M. Patricia Juárez

Control of Pyrethroid-Resistant Chagas Disease Vectors with Entomopathogenic Fungi

Background Triatoma infestans-mediated transmission of Tripanosoma cruzi, the causative agent of Chagas disease, remains as a major health issue in southern South America. Key factors of T. infestans prevalence in specific areas of the geographic Gran Chaco region—which extends through northern Argentina, Bolivia, and Paraguay—are both recurrent reinfestations after insecticide spraying and emerging pyrethroid-resistance over the past ten years. Among alternative control tools, the pathogenicity of entomopathogenic fungi against triatomines is already known; furthermore, these fungi have the ability to fully degrade hydrocarbons from T. infestans cuticle and to utilize them as fuel and for incorporation into cellular components. Methodology and Findings Here we provide evidence of resistance-related cuticle differences; capillary gas chromatography coupled to mass spectrometry analyses revealed that pyrethroid-resistant bugs have significantly larger amounts of surface hydrocarbons, peaking 56.2±6.4% higher than susceptible specimens. Also, a thicker cuticle was detected by scanning electron microscopy (32.1±5.9 µm and 17.8±5.4 µm for pyrethroid-resistant and pyrethroid-susceptible, respectively). In laboratory bioassays, we showed that the virulence of the entomopathogenic fungi Beauveria bassiana against T. infestans was significantly enhanced after fungal adaptation to grow on a medium containing insect-like hydrocarbons as the carbon source, regardless of bug susceptibility to pyrethroids. We designed an attraction-infection trap based on manipulating T. infestans behavior in order to facilitate close contact with B. bassiana. Field assays performed in rural village houses infested with pyrethroid-resistant insects showed 52.4% bug mortality. Using available mathematical models, we predicted that further fungal applications could eventually halt infection transmission. Conclusions This low cost, low tech, ecologically friendly methodology could help in controlling the spread of pyrethroid-resistant bugs.

Cytochrome P450 associated with insecticide resistance catalyzes cuticular hydrocarbon production in Anopheles gambiae

Significance Malaria incidence has halved since 2000, with 80% of the reduction attributable to the use of insecticides, which now are under threat of resistance. Understanding the mechanisms of insecticide resistance is a key step in delaying and tackling the phenomenon. This study provides evidence of a cuticular mechanism that slows the uptake of pyrethroids, contributing to the resistance phenotype and potentially broadening resistance to multiple insecticide classes, thus providing additional challenges to resistance management. Quantitative modification of cuticular hydrocarbons is associated with increased expression of a 4G cytochrome P450 enzyme, CYP4G16, which catalyzes epicuticular hydrocarbon biosynthesis. This work improves our understanding of insecticide resistance and may facilitate the development of insecticides with greater specificity to mosquitoes and greater potency. The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of 14C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An. gambiae.

Tenebrionid secretions and a fungal benzoquinone oxidoreductase form competing components of an arms race between a host and pathogen

Significance Although entomopathogenic fungi and their invertebrate hosts share a >300 million year co-evolutionary history, little is known concerning the biochemical and genetic basis of insect defensive tactics and the countermeasures evolved and evolving by the pathogen to thwart these defenses. Our results provide a molecular mechanism to help explain why some insects are more resistant to broad host-range entomopathogenic fungi. We identify beetle cuticular secretions and a fungal detoxifying enzyme as components of an arms race between insects and the fungal pathogen, suggesting an evolving role for the quinone reductase enzyme as a specific virulence factor for host quinone detoxification. As races have winners and losers, this paper captures a snapshot where the host is leading the race. Entomopathogenic fungi and their insect hosts represent a model system for examining invertebrate-pathogen coevolutionary selection processes. Here we report the characterization of competing components of an arms race consisting of insect protective antimicrobial compounds and evolving fungal mechanisms of detoxification. The insect pathogenic fungus Beauveria bassiana has a remarkably wide host range; however, some insects are resistant to fungal infection. Among resistant insects is the tenebrionid beetle Tribolium castaneum that produces benzoquinone-containing defensive secretions. Reduced fungal germination and growth was seen in media containing T. castaneum dichloromethane extracts or synthetic benzoquinone. In response to benzoquinone exposure, the fungus expresses a 1,4-benzoquinone oxidoreductase, BbbqrA, induced >40-fold. Gene knockout mutants (ΔBbbqrA) showed increased growth inhibition, whereas B. bassiana overexpressing BbbqrA (Bb::BbbqrAO) displayed increased resistance to benzoquinone compared with wild type. Increased benzoquinone reductase activity was detected in wild-type cells exposed to benzoquinone and in the overexpression strain. Heterologous expression and purification of BbBqrA in Escherichia coli confirmed NAD(P)H-dependent benzoquinone reductase activity. The ΔBbbqrA strain showed decreased virulence toward T. castaneum, whereas overexpression of BbbqrA increased mortality versus T. castaneum. No change in virulence was seen for the ΔBbbqrA or Bb::BbbqrAO strains when tested against the greater wax moth Galleria mellonella or the beetle Sitophilus oryzae, neither of which produce significant amounts of cuticular quinones. The observation that artificial overexpression of BbbqrA results in increased virulence only toward quinone-secreting insects implies the lack of strong selection or current failure of B. bassiana to counteradapt to this particular host defense throughout evolution.

