Multifaceted Beauveria bassiana and Other Insect-Related Fungi.

Since Agostino Bassi first isolated the fungal pathogenic agent of the white muscardine in insects (later named Beauveria bassiana in his honor), and Ilya Mechnikov cultivated Metarhizium anisopliae as a first approach to use fungi as pest control agents, many other entomopathogenic fungi have been studied over the last two centuries [...].

Beauveria bassiana transcriptomics reveal virulence-associated shifts during insect lipid assimilation.

Insect cuticular lipids, especially epicuticular hydrocarbons (CHC), have a significant role in insect ecology and interactions with other organisms, including fungi. The CHC composition of a specific insect species may influence the outcome of the interaction with a specific fungal strain. Some insects, such as Piezodorus guildinii, have low susceptibility towards fungal infections seemingly due to their CHC composition. The entomopathogenic fungus Beauveria bassiana can assimilate CHC and incorporate them as building blocks via cytochrome P450 monooxygenases (CYPs). However, little is known about other enzymes that promote the degradation/assimilation of these cuticular components. In this study, we performed a transcriptomic analysis to evaluate the in vitro response of two virulence-contrasting B. bassiana strains when grown on three different P. guildinii CHC sources. We found a different expression profile of virulence-related genes, as well as different GO and KEGG parameters enriched at 4 days post-inoculation, which could help account for the intrinsic virulence and for an alkane-priming virulence enhancement effect. The hypovirulent strain predominantly showed higher expression of cuticle penetration genes, including chitinases, proteases, and CYPs, with GO term categories of "heme binding," "monooxygenase activity," and "peroxisome" pathways enriched. The hypervirulent strain showed higher expression of cell wall remodeling and cell cycle genes, and cuticle adhesion and a distinct set of CYPs, with GO categories of "DNA-binding transcription factor activity" and KEGG pathways corresponding to "meiosis-yeast" and "cell cycle" enriched. These results suggest a delay and alternate routes in pathogenicity-related metabolism in the hypovirulent strain in comparison with the hypervirulent strain. KEY POINTS: •Transcriptomics of two B. bassiana strains grown in P. guildinii cuticular components •Virulence-related genes correlated with virulence enhancement towards P. guildinii •Differentially expressed genes, GOs and KEGGs showed different metabolic timelines associated with virulence.

Pathogenicity of microsclerotia from Metarhizium robertsii against Aedes aegypti larvae and antimicrobial peptides expression by mosquitoes during fungal-host interaction.

Aedes aegypti is a vector of various disease-causing arboviruses. Chemical insecticide-based methods for mosquito control have increased resistance in different parts of the world. Thus, alternative control agents such as the entomopathogenic fungi are excellent candidates to control mosquitoes as part of an ecofriendly strategy. There is evidence of the potential of entomopathogenic fungal conidia and blastospores for biological control of eggs, larval and adult stages, as well as the pathogenicity of fungal microsclerotia against adults and eggs. However, there are no studies on the pathogenicity of microsclerotia against either aquatic insects or insects that develop part of their life cycle in the water, such as the A. aegypti larvae. In this study, we assayed the production of microsclerotia and their pathogenicity against A. aegypti larvae of two isolates of Metarhizium robertsii, i.e., CEP 423 isolated in La Plata, Argentina, and the model ARSEF 2575. Both isolates significantly reduced the survival of A. aegypti exposed to their microsclerotia. The fungus-larva interaction resulted in a delayed response in the host. This was evidenced by the expression of some humoral immune system genes such as defensins and cecropin on the 9th day post-infection, when the fungal infection was consolidated as a successful process that culminates in larvae mortality. In conclusion, M. robertsii microsclerotia are promising propagules to be applied as biological control agents against mosquitoes since they produce pathogenic conidia against A. aegypti larvae.

Dark Pigments in Entomopathogenic Fungal Microsclerotia: Preliminary Evidence of a 1,8-Dihydroxynaphthalene-melanin-like Compound in Metarhizium robertsii.

