Volatile Organic Compounds: A Promising Tool for Bed Bug Detection.

The recent decades' resurgence of bed bugs as a public health concern in industrialized countries has driven an increased interest on new sustainable insecticide-free methods to monitor and control these ectoparasites. Current methods of detection rely mainly on visual inspection or canine scent detection, which are methods that are time-consuming, require experience, are non-specific or require costly mission repetitions. Volatile organic compounds (VOCs) are considered an environmentally friendly alternative and a promising approach for bed bug detection. An overview of the released literature on VOCs, their chemical characteristics and their role in bed bugs' intra- and inter-species communications allowed us to highlight the identification of 49 VOCs in Cimex lectularius (23 molecules) and C. hemipterus (26), which are emitted by both sexes during diverse compartments including aggregation (46), mating (11), defense (4), etc., and all life stages including exuviae or dead bed bugs as a principal indicator of infestation. The latter has a great importance for application of these semiochemicals in successful detection and control management of bed bugs and to prevent their further dispersion. This approach has the advantage of more reliability compared to conventional detection methods with no need for repeated inspections, household furniture moving or resident rehousing for bed bugs' VOC detection, which are commonly performed by active or passive sampling with absorbing tubes and analyzed by gas chromatography-based analytical platforms.

GUN Mutants: New Weapons To Unravel Ascospore Germination Regulation in the Model Fungus Podospora anserina.

In Podospora anserina as in many other Ascomycetes, ascospore germination is a regulated process that requires the breaking of dormancy. Despite its importance in survival and dispersal, ascospore germination in filamentous fungi has been poorly investigated, and little is known about its regulation and genetic control. We have designed a positive genetic screen that led to the isolation of mutants showing uncontrolled germination, the GUN (Germination UNcontrolled) mutants. Here, we report on the characterization of the gun1SG (Spontaneous Germination) mutant. We show that gun1SG is mutated in Pa_6_1340, the ortholog of Magnaporthe oryzae Pth2, which encodes a carnitine-acetyltransferase (CAT) involved in the shuttling of acetyl coenzyme A between peroxisomes and mitochondria and which is required for appressorium development. Bioinformatic analysis revealed that the mutated residue (I441) is highly conserved among Fungi and that the mutation has a deleterious impact on the protein function. We show that GUN1 is essential for ascospore germination and that the protein is localized both in mitochondria and in peroxisomes. Finally, epistasis studies allowed us to place GUN1 together with the PaMpk2 MAPK pathway upstream of the PaNox2/PaPls1 complex in the regulation of ascospore germination. In addition, we show that GUN1 plays a role in appressorium functioning. The pivotal role of GUN1, the ortholog of Pth2, in ascospore germination and in appressorium functioning reinforces the idea of a common genetic regulation governing both appressorium development and melanized ascospore germination. Furthermore, we characterize the second CAT encoded in P. anserina genome, Pa_3_7660/GUP1, and we show that the function of both CATs is conserved in P. anserina. IMPORTANCE The regulation of ascospore germination in filamentous fungi has been poorly investigated so far. To unravel new genes involved in this regulation pathway, we conducted a genetic screen in Podospora anserina, and we isolated 57 mutants affected in ascospore germination. Here, we describe the Germination UNcontrolled One (gun1SG) mutant, and we characterize the gene affected. GUN1 is a peroxisomal/mitochondrial carnitine-acetyltransferase required for acetyl coenzyme A shuttling between both organelles, and we show that GUN1 is a pleiotropic gene also involved in appressorium functioning similarly to its ortholog, the pathogenesis factor Pth2, in the plant pathogen Magnaporthe oryzae. Given the similarities in the regulation of appressorium development and ascospore germination, we speculate that discovering new genes controlling ascospore germination in P. anserina may lead to the discovery of new pathogenesis factors in pathogenic fungi. The characterization of GUN1, the ortholog of M. oryzae Pth2, represents a proof of concept.

Cyclooxygenases and lipoxygenases are used by the fungus Podospora anserina to repel nematodes.

Oxylipins are secondary messengers used universally in the living world for communication and defense. The paradigm is that they are produced enzymatically for the eicosanoids and non-enzymatically for the isoprostanoids. They are supposed to be degraded into volatile organic compounds (VOCs) and to participate in aroma production. Some such chemicals composed of eight carbons are also envisoned as alternatives to fossil fuels. In fungi, oxylipins have been mostly studied in Aspergilli and shown to be involved in signalling asexual versus sexual development, mycotoxin production and interaction with the host for pathogenic species. Through targeted gene deletions of genes encoding oxylipin-producing enzymes and chemical analysis of oxylipins and volatile organic compounds, we show that in the distantly-related ascomycete Podospora anserina, isoprostanoids are likely produced enzymatically. We show the disappearance in the mutants lacking lipoxygenases and cyclooxygenases of the production of 10-hydroxy-octadecadienoic acid and that of 1-octen-3-ol, a common volatile compound. Importantly, this was correlated with the inability of the mutants to repel nematodes as efficiently as the wild type. Overall, our data show that in this fungus, oxylipins are not involved in signalling development but may rather be used directly or as precursors in the production of odors against potential agressors. SIGNIFICANCE: We analyzse the role in inter-kingdom communication of lipoxygenase (lox) and cyclooxygenase (cox) genes in the model fungus Podospora anserina. Through chemical analysis we define the oxylipins and volatile organic compounds (VOCs)produce by wild type and mutants for cox and lox genes, We show that the COX and LOX genes are required for the production of some eight carbon VOCs. We show that COX and LOX genes are involved in the production of chemicals repelling nematodes. This role is very different from the ones previously evidenced in other fungi.

Identification of NoxD/Pro41 as the homologue of the p22phox NADPH oxidase subunit in fungi.

NADPH oxidases (Nox) are membrane complexes that produce O2(-). Researches in mammals, plants and fungi highlight the involvement of Nox-generated ROS in cell proliferation, differentiation and defense. In mammals, the core enzyme gp91(phox)/Nox2 is associated with p22(phox) forming the flavocytochrome b558 ready for activation by a cytosolic complex. Intriguingly, no homologue of the p22(phox) gene has been found in fungal genomes, questioning how the flavoenzyme forms. Using whole genome sequencing combined with phylogenetic analysis and structural studies, we identify the fungal p22(phox) homologue as being mutated in the Podospora anserina mutant IDC(509). Functional studies show that the fungal p22(phox), PaNoxD, acts along PaNox1, but not PaNox2, a second fungal gp91(phox) homologue. Finally, cytological analysis of functional tagged versions of PaNox1, PaNoxD and PaNoxR shows clear co-localization of PaNoxD and PaNox1 and unravel a dynamic assembly of the complex in the endoplasmic reticulum and in the vacuolar system.

Characterisation of exposure to airborne fungi: measurement of ergosterol.

In order to gain a clearer understanding of the level of fungal air contamination in indoor environments, we have adapted and tested a method to evaluate fungal biomass. Liquid phase chromatography (HPLC) of ergosterol, a component of the cell membrane of microscopic fungi, was employed. This method permits the detection and identification of ergosterol molecules at a concentration of 40 microg/ml (n=33, sigma=5). By combining this assay with a rotating cup collection apparatus, it was possible to measure fungal flora levels with a limit of quantification of 0.4 ng/m3 or a theoretical value of 150 spores per cubic meter (m3). Measurements of ergosterol levels performed on different sites showed that this method reflected the different situations of exposure of occupants to airborne fungal flora.