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Os padrões do comportamento ingestivo foram avaliados usando nove ovinos 1/2 Texel +1/2 Ile-de-France durante seis meses. Os ovinos receberam a dieta às 9 e 16 h, composta de 250 g de concentrado (15,45% PB, 36,54% FDN) e feno de gramíneas à vontade (6,69% PB, 69,10% FDN). O comportamento ingestivo foi medido continuamente durante cinco dias por período, suas atividades foram classificadas como ingestão, ruminação ou descanso eregistradas seqüencialmente. O cálculo das probabilidades de estado abrangeu três tipos: as probabilidades de estar em determinado estado, de permanecer neste estado e de mudar de um estado para outro. Foi aplicada a transformação finita de Fourier sobre as séries de probabilidades estimadas a cada cinco minutos, durante 22 horas por dia. No estudo da evolução nictemeral das probabilidades, foram utilizadas as análises de periodogramas, diagramas de fase e análise da variância sobre os valores transformados das séries de probabilidades. A análise de variância mostrou que todos os tipos de probabilidades seguiram uma distribuição nictemeral, influenciada significativamente pelos períodos e animais experimentais, em diversas freqüências. A análise dos periodogramas e diagramas de fase mostrou que as diferenças entre os períodos e animais experimentais foi especialmente devido às diferenças de amplitude das funções trigonométricas que descreveram a distribuição nictemeral das probabilidades de permanecer ou estar em determinado estado. O momento do dia, o período experimental e os indivíduos influenciaram a distribuição nictemeral das probabilidades de transição de estado.

Abstract Background Bacillus thuringiensis var. israelensis (Bti) is a natural larval mosquito pathogen producing pore-forming toxins targeting the midgut of Diptera larvae. It is used worldwide for mosquito control. Resistance mechanisms of an Aedes aegypti laboratory strain selected for 30 generations with field-collected leaf litter containing Bti toxins were investigated in larval midguts at two levels: 1. gene transcription using DNA microarray and RT-qPCR and 2. differential expression of brush border membrane proteins using DIGE (Differential In Gel Electrophoresis). Results Several Bti Cry toxin receptors including alkaline phosphatases and N-aminopeptidases and toxin-binding V-ATPases exhibited altered expression levels in the resistant strain. The under-expression of putative Bti-receptors is consistent with Bt-resistance mechanisms previously described in Lepidoptera. Four soluble metalloproteinases were found under-transcribed together with a drastic decrease of metalloproteinases activity in the resistant strain, suggesting a role in resistance by decreasing the amount of activated Cry toxins in the larval midgut. Conclusions By combining transcriptomic and proteomic approaches, we detected expression changes at nearly each step of the ingestion-to-infection process, providing a short list of genes and proteins potentially involved in Bti-resistance whose implication needs to be validated. Collectively, these results open the way to further functional analyses to better characterize Bti-resistance mechanisms in mosquitoes.

Abstract Background AFLP markers are widely used in evolutionary genetics and ecology. However the frequent occurrence of non-homologous co-migrating fragments (homoplasy) both at the intra- and inter-individual levels in AFLP data sets is known to skew key parameters in population genetics. Geneticists can take advantage of the growing number of full genome sequences available for model species to study AFLP homoplasy and to predict it in non-model species. Results In this study we performed in silico AFLPs on the complete genome of three model species to predict intra-individual homoplasy in a prokaryote (Bacillus thuringiensis ser. konkukian), a plant (Arabidopsis thaliana) and an animal (Aedes aegypti). In addition, we compared in silico AFLPs to empirical data obtained from three related non-model species (Bacillus thuringiensis ser. israelensis, Arabis alpina and Aedes rusticus). Our results show that homoplasy rate sharply increases with the number of peaks per profile. However, for a given number of peaks per profile, genome size or taxonomical range had no effect on homoplasy. Furthermore, the number of co-migrating fragments in a single peak was dependent on the genome richness in repetitive sequences: we found up to 582 co-migrating fragments in Ae. aegypti. Finally, we show that in silico AFLPs can help to accurately predict AFLP profiles in related non-model species. Conclusions These predictions can be used to tackle current issues in the planning of AFLP studies by limiting homoplasy rate and population genetic estimation bias. ISIF (In SIlico Fingerprinting) program is freely available at http://www-leca.ujf-grenoble.fr/logiciels.htm.

Abstract Background The evolutionary success of phytophagous insects could result from their adaptation to different host-plants. Alternatively, the diversification of widespread species might be driven by adaptation along environmental gradients. To disentangle the respective roles of host-plant versus abiotic environmental variables acting on the genome of an oligophagous insect, we performed a genome scan using 83 unlinked AFLP markers on larvae of the large pine weevil collected on two host-plants (pine and spruce) in four forestry regions across Europe. Results At this large geographic scale, the global genetic differentiation was low and there was no isolation by distance pattern, suggesting that migration is overwhelming genetic drift in this species. In this context, the widely used frequentist methods to detect outliers (e.g. Dfdist), which assume migration - drift equilibrium are not the most appropriate approach. The implementation of a recently developed Bayesian approach, conceived to detect outliers even in non-equilibrium situations, consistently detected 9 out of 83 loci as outliers. Eight of these were validated as outliers by multiple logistic regressions: six correlated with environmental variables, one with host-plant and one with the interaction between environmental variables and host-plant. Conclusion These results suggest a relatively greater importance of abiotic environmental variables, as opposed to factors linked with the host-plant, in shaping genetic differentiation across the genome in this species. Logistic regression allows the nature of factors involved in locus-specific selection to be precisely identified and represents another step forward in the process of identifying adaptive loci.