Metarhizium robertsii microsclerotia are fungal aggregates composed of compacted, pigmented hyphae. As they are highly tolerant to desiccation and produce infective conidia, they are promising candidates to be formulated as bioinsecticides. Despite this potential, the nature of the pigments within these structures remains unclear. In this study, routine culture media used for the differentiation of M. robertsii microsclerotia were supplemented with four melanin inhibitors, and the resulting propagules were characterized. Inhibitors of the 1,8-dihydroxynaphthalene (DHN)-melanin biosynthetic pathway such as tricyclazole and guaiacol induced significant phenotypic and molecular modifications in the obtained M. robertsii propagules, which exhibited a more spherical shape, reduced size, and increased susceptibility to desiccation, heat, and oxidative stress than microsclerotia obtained without inhibitors. Additionally, genes encoding for a polyketide synthase (Mrpks2) and a putative 1,3,6,8-tetrahydroxynaphthalene reductase (Mrthnr), potentially involved in the DHN-melanin biosynthetic pathway, were upregulated in fungi grown in the inhibitor-added media. In conclusion, M. robertsii microsclerotia contain melanins of type DHN that might play a role in both microsclerotia differentiation and environmental stress tolerance.

Transcriptomic landscape of the interaction between the entomopathogenic fungus Beauveria bassiana and its tolerant host Tribolium castaneum revealed by dual RNA-seq.

Entomopathogenic fungi such as Beauveria bassiana are the only insect pathogens able to start the infection process by penetrating through the host cuticle. However, some insects try to avoid fungal infection by embedding their cuticle with antifungal compounds. This is the case of the red flour beetle Tribolium castaneum, which generates economical loss of great significance in stored product environments worldwide. In this study, T. castaneum adults were fed during different time periods (from 3 to 72 h) on B. bassiana conidia-covered corn kernels. The progression of fungal infection was monitored using the dual RNA-seq technique to reconstruct the temporal transcriptomic profile and to perform gene enrichment analyses in both interacting organisms. After mapping the total reads with the B. bassiana genome, 904 genes were identified during this process. The more expressed fungal genes were related to carbon catabolite repression, cation binding, peptidase inhibition, redox processes, and stress response. Several immune-related genes from Toll, IMD, and JNK pathways, as well as genes related to chitin modification, were found to be differentially expressed in fungus-exposed T. castaneum. This study represents the first dual transcriptomic approach to help understand the interaction between the entomopathogenic fungus B. bassiana and its tolerant host T. castaneum.

Asphyxiation of Metarhizium robertsii during mycelial growth produces conidia with increased stress tolerance via increased expression of stress-related genes.

Little is known about the impact of hypoxia and anoxia during mycelial growth on tolerance to different stress conditions of developing fungal conidia. Conidia of the insect-pathogenic fungus Metarhizium robertsii were produced on potato dextrose agar (PDA) medium under normoxia (control = normal oxygen concentrations), continuous hypoxia, and transient anoxia, as well as minimal medium under normoxia. The tolerance of the conidia produced under these different conditions was evaluated in relation to wet heat (heat stress), menadione (oxidative stress), potassium chloride (osmotic stress), UV radiation, and 4-nitroquinoline-1-oxide (=4-NQO genotoxic stress). Growth under hypoxic condition induced higher conidial tolerance of M. robertsii to menadione, KCl, and UV radiation. Transient anoxic condition induced higher conidial tolerance to KCl and UV radiation. Nutritional stress (i.e., minimal medium) induced higher conidial tolerance to heat, menadione, KCl, and UV radiation. However, neither of these treatments induced higher tolerance to 4-NQO. The gene hsp30 and hsp101 encoding a heat shock protein was upregulated under anoxic condition. In conclusion, growth under hypoxia and anoxia produced conidia with higher stress tolerances than conidia produced in normoxic condition. The nutritive stress generated by minimal medium, however, induced much higher stress tolerances. This condition also caused the highest level of gene expression in the hsp30 and hsp101 genes. Thus, the conidia produced under nutritive stress, hypoxia, and anoxia had greater adaptation to stress.

Microencapsulation of a Native Strain of the Entomopathogenic Fungus Beauveria bassiana and Bioinsecticide Activity against Pyrethroid-Resistant Triatoma infestans to Vector Control of Chagas Disease in the Argentine Gran Chaco Region.

