The impact of agrochemical pollutant mixtures on the selection of insecticide resistance in the malaria vector Anopheles gambiae: insights from experimental evolution and transcriptomics.

BACKGROUND: There are several indications that pesticides used in agriculture contribute to the emergence and spread of resistance of mosquitoes to vector control insecticides. However, the impact of such an indirect selection pressure has rarely been quantified and the molecular mechanisms involved are still poorly characterized. In this context, experimental selection with different agrochemical mixtures was conducted in Anopheles gambiae. The multi-generational impact of agrochemicals on insecticide resistance was evaluated by phenotypic and molecular approaches. METHODS: Mosquito larvae were selected for 30 generations with three different agrochemical mixtures containing (i) insecticides, (ii) non-insecticides compounds, and (iii) both insecticide and non-insecticide compounds. Every five generations, the resistance of adults to deltamethrin and bendiocarb was monitored using bioassays. The frequencies of the kdr (L995F) and ace1 (G119S) target-site mutations were monitored every 10 generations. RNAseq was performed on all lines at generation 30 in order to identify gene transcription level variations and polymorphisms associated with each selection regime. RESULTS: Larval selection with agrochemical mixtures did not affect bendiocarb resistance and did not select for ace1 mutation. Contrastingly, an increased deltamethrin resistance was observed in the three selected lines. Such increased resistance was not majorly associated with the presence of kdr L995F mutation in selected lines. RNA-seq identified 63 candidate resistance genes over-transcribed in at least one selected line. These include genes coding for detoxification enzymes or cuticular proteins previously associated with insecticide resistance, and other genes potentially associated with chemical stress response. Combining an allele frequency filtering with a Bayesian FST-based genome scan allowed to identify genes under selection across multiple genomic loci, supporting a multigenic adaptive response to agrochemical mixtures. CONCLUSION: This study supports the role of agrochemical contaminants as a significant larval selection pressure favouring insecticide resistance in malaria vectors. Such selection pressures likely impact kdr mutations and detoxification enzymes, but also more generalist mechanisms such as cuticle resistance, which could potentially lead to cross-tolerance to unrelated insecticide compounds. Such indirect effect of global landscape pollution on mosquito resistance to public health insecticides deserves further attention since it can affect the nature and dynamics of resistance alleles circulating in malaria vectors and impact the efficacy of control vector strategies.

Deltamethrin and transfluthrin select for distinct transcriptomic responses in the malaria vector Anopheles gambiae.

BACKGROUND: The widespread use of pyrethroid insecticides in Africa has led to the development of strong resistance in Anopheles mosquitoes. Introducing new active ingredients can contribute to overcome this phenomenon and ensure the effectiveness of vector control strategies. Transfluthrin is a polyfluorinated pyrethroid whose structural conformation was thought to prevent its metabolism by cytochrome P450 monooxygenases in malaria vectors, thus representing a potential alternative for managing P450-mediated resistance occurring in the field. In this study, a controlled selection was used to compare the dynamics of resistance between transfluthrin and the widely used pyrethroid deltamethrin in the mosquito Anopheles gambiae. Then, the associated molecular mechanisms were investigated using target-site mutation genotyping and RNA-seq. METHODS: A field-derived line of An. gambiae carrying resistance alleles at low frequencies was used as starting material for a controlled selection experiment. Adult females were selected across 33 generations with deltamethrin or transfluthrin, resulting in three distinct lines: the Delta-R line (selected with deltamethrin), the Transflu-R line (selected with transfluthrin) and the Tiassale-S line (maintained without selection). Deltamethrin and transfluthrin resistance levels were monitored in each selected line throughout the selection process, as well as the frequency of the L1014F kdr mutation. At generation 17, cross-resistance to other public health insecticides was investigated and transcriptomes were sequenced to compare gene transcription variations and polymorphisms associated with adaptation to each insecticide. RESULTS: A rapid increase in resistance to deltamethrin and transfluthrin was observed throughout the selection process in each selected line in association with an increased frequency of the L1014F kdr mutation. Transcriptomic data support a broader response to transfluthrin selection as compared to deltamethrin selection. For instance, multiple detoxification enzymes and cuticle proteins were specifically over-transcribed in the Transflu-R line including the known pyrethroid metabolizers CYP6M2, CYP9K1 and CYP6AA1 together with other genes previously associated with resistance in An. gambiae. CONCLUSION: This study confirms that recurrent exposure of adult mosquitoes to pyrethroids in a public health context can rapidly select for various resistance mechanisms. In particular, it indicates that in addition to target site mutations, the polyfluorinated pyrethroid transfluthrin can select for a broad metabolic response, which includes some P450s previously associated to resistance to classical pyrethroids. This unexpected finding highlights the need for an in-depth study on the adaptive response of mosquitoes to newly introduced active ingredients in order to effectively guide and support decision-making programmes in malaria control.

