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Context: 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 indirect selection pressure has rarely been quantified and the molecular mechanisms involved are still poorly characterised. In this context, experimental selection with different agrochemical mixtures was conducted in An. gambiae and the impact on insecticide resistance was evaluated by phenotypic and molecular approaches. Methods: Mosquito larvae were selected for 30 generations with three different mixtures of agrochemicals containing i) insecticides, ii) non-insecticides compounds and iii) both agrochemical types. Each five generations, the resistance of adults to deltamethrin and bendiocarb was monitored using bioassays. The frequencies of the kdr west (L1014F) and Ace1 (G119S) target-site mutations were evaluated every 10 generations. Gene expression and polymorphism variations associated with each selection regime were investigated after 30 generations by RNA-seq. 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 as compared to the non-selected line. Such increased resistance was associated with an increased frequency of the Kdr west mutation in the insecticide and non-insecticide selected lines. RNA-seq identified 63 candidate resistance genes over-transcribed in at least one selected line as compared to the non-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. Polymorphism analyses identified several genes under selection in each line 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 impact Kdr mutations, detoxification enzymes but also other more generalist resistance mechanisms 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.

By altering gene expression and creating paralogs, genomic amplifications represent a key component of short-term adaptive processes. In insects, the use of insecticides can select gene amplifications causing an increased expression of detoxification enzymes, supporting the usefulness of these DNA markers for monitoring the dynamics of resistance alleles in the field. In this context, the present study aims to characterise a genomic amplification event associated with resistance to organophosphate insecticides in the mosquito Aedes aegypti and to develop a molecular assay to monitor the associated resistance alleles in the field. An experimental evolution experiment using a composite population from Laos supported the association between the over-transcription of multiple contiguous carboxylesterase genes on chromosome 2 and resistance to multiple organophosphate insecticides. Combining whole genome sequencing and qPCR on specific genes confirmed the presence of a ~100 Kb amplification spanning at least five carboxylesterase genes at this locus with the co-existence of multiple structural duplication haplotypes. Field data confirmed their circulation in South-East Asia and revealed high copy number polymorphism among and within populations suggesting a trade-off between this resistance mechanism and associated fitness costs. A dual-colour multiplex TaqMan assay allowing the rapid detection and copy number quantification of this amplification event in Ae. aegypti was developed and validated on field populations. The routine use of this novel assay will improve the tracking of resistance alleles in this major arbovirus vector.

BackgroundThe introduction of neonicotinoids for managing insecticide resistance in mosquitoes is of high interest as they interact with a biochemical target not previously used in public health. In this concern, Bayer developed a combination of the neonicotinoid clothianidin and the pyrethroid deltamethrin (brand name Fludora® Fusion) as a new vector control tool. Although this combination proved to be efficient against pyrethroid-resistant mosquitoes, its ability to prevent the selection of pyrethroid and neonicotinoid resistance alleles was not investigated. In this context, the objective of this work was to study the dynamics and the molecular mechanisms of resistance of An. gambiae to the separated or combined components of this combination. A field-derived An. gambiae line carrying resistance alleles to multiple insecticides at low frequencies was used as a starting for 33 successive generations of controlled selection. Resistance levels to each insecticide and target site mutation frequencies were monitored throughout the selection process. Cross resistance to other public health insecticides were also investigated. RNA-seq was used to compare gene transcription variations and polymorphisms across all lines.ResultsThis study confirmed the potential of this insecticide combination to impair the selection of resistance as compared to its two separated components. Deltamethrin selection led to the rapid enrichment of the kdr L1014F target-site mutation while clothianidin selection led to the over-transcription of multiple cytochrome P450s including some showing high homology with the ones conferring neonicotinoid resistance in other insects. A strong selection signature associated with clothianidin selection was observed on a cytochrome P450 gene cluster previously associated with resistance. Within this cluster, the gene CYP6M1 showed the highest selection signature together with a transcription profile supporting a role in clothianidin resistance. Modelling the impact of point mutations selected by clothianidin on CYP6M1 protein structure suggested that the selection of variants affecting its active site can enhance clothianidin metabolism.ConclusionsIn the context of the recent deployment of neonicotinoids for mosquito control and their frequent usage in agriculture, the present study highlights the benefit of combining them with other insecticides for preventing the selection of resistance and sustaining vector control activities.

