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

Effect of methylisothiazolinone on biological treatments: Efficiency of SBRs and Bioindicative Studies

Methylisothiazolinone (MIT) is widely used as biocide in hair and skin care products and in high concentrations, more than 150 mg/L, in cooling towers in the paper mill and refinery industry. This product has been recognized as a potent contact allergen and is discharged to the wastewater treatment plants (WWTP) from industrial processes or domestic usages. However, the role of MIT in biological treatment has not been characterized, and therefore, the effect of MIT on the composition and performance of activated sludge has been analyzed. For this purpose, a sequential batch reactor was fed with a synthetic solution containing 5 mg/L of MIT. Addition of MIT had shown no significant effect on organic matter metabolization (dissolved organic carbon [DOC] removal remained constant at 80-90%). On the contrary, a loss of efficiency in the nitrification process occurred (ammonium removal decreased from 90% to 20% per cycle throughout the experiment), first affecting the nitrite oxidizing bacteria and, after around 40 cycles, also the ammonium-oxidizing bacteria disappeared, as shown by fluorescence in situ hybridization analysis. Bioindicative studies based on counts of protozoa and sludge biotic index indicated that, at the end of the process, a stable and well-colonized protozoa community was obtained. Finally, respirometric tests indicated some acclimatization of the heterotrophic bacteria to MIT, as shown by shorter lag periods when the sludge was previously fed with MIT. The role of MIT in biological treatment has been characterized to increase the knowledge about this pollutant effects on activated sludge to improve WWTP performance.We would like to thank the financial support of the Spanish Ministerio de Economia y Competitividad AQUAFOTOX (CTQ2012-38754-C03-02).Amat Payá, AM.; Arques Sanz, A.; López Pérez, MF.; Nácher Pastor, M.; Palacios Guillem, S. (2015). Effect of methylisothiazolinone on biological treatments: Efficiency of SBRs and Bioindicative Studies. Environmental Engineering Science. 32(6):479-485. https://doi.org/10.1089/ees.2014.0283S47948532

The whole genome sequence of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), reveals insights into the biology and adaptive evolution of a highly invasive pest species

31 p.-11 fig.-2 tab.+ Erratum (2 p.) Papanikolaou, Alexie et al.Background: The Mediterranean fruit fly (medfly), Ceratitis capitata, is a major destructive insect pest due to its broad host range, which includes hundreds of fruits and vegetables. It exhibits a unique ability to invade and adapt to ecological niches throughout tropical and subtropical regions of the world, though medfly infestations have been prevented and controlled by the sterile insect technique (SIT) as part of integrated pest management programs (IPMs). The genetic analysis and manipulation of medfly has been subject to intensive study in an effort to improve SIT efficacy and other aspects of IPM control.Results: The 479 Mb medfly genome is sequenced from adult flies from lines inbred for 20 generations. A highquality assembly is achieved having a contig N50 of 45.7 kb and scaffold N50 of 4.06 Mb. In-depth curation of more than 1800 messenger RNAs shows specific gene expansions that can be related to invasiveness and host adaptation, including gene families for chemoreception, toxin and insecticide metabolism, cuticle proteins, opsins, and aquaporins. We identify genes relevant to IPM control, including those required to improve SIT.Conclusions: The medfly genome sequence provides critical insights into the biology of one of the most serious and widespread agricultural pests. This knowledge should significantly advance the means of controlling the size and invasive potential of medfly populations. Its close relationship to Drosophila, and other insect species important to agriculture and human health, will further comparative functional and structural studies of insect genomes that should broaden our understanding of gene family evolutionSupport of this project was provided by the U.S. Department of Agriculture(USDA), Agricultural Research Service (ARS), Animal and Plant Health Inspection Service (APHIS), and National Institute of Food and Agriculture(NIFA)-Biotechnology Risk Assessment Grants Program (grant #2011-39211-30769 to AMH) for funding the initial phase of this project, and to the National Institutes of Health (NIH)-National Human Genome Research Institute (NHGRI) for funding the medfly genome sequencing, assembly and Maker 2.0 automated annotation as part of the i5K 30 genome pilot project (grant #U54 HG003273 to RAG). The NIH Intramural Research Program, National Library of Medicine funded the NCBI Gnomon annotation and the USDA-National Agricultural Library (NAL) provided support for the WebApollo curation website, with support for manual curation training (to MM-T) provided by NIGMS (grant #5R01GM080203),NHGRI (grant #5R01HG004483), and the U.S. Department of Energy(contract #DE-AC02-05CH11231). Support was provided for: toxin metabolism and insecticide resistance gene studies from MINECO,Spain (AGL2013-42632-R to FO and PH-C); microRNAs, horizontal gene transfer and bacterial contaminant studies from the European Social Fund and National Strategic Reference Framework-THALES (MIS375869 to KB, GT, AGH, and KM) and the U.S. National Science Foundation(DEB 1257053 to JHW); cuticle protein gene studies from USDA-NIFA(grant #2016-67012-24652 to AJR); sex-determination studies from L.R. Campania (grant 5/02, 2008 to GS); male reproduction and sexual differentiation studies from the FAO/IAEA (Technical Contract No: 16966 to GGa) and Cariplo IMPROVE (to FS); and programmed cell death gene studies and genomic data analysis (to MFS) from the Emmy Noether program, DFG(SCHE 1833/1-1) and the LOEWE Center for Insect Biotechnology & Bioresources grant of the Hessen State Ministry of Higher Education, Research and the Arts(HMWK), Germany and from the USDA-NIFA-Biotechnology Risk Assessment Grants Program (grant #2015-33522-24094 to AMH).Peer reviewe