Emerging concepts and opportunities for endocrine disruptor screening of the non-EATS modalities.

Endocrine disrupting chemicals (EDCs) are ubiquitous in the environment and involve diverse chemical-receptor interactions that can perturb hormone signaling. The Organization for Economic Co-operation and Development has validated several EDC-receptor bioassays to detect endocrine active chemicals and has established guidelines for regulatory testing of EDCs. Focus on testing over the past decade has been initially directed to EATS modalities (estrogen, androgen, thyroid, and steroidogenesis) and validated tests for chemicals that exert effects through non-EATS modalities are less established. Due to recognition that EDCs are vast in their mechanisms of action, novel bioassays are needed to capture the full scope of activity. Here, we highlight the need for validated assays that detect non-EATS modalities and discuss major international efforts underway to develop such tools for regulatory purposes, focusing on non-EATS modalities of high concern (i.e., retinoic acid, aryl hydrocarbon receptor, peroxisome proliferator-activated receptor, and glucocorticoid signaling). Two case studies are presented with strong evidence amongst animals and human studies for non-EATS disruption and associations with wildlife and human disease. This includes metabolic syndrome and insulin signaling (case study 1) and chemicals that impact the cardiovascular system (case study 2). This is relevant as obesity and cardiovascular disease represent two of the most significant health-related crises of our time. Lastly, emerging topics related to EDCs are discussed, including recognition of crosstalk between the EATS and non-EATS axis, complex mixtures containing a variety of EDCs, adverse outcome pathways for chemicals acting through non-EATS mechanisms, and novel models for testing chemicals. Recommendations and considerations for evaluating non-EATS modalities are proposed. Moving forward, improved understanding of the non-EATS modalities will lead to integrated testing strategies that can be used in regulatory bodies to protect environmental, animal, and human health from harmful environmental chemicals.

A cross-species comparative approach to assessing multi- and transgenerational effects of endocrine disrupting chemicals.

A wide range of chemicals have been identified as endocrine disrupting chemicals (EDCs) in vertebrate species. Most studies of EDCs have focused on exposure of both male and female adults to these chemicals; however, there is clear evidence that EDCs have dramatic effects when mature or developing gametes are exposed, and consequently are associated with in multigenerational and transgenerational effects. Several publications have reviewed such actions of EDCs in subgroups of species, e.g., fish or rodents. In this review, we take a holistic approach synthesizing knowledge of the effects of EDCs across vertebrate species, including fish, anurans, birds, and mammals, and discuss the potential mechanism(s) mediating such multi- and transgenerational effects. We also propose a series of recommendations aimed at moving the field forward in a structured and coherent manner.

Unexpected metabolic disorders induced by endocrine disruptors in Xenopus tropicalis provide new lead for understanding amphibian decline.

Despite numerous studies suggesting that amphibians are highly sensitive to endocrine disruptors (EDs), both their role in the decline of populations and the underlying mechanisms remain unclear. This study showed that frogs exposed throughout their life cycle to ED concentrations low enough to be considered safe for drinking water, developed a prediabetes phenotype and, more commonly, a metabolic syndrome. Female Xenopus tropicalis exposed from tadpole stage to benzo(a)pyrene or triclosan at concentrations of 50 ng⋅L-1 displayed glucose intolerance syndrome, liver steatosis, liver mitochondrial dysfunction, liver transcriptomic signature, and pancreatic insulin hypersecretion, all typical of a prediabetes state. This metabolic syndrome led to progeny whose metamorphosis was delayed and occurred while the individuals were both smaller and lighter, all factors that have been linked to reduced adult recruitment and likelihood of reproduction. We found that F1 animals did indeed have reduced reproductive success, demonstrating a lower fitness in ED-exposed Xenopus Moreover, after 1 year of depuration, Xenopus that had been exposed to benzo(a)pyrene still displayed hepatic disorders and a marked insulin secretory defect resulting in glucose intolerance. Our results demonstrate that amphibians are highly sensitive to EDs at concentrations well below the thresholds reported to induce stress in other vertebrates. This study introduces EDs as a possible key contributing factor to amphibian population decline through metabolism disruption. Overall, our results show that EDs cause metabolic disorders, which is in agreement with epidemiological studies suggesting that environmental EDs might be one of the principal causes of metabolic disease in humans.