Epicuticle Lipids Mediate Mate Recognition in Triatoma infestans

Epicuticular lipids are contact cues in intraspecific chemical communication in insects, both for aggregation and sexual behavior. Triatomine bugs are vectors of the parasite Trypanosoma cruzi, the cause of Chagas disease. In Triatoma infestans, the major epicuticular lipids are hydrocarbons, fatty alcohols, and free and esterified fatty acids. Previously, we found that epicuticular lipid extracts, or selected fatty acid components, trigger aggregation and arrestment behavior in this bug. Using headspace solid phase microextraction, we found no sexual dimorphism in epicuticular hydrocarbons, but found female-specific fatty alcohols (eicosanol and docosanol). The role of epicuticular lipids in T. infestans copulation behavior was tested by observing male responses to live or various treatments of freeze-killed females. We report that hexane-soluble contact cues on females trigger copulation by males. Freeze-killed intact females were attractive to males, but no response was observed when males were exposed to hexane-washed females. Responses were partially recovered when epicuticular extract was applied to the dorsal surface of dead, hexane-washed females. One female equivalent of docosanol, evoked similar responses.

Cuticular Hydrocarbons of Triatoma dimidiata (Hemiptera: Reduviidae): Intraspecific Variation and Chemotaxonomy

ABSTRACT Triatoma dimidiata Latreille is a major vector of Chagas disease with an extensive geographic distribution from Central Mexico, through Central America, to northern South America. As a result of its variability in phenetic and genetic characters, disagreement concerning its taxonomic status has been raised. In this study, the cuticular hydrocarbon pattern of T. dimidiata populations from Mexico, Belize, Guatemala, Honduras, Costa Rica, and Colombia was analyzed by capillary gas chromatography coupled to mass spectrometry; linear discriminant analysis was used to help elucidate population structure. Vector populations segregated into five distinct groups; specimens from Yucatan Peninsula, together with those from Central Mexico, Central America, and Colombia corresponded to different T. dimidiata subspecies, a putative different species comprising insects from Belize, together with an isolated population collected at bat caves in Guatemala. The analysis revalidates the earlier division of T. dimidiata into three subspecies, T. d. maculipennis, T. d. dimidiata, and T. d. capitata; and an additional subspecies and a distinct species are proposed.

Contribution of the horizontal transmission of the entomopathogenic fungus Beauveria bassiana to the overall performance of a fungal powder formulation against Triatoma infestans

Control of domiciliated Triatoma infestans, the major Chagas disease vector in southern South America, is currently achieved by indoor residual spraying of infested houses with chemical insecticides. However, in recent years this strategy has been threatened by the emergence of pyrethroid-resistant bug populations. As an alternative approach, we have previously demonstrated the efficacy of the entomopathogenic fungus Beauveria bassiana to control T. infestans bugs regardless of their pyrethroid susceptibility. In this work, we tested the virulence and residual activity of a powdered fungal formulation, and studied the significance of the horizontal transmission process (autodissemination) to fungal infection of bugs. The B. bassiana-based formulation was highly virulent against all T. infestans stages, and maintained its insecticidal capability for at least 5 months under natural ambient conditions. We showed that horizontal transmission of conidia is associated to bug density, and contributes significantly to the overall population infection event.

