Inheritance, Fitness Cost, and Management of Lambda-Cyhalothrin Resistance in a Laboratory-Selected Strain of Ceratitis capitata (Wiedemann)

The Mediterranean fruit fly (medfly), Ceratitis capitata, is considered one of the most destructive and economically damaging pests of citrus and other fruit crops worldwide. Current control practices in Spain rely on the use of insecticides (mainly lambda-cyhalothrin, spinosad, and deltamethrin) and the release of sterile males. However, the sustainability of medfly control programs is threatened by reports of resistance to lambda-cyhalothrin in field populations. In this work, we used a laboratory-selected lambda-cyhalothrin-resistant strain to study key factors required for devising effective insecticide resistance management strategies. Specifically, we have (1) determined that the inheritance of resistance is autosomic (non-associated to the sexual chromosome), completely dominant (a single copy of the gene is enough to confer resistance), and polygenic (controlled by more than one gene); (2) observed that resistant individuals present fitness alterations in regard to biological parameters (lower survival in the first growth stages, a slower developmental time, and higher adults’ weight and longevity); and (3) shown under laboratory conditions that the alternation of lambda-cyhalothrin with spinosad helped delay the development of resistance. Taken together, our results indicate that it would be advisable to encourage the rotation of these insecticides to manage the resistance problem

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

[EN] 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 alpha 6 subunit of the nicotinic acetylcholine receptor (Cc alpha 6) including an isoform-specific truncation allele, Cc alpha 6(3aQ68*). Using the GAL4 > UAS system in Drosophila melanogaster to demonstrate expression of this truncated alpha 6 subunit, in a d alpha 6 loss-of-function genetic background, does not rescue susceptibility to spinosad, while the expression of Cc alpha 6 wild-type isoforms does. We have also generated C. capitata isolines from JW-100s homozygous for: (1) the Cc alpha 6(3aQ68*Delta 3b-4) allele, which contains the mutation 3aQ68*, and (2) the Cc alpha 6(3aQ68*-K352*) allele, which contains the mutations 3aQ68* and K352*. Neither of these produce complete Cc alpha 6 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 Cc alpha 6(3aQ68*Delta 3b-4) males to detect the parapheromone and to mate with females carrying the Cc alpha 6(3aQ68*-K352*) allele in competition experiments. Thus, not only the potential for spontaneous resistant mutations to arise in Cc alpha 6 but also their fitness costs must be considered when planning resistance management strategies for C. capitata.This work received financial support from CICYT (AGL2016-76516-R). The Spanish MINECO granted A. Guillem-Amat a predoc (BES-C-2014-068937) and a mobility (EEBB-I-16-11336) fellowships. We gratefully acknowledge Maria Torne (Dow Agro-Science Iberica) for providing technical grade spinosad, Charles Robin (University of Melbourne) for assisting with bureaucratic issues with the Australian Government, Tinna Yang (University of Melbourne) for the keeping and shipping of the flies and Sandra Vacas (Universitat Politecnica de Valencia) for the scientific advice on electroantennography.Guillem-Amat, A.; Ureña, E.; López-Errasquín, E.; Navarro-Llopis, V.; Batterham, P.; Sánchez, L.; Perry, T.... (2020). Functional characterization and fitness cost of spinosad-resistant alleles in Ceratitis capitata. Journal of Pest Science. 93(3):1043-1058. https://doi.org/10.1007/s10340-020-01205-xS10431058933Abbas N, Mansoor MM, Shad SA et al (2014) Fitness cost and realized heritability of resistance to spinosad in Chrysoperla carnea (Neuroptera: Chrysopidae). 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Current situation and forecasting of resistance evolution to lambda-cyhalothrin in Spanish medfly populations

43 p.-4 fig.-3 tab.-1 graph. abst.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.This work received financial support from CICYT (AGL2016-76516-R) and MCIN/AEI (PID2019-104578RB-100). The Spanish MINECO granted a predoc fellowship to A. Guillem-Amat (BES-C-2014-068937) and Javier Castells-Sierra (BES-2017-081609).Peer reviewe

First detection of resistance to deltamethrin in Spanish populations of the Mediterranean fruit fly, Ceratitis capitata