Identifying genomic changes associated with insecticide resistance in the dengue mosquito Aedes aegypti by deep targeted sequencing.

The capacity of mosquitoes to resist insecticides threatens the control of diseases such as dengue and malaria. Until alternative control tools are implemented, characterizing resistance mechanisms is crucial for managing resistance in natural populations. Insecticide biodegradation by detoxification enzymes is a common resistance mechanism; however, the genomic changes underlying this mechanism have rarely been identified, precluding individual resistance genotyping. In particular, the role of copy number variations (CNVs) and polymorphisms of detoxification enzymes have never been investigated at the genome level, although they can represent robust markers of metabolic resistance. In this context, we combined target enrichment with high-throughput sequencing for conducting the first comprehensive screening of gene amplifications and polymorphisms associated with insecticide resistance in mosquitoes. More than 760 candidate genes were captured and deep sequenced in several populations of the dengue mosquito Ae. aegypti displaying distinct genetic backgrounds and contrasted resistance levels to the insecticide deltamethrin. CNV analysis identified 41 gene amplifications associated with resistance, most affecting cytochrome P450s overtranscribed in resistant populations. Polymorphism analysis detected more than 30,000 variants and strong selection footprints in specific genomic regions. Combining Bayesian and allele frequency filtering approaches identified 55 nonsynonymous variants strongly associated with resistance. Both CNVs and polymorphisms were conserved within regions but differed across continents, confirming that genomic changes underlying metabolic resistance to insecticides are not universal. By identifying novel DNA markers of insecticide resistance, this study opens the way for tracking down metabolic changes developed by mosquitoes to resist insecticides within and among populations.

Multiscale Approach to Deciphering the Molecular Mechanisms Involved in the Direct and Intergenerational Effect of Ibuprofen on Mosquito Aedes aegypti.

The anti-inflammatory ibuprofen is a ubiquitous surface water contaminant. However, the chronic impact of this pharmaceutical on aquatic invertebrate populations remains poorly understood. In model insect Aedes aegypti, we investigated the intergenerational consequences of parental chronic exposure to an environmentally relevant concentration of ibuprofen. While exposed individuals did not show any phenotypic changes, their progeny showed accelerated development and an increased tolerance to starvation. In order to understand the mechanistic processes underpinning the direct and intergenerational impacts of ibuprofen, we combined transcriptomic, metabolomics, and hormone kinetics studies at several life stages in exposed individuals and their progeny. This integrative approach revealed moderate transcriptional changes in exposed larvae consistent with the pharmacological mode of action of ibuprofen. Parental exposure led to lower levels of several polar metabolites in progeny eggs and to major transcriptional changes in the following larval stage. These transcriptional changes, most likely driven by changes in the expression of numerous transcription factors and epigenetic regulators, led to ecdysone signaling and stress response potentiation. Overall, the present study illustrates the complexity of the molecular basis of the intergenerational pollutant response in insects and the importance of considering the entire life cycle of exposed organisms and of their progeny in order to fully understand the mode of action of pollutants and their impact on ecosystems.

Gene expression patterns and sequence polymorphisms associated with mosquito resistance to Bacillus thuringiensis israelensis toxins.

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.

Comparative analysis of response to selection with three insecticides in the dengue mosquito Aedes aegypti using mRNA sequencing.