Abstract Background Mutualisms are inherently conflictual as one partner always benefits from reducing the costs imposed by the other. Despite the widespread recognition that mutualisms are essentially reciprocal exploitation, there are few documented examples of traits that limit the costs of mutualism. In plant/seed-eating pollinator interactions the only mechanisms reported so far are those specific to one particular system, such as the selective abortion of over-exploited fruits. Results This study shows that plant chemical defence against developing larvae constitutes another partner sanction mechanism in nursery mutualisms. It documents the chemical defence used by globeflower Trollius europaeus L. (Ranunculaceae) against the seed-eating larvae of six pollinating species of the genus Chiastocheta Pokorny (Anthomyiidae). The correlative field study carried out shows that the severity of damage caused by Chiastocheta larvae to globeflower fruits is linked to the accumulation in the carpel walls of a C-glycosyl-flavone (adonivernith), which reduces the larval seed predation ability per damaged carpel. The different Chiastocheta species do not exploit the fruit in the same way and their interaction with the plant chemical defence is variable, both in terms of induction intensity and larval sensitivity to adonivernith. Conclusion Adonivernith accumulation and larval predation intensity appear to be both the reciprocal cause and effect. Adonivernith not only constitutes an effective chemical means of partner control, but may also play a key role in the sympatric diversification of the Chiastocheta genus.

Abstract Background Genome scans are becoming an increasingly popular approach to study the genetic basis of adaptation and speciation, but on their own, they are often helpless at identifying the specific gene(s) or mutation(s) targeted by selection. This shortcoming is hopefully bound to disappear in the near future, thanks to the wealth of new genomic resources that are currently being developed for many species. In this article, we provide a foretaste of this exciting new era by conducting a genome scan in the mosquito Aedes aegypti with the aim to look for candidate genes involved in resistance to Bacillus thuringiensis subsp. israelensis (Bti) insecticidal toxins. Results The genome of a Bti-resistant and a Bti-susceptible strains was surveyed using about 500 MITE-based molecular markers, and the loci showing the highest inter-strain genetic differentiation were sequenced and mapped on the Aedes aegypti genome sequence. Several good candidate genes for Bti-resistance were identified in the vicinity of these highly differentiated markers. Two of them, coding for a cadherin and a leucine aminopeptidase, were further examined at the sequence and gene expression levels. In the resistant strain, the cadherin gene displayed patterns of nucleotide polymorphisms consistent with the action of positive selection (e.g. an excess of high compared to intermediate frequency mutations), as well as a significant under-expression compared to the susceptible strain. Conclusion Both sequence and gene expression analyses agree to suggest a role for positive selection in the evolution of this cadherin gene in the resistant strain. However, it is unlikely that resistance to Bti is conferred by this gene alone, and further investigation will be needed to characterize other genes significantly associated with Bti resistance in Ae. aegypti. Beyond these results, this article illustrates how genome scans can build on the body of new genomic information (here, full genome sequence and MITE characterization) to finally hold their promises and help pinpoint candidate genes for adaptation and speciation.

Abstract Background For most organisms, developing hundreds of genetic markers spanning the whole genome still requires excessive if not unrealistic efforts. In this context, there is an obvious need for methodologies allowing the low-cost, fast and high-throughput genotyping of virtually any species, such as the Diversity Arrays Technology (DArT). One of the crucial steps of the DArT technique is the genome complexity reduction, which allows obtaining a genomic representation characteristic of the studied DNA sample and necessary for subsequent genotyping. In this article, using the mosquito Aedes aegypti as a study model, we describe a new genome complexity reduction method taking advantage of the abundance of miniature inverted repeat transposable elements (MITEs) in the genome of this species. Results Ae. aegypti genomic representations were produced following a two-step procedure: (1) restriction digestion of the genomic DNA and simultaneous ligation of a specific adaptor to compatible ends, and (2) amplification of restriction fragments containing a particular MITE element called Pony using two primers, one annealing to the adaptor sequence and one annealing to a conserved sequence motif of the Pony element. Using this protocol, we constructed a library comprising more than 6,000 DArT clones, of which at least 5.70% were highly reliable polymorphic markers for two closely related mosquito strains separated by only a few generations of artificial selection. Within this dataset, linkage disequilibrium was low, and marker redundancy was evaluated at 2.86% only. Most of the detected genetic variability was observed between the two studied mosquito strains, but individuals of the same strain could still be clearly distinguished. Conclusion The new complexity reduction method was particularly efficient to reveal genetic polymorphisms in Ae. egypti. Overall, our results testify of the flexibility of the DArT genotyping technique and open new prospects as regards its application to a wider range of species, including animals which have been refractory to it so far. DArT has also a role to play in the current burst of whole-genome scans carried out in various organisms, which track signatures of selection in order to unravel the basis of genetic adaptation.

Phylogenetic inference is challenged by genealogical heterogeneity amongst molecular markers. Such discordance is driven predominantly by incomplete lineage sorting (ILS) and interspecific gene flow, and bears attendant consequences for the accurate resolution of species relationships. Understanding the distribution of gene conflict in organismal genomes is, therefore, a key aspect of phylogenetic analysis. In this study, three large phylogenomic datasets (i.e., whole mitogenomes, conserved nuclear protein-coding loci, and genomic windows) are used to probe the extent to which discordance pervades the unresolved phylogeny of Coenonympha (Nymphalidae) butterflies. Gene tree discordance is found to be elevated at multiple historically recalcitrant phylogenetic positions. In particular, species relationships near the crown of Coenonympha and within a rapidly diversifying subclade (the hero group) remain difficult to resolve, suggesting that ILS and gene flow have obscured the evolution of this genus. These findings have implications for the taxonomy of this butterfly group and the study of its diversification history. In addition, this work lends support to a growing body of evidence that gene conflict driven by biological processes stands to confound phylogeny, even when extensive data are used., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