The blood-sucking bug Triatoma infestans is the main Chagas disease vector in the Southern Cone of Latin America. Populations resistant to pyrethroid insecticides have been detected in the early 2000s and then expanded to the endemic area of northern Salta province, Argentina. In this context, the entomopathogenic fungus Beauveria bassiana has been shown to be pathogenic to pyrethroid-resistant T. infestans. In this study, both the bioinsecticidal activity and the residual effect of an alginate-based microencapsulation of a native strain of B. bassiana (Bb-C001) were tested under semi-field conditions against pyrethroid-resistant T. infestans nymphs. Fungal microencapsulated formulation caused higher nymph mortality than the unmicroencapsulated fungus and contributed to maintaining the conidial viability throughout the period evaluated under the tested conditions. These results suggest that alginate microencapsulation is an effective, simple, low-cost method that could be incorporated into the formulation of a bioinsecticide as a strategy to reduce the vector transmission of Chagas disease.

Secretion and Detection of Defensive Compounds by the Red Flour Beetle Tribolium castaneum Interacting with the Insect Pathogenic Fungus Beauveria bassiana.

Entomopathogenic fungi such as Beauveria bassiana are extensively used for the control of insect pests worldwide. They infect mostly by adhesion to the insect surface and penetration through the cuticle. However, some insects, such as the red flour beetle Tribolium castaneum (Herbst), have evolved resistance by embedding their cuticle with antifungal compounds. Thus, they avoid fungal germination on the cuticle, which result in low susceptibility to entomopathogenic fungi. In adult T. castaneum, these antifungals are the well-known defensive compounds methyl-1,4- and ethyl-1,4-benzoquinone. In this study, we added B. bassiana conidia on the diet of adult beetles to study the effect of the entomopathogen on the secretion and detection of the beetle volatile blend containing both benzoquinones. The compounds were analyzed by solid phase microextraction coupled to gas chromatography-flame ionization detection, and were detected by electroantennography. In addition, we measured the expression level of four genes encoding for two odorant-binding proteins (OBP), one chemosensory protein (CSP), and one odorant receptor (OR) in both healthy and fungus-treated insects. Significant alterations in the secretion of both benzoquinones, as well as in the perception of methyl-1,4-benzoquinone, were found in fungus-treated insects. TcOBP7D, TcOBP0A and TcCSP3A genes were down-regulated in insects fed conidia for 12 and 48 h, and the latter gene was up-regulated in 72 h samples. TcOR1 expression was not altered at the feeding times studied. We conclude that fungus-treated insects alter both secretion and perception of benzoquinones, but additional functional and genetic studies are needed to fully understand the effects of fungal infection on the insect chemical ecology.

The Entomopathogenic Fungus Beauveria bassiana Shows Its Toxic Side within Insects: Expression of Genes Encoding Secondary Metabolites during Pathogenesis.

Entomopathogenic fungi are extensively used for the control of insect pests worldwide. Among them, Beauveria bassiana (Ascomycota: Hypocreales) produce a plethora of toxic secondary metabolites that either facilitate fungal invasion or act as immunosuppressive compounds. These toxins have different chemical natures, such as nonribosomal peptides and polyketides. Even though their precise role is poorly understood, they are usually linked to virulence. These fungal secondary metabolites are produced by the expression of gene clusters encoding the various proteins needed for their biosynthesis. Each cluster includes synthetases for nonribosomal peptides (NRPS), polyketides (PKS), or hybrid NRPS-PKS genes. The aim of this review is to summarize the information available from transcriptomics and quantitative PCR studies related to the expression of B. bassiana NRPS and PKS genes inside different insects as the infection progresses; as for the host immune response, to help understand the mechanisms that these toxins trigger as virulence factors, antimicrobials, or immunosuppressives within the context of a fungus-insect interaction.

Alkane-priming of Beauveria bassiana strains to improve biocontrol of the redbanded stink bug Piezodorus guildinii and the bronze bug Thaumastocoris peregrinus.

