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.

Estrogen-related receptor γ is an in vivo receptor of bisphenol A.

Bisphenol A (BPA) is an endocrine disruptor that displays estrogenic activity. Several reports suggest that BPA may have estrogen receptor-independent effects. In zebrafish, 50 µM BPA exposure induces otic vesicle abnormalities, including otolith aggregation. The purpose of this study was to test if BPA action was mediated in vivo during zebrafish development by the orphan nuclear estrogen related receptor (ERR) γ. Combining pharmacological and functional approaches, we demonstrate that the zebrafish ERRγ mediates BPA-induced malformations in otoliths. Using different bisphenol derivatives, we show that different compounds can induce a similar otolith phenotype than BPA and that the binding affinity of these derivatives to the zebrafish ERRγ correlates with their ability to induce otolith malformations. Morpholino knockdown of ERRγ function suppresses the BPA effect on otoliths whereas overexpression of ERRγ led to a BPA-like otolith phenotype. Moreover, a subphenotypical dose of BPA (1 µM) combined with ERRγ overexpression led to a full-dose (50 µM) BPA otolith phenotype. We therefore conclude that ERRγ mediates the otic vesicle phenotype generated by BPA. Our results suggest that the range of pathways perturbed by this compound and its potential harmful effect are larger than expected.-Tohmé, M., Prud'homme, S. M., Boulahtouf, A., Samarut, E., Brunet, F., Bernard, L., Bourguet, W., Gibert, Y., Balaguer, P., Laudet, V. Estrogen-related receptor γ is an in vivo receptor of bisphenol A.

Soil sampling and isolation of extracellular DNA from large amount of starting material suitable for metabarcoding studies.

DNA metabarcoding refers to the DNA-based identification of multiple species from a single complex and degraded environmental sample. We developed new sampling and extraction protocols suitable for DNA metabarcoding analyses targeting soil extracellular DNA. The proposed sampling protocol has been designed to reduce, as much as possible, the influence of local heterogeneity by processing a large amount of soil resulting from the mixing of many different cores. The DNA extraction is based on the use of saturated phosphate buffer. The sampling and extraction protocols were validated first by analysing plant DNA from a set of 12 plots corresponding to four plant communities in alpine meadows, and, second, by conducting pilot experiments on fungi and earthworms. The results of the validation experiments clearly demonstrated that sound biological information can be retrieved when following these sampling and extraction procedures. Such a protocol can be implemented at any time of the year without any preliminary knowledge of specific types of organisms during the sampling. It offers the opportunity to analyse all groups of organisms using a single sampling/extraction procedure and opens the possibility to fully standardize biodiversity surveys.

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.

Impact of micropollutants on the life-history traits of the mosquito Aedes aegypti: On the relevance of transgenerational studies.

Hazard assessment of chemical contaminants often relies on short term or partial life-cycle ecotoxicological tests, while the impact of low dose throughout the entire life cycle of species across multiple generations has been neglected. This study aimed at identifying the individual and population-level consequences of chronic water contamination by environmental concentrations of three organic micropollutants, ibuprofen, bisphenol A and benzo[a]pyrene, on Aedes aegypti mosquito populations in experimental conditions. Life-history assays spanning the full life-cycle of exposed individuals and their progeny associated with population dynamics modelling evidenced life-history traits alterations in unexposed progenies of individuals chronically exposed to 1 µg/L ibuprofen or 0.6 µg/L benzo[a]pyrene. The progeny of individuals exposed to ibuprofen showed an accelerated development while the progeny of individuals exposed to benzo[a]pyrene showed a developmental acceleration associated with an increase in mortality rate during development. These life-history changes due to pollutants exposure resulted in relatively shallow increase of Ae. aegypti asymptotic population growth rate. Multigenerational exposure for six generations revealed an evolution of population response to ibuprofen and benzo[a]pyrene across generations, leading to a loss of previously identified transgenerational effects and to the emergence of a tolerance to the bioinsecticide Bacillus turingiensis israelensis (Bti). This study shed light on the short and long term impact of environmentally relevant doses of ibuprofen and benzo[a]pyrene on Ae. aegypti life-history traits and insecticide tolerance, raising unprecedented perspectives about the influence of surface water pollution on vector-control strategies. Overall, our approach highlights the importance of considering the entire life cycle of organisms, and the necessity to assess the transgenerational effects of pollutants in ecotoxicological studies for ecological risk assessment. Finally, this multi-generational study gives new insight about the influence of surface water pollution on microevolutionary processes.

UV light and urban pollution: bad cocktail for mosquitoes?