We present the first chromosome-level genome assembly and annotation of the pearly heath Coenonympha arcania, generated with a PacBio HiFi sequencing approach and complemented with Hi-C data. We additionally compare synteny, gene, and repeat content between C. arcania and other Lepidopteran genomes. This reference genome will enable future population genomics studies with Coenonympha butterflies, a species-rich genus that encompasses some of the most highly endangered butterfly taxa in Europe., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Bacillus thuringiensis subsp. israelensis (Bti) is the main larvicide used to control mosquitoes worldwide. Although there is accumulating evidence of Bti having environmental effects on non-target fauna, relatively few field studies have documented the fate of Bti spores in the environment. Spore density was quantified over a 6-yr period (2012-2017) in Mediterranean marshes sprayed with Vectobac 12AS (32 ITU/ha) since 2006 to reduce the nuisance caused by Aedes caspius. Bti spores were naturally found in all habitat types. Spore density expressed as colony-forming units per gram of soil (CFU g -1 ) increased significantly at treated sites by a factor of 22 to 500 times relative to control sites, with mean values of 7730 CFU g -1 in halophilous scrubs, 38,000 in reed beds, 49,000 in bulrush beds and 50 000 in rush beds. Spore density varied little in the first months after the spraying season (April-October), but increased sharply in spring, just before the annual launch of mosquito control. Considering that Bti is an insect pathogen that cannot proliferate without a suitable insect host, this unexpected recrudescence in spring could be related to the warming of water that triggers activity and development of benthic organisms such as chironomids, which may contribute to Bti proliferation by ingesting accumulated spores at the surface of sediments. While spore density tends to decrease over time, presumably during the summer period as a result of increased UV exposure, three to four years were necessary for spore density to return to normal levels after mosquito-control interruption. This study is important because it demonstrates that environmental effects of mosquito-control using Bti can far exceed the short period of Bti efficacy against lentic mosquitoes. Considering that Bti is a microbial agent, these long-term effects should be addressed at multiple levels of ecosystem organization from a one-health perspective., Competing Interests: Declaration of Competing Interest The authors declare that they have not been employed by, performed work for, or received funding from a company or organization whose operations relate to the commercialization or use of Bti or any other pesticide. Conflict of interest The authors declare that they have not been employed by, performed work for, or received funding from a company or organization whose operations relate to the commercialization or use of Bti or any other pesticide., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control., (© 2022. The Author(s).)

Climatic variation is a key driver of genetic differentiation and phenotypic traits evolution, and local adaptation to temperature is expected in widespread species. We investigated phenotypic and genomic changes in the native range of the Asian tiger mosquito, Aedes albopictus. We first refine the phylogeographic structure based on genome-wide regions (1,901 double-digest restriction-site associated DNA single nucleotide polymophisms [ddRAD SNPs]) from 41 populations. We then explore the patterns of cold adaptation using phenotypic traits measured in common garden (wing size and cold tolerance) and genotype-temperature associations at targeted candidate regions (51,706 exon-capture SNPs) from nine populations. We confirm the existence of three evolutionary lineages including clades A (Malaysia, Thailand, Cambodia, and Laos), B (China and Okinawa), and C (South Korea and Japan). We identified temperature-associated differentiation in 15 out of 221 candidate regions but none in ddRAD regions, supporting the role of directional selection in detected genes. These include genes involved in lipid metabolism and a circadian clock gene. Most outlier SNPs are differently fixed between clades A and C, whereas clade B has an intermediate pattern. Females are larger at higher latitude yet produce no more eggs, which might favor the storage of energetic reserves in colder climate. Nondiapausing eggs from temperate populations survive better to cold exposure than those from tropical populations, suggesting they are protected from freezing damages but this cold tolerance has a fitness cost in terms of egg viability. Altogether, our results provide strong evidence for the thermal adaptation of A. albopictus across its wide temperature range., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Cold-adapted species escape climate warming by latitudinal and/or altitudinal range shifts, and currently occur in Southern Europe in isolated mountain ranges within "sky islands". Here, we studied the genetic structure of the Apollo butterfly in five such sky islands (above 1,000 m) in France, and infer its demographic history since the last interglacial, using single nucleotide polymorphisms (ddRADseq SNPs). The Auvergne and Alps populations show strong genetic differentiation but not alpine massifs, although separated by deep valleys. Combining three complementary demographic inference methods and species distribution models (SDMs) we show that the LIG period was highly unfavourable for Apollo that probably survived in small population in the highest summits of Auvergne. The population shifted downslope and expanded eastward between LIG and LGM throughout the large climatically suitable Rhône valley between the glaciated summits of Auvergne and Alps. The Auvergne and Alps populations started diverging before the LGM but remained largely connected till the mid-Holocene. Population decline in Auvergne was more gradual but started before (~7 kya vs. 800 ya), and was much stronger with current population size ten times lower than in the Alps. In the Alps, the low genetic structure and limited evidence for isolation by distance suggest a nonequilibrium metapopulation functioning. The core Apollo population experienced cycles of contraction-expansion with climate fluctuations with largely interconnected populations overtime according to a "metapopulation-pulsar" functioning. This study demonstrates the power of combining demographic inferences and SDMs to determine past and future evolutionary trajectories of an endangered species at a regional scale., (© 2021 John Wiley & Sons Ltd.)

Biological invasions, the establishment and spread of non-native species in new regions, can have extensive economic and environmental consequences. Increased global connectivity accelerates introduction rates, while climate and land-cover changes may decrease the barriers to invasive populations spread. A detailed knowledge of the invasion history, including assessing source populations, routes of spread, number of independent introductions, and the effects of genetic bottlenecks and admixture on the establishment success, adaptive potential, and further spread, is crucial from an applied perspective to mitigate socioeconomic impacts of invasive species, as well as for addressing fundamental questions on the evolutionary dynamics of the invasion process. Recent advances in genomics together with the development of geographic information systems provide unprecedented large genetic and environmental datasets at global and local scales to link population genomics, landscape ecology, and species distribution modeling into a common framework to study the invasion process. Although the factors underlying population invasiveness have been extensively reviewed, analytical methods currently available to optimally combine molecular and environmental data for inferring invasive population demographic parameters and predicting further spreading are still under development. In this review, we focus on the few recent insect invasion studies that combine different datasets and approaches to show how integrating genetic, observational, ecological, and environmental data pave the way to a more integrative biological invasion science. We provide guidelines to study the evolutionary dynamics of invasions at each step of the invasion process, and conclude on the benefits of including all types of information and up-to-date analytical tools from different research areas into a single framework., Competing Interests: The authors declare that they have no conflict of interest., (© 2021 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