BACKGROUND: Mosquito control programmes using chemical insecticides are increasingly threatened by the development of resistance. Such resistance can be the consequence of changes in proteins targeted by insecticides (target site mediated resistance), increased insecticide biodegradation (metabolic resistance), altered transport, sequestration or other mechanisms. As opposed to target site resistance, other mechanisms are far from being fully understood. Indeed, insecticide selection often affects a large number of genes and various biological processes can hypothetically confer resistance. In this context, the aim of the present study was to use RNA sequencing (RNA-seq) for comparing transcription level and polymorphism variations associated with adaptation to chemical insecticides in the mosquito Aedes aegypti. Biological materials consisted of a parental susceptible strain together with three child strains selected across multiple generations with three insecticides from different classes: the pyrethroid permethrin, the neonicotinoid imidacloprid and the carbamate propoxur. RESULTS: After ten generations, insecticide-selected strains showed elevated resistance levels to the insecticides used for selection. RNA-seq data allowed detecting over 13,000 transcripts, of which 413 were differentially transcribed in insecticide-selected strains as compared to the susceptible strain. Among them, a significant enrichment of transcripts encoding cuticle proteins, transporters and enzymes was observed. Polymorphism analysis revealed over 2500 SNPs showing > 50% allele frequency variations in insecticide-selected strains as compared to the susceptible strain, affecting over 1000 transcripts. Comparing gene transcription and polymorphism patterns revealed marked differences among strains. While imidacloprid selection was linked to the over transcription of many genes, permethrin selection was rather linked to polymorphism variations. Focusing on detoxification enzymes revealed that permethrin selection strongly affected the polymorphism of several transcripts encoding cytochrome P450 monooxygenases likely involved in insecticide biodegradation. CONCLUSIONS: The present study confirmed the power of RNA-seq for identifying concomitantly quantitative and qualitative transcriptome changes associated with insecticide resistance in mosquitoes. Our results suggest that transcriptome modifications can be selected rapidly by insecticides and affect multiple biological functions. Previously neglected by molecular screenings, polymorphism variations of detoxification enzymes may play an important role in the adaptive response of mosquitoes to insecticides.

Impaired liver function in Xenopus tropicalis exposed to benzo[a]pyrene: transcriptomic and metabolic evidence.

BACKGROUND: Despite numerous studies suggesting that amphibians are highly sensitive to cumulative anthropogenic stresses, the role pollutants play in the decline of amphibian populations remains unclear. Amongst the most common aquatic contaminants, polycyclic aromatic hydrocarbons (PAHs) have been shown to induce several adverse effects on amphibian species in the larval stages. Conversely, adults exposed to high concentrations of the ubiquitous PAH, benzo[a]pyrene (BaP), tolerate the compound thanks to their highly efficient hepatic detoxification mechanisms. Due to this apparent lack of toxic effect on adults, no studies have examined in depth the potential toxicological impact of PAH on the physiology of adult amphibian livers. This study sheds light on the hepatic responses of Xenopus tropicalis when exposed to high environmentally relevant concentrations of BaP, by combining a high throughput transcriptomic approach (mRNA deep sequencing) and a characterization of cellular and physiological modifications to the amphibian liver. RESULTS: Transcriptomic changes observed in BaP-exposed Xenopus were further characterized using a time-dependent enrichment analysis, which revealed the pollutant-dependent gene regulation of important biochemical pathways, such as cholesterol biosynthesis, insulin signaling, adipocytokines signaling, glycolysis/gluconeogenesis and MAPK signaling. These results were substantiated at the physiological level with the detection of a pronounced metabolic disorder resulting in a possible insulin resistance-like syndrome phenotype. Hepatotoxicity induced by lipid and cholesterol metabolism impairments was clearly identified in BaP-exposed individuals. CONCLUSIONS: Our data suggested that BaP may disrupt overall liver physiology, and carbohydrate and cholesterol metabolism in particular, even after short-term exposure. These results are further discussed in terms of how this deregulation of liver physiology can lead to general metabolic impairment in amphibians chronically exposed to contaminants, thereby illustrating the role xenobiotics might play in the global decline in amphibian populations.

Chemical and biological insecticides select distinct gene expression patterns in Aedes aegypti mosquito.

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.

The central role of mosquito cytochrome P450 CYP6Zs in insecticide detoxification revealed by functional expression and structural modelling.

The resistance of mosquitoes to chemical insecticides is threatening vector control programmes worldwide. Cytochrome P450 monooxygenases (CYPs) are known to play a major role in insecticide resistance, allowing resistant insects to metabolize insecticides at a higher rate. Among them, members of the mosquito CYP6Z subfamily, like Aedes aegypti CYP6Z8 and its Anopheles gambiae orthologue CYP6Z2, have been frequently associated with pyrethroid resistance. However, their role in the pyrethroid degradation pathway remains unclear. In the present study, we created a genetically modified yeast strain overexpressing Ae. aegypti cytochrome P450 reductase and CYP6Z8, thereby producing the first mosquito P450-CPR (NADPH-cytochrome P450-reductase) complex in a yeast recombinant system. The results of the present study show that: (i) CYP6Z8 metabolizes PBAlc (3-phenoxybenzoic alcohol) and PBAld (3-phenoxybenzaldehyde), common pyrethroid metabolites produced by carboxylesterases, producing PBA (3-phenoxybenzoic acid); (ii) CYP6Z8 transcription is induced by PBAlc, PBAld and PBA; (iii) An. gambiae CYP6Z2 metabolizes PBAlc and PBAld in the same way; (iv) PBA is the major metabolite produced in vivo and is excreted without further modification; and (v) in silico modelling of substrate-enzyme interactions supports a similar role of other mosquito CYP6Zs in pyrethroid degradation. By playing a pivotal role in the degradation of pyrethroid insecticides, mosquito CYP6Zs thus represent good targets for mosquito-resistance management strategies.