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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BMC Evol Biol 7:1–12. https://doi.org/10.1186/1471-2148-7-98Jones AK, Raymond-Delpech V, Thany SH et al (2006) The nicotinic acetylcholine receptor gene family of the honey bee, Apis mellifera. Genome Res 16:1422–1430. https://doi.org/10.1101/gr.4549206Khan HAA, Akram W, Shad SA (2014) Genetics, cross-resistance and mechanism of resistance to spinosad in a field strain of Musca domestica L. (Diptera: Muscidae). Acta Trop 130:148–154. https://doi.org/10.1016/j.actatropica.2013.11.006Li ZM, Liu SS, Liu YQ, Ye GY (2007) Temperature-related fitness costs of resistance to spinosad in the diamondback moth, Plutella xylostella (Lepidoptera: Plutelidae). Bull Entomol Res 97:627–635. https://doi.org/10.1017/S0007485307005366Li X, Wan Y, Yuan G et al (2017) Fitness trade-off associated with spinosad resistance in Frankliniella occidentalis (Thysanoptera: Thripidae). 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Estudio de la herencia y mapeo de la resistencia de Ceratitis capitata (Wiedemann, 1824) (Diptera, Tephritidae) al insecticida λ-cihalotrina

La mosca mediterránea de la fruta, Ceratitis capitata (Wiedemann, 1824), es una de las principales plagas de cítricos y otros frutales en España. Las actuales estrategias de control, basadas mayoritariamente en el uso de insecticidas, están viendo amenazada su eficacia debido a la aparición de resistencia en poblaciones de campo. Recientemente se ha descrito resistencia metabólica a lambda-cihalotrina mediada por P450s. Con el fin de estudiar el tipo de herencia de la resistencia a lambda-cihalotrina, se realizaron cruzamientos recíprocos entre una línea de laboratorio resistente a este insecticida y una línea susceptible, se obtuvieron la generación F1 y la F2 y se realizó el retrocruce de la F1 con el parental susceptible. Los resultados muestran que la herencia de la resistencia es completamente dominante y autosómica, y que no se ajusta a un modelo mendeliano monogénico. Asimismo, se está realizando un mapeo genético de la resistencia a lambda-cihalotrina a partir de los individuos ensayados en el estudio de la herencia, con el propósito de identificar marcadores moleculares que nos permitan la detección precoz y el seguimiento de la resistencia en campo, facilitando así el control de la plaga. Por último, mediante el simulacro en laboratorio de distintos escenarios de tratamiento se ha podido determinar que la rotación de lambda-cihalotrina con spinosad resulta eficaz para el manejo de la resistencia a lambda-cihalotrina

Solar photocatalytic detoxification of cyanide effluents from metal finishing industry

