Genetics and mechanisms of insecticide resistance in ceratitis capitata and its implications for resistance management

  1. Guillem Amat, Ana
Supervised by:
  1. Félix Ortego Alonso Director

Defence university: Universidad Politécnica de Madrid

Fecha de defensa: 31 October 2019

Committee:
  1. Isabel Díaz Rodríguez Chair
  2. Sandra Vacas González Secretary
  3. Joel González Cabrera Committee member
  4. Maria Dolors Piulachs Baga Committee member
  5. John Vontas Committee member

Type: Thesis

Teseo: 605015 DIALNET

Abstract

ABSTRACT The Mediterranean fruit fly (medfly), Ceratitis capitata, is one of the main insect pests of fruits. Current control practices in citrus crops in the Comunitat Valenciana mainly rely on the use of insecticides (spinosad and lambda-cyhalothrin as bait sprays, and deltamethrin as lure and kill traps) and the release of sterile males. However, the sustainability of medfly management programs is threatened by the reports of resistance to lambda-cyhalothrin in field populations and the potential for spinosad resistance development, which has been obtained under laboratory selection. The aim of this work was to study the mechanisms and genetics of spinosad and lambda-cyhalothrin resistance and their implications for resistance monitoring and management. Spinosad resistance has been associated with different mutant alleles of the α6 subunit of the nicotinic acetylcholine receptor (Ccα6) that lead to loss-of-function phenotypes in the laboratory selected JW-100s strain. Using the GAL4>UAS system in a resistant Drosophila melanogaster strain, lacking the endogenous α6 subunit, we demonstrated that the ectopic expression of Ccα6 wild-type isoforms rescue susceptibility, while the expression of the truncated allele Ccα63aQ68* (carrying the mutation 3aQ68*) does not. These results confirmed that truncated transcripts do not produce a response to spinosad. We also generated C. capitata isolines homozygous for the alleles: 1) Ccα63aQ68*Δ3b-4, which contains the mutation 3aQ68* and a deletion of exons 3b and 4; and 2) Ccα63aQ68*-K352*, which contains the mutations 3aQ68* and K352*. Both were present in JW-100s and neither of them produced complete Ccα6 transcripts. We confirmed that both alleles contributed equally to the resistant phenotype. However, their frequency declined when in competition with individuals carrying the wild-type allele, suggesting a fitness cost associated with them. Through extensive testing of both biological and behavioral fitness traits, we identified a reduced ability of Ccα63aQ68*Δ3b-4 males to detect the parapheromone trimedlure and to mate with females carrying the Ccα63aQ68*-K352* allele in competition experiments. Lambda-cyhalothrin resistance in the laboratory selected W-1Kλ strain had previously been associated with P450-metabolic detoxification mediated by over-expression of the CcCYP6A51 gene. However, our results indicated that other traits must also be involved. Thus, we showed that the expression of several P450s, including CcCYP6A51, was highly variable and induced in some individuals in response to lambda-cyhalothrin treatments in both W-1Kλ and C strains. Besides, we proved that the inheritance pattern is polygenic, dominant and autosomic. A genotyping-by-sequencing analysis confirmed the polygenic character of resistance and identified seven candidate regions associated with the resistant phenotype. However, the subsequent analysis showed that they have a minor contribution with a marked cumulative effect. Moreover, none of the seven candidate regions were at or near P450 gene loci. As a result, we have not found a proper diagnostic marker to confidently monitor lambda-cyhalothrin resistance in field populations of C. capitata. The susceptibility to available insecticides was monitored in Spanish medfly field populations in the period 2015-2017. We have found that the level of resistance to lambda-cyhalothrin (3-8 fold) is similar to that reported in previous years. Resistance to deltamethrin has been detected for the first time in field populations exposed to MagnetMEDTM traps, which are coated with this insecticide. All populations analyzed are susceptible to spinosad, though resistant alleles have been detected at low frequency by the combined use of an F1-screen assay and the search of mutations in the Ccα6 gene. Modelling of spinosad resistance evolution was carried out considering the frequency of resistant alleles in the field, type of inheritance, fitness costs, level of exposition and the use of other insecticides. In addition, laboratory simulation studies were performed to evaluate different insecticide treatment scenarios in a multiresistant laboratory strain, generated by crossing JW-100s and W-1Kλ. The results indicated that the best option for resistance management is the rotation of insecticides with different modes of action and no cross-resistance. In conclusion, medfly control is compromised by the development of resistance to available insecticides. Hence, the implementation of resistance management strategies is required. It is advisable to perform rotations of spinosad and lambda-cyhalothrin, or to use spinosad in those orchards where lure-and-kill traps of deltamethrin are deployed. On the contrary, the simultaneous use of lambda-cyhalothrin and deltamethrin is discouraged, as there is a risk of cross-resistance between them. These strategies must be harmonized with other control methods, such as cultural practices and the sterile insect technique.