Foundation plants shape the composition of local biotic communities and abiotic environments, but the impact of a plant's intraspecific variations on these processes is poorly understood. We examined these links in the alpine cushion moss campion (Silene acaulis) on two neighboring mountain ranges in the French Alps. Genotyping of cushion plants revealed two genetic clusters matching known subspecies. The exscapa subspecies was found on both limestone and granite, while the longiscapa one was only found on limestone. Even on similar limestone bedrock, cushion soils from the two S. acaulis subspecies deeply differed in their impact on soil abiotic conditions. They further strikingly differed from each other and from the surrounding bare soils in fungal community composition. Plant genotype variations accounted for a large part of the fungal composition variability in cushion soils, even when considering geography or soil chemistry, and particularly for the dominant molecular operational taxonomic units (MOTUs). Both saprophytic and biotrophic fungal taxa were related to the MOTUs recurrently associated with a single plant genetic cluster. Moreover, the putative phytopathogens were abundant, and within the same genus (Cladosporium) or species (Pyrenopeziza brassicae), MOTUs showing specificity for each plant subspecies were found. Our study highlights the combined influences of bedrock and plant genotype on fungal recruitment into cushion soils and suggests the coexistence of two mechanisms, an indirect selection resulting from the colonization of an engineered soil by free‐living saprobes and a direct selection resulting from direct plant–fungi interactions.

Foundation plants shape the composition of local biotic communities and abiotic en ‐ vironments, but the impact of a plant’s intraspecific variations on these processes is poorly understood. We examined these links in the alpine cushion moss campion ( Silene acaulis ) on two neighboring mountain ranges in the French Alps. Genotyping of cushion plants revealed two genetic clusters matching known subspecies. The ex ‐ scapa subspecies was found on both limestone and granite, while the longiscapa one was only found on limestone. Even on similar limestone bedrock, cushion soils from the two S. acaulis subspecies deeply differed in their impact on soil abiotic condi ‐ tions. They further strikingly differed from each other and from the surrounding bare soils in fungal community composition. Plant genotype variations accounted for a large part of the fungal composition variability in cushion soils, even when consider ‐ ing geography or soil chemistry, and particularly for the dominant molecular opera ‐ tional taxonomic units (MOTUs). Both saprophytic and biotrophic fungal taxa were related to the MOTUs recurrently associated with a single plant genetic cluster. Moreover, the putative phytopathogens were abundant, and within the same genus ( Cladosporium ) or species ( Pyrenopeziza brassicae ), MOTUs showing specificity for each plant subspecies were found. Our study highlights the combined influences of bedrock and plant genotype on fungal recruitment into cushion soils and suggests the coexistence of two mechanisms, an indirect selection resulting from the coloniza ‐ tion of an engineered soil by free‐living saprobes and a direct selection resulting from direct plant–fungi interactions.

Summary The effect of plant species composition on soil microbial communities was studied at the multiregional level. We compared the soil microbial communities of alpine natural grasslands dominated by Carex curvula and anthropogenic subalpine pastures dominated by Nardus stricta. We conducted paired sampling across the Carpathians and the Alps and used Illumina sequencing to reveal the molecular diversity of soil microbes. We found that bacterial and fungal communities exhibited contrasting regional distributions and that the distribution in each grassland is well discriminated. Beta diversity of microbial communities was much higher in C. curvula grasslands due to a marked regional effect. The composition of grassland-type core microbiomes suggest that C. curvula, and N. stricta to a lesser extent, tend to select a cohort of microbes related to antibiosis/exclusion, pathogenesis and endophytism. We discuss these findings in light of the postglacial history of the studied grasslands, the habitat connectivity and the disturbance regimes. Human-induced disturbance in the subalpine belt of European mountains has led to homogeneous soil microbial communities at large biogeographical scales. Our results confirm the overarching role of the dominant grassland plant species in the distribution of microbial communities and highlight the relevance of biogeographical history.

The control of the stoichiometric balance of alpha- and beta-tubulin is important during microtubule biogenesis. This process involves several tubulin-folding cofactors (TFCs), of which only TFC A is not essential in mammalian in vitro systems or in vivo in yeast. Here, we show that the TFC A gene is important in vivo in plants. The Arabidopsis gene KIESEL (KIS) shows sequence similarity to the TFC A gene. Expression of the mouse TFC A gene under the control of the 35S promoter rescues the kis mutation, indicating that KIS is the Arabidopsis ortholog of TFC A. kis plants exhibit a range of defects similar to the phenotypes associated with impaired microtubule function: plants are reduced in size and show meiotic defects, cell division is impaired, and trichomes are bulged and less branched. Microtubule density was indistinguishable from that of the wild type, but microtubule organization was affected in trichomes and hypocotyl cells of dark-grown kis plants. The kis phenotype was rescued by overexpression of an alpha-tubulin, indicating that KIS is involved in the control of the correct balance of alpha- and beta-tubulin monomers.