[En] Wastewaters from metal fi nishing industry contain, among other pollutants, high amounts of cyanide and heavy metals, which results in a high toxicity of the effl uent. Therefore, it is necessary to detoxify the effl uent before discharging to a sewage treatment plant which commonly consists in a biological treatment. In this study, we analyzed different photocatalytic methods for cyanide removal: photo-Fenton, Fenton-like with metals such as manganese, zinc, silver, cobalt, chromium and copper and photocatalysis with addition of other oxidants such as persulfate. Initially, we performed a study on synthetic water prepared with contaminants found in real wastewater. In order to better simulate real conditions, the possible interferences from ions usually present in water, such as carbonates, sulfates, fl uorides or nitrates has been studied. The best treatment (Fenton-Like) was applied to real wastewaters from a metallurgical industry of the east of Spain, containg high amounts of copper. The global analysis of the treatments determined that the best results were obtained by applying solar photo-Fenton process and photo-Fenton-like processes with copper. The total degradation of cyanide and copper precipitation was achieved, improving the characteristics of the treated effl uent.[ES] Las aguas procedentes de la industria metalúrgica contienen, entre otros contaminantes, grandes cantidades de cianuro y metales pesados que les confieren elevadas toxicidades; por ello se hace necesario detoxificar estos efluentes antes de verterlos a una Estación de Depuración de Aguas Residuales Urbanas (EDARU) con tratamientos biológicos. En este estudio se analizan distintos métodos fotocatalíticos para la eliminación de cianuro: proceso foto-Fenton, Fentonlike con diferentes metales frecuentes en aguas reales, como manganeso, cinc, plata, cobalto, cromo y cobre y fotocatálisis solar con adición de oxidantes adicionales como persulfato. Inicialmente, se realiza un estudio sobre aguas preparadas con contaminantes encontrados en las analíticas de aguas de empresa. Con objeto de acercarnos más a condiciones reales, se determinan posibles interferencias causadas por iones presentes en aguas de forma habitual: carbonatos, sulfatos, fluoruros y nitratos. El tratamiento que proporciona mejores resultados (proceso similares al fotoFenton: ¿foto-Like-Fenton¿) se aplica en las aguas procedentes de una industria metalúrgica de la Comunidad Valenciana, cuyas aguas contienen cobre en cantidad suficiente como para influir de manera beneficiosa en el proceso. El análisis global de los tratamientos aplicados determina que los mejores resultados se obtienen con la aplicación del proceso foto-Fenton solar y procesos fotocatalíticos similares con cobre; se consigue la total degradación del cianuro y la precipitación final del cobre que favorece su eliminación del efluente mediante aplicación de un filtro prensa.Silvestre Mira, M.; Vercher Pérez, RF.; Palacios Guillem, S.; Arqués Sanz, A.; Amat Payá, AM.; Añó Montalvá, EJ. (2012). Detoxificación por fotocatálisis solar de efl uentes cianurados provenientes de una industria metalúrgica. Dyna Ingeniería e Industria. 87(6):698-706. doi:10.6036/4676S69870687

The whole genome sequence of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), reveals insights into the biology and adaptive evolution of a highly invasive pest species

The Mediterranean fruit fly (medfly), Ceratitis capitata, is a major destructive insect pest due to its broad host range, which includes hundreds of fruits and vegetables. It exhibits a unique ability to invade and adapt to ecological niches throughout tropical and subtropical regions of the world, though medfly infestations have been prevented and controlled by the sterile insect technique (SIT) as part of integrated pest management programs (IPMs). The genetic analysis and manipulation of medfly has been subject to intensive study in an effort to improve SIT efficacy and other aspects of IPM control

Dietary α‐Linolenic Acid, Marine ω‐3 Fatty Acids, and Mortality in a Population With High Fish Consumption: Findings From the PREvención con DIeta MEDiterránea (PREDIMED) Study

