Structure and expression of Rhodnius prolixus GH18 chitinases and chitinase-like proteins: Characterization of the physiological role of RpCht7, a gene from subgroup VIII, in vector fitness and reproduction.

Chitinases are enzymes responsible for the hydrolysis of glycosidic linkages within chitin chains. In insects, chitinases are typically members of the multigenic glycoside hydrolase family 18 (GH18). They participate in the relocation of chitin during development and molt, and in digestion in detritivores and predatory insects, and they control the peritrophic membrane thickness. Chitin metabolism is a promising target for developing vector control strategies, and knowledge of the roles of chitinases may reveal new targets and illuminate unique aspects of their physiology and interaction with microorganisms. Rhodnius prolixus is an important vector of Chagas disease, which is caused by the parasite Trypanosoma cruzi. In this study, we performed annotation and structural characterization of nine chitinase and chitinase-like protein genes in the R. prolixus genome. The roles of their corresponding transcripts were studied in more depth; their physiological roles were studied through RNAi silencing. Phylogenetic analysis of coding sequences showed that these genes belong to different subfamilies of GH18 chitinases already described in other insects. The expression patterns of these genes in different tissues and developmental stages were initially characterized using RT-PCR. RNAi screening showed silencing of the gene family members with very different efficiencies. Based on the knockdown results and the general lack of information about subgroup VIII of GH18, the RpCht7 gene was chosen for phenotype analysis. RpCht7 knockdown doubled the mortality in starving fifth-instar nymphs compared to dsGFP-injected controls. However, it did not alter blood intake, diuresis, digestion, molting rate, molting defects, sexual ratio, percentage of hatching, or average hatching time. Nevertheless, female oviposition was reduced by 53% in RpCht7-silenced insects, and differences in oviposition occurred within 14-20 days after a saturating blood meal. These results suggest that RpCht7 may be involved in the reproductive physiology and vector fitness of R. prolixus.

Synchrotron X-ray biosample imaging: opportunities and challenges.

Synchrotron radiation phase-contrast microtomography is sensitive to low attenuating tissues, giving an alternative visualisation of the sample and being useful for investigating microstructure inside biological specimens without staining them with a contrast medium. The phase-contrast technique has been widely used in the scientific community, as it is a technique associated with radiography and microscopy and able to enhance contrast in soft tissues, specifically at the edges, showing details that could not be seen by the absorption technique. This work aims to show the ability of synchrotron-based phase-contrast microtomography for the visualisation of soft tissues and hard internal structures of millimetre-sized biological organisms. Case studies of the anatomy of Rhodnius prolixus head and Thoropa miliaris tadpole are presented to illustrate the imaging technique.

Overview of paratransgenesis as a strategy to control pathogen transmission by insect vectors.

This article presents an overview of paratransgenesis as a strategy to control pathogen transmission by insect vectors. It first briefly summarises some of the disease-causing pathogens vectored by insects and emphasises the need for innovative control methods to counter the threat of resistance by both the vector insect to pesticides and the pathogens to therapeutic drugs. Subsequently, the state of art of paratransgenesis is described, which is a particularly ingenious method currently under development in many important vector insects that could provide an additional powerful tool for use in integrated pest control programmes. The requirements and recent advances of the paratransgenesis technique are detailed and an overview is given of the microorganisms selected for genetic modification, the effector molecules to be expressed and the environmental spread of the transgenic bacteria into wild insect populations. The results of experimental models of paratransgenesis developed with triatomines, mosquitoes, sandflies and tsetse flies are analysed. Finally, the regulatory and safety rules to be satisfied for the successful environmental release of the genetically engineered organisms produced in paratransgenesis are considered.

Ecdysone modulates both ultrastructural arrangement of hindgut and attachment of Trypanosoma cruzi DM 28c to the rectum cuticle of Rhodnius prolixus fifth-instar nymph.

Studies on the effects of azadirachtin treatment, ecdysone supplementation and ecdysone therapy on both the ultrastructural organization of the rectum in 5th-instar nymph of Rhodnius prolixus and the ex vivo attachment behavior of Trypanosoma cruzi under these experimental conditions were carried out. Control insects had a typical and significant organization of the rectum cuticle consisted of four main layers (procuticle, inner epicuticle, outer epicuticle, and wax layer) during the entire period of the experiment. Both azadirachtin treatment and ecdysone supplementation avoid the development of both outer epicuticle and wax layer. Oral therapy with ecdysone partially reversed the altered organization and induce the development of the four main rectal cuticle layers. In the same way, the ex vivo attachment of T. cruzi to rectal cuticle was blocked by azadirachtin treatment but ecdysone therapy also partially recovered the parasite adhesion rates to almost those detected in control insects. These results point out that ecdysone may be a factor responsible - directly or indirectly - by the modulation of rectum ultrastructural arrangement providing a superficial wax layer to the attachment followed by metacyclogenesis of T. cruzi in the rectum of its invertebrate hosts.