Leaf trichomes in Arabidopsis are unicellular epidermal hairs with a branched morphology. They undergo successive endoreduplication rounds early during cell morphogenesis. Mutations affecting trichome nuclear DNA content, such as triptychon or glabra3, alter trichome branching. We isolated new mutants with supernumerary trichome branches, which fall into three unlinked complementation groups: KAKTUS and the novel loci, POLYCHOME and RASTAFARI. They map to chromosomes IV, II, and V, respectively. The trichomes of these mutants presented an increased DNA content, although to a variable extent. The spindly-5 mutant, which displays a constitutive gibberellin response, also produces overbranched trichomes containing more nuclear DNA. We analyzed genetic interactions using double mutants and propose that two independent pathways, defined by SPINDLY and TRIPTYCHON, act to limit trichome growth. KAKTUS and POLYCHOME might have redundant actions mediating gibberellin control via SPINDLY. The overall leaf polysomaty was not notably affected by these mutations, suggesting that they affect the control of DNA synthesis in a tissue- or cell type-specific manner. Wild-type tetraploids also produce overbranched trichomes; they displayed a shifted polysomaty in trichomes and in the whole leaf, suggesting a developmental program controlling DNA increases via the counting of endoreduplication rounds.

International audience; Aims: Climate-induced changes in snow cover are likely to affect cold arctic and alpine ecosystems functioning and major processes such as wintertime plant litter decomposition. However, it remains poorly studied in subalpine systems where the snowpack may be irregular. In this paper we explored the dynamic of the winter plant litter decomposition process, its magnitude and its relationship with the snowpack properties.Methods: In subalpine grasslands of the Central French Alps, we performed a litter bag experiment monitoring over a whole winter the litter decomposition from the exploitative Dactylis glomerata and the conservative Festuca paniculata, under two contiguous experimental sites with snowpacks differing in depth and physical properties.Results: Litter decomposition rates were stable during winter and 3-fold higher under deeper and permanent snowpack with higher thermal resistance. Litter quality appeared only significant under thinner snowpack with higher decomposition rates for the exploitative species. A snowpack with higher thermal resistance created an insulating layer promoting the decomposition process.Conclusion: These results suggest that the temporal (permanence vs. intermittency) and physical (depth and thermal resistance) characteristics of the snowpack should be considered when studying the response of winter ecosystems functioning to global changes.

International audience; The snowpack is a complex photochemical reactor that emits a wide variety of reactive molecules to the atmosphere. In particular, the photolysis of nitrate ions, NO 3-, produces NO, NO 2 , and HONO, which affects the oxidative capacity of the atmosphere. We report measurements in the European High Arctic where we observed for the first time emissions of NO, NO 2 , and HONO by the seasonal snowpack in winter, in the complete or near-complete absence of sunlight and in the absence of melting. We also detected unusually high concentrations of nitrite ions, NO 2-, in the snow. These results suggest that microbial activity in the snowpack is responsible for the observed emissions. Isotopic analysis of NO 2-and NO 3-in the snow confirm that these ions, at least in part, do not have an atmospheric origin and are most likely produced by the microbial oxidation of NH 4 + coming from clay minerals into NO 2-and NO 3-. These metabolic pathways also produce NO. Subsequent dark abiotic reactions lead to NO 2 and HONO production. The snow cover is therefore not only an active photochemical reactor but also a biogeochemical reactor active in the cycling of nitrogen and it can affect atmospheric composition all year round.

Understanding the role of transcription factors (TFs) is essential in reconstructing developmental regulatory networks. The plant-specific GeBP TF family of Arabidopsis thaliana (Arabidopsis) comprises 21 members, all of unknown function. A subset of four members, the founding member GeBP and GeBP-like proteins (GPL) 1, 2, and 3, shares a conserved C-terminal domain. Here we report that GeBP/GPL genes represent a newly defined class of leucine-zipper (Leu-zipper) TFs and that they play a redundant role in cytokinin hormone pathway regulation. Specifically, we demonstrate using yeast, in vitro, and split-yellow fluorescent protein in planta assays that GeBP/GPL proteins form homo- and heterodimers through a noncanonical Leu-zipper motif located in the C-terminal domain. A triple loss-of-function mutant of the three most closely related genes gebp gpl1 gpl2 shows a reduced sensitivity to exogenous cytokinins in a subset of cytokinin responses such as senescence and growth, whereas root inhibition is not affected. We find that transcript levels of type-A cytokinin response genes, which are involved in the negative feedback regulation of cytokinin signaling, are higher in the triple mutant. Using a GPL version that acts as a constitutive transcriptional activator, we show that the regulation of Arabidopsis response regulators (ARRs) is mediated by at least one additional, as yet unknown, repressor acting genetically downstream in the GeBP/GPL pathway. Our results indicate that GeBP/GPL genes encode a new class of unconventional Leu-zipper TF proteins and suggest that their role in the cytokinin pathway is to antagonize the negative feedback regulation on ARR genes to trigger the cytokinin response.

