Functional characterization and fitness cost of spinosad-resistant alleles in Ceratitis capitata

Abstract

The sustainability of control programs for the Mediterranean fruit fly, Ceratitis capitata, for citrus crops in Spain has been threatened by the development of resistance to malathion and lambda-cyhalothrin in recent years. Spinosad is widely used without apparent loss of efficacy. However, a highly resistant strain, JW-100s, has been obtained after laboratory selection. Spinosad resistance in JW-100s has been associated with different mutant alleles of the α6 subunit of the nicotinic acetylcholine receptor (Ccα6) including an isoform-specific truncation allele, Ccα6 3aQ68*. Using the GAL4 > UAS system in Drosophila melanogaster to demonstrate expression of this truncated α6 subunit, in a dα6 loss-of-function genetic background, does not rescue susceptibility to spinosad, while the expression of Ccα6 wild-type isoforms does. We have also generated C. capitata isolines from JW-100s homozygous for: (1) the Ccα6 3aQ68*Δ3b-4 allele, which contains the mutation 3aQ68*, and (2) the Ccα6 3aQ68*-K352* allele, which contains the mutations 3aQ68* and K352*. Neither of these produce complete Ccα6 transcripts. The frequency of resistant alleles declined when in competition with individuals carrying the wild-type allele. Through extensive testing of both biological and behavioral fitness traits, we identified a reduced ability of Ccα6 3aQ68*Δ3b-4 males to detect the parapheromone and to mate with females carrying the Ccα6 3aQ68*-K352* allele in competition experiments. Thus, not only the potential for spontaneous resistant mutations to arise in Ccα6 but also their fitness costs must be considered when planning resistance management strategies for C. capitata.

Field detection and predicted evolution of spinosad resistance in Ceratitis capitata

“…The spinosad‐resistant strain JW‐100s, derived from field individuals collected in Xàbia in 2007, has been maintained under regular selection in the laboratory, as previously reported 6 . At the time the experiments were performed, the strain was constituted by individuals homozygous for the Ccα6 3aQ68*‐K352* allele and heterozygous for the Ccα6 3aQ68*‐K352* and Ccα6 3aQ68*Δ3b‐4 alleles 6,20 . The isoline Q68*‐K352* is homozygous for the allele Ccα6 3aQ68*‐K352* and was obtained by isolating it from JW‐100s strain 20 …”

“…Our previous work determined that the inheritance of spinosad resistance in JW‐100s was autosomic and completely recessive 6 . In addition, we showed that spinosad resistance imposes a fitness cost to resistant individuals compared to susceptible ones, since the resistant Ccα6 3aQ68*‐K352* and Ccα6 3aQ68*Δ3b‐4 alleles were not stable when in competition with the wild‐type allele 20 . Remarkably, we also demonstrated that spinosad resistance could have an impact on the behavior of this species, affecting the ability of males homozygous for the Ccα6 3aQ68*Δ3b‐4 allele to detect the parapheromone trimedlure and to mate in competition experiments 20 .…”

“…In addition, we showed that spinosad resistance imposes a fitness cost to resistant individuals compared to susceptible ones, since the resistant Ccα6 3aQ68*‐K352* and Ccα6 3aQ68*Δ3b‐4 alleles were not stable when in competition with the wild‐type allele 20 . Remarkably, we also demonstrated that spinosad resistance could have an impact on the behavior of this species, affecting the ability of males homozygous for the Ccα6 3aQ68*Δ3b‐4 allele to detect the parapheromone trimedlure and to mate in competition experiments 20 . However, the fitness values of these resistant genotypes have not been estimated, and the frequency of resistance alleles in the field is unknown.…”

“…The participation of mutation Q68* in exon 3a in the resistant phenotype was functionally validated by ectopic gene expression in D. melanogaster using the GAL4 > UAS system. In addition, the generation of two isolines homozygous for each one of the alleles Ccα6 3aQ68*Δ3b‐4 (carrying Q68* and a deletion of exons 3b and 4) and Ccα6 3aQ68*‐K352* (carrying Q68* and K352* in exon 10) allowed the determination that both alleles conferred the same level of resistance 20 . Knowledge about the mutations conferring resistance have permitted the design of molecular markers for spinosad resistance in medfly, enabling the detection of the specific mutations Q68* and K352* observed in the JW‐100s strain 6 .…”

“…In the present study we have (i) developed and implemented a methodology for the detection of resistant alleles in field populations, (ii) reanalyzed the data from the study of the stability of the two resistant genotypes found in the JW‐100s strain 20 to estimate their fitness value and (iii) predicted how resistance would evolve in the field based on evolutionary models.…”