Influence of Serratia marcescens and Rhodococcus rhodnii on the Humoral Immunity of Rhodnius prolixus.

Chagas disease is a human infectious disease caused by Trypanosoma cruzi and can be transmitted by triatomine vectors, such as Rhodnius prolixus. One limiting factor for T. cruzi development is the composition of the bacterial gut microbiota in the triatomine. Herein, we analyzed the humoral immune responses of R. prolixus nymphs treated with antibiotics and subsequently recolonized with either Serratia marcescens or Rhodococcus rhodnii. The treatment with antibiotics reduced the bacterial load in the digestive tract, and the recolonization with each bacterium was successfully detected seven days after treatment. The antibiotic-treated insects, recolonized with S. marcescens, presented reduced antibacterial activity against Staphylococcus aureus and phenoloxidase activity in hemolymph, and lower nitric oxide synthase (NOS) and higher defensin C gene (DefC) gene expression in the fat body. These insects also presented a higher expression of DefC, lower prolixicin (Prol), and lower NOS levels in the anterior midgut. However, the antibiotic-treated insects recolonized with R. rhodnii had increased antibacterial activity against Escherichia coli and lower activity against S. aureus, higher phenoloxidase activity in hemolymph, and lower NOS expression in the fat body. In the anterior midgut, these insects presented higher NOS, defensin A (DefA) and DefC expression, and lower Prol expression. The R. prolixus immune modulation by these two bacteria was observed not only in the midgut, but also systemically in the fat body, and may be crucial for the development and transmission of the parasites Trypanosoma cruzi and Trypanosoma rangeli.

Azadirachtin interferes with basal immunity and microbial homeostasis in the Rhodnius prolixus midgut.

Rhodnius prolixus is an insect vector of two flagellate parasites, Trypanosoma rangeli and Trypanosoma cruzi, the latter being the causative agent of Chagas disease in Latin America. The R. prolixus neuroendocrine system regulates the synthesis of the steroid hormone ecdysone, which is essential for not only development and molting but also insect immunity. Knowledge for how this modulates R. prolixus midgut immune responses is essential for understanding interactions between the vector, its parasites and symbiotic microbes. In the present work, we evaluated the effects of ecdysone inhibition on R. prolixus humoral immunity and homeostasis with its microbiota, using the triterpenoid natural product, azadirachtin. Our results demonstrated that azadirachtin promoted a fast and lasting inhibitory effect on expression of both RpRelish, a nuclear factor kappa B transcription factor (NF-kB) component of the IMD pathway, and several antimicrobial peptide (AMP) genes. On the other hand, RpDorsal, encoding the equivalent NF-kB transcription factor in the Toll pathway, and the defC AMP gene were upregulated later in azadirachtin treated insects. The treatment also impacted on proliferation of Serratia marcescens, an abundant commensal bacterium. The simultaneous administration of ecdysone and azadirachtin in R. prolixus blood meals counteracted the azadirachtin effects on insect molting and also on expression of RpRelish and AMPs genes. These results support the direct involvement of ecdysone in regulation of the IMD pathway in the Rhodnius prolixus gut.

Proteome of the Triatomine Digestive Tract: From Catalytic to Immune Pathways; Focusing on Annexin Expression.

Rhodnius prolixus, Panstrongylus megistus, Triatoma infestans, and Dipetalogaster maxima are all triatomines and potential vectors of the protozoan Trypanosoma cruzi responsible for human Chagas' disease. Considering that the T. cruzi's cycle occurs inside the triatomine digestive tract (TDT), the analysis of the TDT protein profile is an essential step to understand TDT physiology during T. cruzi infection. To characterize the protein profile of TDT of D. maxima, P. megistus, R. prolixus, and T. infestans, a shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach was applied in this report. Most proteins were found to be closely related to metabolic pathways such as gluconeogenesis/glycolysis, citrate cycle, fatty acid metabolism, oxidative phosphorylation, but also to the immune system. We annotated this new proteome contribution gathering it with those previously published in accordance with Gene Ontology and KEGG. Enzymes were classified in terms of class, acceptor, and function, while the proteins from the immune system were annotated by reference to the pathways of humoral response, cell cycle regulation, Toll, IMD, JNK, Jak-STAT, and MAPK, as available from the Insect Innate Immunity Database (IIID). These pathways were further subclassified in recognition, signaling, response, coagulation, melanization and none. Finally, phylogenetic affinities and gene expression of annexins were investigated for understanding their role in the protection and homeostasis of intestinal epithelial cells against the inflammation.