The plastid envelope of higher plant chloroplasts is a focal point of plant metabolism. It is involved in numerous pathways, including tetrapyrrole biosynthesis and protein translocation. Chloroplasts need to import a large number of proteins from the cytosol because most are encoded in the nucleus. Here we report that a loss-of-function mutation in the outer plastid envelope 16-kDa protein ( oep16 ) gene causes a conditional seedling lethal phenotype related to defects in import and assembly of NADPH:protochlorophyllide (Pchlide) oxidoreductase A. In the isolated knockout mutant of Arabidopsis thaliana , excess Pchlide accumulated in the dark operated as photosensitizer and provoked cell death during greening. Our results highlight the essential role of the substrate-dependent plastid import pathway of precursor Pchlide oxidoreductase A for seedling survival and the avoidance of developmentally programmed porphyria in higher plants.

Plants often respond to pathogens by sacrificing cells at the infection site. This type of programmed cell death is mimicked by the constitutive pathogene response5 (cpr5) mutant in Arabidopsis in the absence of pathogens, suggesting a role for CPR5 in programmed cell death control [1]. The analysis of the cellular phenotypes of two T-DNA-tagged cpr5 alleles revealed an additional role for CPR5 in the regulation of endoreduplication and cell division. In cpr5 mutant trichomes, endoreduplication cycles stop after two rounds instead of four, and trichome cells have fewer branches than normal. Eventually, cpr5 trichomes die, the nucleus disintegrates, and the cell collapses. Similarly, leaf growth stops earlier than in wild-type, and, frequently, regions displaying spontaneous cell death are observed. The cloning of the CPR5 gene revealed a novel putative transmembrane protein with a cytosolic domain containing a nuclear-targeting sequence. The dual role of CPR5 in cell proliferation and cell death control suggests a regulatory link between these two processes.

The biogenesis of microtubules comprises several steps, including the correct folding of α- and β-tubulin and heterodimer formation. In vitro studies and the genetic analysis in yeast revealed that, after translation, α- and β-tubulin are processed by several chaperonins [1, 2] and microtubule-folding cofactors (TFCs) to produce assembly-competent α-/β-tubulin heterodimers [3–11]. One of the TFCs, TFC-C, does not exist in yeast, and a potential function of TFC-C is thus based only on the biochemical analysis. In this study and in a very recently published study by Steinborn and coworkers [12], the analysis of the Arabidopsis porcino (por) mutant has shown that TFC-C is important for microtubule function in vivo. The predicted POR protein shares weak amino acid similarity with the human TFC-C (hTFC-C). Our finding that hTFC-C under the control of the ubiquitously expressed 35S promoter can rescue the por mutant phenotype shows that the POR gene encodes the Arabidopsis ortholog of hTFC-C. The analysis of plants carrying a GFP:POR fusion construct showed that POR protein is localized in the cytoplasm and is not associated with microtubules. While, in por mutants, microtubule density was indistinguishable from wild-type, their organization was affected.

Nodule-specific uricase (uricase II) from Phaseolus vulgaris L. was purified to homogeneity by chromatographic methods. Purification data indicated that uricase II is approximately 2% of the total soluble protein from mature nodules. Specific antiserum was raised and used to determine the developmental expression and for immunoselection of polysomes. Uricase II was antigenically detected early in nodule development, 2 to 3 days before nitrogen fixation. Uricase-encoding cDNA clones were isolated by hybridizing a nodule-specific pUC9 cDNA library with labeled mRNA from immunoselected polysomes and a 35,000 molecular weight uricase II-encoding cDNA from soybean. An homologous clone (pNF-UR07) was used to assess the expression pattern of the specific transcript during development. Northern-blot analysis indicated that uricase II mRNA is exclusively expressed in nodule tissue.