Mosquito breeding sites consist of water pools, which can either be large open areas or highly covered ponds with vegetation, thus with different light exposures combined with the presence in water of xenobiotics including polycyclic aromatic hydrocarbons (PAHs) generated by urban pollution. UV light and PAHs are abiotic factors known to both affect the mosquito insecticide resistance status. Nonetheless, their potential combined effects on the mosquito physiology have never been investigated. The present article aims at describing the effects of UV exposure alongside water contamination with two major PAH pollutants (fluoranthene and benzo[a]pyrene) on a laboratory population of the yellow fever mosquito Aedes aegypti. To evaluate the effects of PAH exposure and low energetic UV (UV-A) irradiation on mosquitoes, different parameters were measured including: (1) The PAH localization and its impact on cell mortality by fluorescent microscopy; (2) The detoxification capacities (cytochrome P450, glutathione-S-transferase, esterase); (3) The responses to oxidative stress (Reactive Oxygen Species-ROS) and (4) The tolerance of mosquito larvae to a bioinsecticide (Bacillus thuringiensis subsp. israelensis-Bti) and to five chemical insecticides (DDT, imidacloprid, permethrin, propoxur and temephos). Contrasting effects regarding mosquito cell mortality, detoxification and oxidative stress were observed as being dependent on the pollutant considered, despite the fact that the two PAHs belong to the same family. Moreover, UV is able to modify pollutant effects on mosquitoes, including tolerance to three insecticides (imidacloprid, propoxur and temephos), cell damage and response to oxidative stress. Taken together, our results suggest that UV and pollution, individually or in combination, are abiotic parameters that can affect the physiology and insecticide tolerance of mosquitoes; but the complexity of their direct effect and of their interaction will require further investigation to know in which condition they can affect the efficacy of insecticide-based vector control strategies in the field.

Contrasting patterns of tolerance between chemical and biological insecticides in mosquitoes exposed to UV-A.

Mosquitoes are vectors of major human diseases, such as malaria, dengue or yellow fever. Because no efficient treatments or vaccines are available for most of these diseases, control measures rely mainly on reducing mosquito populations by the use of insecticides. Numerous biotic and abiotic factors are known to modulate the efficacy of insecticides used in mosquito control. Mosquito breeding sites vary from opened to high vegetation covered areas leading to a large ultraviolet gradient exposure. This ecological feature may affect the general physiology of the insect, including the resistance status against insecticides. In the context of their contrasted breeding sites, we assessed the impact of low-energetic ultraviolet exposure on mosquito sensitivity to biological and chemical insecticides. We show that several mosquito detoxification enzyme activities (cytochrome P450, glutathione S-transferases, esterases) were increased upon low-energy UV-A exposure. Additionally, five specific genes encoding detoxification enzymes (CYP6BB2, CYP6Z7, CYP6Z8, GSTD4, and GSTE2) previously shown to be involved in resistance to chemical insecticides were found over-transcribed in UV-A exposed mosquitoes, revealed by RT-qPCR experiments. More importantly, toxicological bioassays revealed that UV-exposed mosquitoes were more tolerant to four main chemical insecticide classes (DDT, imidacloprid, permethrin, temephos), whereas the bioinsecticide Bacillus thuringiensis subsp. israelensis (Bti) appeared more toxic. The present article provides the first experimental evidence of the capacity of low-energy UV-A to increase mosquito tolerance to major chemical insecticides. This is also the first time that a metabolic resistance to chemical insecticides is linked to a higher susceptibility to a bioinsecticide. These results support the use of Bti as an efficient alternative to chemical insecticides when a metabolic resistance to chemicals has been developed by mosquitoes.

Differential effects of bisphenol A and diethylstilbestrol on human, rat and mouse fetal leydig cell function.

Endocrine disruptors (ED) have been incriminated in the current increase of male reproductive alterations. Bisphenol A (BPA) is a widely used weak estrogenic environmental ED and it is debated whether BPA concentrations within the average internal exposure are toxic. In the present study we investigated the effects of 10(-12) to 10(-5) M BPA concentrations on fetal Leydig cell function, as fetal life is a critical period of sensitivity to ED effects on male reproductive function. To this aim, fetal testes from human at 6.5-10.5 gestational weeks (GW) or from rat and mouse at a comparable critical period of development (14.5 days post-coitum (dpc) for rat and 12.5 dpc for mouse) were explanted and cultured using our validated organotypic culture system in the presence or absence of BPA for 1-3 days. BPA concentrations as low as 10(-8) M reduced testosterone secretion by human testes from day 1 of culture onwards, but not by mouse and rat testes where concentrations equal to 10(-5) M BPA were required. Similarly, 10(-8) M BPA reduced INSL3 mRNA levels only in human cultured testes. On the contrary, 10(-5) and 10(-6) M diethylstilbestrol (DES), a classical estrogenic compound, affected testosterone secretion only in rat and mouse testis cultures, but not in human testis cultures. Lastly, contrarily to the DES effect, the negative effect of BPA on testosterone produced by the mouse fetal testis was maintained after invalidation of estrogen receptor α (ERα). In conclusion, these results evidenced i) a deleterious effect of BPA on fetal Leydig cells function in human for concentrations from 10(-8) M upwards, ii) species-specific differences raising concerns about extrapolation of data from rodent studies to human risk assessment, iii) a specific signaling pathway for BPA which differs from the DES one and which does not involve ERα.