Environmental features impacting the spread of invasive species after introduction can be assessed using population genetic structure as a quantitative estimation of effective dispersal at the landscape scale. However, in the case of an ongoing biological invasion, deciphering whether genetic structure represents landscape connectivity or founder effects is particularly challenging. We examined the modes of dispersal (natural and human-aided) and the factors (landscape or founders history) shaping genetic structure in range edge invasive populations of the Asian tiger mosquito, Aedes albopictus, in the region of Grenoble (Southeast France). Based on detailed occupancy-detection data and environmental variables (climatic, topographic and land-cover), we modelled A. albopictus potential suitable area and its expansion history since first introduction. The relative role of dispersal modes was estimated using biological dispersal capabilities and landscape genetics approaches using genome-wide SNP dataset. We demonstrate that both natural and human-aided dispersal have promoted the expansion of populations. Populations in diffuse urban areas, representing highly suitable habitat for A. albopictus, tend to disperse less, while roads facilitate long-distance dispersal. Yet, demographic bottlenecks during introduction played a major role in shaping the genetic variability of these range edge populations. The present study is one of the few investigating the role of founder effects and ongoing expansion processes in shaping spatial patterns of genetic variation in an invasive species at the landscape scale. The combination of several dispersal modes and large proportions of continuous suitable habitats for A. albopictus promoted range filling of almost its entire potential distribution in the region of Grenoble only few years after introduction., (© 2020 British Ecological Society.)

Bacillus thuringiensis subsp. israelensis (Bti) has been used in mosquito control programs to reduce nuisance in Europe for decades and is generally considered an environmentally-safe, effective and target-specific biocide. However, the use of Bti is not uncontroversial. Target mosquitoes and affected midges represent an important food source for many aquatic and terrestrial predators and reduction of their populations is likely to result in food-web effects at higher trophic levels. In the context of global biodiversity loss, this appears particularly critical since treated wetlands are often representing conservation areas. In this review, we address the current large-scale use of Bti for mosquito nuisance control in Europe, provide a description of its regulation followed by an overview of the available evidence on the parameters that are essential to evaluate Bti use in mosquito control. Bti accumulation and toxin persistence could result in a chronic expose of mosquito populations ultimately affecting their susceptibility, although observed increase in resistance to Bti in mosquito populations is low due to the four toxins involved. A careful independent monitoring of mosquito susceptibility, using sensitive bioassays, is mandatory to detect resistance development timely. Direct Bti effects were documented for non-target chironomids and other invertebrate groups and are discussed for amphibians. Field studies revealed contrasting results on possible impacts on chironomid abundances. Indirect, food-web effects were rarely studied in the environment. Depending on study design and duration, Bti effects on higher trophic levels were demonstrated or not. Further long-term field studies are needed, especially with observations of bird declines in Bti-treated wetland areas. Socio-economic relevance of mosquito control requires considering nuisance, vector-borne diseases and environmental effects jointly. Existing studies indicate that a majority of the population is concerned regarding potential environmental effects of Bti mosquito control and that they are willing to pay for alternative, more environment-friendly techniques., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Cyt1Aa is the one of four crystalline protoxins produced by mosquitocidal bacterium Bacillus thuringiensis israelensis (Bti) that has been shown to delay the evolution of insect resistance in the field. Limiting our understanding of Bti efficacy and the path to improved toxicity and spectrum has been ignorance of how Cyt1Aa crystallizes in vivo and of its mechanism of toxicity. Here, we use serial femtosecond crystallography to determine the Cyt1Aa protoxin structure from sub-micron-sized crystals produced in Bti. Structures determined under various pH/redox conditions illuminate the role played by previously uncharacterized disulfide-bridge and domain-swapped interfaces from crystal formation in Bti to dissolution in the larval mosquito midgut. Biochemical, toxicological and biophysical methods enable the deconvolution of key steps in the Cyt1Aa bioactivation cascade. We additionally show that the size, shape, production yield, pH sensitivity and toxicity of Cyt1Aa crystals grown in Bti can be controlled by single atom substitution.

Identifying the mechanisms involved in the formation and maintenance of species is a central question in evolutionary biology, and distinguishing the selective drivers of populations' divergence from demographic processes is of particular interest to better understand the speciation process. Hybrid zones are recognized to provide ideal places to investigate the genetic architecture of speciation and to identify the mechanisms allowing diverging species to maintain their integrity in the face of gene flow. Here, we studied two alpine butterfly species, Coenonympha macromma and C. gardetta, which can be found flying together and hybridizing in narrow contact zones in the southern French Alps. We characterized the genomic composition of individuals, their morphology and their local habitat requirements, within and around a hybrid zone. Genetic diversity analysis at 794 SNPs revealed that all individuals within the hybrid zone were highly admixed, which was not the case outside the hybrid zone. Cline analysis showed that, despite ongoing hybridization, 56 out of 122 loci differentially fixed or nearly so between the two species were impermeable to introgression across the sharp hybrid zone (9 km wide). We also found concordance in cline position and width among genetic, morphological and environmental variation, suggesting a coupling of different reproductive barriers. Habitat characteristics such as the presence of trees and shrubs and the start of the growing season were strongly associated with the genetic variation, and we found evidence of divergence at genetic markers associated with morphology and physiology, putatively involved in visual or environmental reproductive isolation. We discuss the various behavioural and ecological factors that might interplay to maintain current levels of divergence and gene flow between this species pair., Competing Interests: None declared., (© 2020 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

Invasive species can encounter environments different from their source populations, which may trigger rapid adaptive changes after introduction (niche shift hypothesis). To test this hypothesis, we investigated whether postintroduction evolution is correlated with contrasting environmental conditions between the European invasive and source ranges in the Asian tiger mosquito Aedes albopictus . The comparison of environmental niches occupied in European and source population ranges revealed more than 96% overlap between invasive and source niches, supporting niche conservatism. However, we found evidence for postintroduction genetic evolution by reanalyzing a published ddRADseq genomic dataset from 90 European invasive populations using genotype-environment association (GEA) methods and generalized dissimilarity modeling (GDM). Three loci, among which a putative heat-shock protein, exhibited significant allelic turnover along the gradient of winter precipitation that could be associated with ongoing range expansion. Wing morphometric traits weakly correlated with environmental gradients within Europe, but wing size differed between invasive and source populations located in different climatic areas. Niche similarities between source and invasive ranges might have facilitated the establishment of populations. Nonetheless, we found evidence for environmental-induced adaptive changes after introduction. The ability to rapidly evolve observed in invasive populations (genetic shift) together with a large proportion of unfilled potential suitable areas (80%) pave the way to further spread of Ae. albopictus in Europe., Competing Interests: None declared., (© 2019 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Adaptation to environmental conditions within the native range of exotic species can condition the invasion success of these species outside their range. The striking success of the Asian tiger mosquito, Aedes albopictus, to invade temperate regions has been attributed to the winter survival of diapause eggs in cold environments. In this study, we evaluate genetic polymorphisms (SNPs) and wing morphometric variation among three biogeographical regions of the native range of A. albopictus. Reconstructed demographic histories of populations show an initial expansion in Southeast Asia and suggest that marine regression during late Pleistocene and climate warming after the last glacial period favored expansion of populations in southern and northern regions, respectively. Searching for genomic signatures of selection, we identified significantly differentiated SNPs among which several are located in or within 20 kb distance from candidate genes for cold adaptation. These genes involve cellular and metabolic processes and several of them have been shown to be differentially expressed under diapausing conditions. The three biogeographical regions also differ for wing size and shape, and wing size increases with latitude supporting Bergmann's rule. Adaptive genetic and morphometric variation observed along the climatic gradient of A. albopictus native range suggests that colonization of northern latitudes promoted adaptation to cold environments prior to its worldwide invasion., (© 2019 The Author(s). Evolution © 2019 The Society for the Study of Evolution.)