Evolution of pace-of-life syndrome under conditions of maternal PCB contamination and global warming in early life stages of cold stenothermic fish (Arctic char).

The end of the 20th century was characterised by rapid modifications of ecosystem functioning under different pressures (such as eutrophication and toxic pollution). Increasing temperatures in the context of global warming could have indirect consequences, such as increased bioavailability of hydrophobic organic pollutants amongst aquatic species. According to the "pace-of-life syndrome" (POLS) theory, these stressors could lead to covariations in many life traits. Lake Bourget is the largest natural lake in France and has been highly polluted from the fifties to the eighties both with a high load of nutrients (wastewater discharge) and polychlorinated biphenyls (PCBs) (industrial effluent discharge). Despite improvements in water quality since the 21st century, PCB levels are still higher than the United States Environmental Protection Agency cut-off for wildlife protection. The population of Arctic char, a cold stenothermic salmonid, has remained low in Lake Bourget for the last ten years despite restocking efforts and complete re-oligotrophication. We hypothesised that PCB pollution can affect the Arctic char population and that the increase in water temperature could magnify the effects of PCB. Thus, this study aimed to investigate the effects of maternal PCB contamination on offspring using a multiparametric and multiscale approach. Female Arctic char were contaminated with PCB before spawning, and each fertilised spawn was incubated at two temperatures (4 and 8.5 °C). The results showed that co-exposure to increased temperature and maternal PCB contamination influenced biodemographic, physiological, and behavioural parameters. The effects were highly dependant on the developmental stage. Based on the POLS theory, a continuum of life traits that may reflect potential physiological and behavioural modifications in response to these concurrent stressors is highlighted.

Decreased toxicity of Bacillus thuringiensis subsp. israelensis to mosquito larvae after contact with leaf litter.

Bacillus thuringiensis subsp. israelensis is a bacterium producing crystals containing Cry and Cyt proteins, which are toxic for mosquito larvae. Nothing is known about the interaction between crystal toxins and decaying leaf litter, which is a major component of several mosquito breeding sites and represents an important food source. In the present work, we investigated the behavior of B. thuringiensis subsp. israelensis toxic crystals sprayed on leaf litter. In the presence of leaf litter, a 60% decrease in the amount of Cyt toxin detectable by immunology (enzyme-linked immunosorbent assays [ELISAs]) was observed, while the respective proportions of Cry toxins were not affected. The toxicity of Cry toxins toward Aedes aegypti larvae was not affected by leaf litter, while the synergistic effect of Cyt toxins on all B. thuringiensis subsp. israelensis Cry toxins was decreased by about 20% when mixed with leaf litter. The toxicity of two commercial B. thuringiensis subsp. israelensis strains (VectoBac WG and VectoBac 12AS) and a laboratory-produced B. thuringiensis subsp. israelensis strain decreased by about 70% when mixed with leaf litter. Taken together, these results suggest that Cyt toxins interact with leaf litter, resulting in a decreased toxicity of B. thuringiensis subsp. israelensis in litter-rich environments and thereby dramatically reducing the efficiency of mosquitocidal treatments.

Fate of Bacillus thuringiensis subsp. israelensis in the field: evidence for spore recycling and differential persistence of toxins in leaf litter.