Insect epicuticle hydrocarbons (CHC) are known to be important determinants in the susceptibility degree of insects to fungal entomopathogens. Five Beauveria bassiana (Balsamo) Vuillemin (Hypocreales; Clavicipitaceae) strains were phenotypically analyzed regarding their response to CHC nutrition and their pathogenicity and virulence towards high fungal-susceptible Thaumastocoris peregrinus (Carpintero and Dellapé) (Heteroptera: Thaumastocoridae) and low fungal-susceptible Piezodorus guildinii (Westwood) (Hemiptera: Pentatomidae), which are important hemipteran pests in eucalyptus and soybean plantations, respectively. Two of these strains, which were the most (ILB308) and the least (ILB299) virulent to P. guildinii, were also evaluated at gene expression level after growth on n-pentadecane, a P. guildinii epicuticular hydrocarbon. Beauveria bassiana hypervirulent strain ILB308 showed the lowest growth on most evaluated CHC media. However, this strain distinctively induced most of the analyzed genes involved in CHC assimilation, cuticle degradation and stress tolerance. Virulence towards low susceptibility P. guildinii was enhanced in both hypervirulent ILB308 and hypovirulent ILB299 strains after growth on n-pentadecane as the sole carbon source, whereas virulence enhancement towards high susceptibility T. peregrinus was only observed in the hypervirulent strain. Virulence enhancement towards P. guildinii could be mostly explained by a priming effect produced by CHC on the induction of some genes related to hydrocarbon assimilation in ILB299 and ILB308, such as cytochrome P450 genes (BbCyp52g11 and BbCyp52x1), together with adhesion and stress tolerance genes, such as hydrophobin (Bbhyd2) and catalase (Bbcatc) and glutathione peroxidase (Bbgpx), respectively.

Epicuticular hydrocarbons of the redbanded stink bug Piezodorus guildinii (Heteroptera: Pentatomidae): sexual dimorphism and alterations in insects collected in insecticide-treated soybean crops.

BACKGROUND: The redbanded stink bug Piezodorus guildinii (Heteroptera: Pentatomidae) is one of the most important species affecting soybean crops in southern South America. Capillary gas chromatography coupled to mass spectrometry was used to characterize the epicuticular hydrocarbon profiles of field-collected insects, and to identify differences in their composition between fifth-instar nymphs and adults, males and females, and between bugs collected in insecticide-treated and insecticide-free soybean crops. RESULTS: Straight chain saturated n-C27 and n-C29, and monomethyl and dimethyl chains of C31 and C33 were the most abundant compounds. A group of volatile hydrocarbons with n-C13 and n-C15 as the predominant compounds were also detected. The hydrocarbon pattern was different between nymphs and adults, either males or females. Heneicosene was almost exclusively detected in adult males and was the most important component to differentiate between both sexes, followed by tricosadiene. The total hydrocarbon amount was significantly higher in nymphs, males and females collected in insecticide-treated fields compared with insects obtained from untreated fields. CONCLUSION: Differences were found in the epicuticular hydrocarbon pattern among nymphs and adults, as well as sexual dimorphism in adult stink bugs. Interestingly, an alteration was also found in the hydrocarbon profile of insects collected in insecticide-treated soybean crops and its relevance is discussed within a pest management context.

Knockdown of CYP4PR1, a cytochrome P450 gene highly expressed in the integument tissue of Triatoma infestans, increases susceptibility to deltamethrin in pyrethroid-resistant insects.

Metabolic resistance to chemical insecticides implies a greater capacity to detoxify insecticides due to an increase in the expression of genes and/or in the activity of enzymes related to detoxification metabolism. The insect integument is known to participate as the cuticular penetration factor of resistance, but recently this tissue was also linked with metabolic resistance due to P450-dependent detoxification in the Chagas disease vector Triatoma infestans. The objectives of this study were i) to name and classify all P450s known to date in T. infestans, ii) to characterise one of them, CYP4PR1, representing the first member of a new cytochrome P450 subfamily described in insects, and iii) to investigate the potential role of CYP4PR1 in metabolic resistance to deltamethrin in T. infestans. We found that CYP4PR1 is expressed almost exclusively in the integument tissue, and its expression was not induced by deltamethrin. Knockdown of CYP4PR1 by RNA interference in pyrethroid-resistant nymphs caused a significant increment in insect mortality after topical application of two different doses of deltamethrin. These results support the role of the integument on metabolic resistance and suggest that CYP4PR1 might contribute to resistance in integument tissue of T. infestans.

Tolerance to Abiotic Factors of Microsclerotia and Mycelial Pellets From Metarhizium robertsii, and Molecular and Ultrastructural Changes During Microsclerotial Differentiation.