Purpose: To compare bacteria recovered by standard cultures and metataxonomics, particularly with regard to ventilator-associated pneumonia (VAP) pathogens, and to determine if the presence of particular bacteria or microbiota in tracheal and oropharyngeal secretions during the course of intubation was associated with the development of VAP., Methods: In this case-control study, oropharyngeal secretions and endotracheal aspirate were collected daily in mechanically ventilated patients. Culture and metataxonomics (16S rRNA gene-based taxonomic profiling of bacterial communities) were performed on serial upper respiratory samples from patients with late-onset definite VAP and their respective controls., Results: Metataxonomic analyses showed that a low relative abundance of Bacilli at the time of intubation in the oropharyngeal secretions was strongly associated with the subsequent development of VAP. On the day of VAP, the quantity of human and bacterial DNA in both tracheal and oropharyngeal secretions was significantly higher in patients with VAP than in matched controls with similar ventilation times. Molecular techniques identified the pathogen(s) of VAP found by culture, but also many more bacteria, classically difficult to culture, such as Mycoplasma spp. and anaerobes., Conclusions: Molecular analyses of respiratory specimens identified markers associated with the development of VAP, as well as important differences in the taxa abundance between VAP and controls. Further prospective trials are needed to test the predictive value of these markers, as well as the relevance of uncultured bacteria in the pathogenesis of VAP.

Until complete reproductive isolation is achieved, the extent of differentiation between two diverging lineages is the result of a dynamic equilibrium between genetic isolation and mixing. This is especially true for hybrid taxa, for which the degree of isolation in regard to their parental species is decisive in their capacity to rise as a new and stable entity. In this work, we explored the past and current patterns of hybridization and divergence within a complex of closely related butterflies in the genus Coenonympha in which two alpine species, C. darwiniana and C. macromma , have been shown to result from hybridization between the also alpine C. gardetta and the lowland C. arcania . By testing alternative scenarios of divergence among species, we show that gene flow has been uninterrupted throughout the speciation process, although leading to different degrees of current genetic isolation between species in contact zones depending on the pair considered. Nonetheless, at broader geographic scale, analyses reveal a clear genetic differentiation between hybrid lineages and their parental species, pointing out to an advanced stage of the hybrid speciation process. Finally, the positive correlation observed between ecological divergence and genetic isolation among these butterflies suggests a potential role for ecological drivers during their speciation processes., Competing Interests: None declared.

Multiple introductions are key features for the establishment and persistence of introduced species. However, little is known about the contribution of genetic admixture to the invasive potential of populations. To address this issue, we studied the recent invasion of the Asian tiger mosquito (Aedes albopictus) in Europe. Combining genome-wide single nucleotide polymorphisms and historical knowledge using an approximate Bayesian computation framework, we reconstruct the colonization routes and establish the demographic dynamics of invasion. The colonization of Europe involved at least three independent introductions in Albania, North Italy and Central Italy that subsequently acted as dispersal centres throughout Europe. We show that the topology of human transportation networks shaped demographic histories with North Italy and Central Italy being the main dispersal centres in Europe. Introduction modalities conditioned the levels of genetic diversity in invading populations, and genetically diverse and admixed populations promoted more secondary introductions and have spread farther than single-source invasions. This genomic study provides further crucial insights into a general understanding of the role of genetic diversity promoted by modern trade in driving biological invasions., (© 2019 John Wiley & Sons Ltd.)

Understanding the interactions between pathogens sharing the same host can be complicated for holometabolous animals when larval and adult stages are exposed to distinct pathogens. In medically important insect vectors, the effect of pathogen exposure at the larval stage may influence susceptibility to human pathogens at the adult stage. We addressed this hypothesis in the mosquito Aedes aegypti , a major vector of arthropod-borne viruses (arboviruses), such as the dengue virus (DENV) and the chikungunya virus (CHIKV). We experimentally assessed the consequences of sub-lethal exposure to the bacterial pathogen Bacillus thuringiensis subsp. israelensis ( Bti ), during larval development, on arbovirus susceptibility at the adult stage in three Ae. aegypti strains that differ in their genetic resistance to Bti . We found that larval exposure to Bti significantly increased DENV susceptibility, but not CHIKV susceptibility, in the Bti -resistant strains. However, there was no major difference in the baseline arbovirus susceptibility between the Bti -resistant strains and their Bti -susceptible parental strain. Although the generality of our results remains to be tested with additional arbovirus strains, this study supports the idea that the outcome of an infection by a pathogen depends on other pathogens sharing the same host even when they do not affect the same life stage of the host. Our findings may also have implications for Bti as a mosquito biocontrol agent, indicating that the sub-optimal Bti efficacy may have counter-productive effects by increasing vector competence, at least for some combinations of arbovirus and mosquito strains.