Bacillus thuringiensis subsp. israelensis is a bioinsecticide increasingly used worldwide for mosquito control. Despite its apparent low level of persistence in the field due to the rapid loss of its insecticidal activity, an increasing number of studies suggested that the recycling of B. thuringiensis subsp. israelensis can occur under specific, unknown conditions. Decaying leaf litters sampled in mosquito breeding sites in the French Rhône-Alpes region several months after a treatment were shown to exhibit a high level of larval toxicity and contained large amounts of spores. In the present article, we show that the high concentration of toxins found in these litters is consistent with spore recycling in the field, which gave rise to the production of new crystal toxins. Furthermore, in these toxic leaf litter samples, Cry4Aa and Cry4Ba toxins became the major toxins instead of Cyt1Aa in the commercial mixture. In a microcosm experiment performed in the laboratory, we also demonstrated that the toxins, when added in their crystal form to nontoxic leaf litter, exhibited patterns of differential persistence consistent with the proportions of toxins observed in the field-collected toxic leaf litter samples (Cry4 > Cry11 > Cyt). These results give strong evidence that B. thuringiensis subsp. israelensis recycled in specific breeding sites containing leaf litters, and one would be justified in asking whether mosquitoes can become resistant when exposed to field-persistent B. thuringiensis subsp. israelensis for several generations.

Transcription profiling of resistance to Bti toxins in the mosquito Aedes aegypti using next-generation sequencing.

The control of mosquitoes transmitting infectious diseases relies mainly on the use of chemical insecticides. However, resistance to most chemical insecticides threatens mosquito control programs. In this context, the spraying of toxins produced by the bacteria Bacillus thuringiensis subsp. israelensis (Bti) in larval habitats represents an alternative to chemical insecticides and is now widely used for mosquito control. Recent studies suggest that resistance of mosquitoes to Bti toxin may occur locally but mechanisms have not been characterized so far. In the present study, we investigated gene transcription level variations associated with Bti toxin resistance in the mosquito Aedes aegypti using a next-generation sequencing approach. More than 6 million short cDNA tags were sequenced from larvae of two strains sharing the same genetic background: a Bti toxins-resistant strain and a susceptible strain. These cDNA tags were mapped with a high coverage (308 reads per position in average) to more than 6000 genes of Ae. aegypti genome and used to quantify and compare the transcription level of these genes between the two mosquito strains. Among them, 86 genes were significantly differentially transcribed more than 4-fold in the Bti toxins resistant strain comparatively to the susceptible strain. These included gene families previously associated with Bti toxins resistance such as serine proteases, alkaline phosphatase and alpha-amylase. These results are discussed in regards of potential Bti toxins resistance mechanisms in mosquitoes.

An amphibian high fat diet model confirms that endocrine disruptors can induce a metabolic syndrome in wild green frogs (Pelophylax spp. complex).

A pre-diabetes syndrome induced by endocrine disruptors (ED) was recently demonstrated in the model amphibian Silurana (Xenopus) tropicalis and was suggested to be a potential cause of amphibian population decline. However, such effects have not been found in wild type frogs exposed to ED and the capacity of amphibians to physiologically develop diabetes under natural conditions has not been confirmed. This study showed that a high fat diet (HFD) model displaying the important characteristics of mammal HFD models including glucose intolerance, insulin resistance and nonalcoholic fatty liver disease (NAFLD) can be developed with green frogs (Pelophylax spp.). Wild green frogs exposed to 10 µg L-1 benzo [a]pyrene (BaP) for 18 h also displayed several characteristics of the pre-diabetes phenotype previously observed in Xenopus including glucose intolerance, gluconeogenesis activation and insulin resistance. The study results confirmed that metabolic disorders induced by ED in wild green frogs are typical of the pre-diabetes phenotype and could serve as a starting point for field studies to determine the role of ED in the decline of amphibian populations. From an environmental perspective, the response of wild green frogs to different ED (10 µg L-1) suggests that a simple glucose-tolerance test could be used on wild anurans to identify bodies of water polluted with metabolic disruptors that could affect species fitness.

Exposure to a mixture of benzo[a]pyrene and triclosan induces multi-and transgenerational metabolic disorders associated with decreased female investment in reproduction in Silurana (Xenopus) tropicalis.

