Deciphering the role of Rhodnius prolixus CYP4G genes in straight and methyl-branched hydrocarbon formation and in desiccation tolerance.

Insect cuticle hydrocarbons are involved primarily in waterproofing the cuticle, but also participate in chemical communication and regulate the penetration of insecticides and microorganisms. The last step in insect hydrocarbon biosynthesis is carried out by an insect-specific cytochrome P450 of the 4G subfamily (CYP4G). Two genes (CYP4G106 and CYP4G107) have been reported in the triatomines Rhodnius prolixus and Triatoma infestans. In this work, their molecular and functional characterization is carried out in R. prolixus, and their relevance to insect survival is assessed. Both genes are expressed almost exclusively in the integument and have an expression pattern dependent on the developmental stage and feeding status. CYP4G106 silencing diminished significantly the straight-chain hydrocarbon production while a significant reduction - mostly of methyl-branched chain hydrocarbons - was observed after CYP4G107 silencing. Molecular docking analyses using different aldehydes as hydrocarbon precursors predicted a better fit of straight-chain aldehydes with CYP4G106 and methyl-branched aldehydes with CYP4G107. Survival bioassays exposing the silenced insects to desiccation stress showed that CYP4G107 is determinant for the waterproofing properties of the R. prolixus cuticle. This is the first report on the in vivo specificity of two CYP4Gs to make mostly straight or methyl-branched hydrocarbons, and also on their differential contribution to insect desiccation.

A fatty acid synthase gene (FASN3) from the integument tissue of Rhodnius prolixus contributes to cuticle water loss regulation.

Fatty acid synthase is a multifunctional enzyme involved in the formation of fatty acids. Despite the role of fatty acids in cell signalling and energy metabolism, and as precursors to pheromones and hydrocarbons that waterproof the cuticle, the insect fatty acid synthases have been scarcely studied. Here we perform the molecular characterization of three fatty acid synthase genes (fatty acid synthase RPRC000123, RPRC000269 and RPRC002909) in the Chagas disease vector, Rhodnius prolixus. Gene expression screening by reverse transcription quantitative PCR showed that RPRC000123 and RPRC002909 are expressed almost exclusively in the integument tissue whilst RPRC000269 is mostly expressed in the fat body and also in several body organs. Phylogenetic analysis, together with gene expression results, showed that RPRC000269, RPRC002909 and RPRC000123 are orthologues of Drosophila melanogaster fatty acid synthase 1 (FASN1), FASN2 and FASN3 genes, respectively. After RNA interference-mediated knockdown of RPRC000123, insects died immediately after moulting to the next developmental stage. However, mortality was prevented by placing the insects under saturated humidity conditions, suggesting that dehydration might play a role in the insects' death. Lipid analyses in RPRC000123-silenced insects showed reduced amounts of integument fatty acids and methyl-branched hydrocarbons, compared to controls. These data support an important role for FASN3 in the biosynthesis of the precursors to hydrocarbons that waterproof the insect cuticle.

Deltamethrin Binding to Triatoma infestans (Hemiptera: Reduviidae) Lipoproteins. Analysis by Solvent Bar Microextraction Coupled to Gas Chromatography.

The binding of deltamethrin (DLM) to the hemipteran Triatoma infestans (Klug) hemolymph lipoproteins was evaluated in vitro. After DLM incubation with the insect hemolymph, lipoproteins were fractioned by ultracentrifugation. DLM binding was analyzed by a microextractive technique-solvent bar microextraction-a solventless methodology to extract DLM from each lipoprotein fraction. This is a novel use of the technique applied to extract an insecticide from an insect fluid. Capillary gas chromatography with microelectron capture detection was used to detect DLM bound by the T. infestans hemolymph lipoproteins and to identify the preferred DLM carrier. We show that Lp and VHDLp I lipoproteins are mainly responsible for DLM transport in T. infestans, both in DLM-resistant and DLM-susceptible bugs. Our results also indicate that DLM amounts transported are not related to DLM susceptibility.

Cuticular hydrocarbon pattern as a chemotaxonomy marker to assess intraspecific variability in Triatoma infestans, a major vector of Chagas' disease.

Triatoma infestans Klug (Hemiptera: Reduviidae) populations were sampled in various localities throughout most of the species' geographic range of distribution in Argentina, Bolivia, Paraguay and Peru. In order to contribute to understanding of the diversity and population structure of this major vector of Chagas' disease, cuticular hydrocarbon (CHC) profiles were analysed by capillary gas chromatography and variations evaluated by statistical methods of classification and ordination. High levels of intrapopulation variation were detected, along with low levels of variability among populations. Based on relative amounts of the major odd-numbered straight-chain hydrocarbons n-C27 to n-C33, two hydrocarbon phenotypes were evident, unequally distributed along the species' geographic range. Analysis of CHC patterns showed that T. infestans populations segregate into two major groups consisting of an Andean group, which comprises specimens from Peru and most parts of Bolivia, and a non-Andean group, which includes all specimens from Argentina and Paraguay, together with those from Tarija (Bolivia). Pyrethroid-resistant and -susceptible specimens were differentiated based on relative amounts of some straight and monomethyl-branched hydrocarbon components.