Background: Insect microbiota is a dynamic microbial community that can actively participate in defense against pathogens. Bacillus thuringiensis (Bt) is a natural entomopathogen widely used as a bioinsecticide for pest control. Although Bt's mode of action has been extensively studied, whether the presence of microbiota is mandatory for Bt to effectively kill the insect is still under debate. An association between a higher tolerance and a modified microbiota was already evidenced but a critical point remained to be solved: is the modified microbiota a cause or a consequence of a higher tolerance to Bt?, Methods: In this study we focused on the mosquito species Aedes aegypti, as no work has been performed on Diptera on this topic to date, and on B. thuringiensis israelensis (Bti), which is used worldwide for mosquito control. To avoid using antibiotics to cure bacterial microbiota, mosquito larvae were exposed to an hourly increasing dose of Bti during 25 hours to separate the most susceptible larvae dying quickly from more tolerant individuals, with longer survival., Results: Denaturing gradient gel electrophoresis (DGGE) fingerprinting revealed that mosquito larval bacterial microbiota was strongly affected by Bti infection after only a few hours of exposure. Bacterial microbiota from the most tolerant larvae showed the lowest diversity but the highest inter-individual differences. The proportion of Bti in the host tissue was reduced in the most tolerant larvae as compared to the most susceptible ones, suggesting an active control of Bti infection by the host., Conclusions: Here we show that a modified microbiota is associated with a higher tolerance of mosquitoes to Bti, but that it is rather a consequence of Bti infection than the cause of the higher tolerance. This study paves the way to future investigations aiming at unraveling the role of host immunity, inter-species bacterial competition and kinetics of host colonization by Bti that could be at the basis of the phenotype observed in this study.

The Asian tiger mosquito Aedes albopictus, native to South-eastern Asia, is currently the most invasive mosquito in the world. The spatio-temporal dynamics of its expansion through the genetic characterization of invasive populations has been challenged so far by the limited number of genetic markers variable enough to infer the genetic structure in recently invaded areas. Here we applied the double-digest Restriction-site Associated DNA sequencing method (ddRADseq) to mosquitoes collected in two invaded areas, Reunion Island (12 localities) and Europe (18 localities). Analyses of genetic diversity, Bayesian clustering, Maximum Likelihood inference and isolation-by-distance tests based on 1561 genome-wide distributed Single Nucleotide Polymorphisms (SNPs) revealed that Reunion Island and Europe form two distinct genetic clusters, supporting no contemporary gene flow and suggesting two different and independent invasion histories. Long-established populations (Reunion Island) were more genetically diverse than recently introduced European populations. The largest part of genetic variance was found at the intra-individual level (>85%) and most F IS values were positive, suggesting inbreeding at the local scale. The two invaded areas showed contrasting patterns of genetic structure. Significant isolation-by-distance was found among Reunion Island populations, suggesting that these populations are at the drift-migration equilibrium. In contrast, long-distance human-assisted transport is probably the main dispersal mechanism in Europe., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Human-driven global environmental changes have considerably increased the risk of biological invasions, especially the spread of human parasites and their vectors. Among exotic species that have major impacts on public health, the dengue fever mosquito Aedes aegypti originating from Africa has spread worldwide during the last three centuries. Although considerable progress has been recently made in understanding the history of this invasion, the respective roles of human and abiotic factors in shaping patterns of genetic diversity remain largely unexplored. Using a genome-wide sample of genetic variants (3,530 ddRAD SNPs), we analyzed the genetic structure of Ae. aegypti populations in the Caribbean, the first introduced territories in the Americas. Fourteen populations were sampled in Guyane and in four islands of the Antilles that differ in climatic conditions, intensity of urbanization, and vector control history. The genetic diversity in the Caribbean was low (He = 0.14-0.17), as compared with a single African collection from Benin (He = 0.26) and site-frequency spectrum analysis detected an ancient bottleneck dating back ∼300 years ago, supporting a founder event during the introduction of Ae. aegypti. Evidence for a more recent bottleneck may be related to the eradication program undertaken on the American continent in the 1950s. Among 12 loci detected as FST-outliers, two were located in candidate genes for insecticide resistance (cytochrome P450 and voltage-gated sodium channel). Genome-environment association tests identified additional loci associated with human density and/or deltamethrin resistance. Our results highlight the high impact of human pressures on the demographic history and genetic variation of Ae. aegypti Caribbean populations., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

We examined how butterfly species richness is affected by human impact and elevation, and how species ranges are distributed along the elevational gradient (200-2700 m) in the Isère Department (French Alps). A total of 35,724 butterfly observations gathered in summer (May-September) between 1995 and 2015 were analyzed. The number of estimated species per 100-m elevational band was fitted to the elevational gradient using a generalized additive model. Estimations were also performed on a 500 m × 500 m grid at low altitude (200-500 m) to test for the human impact on species richness using generalized least squares regression models. Each species elevational range was plotted against the elevational gradient. Butterfly richness along the elevational gradient first increased (200-500 m) to reach a maximum of 150 species at 700 m and then remained nearly constant till a sharp decrease after 1900 m, suggesting that after some temperature threshold, only few specialized species can survive. At low elevation, urbanization and arable lands had a strongly negative impact on butterfly diversity, which was buffered by a positive effect of permanent crops. Butterfly diversity is exceptionally high (185 species) in this alpine department that represents less than 5% of the French territory and yet holds more than 70% of all the Rhopalocera species recorded in France. Both climate and habitat shape the distribution of species, with a negative effect of anthropization at low altitude and strong climatic constraints at high altitude.