14 p.-1 fig.-7 tab.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.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work received financial support from MCIN/AEI (PID2019-104578RB-100) and CICYT (AGL2016-76516-R). The Spanish Ministry of Science and Innovation Granted predoc fellowships to J. Castells-Sierra (BES-2017–081609) and A. Guillem-Amat (BES-C-2014–068937).Peer reviewe

Running title: Detection and evolution of spinosad resistance

9 p.-3 fig.-3 tab.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 to the presence of different mutations causing truncated transcripts of the α6 subunit of the nicotinic acetylcholine receptor (nAChRα6).RESULTS:A F1‐screen assay followed by the molecular characterization of surviving flies has been used to search for spinosad resistant alleles in field populations. Two different resistant alleles giving rise to truncated isoforms of Ccα6 have been identified, which corresponds to an estimated allelic frequency of at least 0.0023‐0.0046. The fitness values of the resistant nAChRα6 alleles found in the laboratory strain JW‐100s were estimated and resulted 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 than the 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.CONLUSION: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.This study was funded by the Spanish MINECO (grant AGL2016-76516-R).Peer reviewe

Genetic analysis of medfly populations in an area of sterile insect technique applications

15 p.-2 fig.-3 tab.The sterile insect technique (SIT) is widely used in integrated pest management programs for the control of the Mediterranean fruit fly (medfly), Ceratitis capitata. The genetic interactions between the released individuals from the genetic sexing strains (GSS), used for SIT applications worldwide, and wild individuals have not been studied. Under the hypothesis that a number of Vienna GSS individuals released to the field might not be completely sterile and may produce viable offspring, we have analyzed medfly Spanish field populations to evaluate the presence of Vienna strain genetic markers. To this goal, we have used contrasted nuclear and mitochondrial genetic markers, and two novel sets of nuclear polymorphisms with the potential to be markers to discriminate between Vienna and wild individuals. Nuclear Vienna markers located on the 5th chromosome of Vienna males have been found in 2.2% (19 from 875) of the Spanish wild medfly females captured at the area where SIT is applied. In addition, a female-inherited mitochondrial Vienna marker has been found in two from the 19 females showing nuclear Vienna markers. The detection of several of these markers in single individuals represents evidence of the introgression of Vienna strain into natural populations. However, alternative explanations as their presence at low frequency in wild populations in the studied areas cannot be fully discarded. The undesired release of non-fully sterile irradiated GSS individuals into the field and their interactions with wild flies, and the potential environmental implications should be taken into account in the application of the SIT.This work received financial support from the Spanish MINECO (AGL2016-76516-R).Peer reviewe

Immunodetection of Truncated Forms of the α6 Subunit of the nAChR in the Brain of Spinosad Resistant <i>Ceratitis capitata</i> Phenotypes

The α6 subunit of the nicotinic acetylcholine receptor (nAChR) has been proposed as the target for spinosad in insects. Point mutations that result in premature stop codons in the α6 gene of Ceratitis capitata flies have been previously associated with spinosad resistance, but it is unknown if these transcripts are translated and if so, what is the location of the putative truncated proteins. In this work, we produced a specific antibody against C. capitata α6 (Ccα6) and validated it by ELISA, Western blotting and immunofluorescence assays in brain tissues. The antibody detects both wild-type and truncated forms of Ccα6 in vivo, and the protein is located in the cell membrane of the brain of wild-type spinosad sensitive flies. On the contrary, the shortened transcripts present in resistant flies generate putative truncated proteins that, for the most part, fail to reach their final destination in the membrane of the cells and remain in the cytoplasm. The differences observed in the locations of wild-type and truncated α6 proteins are proposed to determine the susceptibility or resistance to spinosad

Multiple mutations in the nicotinic acetylcholine receptor Ccα6 gene associated with resistance to spinosad in medfly

12 p.-6 tab.-1 fig.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) Ccα63aQ68* containing a point mutation that generates a premature stop codon on exon 3a (3aQ68*); (ii) Ccα63aAG>AT containing a point mutation in the 5′ splicing site of exon 3a (3aAG > AT); and (iii) Ccα63aQ68*-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.This study was funded by the Spanish Ministerio de Economía, Industria y Competitividad (grants AGL2013-42632-R and AGL2016-76516-R).Peer reviewe