Beauveria bassiana infection alters colony development and defensive secretions of the beetles Tribolium castaneum and Ulomoides dermestoides (Coleoptera: Tenebrionidae).

We studied the effect of the entomopathogenic fungus Beauveria bassiana strain GHA on a) colony development of the beetles Tribolium castaneum (Herbst) and Ulomoides dermestoides (Fairmaire) (Coleoptera: Tenebrionidae) under laboratory conditions; and 2) the volatile blend released by both beetles, containing defensive pheromones, by using the solid phase microextraction technique. Colony development of both species was strongly altered 3 mo after treatment with B. bassiana, showing a significant reduction in progeny of 37.5% for T. castaneum and 50.0% for U. dermestoides. We also showed that the volatiles released by T. castaneum diminished close to 20% compared with those of healthy beetles, whereas in U. dermestoides secretions dramatically dropped to 5%, 7 d after immersion in 1 x 10(9) conidia per ml. These results suggest that after infection events take place, fungus-induced diminished secretion of the defensive pheromones may be a physiologic clue for behavioral changes in infected beetles.

Volatile organic compounds released by the entomopathogenic fungus Beauveria bassiana.

The composition of volatile organic compounds (VOC) released by the entomopathogenic fungus Beauveria bassiana (Hyphomycete: Deuteromycotina) utilizing two different carbon sources was investigated. Analyses were performed by solid-phase microextraction (SPME) coupled to capillary gas chromatography (CGC) and CGC-mass spectrometry (MS). Major components in glucose-grown cultures were diisopropyl naphthalenes, ethanol, and sesquiterpenes. Alkane-grown fungal VOC switched to a fingerprint with prevalence of n-decane. This is the first report on the volatiles released by entomopathogenic fungi.

Cuticular hydrocarbons of triatomines.

Triatomine insects (Hemiptera) are the vectors of Chagas disease. Their cuticular surface is covered by a thin layer of lipids, mainly hydrocarbons, wax esters, fatty alcohols, and free or esterified fatty acids. These lipids play a major role in preventing a lethal desiccation, altering the absorption of chemicals and microorganism penetration, they also participate in chemical communication events. Lipid components are biosynthetically related, the synthesis of long chain and very long chain fatty acids was first shown in the integument of Triatoma infestans through the concerted action of fatty acid synthases (FAS's) and fatty acyl-CoA elongases. A final decarboxylation step produces the corresponding hydrocarbon. Capillary gas chromatography coupled to mass spectrometry analyses showed that cuticular hydrocarbons of Triatominae comprise saturated straight and methyl-branched chains, from 18 to more than 43 carbon atoms. Odd-chain hydrocarbons, mostly from 27 to 33 carbons, are the major straight chains. Different isomers of mono, di, tri, and tetramethylcomponents, mostly from 29 to 39 atoms in the carbon skeleton, account for the major methyl-branched hydrocarbons. The presence, absence, and relative quantities of these hydrocarbons represent characters for their chemical phenotype, and are useful for differentiating genera, species and populations. In this review, we will discuss the metabolic pathways involved in hydrocarbon formation, and their structure, together with their role in insect survival. We will also review the utility of cuticular hydrocarbon fingerprints in chemotaxonomy.

Cuticular hydrocarbon variability among Triatoma dimidiata (Hemiptera: Reduviidae) populations from Mexico and Guatemala.

The geographic variation in the cuticular hydrocarbon pattern among 11 populations of Triatoma dimidiata Latreille (Hemiptera: Reduviidae: Triatominae) from different regions of Mexico and Guatemala, was studied using capillary gas chromatography. T. dimidiata populations were differentiated based on the relative amounts of 71 hydrocarbon components. Insect population classification was mostly in agreement with their geographical vicinity; Mexican populations from the Yucatan peninsula grouped together with those from northern Guatemala, insects from the Mexican Gulf coast states were closely related to those collected from northern Oaxaca, and to a lesser extent, to insects from Chiapas. Insects from southern Oaxaca were clustered together with those from southern Guatemala. All these populations were clearly separated from Guatemalan specimens collected in caves from Alta Verapaz.

Intraspecific variability in Triatoma dimidiata (Hemiptera: Reduviidae) populations from Guatemala based on chemical and morphometric analyses.