Nitric oxide effects on Rhodnius prolixus's immune responses, gut microbiota and Trypanosoma cruzi development.

The immune system of Rhodnius prolixus comprehends the synthesis of different effectors that modulate the intestinal microbiota population and the life cycle of the parasite Trypanosoma cruzi inside the vector midgut. One of these immune responses is the production of reactive nitrogen species (RNS) derived by the action of nitric oxide synthase (NOS). Therefore, we investigated the effects of L-arginine, the substrate for nitric oxide (NO) production and Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of NOS, added in the insect blood meal. We analyzed the impact of these treatments on the immune responses and development of intestinal bacteria and parasites on R. prolixus nymphs. The L-arginine treatment in R. prolixus nymphs induced a higher NOS gene expression in the fat body and increased NO production, but reduced catalase and antimicrobial activities in the midgut. As expected, L-NAME treatment reduced NOS gene expression in the fat body. In addition, L-NAME treatment diminished catalase activity in the hemolymph and posterior midgut reduced phenoloxidase activity in the anterior midgut and increased the antimicrobial activity in the hemolymph. Both treatments caused a reduction in the cultivatable intestinal microbiota, especially in insects treated with L-NAME. However, T. cruzi development in the insect's digestive tract was suppressed after L-arginine treatment and the opposite was observed with L-NAME, which resulted in higher parasite counts. Therefore, we conclude that induction and inhibition of NOS and NO production are associated with other R. prolixus humoral immune responses, such as catalase, phenoloxidase, and antibacterial activities in different insect organs. These alterations reflect on intestinal microbiota and T. cruzi development.

Determination of Chitin Content in Insects: An Alternate Method Based on Calcofluor Staining.

Chitin is an aminopolysaccharide present in yeast cells and arthropod cuticle and is one of the most abundant biopolymers. The conventional methods for the quantitation of chitin content in biological samples are based on its hydrolysis (acid or enzymatic), and the assessment of the byproduct, glucosamine. However, previously described methodologies are time-consuming, laborious, low throughput, and not applicable to insect samples in many cases. Here we describe a new approach to chitin content quantitation based on calcofluor fluorescent brightener staining of samples, followed by microplate fluorescence readings. Calcofluor is a specific chitin stain commonly used for topological localization of the polymer. The protocol was tested in three important disease vector species, namely Lutzomyia longipalpis, Aedes aegypti, and Rhodnius prolixus, and then compared to a classic colorimetric chitin assessment method. Results show that chitin content in the tested insects can vary largely in a range of 8-4600 micrograms of chitin per insect, depending on species, sex, and instar. Comparisons between measurements from the previous protocol and calcofluor method showed statistically significant differences in some samples. However, the difference might be due to interference in the classic method from non-chitin sources of glucosamine and reducing agents. Furthermore, chitinase hydrolysis reduces the total chitin mass estimated between 36 and 74%, consolidating the fluorescent measurements as actual stained chitin in the same extent that was observed with the standard protocol. Therefore, the calcofluor staining method revealed to be a fast and reliable technique for chitin quantitation in homogenized insect samples.

Characterization of the Temporal Pattern of Blood Protein Digestion in Rhodnius prolixus: First Description of Early and Late Gut Cathepsins.

Rhodnius prolixus is one important vector for the parasite Trypanosoma cruzi in Latin America, where Chagas disease is a significant health issue. Although R. prolixus is a model for investigations of vector-parasite interaction and transmission, not much has been done recently to further comprehend its protein digestion. In this work, gut proteolysis was characterized using new fluorogenic substrates, including optimum pH, inhibition profiles, and tissue and temporal expression patterns. Each protease possessed a particular tissue prevalence and activity cycle after feeding. Cathepsin L had a higher activity in the posterior midgut lumen, being characterized by a plateau of high activities during several days in the intermediate phase of digestion. Cathepsin D showed high activity levels in the tissue homogenates and in the luminal content of the posterior midgut, with a single peak 5 days after blood feeding. Aminopeptidases are highly associated with the midgut wall, where the highest activity is located. Assays with proteinaceous substrates as casein, hemoglobin, and serum albumin revealed different activity profiles, with some evidence of biphasic temporal proteolytic patterns. Cathepsin D genes are preferentially expressed in the anterior midgut, while cathepsin L genes are mainly located in the posterior portion of the midgut, with specific sets of genes being differently expressed in the initial, intermediate, or late phases of blood digestion. Significance Statement This is the first description in a non-dipteran hematophagous species of a sequential protease secretion system based on midgut cathepsins instead of the most common insect digestive serine proteases (trypsins and chymotrypsins). The midgut of R. prolixus (Hemiptera) shows a different temporal expression of proteases in the initial, intermediate, and late stages of blood digestion. In this respect, a different timing in protease secretion may be an example of adaptative convergence in blood-sucking vectors from different orders. Expanding the knowledge about gut physiology in triatomine vectors may contribute to the development of new control strategies, aiming the blocking of parasite transmission.

