance. LG-Z is the sex chromosome.

Figure 2. Recombinational map of the Plutella xylostella spinosad resistance locus, PxSpinR.

Genes flanking nAChR Pxα6 were chosen for genotyping based on relative position within the B. mori genome (distances are shown). Susceptible and resistant alleles were inherited in untreated controls at a ∼1∶1 ratio. The 3′ end of nAChR Pxα6 was completely linked to the spinosad resistance locus.

Figure 3. nAChR Pxα6 gene and coding sequence.

(A) Intron distances and relative exon sizes of nAChR Pxα6 from Geneva 88 BAC clone Px8d14. Exon variants 3a, 3b, 8b, and 8c are shown. Scale bars differ for intron length and exon size. Introns 1 and 4 contain sequencing gaps. (B) nAChR Pxα6 coding sequence, containing exons 3a and 8b. The predicted N-terminal signal leader peptide (probability = 0.988) is shown with a dashed line. The four transmembrane domains are underlined in bold (TM1-4), signature cysteines of nAChR alpha subunits are double underlined and neurotransmitter-gated ion-channels signature of cysteines, separated by 13 amino acids, shown with stars. Intron positions are shown in numbered boxes. PCR primers in the 5′ and 3′ UTRs (shaded boxes) amplified a product from cDNA of Geneva88 4^th instar larvae (GenBank GU207835).

Figure 4. Truncating mutation of nAChR Pxα6 in spinosad resistant Plutella xylostella.

(A) Schematic diagram of the four nAChR transmembrane domains (TM1-4). A premature stop codon in the resistant BCS3-Pearl strain is denoted with a star in the intracellular loop. The subunit region predicted to be missing is shown with dashed lines. (B) DNA sequence of intron 9 from the susceptible strain (first 45 bp), and equivalent region in the resistant strain. Intronic GT splice sites are boxed. A G→A point mutation in the resistant strain (bold) results in a mis-splicing event that introduces this 40 bp sequence into mRNA and introduces a premature stop codon. (C) The peptide sequence between exon 9 and 10 of the susceptible strain and truncated product of the resistant strain. Conserved bases or amino acids are shown with an asterisk (*).

Figure 5. Summary of nAChR Pxα6 splice variation in resistant and susceptible Plutella xylostella larvae.

(A) Schematic of a full-length transcript, with four transmembrane domains. Two exon 3 variants, 3a or 3b, were observed through cloning. (B) Summary of transcripts observed from PCR amplification between exons 2 and 12. PCR 1 was performed with primers Pxα6_ex2_F and Pxα6_ex12_R3, products excised from agarose gels and reamplified with nested PCR 2 using primers Pxα6_ex2_F and Pxα6_ex12_R2. Amplicon sizes are shown in base pairs (bp). Isoform names are provided in general accordance with Rinkevich and Scott or new isoform numbers assigned. (C) Summary of transcripts from PCR between exons 7 and 11. PCR 3 was amplified with Pxα6_ex6F and Pxα6_ex12R, products column purified and reamplified with PCR 4, Pxα6_ex7_F and Pxα6_ex11_R. All clones sequenced from the resistant strain contained premature stop codons (black triangles). There were no stop codons or change in reading frame observed in clones from the susceptible strain. Insertions of 30, 40, or 4 base pairs are shown with dashed lines.