The intraspecific variability of Triatoma dimidiata Latreille, a major vector of Chagas disease, was studied in four departments of Guatemala. Insects were collected from either domestic and sylvatic habitats, and their cuticular hydrocarbon pattern and head morphology were analyzed using ordination and classification techniques. A significant discrimination was obtained both with morphometric and hydrocarbon analyses. Insects from northern departments were easily differentiated from southern conspecifics. Distinctive hydrocarbon pattern and head shape were detected for insects collected from caves in the north central region of the country, posing concern about their taxonomic status.

Characterization and carbon metabolism in fungi pathogenic to Triatoma infestans, a chagas disease vector.

The pathogenicity of Metarhizium anisopliae (Ma) and Beauveria bassiana (Bb) isolates against Triatoma infestans, the major vector of Chagas disease in Argentina is reported. A 100% mortality was achieved with mean lethal times varying form 5.8 (Ma6) to 7.7 (Bb5) or 11.1 days (Bb10). The fatty acid, hydrocarbon, and total lipid patterns were compared for glucose-grown and alkane-grown Bb10 cultures. The alkane-grown cells showed a lipid pattern different from that of glucose-grown cells, with triacylglyercol as the major lipid fraction, whereas sterols prevailed in the glucose-grown cells. A significant reduction in the relative amounts of linoleic acid diminished the unsaturated/saturated fatty acid ratio for alkane-grown cells; in addition, large amounts of heptacosanoic and eicosanoic acids were detected in the saturated fraction. The hydrocarbon profile of Bb10 showed a saturated chain length distribution,with a marked prevalence for straight chains, ranging from n-C18 to n-C37 in the carbon skeleton, with n-C22 as the major component. Alkane-grown cells showed no qualitative changes in their hydrocarbon fraction, but a similar ratio for odd/even carbon chains. After 48-h incubation assays,[1-(14)C]acetate uptake was largely diminished following a period of alkane growth induction. Glucose-grown cells readily incorporated 19% of the labelinto phospholipids, hydrocarbons, triacylglycerols, and free fatty acids. In contrast, incorporation was reduced to 5.3% for alkane-grown cells, accounting only for phospholipid synthesis.

Effects of organic acids on lipid synthesis and ecdysis in Triatoma infestans eggs.

Medium chain fatty acids are known inhibitors of the activity of lipogenic enzymes at the transcriptional level. Mature Triatoma infestans eggs incorporate [14C]-acetate into phospholipid (PL), triacylglycerol (TG), and free fatty acid (FFA) fractions. In the presence of sodium octanoate (SO) or sodium phenylethylpropionate (PHEP), lipid synthesis is inhibited in a dose-dependent manner. When eggs are incubated in vivo with [14C]-acetate, the usual fatty acid products are largely palmitic and oleic acids. However, in the presence of SO (5-10 mM), the elongation of [14C]-acetate units is interrupted at [14C] eight total carbons. Eggs incubated in vivo with [14C]-SO, accumulate most of the label in the FFA fraction. SO (> 0.1 mM) but not sodium hexanoate (SH), inhibits the activity of microsomal and cytosolic fatty acid synthetases (FAS's), measured as [14C]-malonyl-CoA incorporation. PHEP (1 mM) and SO (10 mM) also produce major alterations in egg hatching and survival of the emerged insects, after a 1 h immersion bioassay.

Entomopathogenous fungi degrade epicuticular hydrocarbons of Triatoma infestans.

Studies were undertaken to analyze the ability of entomopathogenous fungi to degrade insect hydrocarbons. Strains of Beauveria bassiana and Metarhizium anisopliae pathogenic to the blood-sucking bug Triatoma infestans were grown on hydrocarbon and non-hydrocarbon insect lipid extracts and on synthetic hydrocarbon-enriched media as the sole carbon source. Entomopathogenous fungi were shown to utilize hydrocarbons as the only carbon source for their growth. Insect-derived hydrocarbons served more efficiently as metabolic fuel rather than synthetic compounds of similar structure. [3H]n-Pentacosane, [11,12-3H]3,11-dimethylnonacosane, and [14C]n-hexadecane were catabolized into different amounts of polar lipids, free fatty acids, and acylglycerols. In experiments using the branched alkane, labeled hydrocarbons of different chain length than the precursor were also synthesized. Evidence of complete catabolism was obtained by a significant release of 14CO2 from [1-14C]n-hexadecane. 14CO2 production might be used as a simple method to compare hydrocarbon utilization by fungal strains. These data demonstrate that entomopathogenous fungi are able to transform a variety of hydrocarbon structures into different lipid products, part of which may be subsequently utilized for energy production and for the biosynthesis of cellular components. These data are the first evidence of hydrocarbon catabolism and synthesis in entomopathogenous fungi.