Background: Epidemiological evidence suggests a cardioprotective role of α‐linolenic acid (ALA), a plant‐derived ω‐3 fatty acid. It is unclear whether ALA is beneficial in a background of high marine ω‐3 fatty acids (long‐chain n‐3 polyunsaturated fatty acids) intake. In persons at high cardiovascular risk from Spain, a country in which fish consumption is customarily high, we investigated whether meeting the International Society for the Study of Fatty Acids and Lipids recommendation for dietary ALA (0.7% of total energy) at baseline was related to all‐cause and cardiovascular disease mortality. We also examined the effect of meeting the society's recommendation for long‐chain n‐3 polyunsaturated fatty acids (≥500 mg/day). Methods and Results: We longitudinally evaluated 7202 participants in the PREvención con DIeta MEDiterránea (PREDIMED) trial. Multivariable‐adjusted Cox regression models were fitted to estimate hazard ratios. ALA intake correlated to walnut consumption (r=0.94). During a 5.9‐y follow‐up, 431 deaths occurred (104 cardiovascular disease, 55 coronary heart disease, 32 sudden cardiac death, 25 stroke). The hazard ratios for meeting ALA recommendation (n=1615, 22.4%) were 0.72 (95% CI 0.56–0.92) for all‐cause mortality and 0.95 (95% CI 0.58–1.57) for fatal cardiovascular disease. The hazard ratios for meeting the recommendation for long‐chain n‐3 polyunsaturated fatty acids (n=5452, 75.7%) were 0.84 (95% CI 0.67–1.05) for all‐cause mortality, 0.61 (95% CI 0.39–0.96) for fatal cardiovascular disease, 0.54 (95% CI 0.29–0.99) for fatal coronary heart disease, and 0.49 (95% CI 0.22–1.01) for sudden cardiac death. The highest reduction in all‐cause mortality occurred in participants meeting both recommendations (hazard ratio 0.63 [95% CI 0.45–0.87]). Conclusions: In participants without prior cardiovascular disease and high fish consumption, dietary ALA, supplied mainly by walnuts and olive oil, relates inversely to all‐cause mortality, whereas protection from cardiac mortality is limited to fish‐derived long‐chain n‐3 polyunsaturated fatty acids. Clinical Trial Registration URL: http://www.Controlled-trials.com/. Unique identifier: ISRCTN35739639