Metarhizium species fungi are able to produce resistant structures termed microsclerotia, formed by compact and melanized threads of hyphae. These propagules are tolerant to desiccation and produce infective conidia; thus, they are promising candidates to use in biological control programs. In this study, we investigated the tolerance to both ultraviolet B (UV-B) radiation and heat of microsclerotia of Metarhizium robertsii strain ARSEF 2575. We also adapted the liquid medium and culture conditions to obtain mycelial pellets from the same isolate in order to compare these characteristics between both types of propagules. We followed the peroxisome biogenesis and studied the oxidative stress during differentiation from conidia to microsclerotia by transmission electron microscopy after staining with a peroxidase activity marker and by the expression pattern of genes potentially involved in these processes. We found that despite their twice smaller size, microsclerotia exhibited higher dry biomass, yield, and conidial productivity than mycelial pellets, both with and without UV-B and heat stresses. From the 16 genes measured, we found an induction after 96-h differentiation in the oxidative stress marker genes MrcatA, MrcatP, and Mrgpx; the peroxisome biogenesis factors Mrpex5 and Mrpex14/17; and the photoprotection genes Mrlac1 and Mrlac2; and Mrlac3. We concluded that an oxidative stress scenario is induced during microsclerotia differentiation in M. robertsii and confirmed that because of its tolerance to desiccation, heat, and UV-B, this fungal structure could be an excellent candidate for use in biological control of pests under tropical and subtropical climates where heat and UV radiation are detrimental to entomopathogenic fungi survival and persistence.

Isolation of Beauveria bassiana from the Chagas Disease Vector Triatoma infestans in the Gran Chaco Region of Argentina: Assessment of Gene Expression during Host-Pathogen Interaction.

A native strain of the entomopathogenic fungus Beauveria bassiana (Bb-C001) was isolated from a naturally infected Triatoma infestans, Klug (Hemiptera: Reduviidae) adult cadaver in the Gran Chaco region, Salta province, Argentina. The isolate was both phenotypic and molecularly characterized in a context of fungus-insect interaction, by measuring the expression pattern of toxin genes during infection and immune response of T. infestans. The commercial strain GHA of B. bassiana, which was previously used in field interventions to control these vectors, was used as reference in this study. The phylogenetic trees based on both ribosomal internal transcribed spacer (ITS) and elongation factor 1-alpha (EF1-α) indicated that Bb-C001 fits into a B. bassiana cluster, and the sequence-characterized amplified regions (SCAR) showed that Bb-C001 is different from the GHA strain. There were no differences between both strains regarding viability, radial growth, and conidia production, either in the median survival time or insect mortality. However, Bb-C001 showed a higher expression than GHA of the bassianolide synthetase gene (BbbslS) during infection, and similar levels of the beauvericin synthetase gene (BbbeaS). Immune-related genes of T. infestans nymphs (limpet-2 and defensin-1, -2, and -6) were later expressed and thus insects failed to stop the infection process. These results showed that B. bassiana Bb-C001 is a promised fungal strain to be incorporated in the current biological control programs of T. infestans in Salta province, Argentina.

Outcome of blue, green, red, and white light on Metarhizium robertsii during mycelial growth on conidial stress tolerance and gene expression.

Fungi sense light and utilize it as a source of environmental information to prepare against many stressful conditions in nature. In this study, Metarhizium robertsii was grown on: 1) potato dextrose agar medium (PDA) in the dark (control); 2) under nutritive stress in the dark; and 3) PDA under continuous (A) white light; (B) blue light lower irradiance = LI; (C) blue light higher irradiance = HI; (D) green light; and (E) red light. Conidia produced under these treatments were tested against osmotic stress and UV radiation. In addition, a suite of genes usually involved in different stress responses were selected to study their expression patterns. Conidia produced under nutritive stress in the dark were the most tolerant to both osmotic stress and UV radiation, and the majority of their stress- and virulence-related genes were up-regulated. For osmotic stress tolerance, conidia produced under white, blue LI, and blue HI lights were the second most tolerant, followed by conidia produced under green light. Conidia produced under red light were the least tolerant to osmotic stress and less tolerant than conidia produced on PDA medium in the dark. For UV tolerance, conidia produced under blue light LI were the second most tolerant to UV radiation, followed by the UV tolerances of conidia produced under white light. Conidia produced under blue HI, green, and red lights were the least UV tolerant and less tolerant than conidia produced in the dark. The superoxide dismutases (sod1 and sod2), photolyases (6-4phr and CPDphr), trehalose-phosphate synthase (tps), and protease (pr1) genes were highly up-regulated under white light condition, suggesting a potential role of these proteins in stress protection as well as virulence after fungal exposure to visible spectrum components.