Animals must partition limited resources between their own growth and subsequent reproduction. Endocrine disruptors (ED) may cause maternal metabolic disorders that decrease successful reproduction and might be responsible for multi- and transgenerational effects in amphibians. We found that the frog Silurana (Xenopus) tropicalis, exposed to environmentally relevant concentrations of benzo[a]pyrene and triclosan throughout its life cycle, produced F1 females with delayed sexual maturity and decreased size and weight. These F1 females displayed a marked metabolic syndrome associated with decreased fasting plasma cholesterol and triglyceride concentrations and decreased gonadal development. F1 females from F0 exposed animals also had decreased reproductive investment highlighted by a decrease of oocyte lipid reserves associated with significant F2-tadpole mortality. F2 females from F0 exposed animals also displayed a marked metabolic syndrome but were able to correctly direct liver lipid metabolism to the constitution of fat bodies and oocyte yolk stores. In addition, the F2 females produced progeny that had normal mortality levels at 5 days post hatching compared to the controls suggesting a good reproductive investment. Our data confirmed that these ED, at concentrations often found in natural ponds, can induce multi- and transgenerational metabolic disorders in the progeny of amphibians that are not directly exposed. We present a hypothesis to explain the transmission of the metabolic syndrome across generations through modification of egg reserves. However, when high mortality occurred at the tadpole stage, surviving females were able to cope with metabolic costs and produce viable progeny through sufficient investment in the contents of oocyte reserves.

Exposure of Anopheles gambiae larvae to a sub-lethal dose of an agrochemical mixture induces tolerance to adulticides used in vector control management.

The heavy use of pesticides in agricultural areas often leads to the contamination of nearby mosquito larvae breeding sites. Exposure to complex mixtures of agrochemicals can affect the insecticide sensitivity of mosquito larvae. Our study objective was to determine whether agrochemical residues in Anopheline larval breeding sites can affect the tolerance of adults to commonly used adulticides. We focussed on Fludora® Fusion, a vector control insecticide formulation combining two insecticides (deltamethrin and clothianidin) with different modes of action. An. gambiae larvae were exposed to a sub-lethal dose of a mixture of agrochemical pesticides used in a highly active agricultural area on the Ivory Coast. Comparative bioassays with Fludora Fusion mixture and its two insecticide components (deltamethrin and clothianidin) were carried out between adult mosquitoes exposed or not to the agrochemicals at the larval stage. A transcriptomic analysis using RNA sequencing was then performed on larvae and adults to study the molecular mechanisms underlying the phenotypic changes observed. Bioassays revealed a significantly increased tolerance of adult females to clothianidin (2.5-fold) and Fludora Fusion mixture (2.2-fold) following larval exposure to agrochemicals. Significantly increased tolerance to deltamethrin was not observed suggesting that insecticide exposure affects the adult efficacy of the Fludora Fusion mixture mainly through mechanisms acting on clothianidin. Transcriptomic analysis revealed the potential of agrochemicals to induce various resistance mechanisms including cuticle proteins, detoxification action and altered insecticide sequestration. These results suggest that although the Fludora Fusion mixture is effective for adult vector control, its efficacy may be locally affected by the ecological context. The present study also suggests that, although the complex interactions between the use of agrochemicals and vector control insecticides are difficult to decipher in the field, they still must be considered in the context of insecticide resistance management programmes.

Molecular bases of P450-mediated resistance to the neonicotinoid insecticide imidacloprid in the mosquito Ae. aegypti.

Resistance to chemical insecticides including pyrethroids, the main insecticide class used against mosquitoes, has re-kindled interest in the use of neonicotinoids. In this context, the present study aimed to characterize the molecular basis of neonicotinoid resistance in the mosquito Aedes aegypti. Resistance mechanisms were studied by combining transcriptomic and genomic data obtained from a laboratory strain selected at the larval stage after 30 generations of exposure to imidacloprid (Imida-R line). After thirty generations of selection, larvae of the Imida-R line showed an 8-fold increased resistance to imidacloprid and a significant cross-tolerance to the pyrethroids permethrin and deltamethrin. Cross-resistance to pyrethroids was only observed in adults when larvae were previously exposed to imidacloprid suggesting a low but inducible expression of resistance alleles at the adult stage. Resistance of the Imida-R line was associated with a slower larval development time in females. Multiple detoxification enzymes were over-transcribed in larvae in association with resistance including the P450s CYP6BB2, CYP9M9 and CYP6M11 previously associated with pyrethroid resistance. Some of them together with their redox partner NADPH P450 reductase were also affected by non-synonymous mutations associated with resistance. Combining genomic and transcriptomic data allowed identifying promoter variations associated with the up-regulation of CYP6BB2 in the resistant line. Overall, these data confirm the key role of P450s in neonicotinoid resistance in Ae. aegypti and their potential to confer cross-resistance to pyrethroids, raising concerns about the use of neonicotinoids for resistance management in this mosquito species.