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

La mosca mediterránea de la fruta, Ceratitis capitata, es uno de los principales insectos plaga de los frutales. Las prácticas actuales de control en los campos de cítricos de la Comunitat Valenciana se basan principalmente en el uso de insecticidas (spinosad y lambda-cihalotrina mediante pulverización-cebo, y deltametrina en trampas de atracción y muerte) y la suelta de machos estériles. Sin embargo, la sostenibilidad de los programas de manejo está amenazada por la detección de resistencia a lambda-cihalotrina en poblaciones de campo y por la capacidad de esta especie para desarrollar resistencia a spinosad bajo presión de selección en laboratorio. El objetivo de este trabajo fue estudiar los mecanismos y la genética de la resistencia a spinosad y lambda-cihalotrina y su implicación en el monitoreo y manejo de la resistencia. La resistencia a spinosad ha sido asociada en la línea seleccionada de laboratorio JW-100s con diferentes alelos mutantes de la subunidad α6 del receptor nicotínico de acetilcolina (Ccα6), que da lugar a fenotipos de pérdida de función. Con la implementación del sistema GAL4>UAS en una línea resistente de Drosophila melanogaster, que carecía de la subunidad α6 endógena, demostramos que la expresión ectópica de isoformas silvestres de Ccα6 restablecen la susceptibilidad, mientras que la expresión del alelo truncado Ccα63aQ68* (que porta la mutación 3aQ68*) no lo hace. Estos resultados confirmaron que los transcritos truncados no producen una respuesta a spinosad. También generamos isolíneas de C. capitata homocigotas para los alelos: 1) Ccα63aQ68*Δ3b-4, que contiene la mutación 3aQ68* y una deleción de los exones 3b y 4; y 2) Ccα63aQ68*-K352*, que contiene las mutaciones 3aQ68* y K352*. Ambos estaban presentes en JW-100s y ninguno de ellos producía transcritos completos de Ccα6. Confirmamos que ambos alelos contribuyen de igual manera al fenotipo resistente. Sin embargo, su frecuencia descendía cuando se encontraban en competencia con individuos que portaban el alelo silvestre, sugiriendo la existencia de un coste biológico. Diferentes caracteres biológicos y comportamentales fueron analizados, identificándose una reducción en la habilidad de los machos Ccα63aQ68*Δ3b-4 para detectar la paraferomona trimedlure y para aparearse en experimentos de competencia con hembras que portan el alelo Ccα63aQ68*-K352*. La resistencia a lambda-cihalotrina en la línea seleccionada de laboratorio W-1Kλ ha sido previamente asociada con detoxificación metabólica mediada por sobreexpresión del gen CcCYP6A51. Sin embargo, nuestros resultados indicaron que otros caracteres debían también estar implicados. Así, mostramos que la expresión de varios P450, incluyendo CcCYP6A51, era altamente variable e inducida en algunos individuos en respuesta al tratamiento con lambda-cihalotrina, tanto en W-1Kλ como en la línea control C. Además, demostramos que el patrón de herencia es poligénico, dominante y autosómico. Un análisis de genotipado por secuenciación confirmó el carácter poligénico de la resistencia, identificándose siete regiones candidatas asociadas con el fenotipo resistente. Sin embargo, el análisis posterior mostró que tenían una contribución menor con un marcado efecto aditivo. Además, ninguna de las regiones candidatas se encontraba dentro ni cerca de los genes P450. Como resultado, no hemos encontrado un marcador de diagnóstico robusto para monitorizar la resistencia a lambda-cihalotrina en poblaciones de campo de C. capitata. La susceptibilidad a los insecticidas utilizados actualmente se monitorizó en poblaciones españolas de C. capitata durante el periodo 2015-2017. Hemos encontrado que el nivel de resistencia a lambda-cihalotrina (3-8 veces) es similar a la encontrada en años anteriores. La resistencia a deltametrina se detectó por primera vez en poblaciones de campo expuestas a trampas MagnetMEDTM, recubiertas con este insecticida. Todas las poblaciones analizadas permanecen susceptibles a spinosad, aunque se han detectados alelos de resistencia a baja frecuencia mediante el uso combinado de ensayos “F1-screen” y la búsqueda de mutaciones en el gen Ccα6. La evolución de resistencia a spinosad fue modelizada considerando la frecuencia de los alelos en el campo, tipo de herencia, coste biológico, nivel de exposición y el uso de otros insecticidas. Además, se realizó un estudio de simulación en laboratorio para evaluar diferentes escenarios de tratamiento con insecticidas con una línea de laboratorio multirresistente, generada mediante el cruce entre JW-100s y W-1Kλ. Los resultados indicaron que la rotación de insecticidas con diferentes mecanismos de acción y sin resistencia-cruzada es la mejor opción para el manejo de la resistencia. En conclusión, el control de C. capitata está comprometido por el desarrollo de resistencia a los insecticidas disponibles, siendo necesaria la implementación de estrategias de manejo de la resistencia. Es recomendable realizar rotaciones entre spinosad y lambda-cihalotrina, o utilizar spinosad en aquellos cultivos en los que se emplean trampas de atracción y muerte con deltametrina. Por el contrario, el uso simultáneo de lambda-cihalotrina y deltametrina está desaconsejado, ya que existe riesgo de resistencia cruzada entre ambos. Estas estrategias deben de ser armonizadas con otros métodos de control, tales como las prácticas culturales y la técnica del insecto estéril. ----------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