Is the Insect Cuticle the only Entry Gate for Fungal Infection? Insights into Alternative Modes of Action of Entomopathogenic Fungi.

Entomopathogenic fungi are the only insect pathogens able to infect their host by adhesion to the surface and penetration through the cuticle. Although the possibility of fungal infection per os was described almost a century ago, there is an information gap of several decades regarding this topic, which was poorly explored due to the continuous elucidation of cuticular infection processes that lead to insect death by mycosis. Recently, with the advent of next-generation sequencing technologies, the genomes of the main entomopathogenic fungi became available, and many fungal genes potentially useful for oral infection were described. Among the entomopathogenic Hypocreales that have been sequenced, Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Cordycipitaceae) is the main candidate to explore this pathway since it has a major number of shared genes with other non-fungal pathogens that infect orally, such as Bacillus thuringiensis Berliner (Bacillales: Bacillaceae). This finding gives B. bassiana a potential advantage over other entomopathogenic fungi: the possibility to infect through both routes, oral and cuticular. In this review, we explore all known entry gates for entomopathogenic fungi, with emphasis on the infection per os. We also set out the fungal infection process in a more integral approach, as a need to exploit its full potential for insect control, considering all of its virulence factors and the conditions needed to improve its virulence against insect that might offer some resistance to the common infection through the cuticle.

The limpet transcription factors of Triatoma infestans regulate the response to fungal infection and modulate the expression pattern of defensin genes.

As part of the innate humoral response to microbial attack, insects activate the expression of antimicrobial peptides (AMP). Understanding the regulatory mechanisms of this response in the Chagas disease vector Triatoma infestans is important since biological control strategies against pyrethroid-resistant insect populations were recently addressed by using the entomopathogenic fungus Beauveria bassiana. By bioinformatics, gene expression, and silencing techniques in T. infestans nymphs, we achieved sequence and functional characterization of two variants of the limpet transcription factor (Tilimpet) and studied their role as regulators of the AMP expression, particularly defensins, in fungus-infected insects. We found that Tilimpet variants may act differentially since they have divergent sequences and different relative expression ratios, suggesting that Tilimpet-2 could be the main regulator of the higher expressed defensins and Tilimpet-1 might play a complementary or more general role. Also, the six defensins (Tidef-1 to Tidef-6) exhibited different expression levels in fungus-infected nymphs, consistent with their phylogenetic clustering. This study aims to contribute to a better understanding of T. infestans immune response in which limpet is involved, after challenge by B. bassiana infection.

The entomopathogenic fungus Beauveria bassiana produces microsclerotia-like pellets mediated by oxidative stress and peroxisome biogenesis.

Several filamentous fungi are known to produce macroscopic pigmented hyphal aggregates named sclerotia. In recent years, some entomopathogenic fungi were reported to produce small sclerotia termed 'microsclerotia', becoming new potential propagules for biocontrol strategies. In this study, we described the production of microsclerotia-like pellets by the entomopathogenic fungus Beauveria bassiana. The carbon: nitrogen ratio equal to or higher than 12.5:1 amended with Fe2+ induced the germination of conidia, producing hyphal aggregate that formed sclerotial structures in submerged liquid cultures. These aggregates were able to tolerate desiccation as they germinated and subsequently produced viable conidia. Conidia derived from microsclerotial aggregates formulated with diatomaceous earth effectively kill Tribolium castaneum larvae. Optical and transmission microscopical imaging, qPCR and spectrophotometric analysis revealed that an oxidative stress scenario is involved in conidial differentiation into microsclerotia-like pellets, inducing fungal antioxidant response with high peroxidase activity - mainly detected in peroxisomes and mitochondria - and progress with active peroxisome proliferation. The results provide clues about B. bassiana microsclerotial differentiation and indicate that these pigmented aggregates are promising propagules for production, formulation and potentially application in the control of soil-inhabiting arthropod pests.

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.