Bacillus thuringiensis israelensis (Bti) toxins are increasingly used for mosquito control, but little is known about the precise mode of action of each of these toxins, and how they interact to kill mosquito larvae. By using RNA sequencing, we investigated change in gene transcription level and polymorphism variations associated with resistance to each Bti Cry toxin and to the full Bti toxin mixture in the dengue vector Aedes aegypti. The up-regulation of genes related to chitin metabolism in all selected strain suggests a generalist, non-toxin-specific response to Bti selection in Aedes aegypti. Changes in the transcription level and/or protein sequences of several putative Cry toxin receptors (APNs, ALPs, α-amylases, glucoside hydrolases, ABC transporters) were specific to each Cry toxin. Selective sweeps associated with Cry4Aa resistance were detected in 2 ALP and 1 APN genes. The lack of selection of toxin-specific receptors in the Bti-selected strain supports the hypothesis that Cyt toxin acts as a receptor for Cry toxins in mosquitoes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Bacillus thuringiensis subsp. israelensis (Bti) is a natural pathogen of dipterans widely used as a biological insecticide for mosquito control. To characterize the response of mosquitoes to intoxication with Bti, the transcriptome profile of Bti-exposed susceptible Aedes aegypti larvae was analysed using Illumina RNA-seq. Gene expression of 11 alkaline phosphatases (ALPs) was further investigated by real time quantitative polymerase chain reaction and ALP activity was measured in the susceptible strain and in four strains resistant to a single Bti Cry toxin or to Bti. These strains were unexposed or exposed to their toxin of selection. Although all resistant strains constitutively exhibited a higher level of transcription of ALP genes than the susceptible strain, they showed a lower total ALP activity. The intoxication with different individual Cry toxins triggered a global pattern of ALP gene under-transcription in all the one-toxin-resistant strains but involving different specific sets of ALPs in each resistant phenotype. Most of the ALPs involved are not known Cry-binding proteins. RNA interference experiment demonstrated that reducing ALP expression conferred increased the survival of larvae exposed to Cry4Aa, confirming the involvement of ALP in Cry4Aa toxicity., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Hybridization has become a central element in theories of animal evolution during the last decade. New methods in population genomics and statistical model testing now allow the disentangling of the complexity that hybridization brings into key evolutionary processes such as local adaptation, colonization of new environments, species diversification and extinction. We evaluated the consequences of hybridization in a complex of three alpine butterflies in the genus Coenonympha, by combining morphological, genetic and ecological analyses. A series of approximate Bayesian computation procedures based on a large SNP data set strongly suggest that the Darwin's Heath (Coenonympha darwiniana) originated through hybridization between the Pearly Heath (Coenonympha arcania) and the Alpine Heath (Coenonympha gardetta) with different parental contributions. As a result of hybridization, the Darwin's Heath presents an intermediate morphology between the parental species, while its climatic niche seems more similar to the Alpine Heath. Our results also reveal a substantial genetic and morphologic differentiation between the two geographically disjoint Darwin's Heath lineages leading us to propose the splitting of this taxon into two different species., (© 2015 John Wiley & Sons Ltd.)

The bacterial insecticide Bacillus thuringiensis subsp. israelensis (Bti) is an increasingly popular alternative to chemical insecticides for controlling mosquito populations. Because Bti toxicity relies on the action of four main toxins, resistance to Bti is very likely a complex phenotype involving several genes simultaneously. Dissecting the underlying genetic basis thus requires associating a quantitative measure of resistance to genetic variation at many loci in a segregating population. Here, we undertake this task using the dengue and yellow fever vector, the mosquito Aedes aegypti, as a study model. We conducted QTL (Quantitative Trait Locus) and admixture mapping analyses on two controlled crosses and on an artificial admixed population, respectively, all obtained from resistant and susceptible lab strains. We detected 16 QTL regions, among which four QTLs were revealed by different analysis methods. These four robust QTLs explained altogether 29.2% and 62.2% of the total phenotypic variance in the two QTL crosses, respectively. They also all showed a dominant mode of action. In addition, we found six loci showing statistical association with Bti resistance in the admixed population. Five of the supercontigs highlighted in this study contained candidate genes as suggested by their function, or by prior evidence from expression and/or outlier analyses. These genomic regions are thus good starting points for fine mapping of resistance to Bti or functional analyses aiming at identifying the underlying genes and mutations. Moreover, for the purpose of this work, we built the first Ae. aegypti genetic map based on markers associated with genes expressed in larvae. This genetic map harbors 229 SNP markers mapped across the three chromosomes for a total length of 311.9cM. It brought to light several assembly discrepancies with the reference genome, suggesting a high level of genome plasticity in Ae. aegypti., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Worldwide evolution of mosquito resistance to chemical insecticides represents a major challenge for public health, and the future of vector control largely relies on the development of biological insecticides that can be used in combination with chemicals (integrated management), with the expectation that populations already resistant to chemicals will not become readily resistant to biological insecticides. However, little is known about the metabolic pathways affected by selection with chemical or biological insecticides. Here we show that Aedes aegypti, a laboratory mosquito strain selected with a biological insecticide (Bacillus thuringiensis israelensis, Bti) evolved increased transcription of many genes coding for endopeptidases while most genes coding for detoxification enzymes were under-expressed. By contrast, in strains selected with chemicals, genes encoding detoxification enzymes were mostly over-expressed. In all the resistant strains, genes involved in immune response were under-transcribed, suggesting that basal immunity might be a general adjustment variable to compensate metabolic costs caused by insecticide selection. Bioassays generally showed no evidence for an increased susceptibility of selected strains towards the other insecticide type, and all chemical-resistant strains were as susceptible to Bti as the unselected parent strain, which is a good premise for sustainable integrated management of mosquito populations resistant to chemicals., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

Background: Despite the intensive use of Bacillus thuringiensis israelensis (Bti) toxins for mosquito control, little is known about the long term effect of exposure to this cocktail of toxins on target mosquito populations. In contrast to the many cases of resistance to Bacillus thuringiensis Cry toxins observed in other insects, there is no evidence so far for Bti resistance evolution in field mosquito populations. High fitness costs measured in a Bti selected mosquito laboratory strain suggest that evolving resistance to Bti is costly. The aim of the present study was to identify transcription level and polymorphism variations associated with resistance to Bti toxins in the dengue vector Aedes aegypti. We used RNA sequencing (RNA-seq) for comparing a laboratory-selected strain showing elevated resistance to Bti toxins and its parental non-selected susceptible strain. As the resistant strain displayed two marked larval development phenotypes (slow and normal), each phenotype was analyzed separately in order to evidence potential links between resistance mechanisms and mosquito life-history traits., Results: A total of 12,458 genes were detected of which 844 were differentially transcribed between the resistant and susceptible strains. Polymorphism analysis revealed a total of 68,541 SNPs of which 12,571 SNPs exhibited more than 40% frequency difference between the resistant and susceptible strains, affecting 2,953 genes. Bti resistance is associated with changes in the transcription level of enzymes involved in detoxification and chitin metabolism. Among previously described Bti-toxin receptors, four alkaline phosphatases (ALPs) were differentially transcribed between resistant and susceptible larvae, and non-synonymous changes affected the protein sequence of one cadherin, six aminopeptidases (APNs) and four α-amylases. Other putative Cry receptors located in lipid rafts, such as flotillin and glycoside hydrolases, were under-transcribed and/or contained non-synonymous substitutions. Finally, immunity-related genes showed contrasted transcription and polymorphisms patterns between the two developmental resistant phenotypes, suggesting the existence of trade-offs between Bti-resistance, life-history traits and immunity., Conclusions: The present study is the first to analyze the whole transcriptome of Bti-resistant mosquitoes by RNA-seq, shedding light on the importance of studying both transcription levels and sequence polymorphism variations to get a comprehensive view of insecticide resistance.