Toxicological and biochemical response of the entomopathogenic fungus Beauveria bassiana after exposure to deltamethrin

BACKGROUND The chemical control of the Chagas disease vector Triatoma infestans is endangered by the emergence of pyrethroid resistance. An effective alternative control tool is the use of the entomopathogenic fungus Beauveria bassiana. The effect of deltamethrin on fungal growth, gene expression and enzyme activity in relation to detoxification, antioxidant response and oxidative stress levels was studied to evaluate fungal tolerance to deltamethrin. RESULTS The mean inhibitory concentration (IC50 ) was 50 µg deltamethrin/cm(2). Cytochrome P450 genes were differentially expressed; cyp52X1 and cyp617N1 transcripts were > 2-fold induced, followed by cyp655C1 (1.8-fold). Minor effects were observed on genes encoding for other P450s, epoxide hydrolase and glutathione S-transferase (GST). Superoxide dismutase (SOD) genes showed induction levels ≤ 2, catalase (CAT) and glutathione peroxidase genes were also induced ∼ 2-3-fold and < 2-fold, respectively. The activities of enzymes participating in the antioxidant defense system and phase II detoxification were also evaluated; SOD, CAT and GST activity showed significant differences with deltamethrin concentration. Lipid peroxidation levels and free proline content were also altered. CONCLUSIONS Beauveria bassiana GHA can be used combined with deltamethrin without significant metabolic detrimental effects. This combination will help optimizing the benefits and increasing the efficacy of vector control tools.

Cuticular hydrocarbons of Chagas disease vectors in Mexico

Capillary gas-liquid chromatography was used to analyse the cuticular hydrocarbons of three triatomine species, Triatoma dimidiata, T. barberi and Dipetalogaster maxima, domestic vectors of Chagas disease in Mexico. Mixtures of saturated hydrocarbons of straight and methyl-branched chains were characteristic of the three species, but quantitatively different. Major methylbranched components mostly corresponded to different saturated isomers of monomethyl, dimethyl and trimethyl branched hydrocarbons ranging from 29 to 39 carbon backbones. Sex-dependent, quantitative differences in certain hydrocarbons were apparent in T. dimidiata.

Tracing the coevolution between Triatoma infestans and its fungal pathogen Beauveria bassiana

The chemical control of Triatoma infestans, the major Chagas disease vector in southern South America, has been threatened in the last years by the emergence of pyrethroid-resistant bug populations. As an alternative approach, the efficacy of the entomopathogenic fungus Beauveria bassiana to control T. infestans populations (regardless their pyrethroid susceptibility) has been demonstrated. Growing research efforts on the interaction between T. infestans and B. bassiana by molecular, ecological, biochemical and behavioral traits has allowed framing such interaction as an evolutionary arms race. This review will focus on the relationships established in this particular host-pathogen system, compiling available data on the relevance of fungal pathogenesis, insect behavior, population dynamics and human intervention to favor fungal dissemination in bug populations. The current snapshot shows the fungus ahead in the evolutionary arms race and predicts a promissory landscape for the biological control of Chagas disease vectors.

Integument CYP genes of the largest genome-wide cytochrome P450 expansions in triatomines participate in detoxification in deltamethrin-resistant Triatoma infestans

Insect resistance to chemical insecticides is attributed to a combination of different mechanisms, such as metabolic resistance, knockdown resistance, and the cuticular resistance or penetration factor. The insect integument offers an efficient barrier against contact insecticides and its role as penetration factor has been previously reported; however, there is no information about its potential function in the metabolic resistance. Cytochrome P450 genes (CYP) are highly expressed in the fat body of several insects and thus play a key role in their metabolic resistance. Here, we describe new members that belong to the highly genome-wide expanded CYP3093A and CYP4EM subfamilies in the Chagas disease vectors Rhodnius prolixus and Triatoma infestans. We modeled the docking of deltamethrin in their active site and detected differences in some amino acids between both species that are critical for a correct interaction with the substrate. We also knocked down the two constitutively most expressed genes in the integument of resistant T. infestans nymphs (CYP3093A11 and CYP4EM10) in order to find clues on their participation in deltamethrin resistance. This is the first report on the role of the insect integument in detoxification events; although these two CYP genes do not fully explain the resistance observed in T. infestans.