Spinosad is an insecticide widely used for the control of insect pest species, including Mediterranean fruit fly, Ceratitis capitata . Its target site is the α6 subunit of the nicotinic acetylcholine receptors, and different mutations in this subunit confer resistance to spinosad in diverse insect species. The insect α6 gene contains 12 exons, with mutually exclusive versions of exons 3 (3a, 3b) and 8 (8a, 8b, 8c). We report here the selection of a medfly strain highly resistant to spinosad, JW-100 s, and we identify three recessive Ccα6 mutant alleles in the JW-100 s population: (i) Ccα6 3aQ68* containing a point mutation that generates a premature stop codon on exon 3a (3aQ68*); (ii) Ccα6 3aAG > AT containing a point mutation in the 5′ splicing site of exon 3a (3aAG > AT); and (iii) Ccα6 3aQ68*-K352* that contains the mutation 3aQ68* and another point mutation on exon 10 (K352*). Though our analysis of the susceptibility to spinosad in field populations indicates that resistance has not yet evolved, a better understanding of the mechanism of action of spinosad is essential to implement sustainable management practices to avoid the development of resistance in field populations.

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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.

BACKGROUND: The sustainable control of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), is compromised by the development of resistance to malathion and lambda-cyhalothrin in Spanish field populations. At present, field populations remain susceptible to spinosad. However, the resistant strain JW-100s has been obtained under laboratory selection with spinosad, and resistance has been associated with the presence of different mutations causing truncated transcripts of the ⊍6 subunit of the nicotinic acetylcholine receptor (nAChR⊍6). RESULTS: An F1 screen assay followed by the molecular characterization of surviving flies has been used to search for spinosadresistant alleles in field populations. Two different resistant alleles giving rise to truncated isoforms of Cc⊍6 have been identified, which corresponds to an estimated allelic frequency of at least 0.0023-0.0046. The fitness values of the resistant nAChR⊍6 alleles found in the laboratory strain JW-100s were estimated to be 0.4 for RR and 0.2 for SR. Mathematical modelling predicted that spinosad-resistant alleles will rapidly decline over time in field populations if their fitness cost was the same as estimated for laboratory-resistant alleles. However, they are predicted to increase in the field if their fitness cost is lower and resistance management strategies are not implemented. CONCLUSION: Spinosad-resistant alleles have been detected in field populations for the first time. Our modelling simulations indicate that the best option to delay the appearance of spinosad resistance would be its rotation with other insecticides without cross-resistance. The integrated F1 screen/molecular genetic analysis presented here can be used for future monitoring studies.

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BACKGROUND The control of the Mediterranean fruit fly Ceratitis capitata (Wiedemann) in Spanish field populations mainly relies on the insecticides lambda‐cyhalothrin and spinosad as bait sprays. However, their sustainable used is compromised by the development of lambda‐cyhalothrin resistance and the detection of spinosad resistant alleles. In addition, the use of lure‐and‐kill traps covered with deltamethrin has increased in the last years. It is thus urgent to predict the impact that the combination of both pyrethroids will have in the evolution of lambda‐cyhalothrin resistance and how they could be combined with spinosad so as to establish proper resistance management programs. RESULTS Toxicity bioassays were performed to analyze the current levels of lambda‐cyhalothrin resistance in field populations, proving that it has remained stable in the last decade. An evolutionary model was established to explore the weight of selected parameters in the evolution of lambda‐cyhalothrin resistance in C. capitata and to forecast resistance development under different resistance management scenarios. Our results highlight the importance of fitness cost and inheritance to fit the experimental results. The analyses predicted that the rotation of lambda‐cyhalothrin and spinosad, when deltamethrin traps are also deployed in the field, will slow down the evolution of resistance, especially when cross‐resistance between both pyrethroids is considered. CONCLUSION Lambda‐cyhalothrin resistance has not increased in the last decade, probably due to the alternation of this insecticide with spinosad. Our modelling results indicate that the best option to avoid an increase in lambda‐cyhalothrin resistant alleles, considering that deltamethrin use is growing, would be to continue combining their use with spinosad. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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The control of the Mediterranean fruit fly (Medfly), Ceratitis capitata, in citrus orchards in Spain is mainly based in three insecticides (spinosad, lambda-cyhalothrin and deltamethrin) and the liberation of sterile males. However, Medfly control is compromised by the development of lambda-cyhalothrin resistance and the detection of spinosad-resistant alleles in field populations. We report here, for the first time, resistance to deltamethrin in populations collected in fields under different management strategies, including MagnetMed™ traps coated with this insecticide and/or spinosad and lambda-cyhalothrin used as bait sprays, and even in populations obtained from non-treated fields. Two deltamethrin-resistant strains (BP-delta and Rfg-delta) were generated from the descendants of some of the field populations that showed lower susceptibility to deltamethrin. Both strains showed low susceptibility to MagnetMed™ traps, moderate susceptibility to Ceratipack traps, and lacked cross-resistance to spinosad and lambda cyhalothrin. Our data suggest that deltamethrin resistance was mediated by P450 enzymes, since bioassays with synergists showed that PBO reverted resistance in a field population and the laboratory strains, whereas the effect of DEF and DEM was minor and no mutations were found in the VGSC gene. The inheritance of resistance for both strains was completely recessive, autosomic and did not fit the mortality expected for a recessive character under a monogenic or digenic model. We also found that deltamethrin resistance presented a fitness cost in terms of males’ weight, males’ and females’ longevity and lifetime fecundity, with a more pronounced effect in the BP-strain than in the Rfg-delta strain. Our results highlight the need to implement insecticide resistance management strategies to prevent control failures.