The bioinsecticide Bacillus thuringiensis subsp. israelensis is a larvicide used worldwide for mosquito control, which contains three Cry toxins and one Cyt toxin. We investigated for the first time in Aedes aegypti (1) the evolution of resistance and cross-resistance of strains selected with each Cry toxin, and (2) the effect of pre-selection with Cry toxin on the evolution of resistance to a mix of Bti toxins. Cross resistance was higher between Cry4Ba and Cry11Aa than between Cry4Aa and either Cry4Ba or Cry11Aa, suggesting both common and specific mechanisms of resistance. Pre-selecting resistance to each Cry toxins facilitated the development of resistance to the full Bti toxins cocktail., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Sprays of commercial preparations of the bacterium Bacillus thuringiensis subsp. israelensis are widely used for the control of mosquito larvae. Despite an abundant literature on B. thuringiensis subsp. israelensis field efficiency on mosquito control, few studies have evaluated the fate of spores in the environment after treatments. In the present article, two complementary experiments were conducted to study the effect of different parameters on B. thuringiensis subsp. israelensis persistence and recycling, in field conditions and in the laboratory. First, we monitored B. thuringiensis subsp. israelensis persistence in the field in two contrasting regions in France: the Rhône-Alpes region, where mosquito breeding sites are temporary ponds under forest cover with large amounts of decaying leaf matter on the ground and the Mediterranean region characterized by open breeding sites such as brackish marshes. Viable B. thuringiensis subsp. israelensis spores can persist for months after a treatment, and their quantity is explained both by the vegetation type and by the number of local treatments. We found no evidence of B. thuringiensis subsp. israelensis recycling in the field. Then, we tested the effect of water level, substrate type, salinity and presence of mosquito larvae on the persistence/recycling of B. thuringiensis subsp. israelensis spores in controlled laboratory conditions (microcosms). We found no effect of change in water level or salinity on B. thuringiensis subsp. israelensis persistence over time (75 days). B. thuringiensis subsp. israelensis spores tended to persist longer in substrates containing organic matter compared to sand-only substrates. B. thuringiensis subsp. israelensis recycling only occurred in presence of mosquito larvae but was unrelated to the presence of organic matter.

Mosquito control is often the main method used to reduce mosquito-transmitted diseases. In order to investigate the genetic basis of resistance to the bio-insecticide Bacillus thuringiensis subsp. israelensis (Bti), we used information on polymorphism obtained from cDNA tag sequences from pooled larvae of laboratory Bti-resistant and susceptible Aedes aegypti mosquito strains to identify and analyse 1520 single nucleotide polymorphisms (SNPs). Of the 372 SNPs tested, 99.2% were validated using DNA Illumina GoldenGate® array, with a strong correlation between the allelic frequencies inferred from the pooled and individual data (r = 0.85). A total of 11 genomic regions and five candidate genes were detected using a genome scan approach. One of these candidate genes showed significant departures from neutrality in the resistant strain at sequence level. Six natural populations from Martinique Island were sequenced for the 372 tested SNPs with a high transferability (87%), and association mapping analyses detected 14 loci associated with Bti resistance, including one located in a putative receptor for Cry11 toxins. Three of these loci were also significantly differentiated between the laboratory strains, suggesting that most of the genes associated with resistance might differ between the two environments. It also suggests that common selected regions might harbour key genes for Bti resistance.

Bacillus thuringiensis subsp. israelensis (Bti) is increasingly used worldwide for mosquito control and is the only larvicide used in the French Rhône-Alpes region since decades. The artificial selection of mosquitoes with field-persistent Bti collected in breeding sites from this region led to a moderate level of resistance to Bti, but to relatively high levels of resistance to individual Bti Cry toxins. Based on this observation, we developed a bioassay procedure using each Bti Cry toxin separately to detect cryptic Bti-resistance evolving in field mosquito populations. Although no resistance to Bti was detected in none of the three mosquito species tested (Aedes rusticus, Aedes sticticus and Aedes vexans), an increased tolerance to Cry4Aa (3.5-fold) and Cry11Aa toxins (8-fold) was found in one Ae. sticticus population compared to other populations of the same species, suggesting that resistance to Bti may be arising in this population. This study confirms previous works showing a lack of Bti resistance in field mosquito populations treated for decades with this bioinsecticide. It also provides a first panorama of their susceptibility status to individual Bti Cry toxins. In combination with bioassays with Bti, bioassays with separate Cry toxins allow a more sensitive monitoring of Bti-resistance in the field.

The bioinsecticide Bacillus thuringiensis var. israelensis (Bti) is increasingly used worldwide for mosquito control. Although no established resistance to Bti has been described in the field so far, a resistant Aedes aegypti strain (LiTOX strain) was selected in the laboratory using field-collected leaf litter containing Bti toxins. This selected strain exhibits a moderate resistance level to Bti, but a high resistance level to individual Cry toxins. As Bti contains four different toxins, generalist resistance mechanisms affecting mosquito tolerance to different toxins were expected in the resistant strain. In the present work, we show that the resistant strain exhibits an increase of various gut proteolytic activities including trypsins, leucine-aminopeptidases, and carboxypeptidase A activities. These elevated proteolytic activities resulted in a faster activation of Cry4Aa protoxins while Cry4Ba or Cry11Aa were not affected. These results suggest that changes in proteolytic activities may contribute to Bti resistance in mosquitoes together with other mechanisms., (© 2012 Wiley Periodicals, Inc.)