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

La mosca mediterránea de la fruta, Ceratitis capitata, es uno de los principales insectos plaga de los frutales. Las prácticas actuales de control en los campos de cítricos de la Comunitat Valenciana se basan principalmente en el uso de insecticidas (spinosad y lambda-cihalotrina mediante pulverización-cebo, y deltametrina en trampas de atracción y muerte) y la suelta de machos estériles. Sin embargo, la sostenibilidad de los programas de manejo está amenazada por la detección de resistencia a lambda-cihalotrina en poblaciones de campo y por la capacidad de esta especie para desarrollar resistencia a spinosad bajo presión de selección en laboratorio. El objetivo de este trabajo fue estudiar los mecanismos y la genética de la resistencia a spinosad y lambda-cihalotrina y su implicación en el monitoreo y manejo de la resistencia. La resistencia a spinosad ha sido asociada en la línea seleccionada de laboratorio JW-100s con diferentes alelos mutantes de la subunidad α6 del receptor nicotínico de acetilcolina (Ccα6), que da lugar a fenotipos de pérdida de función. Con la implementación del sistema GAL4>UAS en una línea resistente de Drosophila melanogaster, que carecía de la subunidad α6 endógena, demostramos que la expresión ectópica de isoformas silvestres de Ccα6 restablecen la susceptibilidad, mientras que la expresión del alelo truncado Ccα63aQ68* (que porta la mutación 3aQ68*) no lo hace. Estos resultados confirmaron que los transcritos truncados no producen una respuesta a spinosad. También generamos isolíneas de C. capitata homocigotas para los alelos: 1) Ccα63aQ68*Δ3b-4, que contiene la mutación 3aQ68* y una deleción de los exones 3b y 4; y 2) Ccα63aQ68*-K352*, que contiene las mutaciones 3aQ68* y K352*. Ambos estaban presentes en JW-100s y ninguno de ellos producía transcritos completos de Ccα6. Confirmamos que ambos alelos contribuyen de igual manera al fenotipo resistente. Sin embargo, su frecuencia descendía cuando se encontraban en competencia con individuos que portaban el alelo silvestre, sugiriendo la existencia de un coste biológico. Diferentes caracteres biológicos y comportamentales fueron analizados, identificándose una reducción en la habilidad de los machos Ccα63aQ68*Δ3b-4 para detectar la paraferomona trimedlure y para aparearse en experimentos de competencia con hembras que portan el alelo Ccα63aQ68*-K352*. La resistencia a lambda-cihalotrina en la línea seleccionada de laboratorio W-1Kλ ha sido previamente asociada con detoxificación metabólica mediada por sobreexpresión del gen CcCYP6A51. Sin embargo, nuestros resultados indicaron que otros caracteres debían también estar implicados. Así, mostramos que la expresión de varios P450, incluyendo CcCYP6A51, era altamente variable e inducida en algunos individuos en respuesta al tratamiento con lambda-cihalotrina, tanto en W-1Kλ como en la línea control C. Además, demostramos que el patrón de herencia es poligénico, dominante y autosómico. Un análisis de genotipado por secuenciación confirmó el carácter poligénico de la resistencia, identificándose siete regiones candidatas asociadas con el fenotipo resistente. Sin embargo, el análisis posterior mostró que tenían una contribución menor con un marcado efecto aditivo. Además, ninguna de las regiones candidatas se encontraba dentro ni cerca de los genes P450. Como resultado, no hemos encontrado un marcador de diagnóstico robusto para monitorizar la resistencia a lambda-cihalotrina en poblaciones de campo de C. capitata. La susceptibilidad a los insecticidas utilizados actualmente se monitorizó en poblaciones españolas de C. capitata durante el periodo 2015-2017. Hemos encontrado que el nivel de resistencia a lambda-cihalotrina (3-8 veces) es similar a la encontrada en años anteriores. La resistencia a deltametrina se detectó por primera vez en poblaciones de campo expuestas a trampas MagnetMEDTM, recubiertas con este insecticida. Todas las poblaciones analizadas permanecen susceptibles a spinosad, aunque se han detectados alelos de resistencia a baja frecuencia mediante el uso combinado de ensayos “F1-screen” y la búsqueda de mutaciones en el gen Ccα6. La evolución de resistencia a spinosad fue modelizada considerando la frecuencia de los alelos en el campo, tipo de herencia, coste biológico, nivel de exposición y el uso de otros insecticidas. Además, se realizó un estudio de simulación en laboratorio para evaluar diferentes escenarios de tratamiento con insecticidas con una línea de laboratorio multirresistente, generada mediante el cruce entre JW-100s y W-1Kλ. Los resultados indicaron que la rotación de insecticidas con diferentes mecanismos de acción y sin resistencia-cruzada es la mejor opción para el manejo de la resistencia. En conclusión, el control de C. capitata está comprometido por el desarrollo de resistencia a los insecticidas disponibles, siendo necesaria la implementación de estrategias de manejo de la resistencia. Es recomendable realizar rotaciones entre spinosad y lambda-cihalotrina, o utilizar spinosad en aquellos cultivos en los que se emplean trampas de atracción y muerte con deltametrina. Por el contrario, el uso simultáneo de lambda-cihalotrina y deltametrina está desaconsejado, ya que existe riesgo de resistencia cruzada entre ambos. Estas estrategias deben de ser armonizadas con otros métodos de control, tales como las prácticas culturales y la técnica del insecto estéril. ----------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