Participation of Trypanosoma cruzi gp63 molecules on the interaction with Rhodnius prolixus.

Trypanosoma cruzi is the causative agent of Chagas disease, a vector-borne disease. The parasite molecules involved in vector interaction have been little investigated. Metallopeptidases and gp63 molecules have been implicated in parasite adhesion of several trypanosomatids to the insect midgut. Although gp63 homologues are highly expanded in the T. cruzi genome, and are implicated in parasite-mammalian host interaction, its role in the insect vector has never been explored. Here, we showed that divalent metal chelators or anti-Tcgp63-I antibodies impaired T. cruzi adhesion to Rhodnius prolixus midgut. Parasites isolated after insect colonization presented a drastic enhancement in the expression of Tcgp63-I. These data highlight, for the first time, that Tcgp63-I and Zn-dependent enzymes contribute to the interaction of T. cruzi with the insect vector.

The NF-κB Inhibitor, IMD-0354, Affects Immune Gene Expression, Bacterial Microbiota and Trypanosoma cruzi Infection in Rhodnius prolixus Midgut.

Rhodnius prolixus is an insect vector of Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. Nuclear factor-κB (NF-κB) transcription factors (TF) are conserved components of the innate immune system in several multicellular organisms including insects. The drug IMD-0354 [N-(3,5-bis-trifluoromethyl-phenyl)-5-chloro-2-hydroxy-benzamide] is a selective inhibitor of IκB kinases. It blocks IκBα phosphorylation thus preventing nuclear translocation of the NF-κb TF. In humans, NF-κB is involved in several biological processes such as inflammation, cell proliferation and immunity. In insects, the activation of the immune system upon microbial challenge can be controlled by signaling pathways such as the immune deficiency (IMD) and Toll, to combat infection. These activated pathways signal to downstream NF-κB TF to stimulate specific immune genes, triggering the synthesis of several molecules such as the antimicrobial peptides. In Drosophila melanogaster, the activation and regulation of NF-κB TF have been elucidated, while in triatomines these mechanisms are not fully understood Therefore, the present study investigated the effects of oral administration of the drug IMD-0354 on the R. prolixus immune response to challenge with bacteria and T. cruzi, as well as the impact on the gut bacterial microbiota. R. prolixus were fed with rabbit blood containing IMD-0354 and Escherichia coli, Staphylococcus aureus, or T. cruzi. The effects of IMD-0354 on insect mortality and antimicrobial activity in insect midgut samples, as well as the relative expression of R. prolixus immune genes were recorded. The bacterial microbiota was analyzed, and viable parasites were counted in insect midgut samples. The IMD-0354 treatment modulated antibacterial activity and the gene expression patterns of defensin A, defensin B, defensin C, and prolixicin, and the genes involved in the IMD and Toll pathways. Additionally, there was an increase of bacterial microbiota in treated insects. Insects treated with IMD-0354 and concomitantly infected with bacteria or T. cruzi through the blood meal had increased mortality, while the T. cruzi population in R. prolixus midgut was reduced. The inhibitory effect of IMD-0354 indicates the importance of NF-κB TF in the innate immune responses involved in the control of bacteria and parasite infections in the R. prolixus midgut.

Lipoproteins from vertebrate host blood plasma are involved in Trypanosoma cruzi epimastigote agglutination and participate in interaction with the vector insect, Rhodnius prolixus.

Chagas disease, infecting ca. 8 million people in Central and South America, is mediated by the protozoan parasite, Trypanosoma cruzi. The parasite is transmitted by the bite of blood sucking triatomine insects, such as Rhodnius prolixus, that had previously fed on parasite-infected vertebrate blood and voided their contaminated feces and urine into the wound. The stages of the parasite life cycle in both the insect vector and human host are well-known, but determinants of infection in the insect gut are complex and enigmatic. This paper examines the possible role of the R. prolixus gut agglutinins in the parasite life cycle. The results, derived from gut extracts made from R. prolixus fed on various diets with different vertebrate blood components, and cross adsorption experiments, showed for the first time that R. prolixus has two distinct gut agglutinins originating from their vertebrate blood meal, one for T. cruzi (the parasite agglutinin, PA) and the other for the erythrocytes (the hemagglutinin, HA). Again, uniquely, the results also demonstrate that these two agglutinins are derived, respectively, from the plasma and erythrocyte components of the vertebrate blood. Subsequent experiments, examining in more detail the nature of the plasma components forming the T. cruzi PA, used fractionated extracts of the vertebrate plasma (high density lipoprotein, HDL; low density lipoprotein, LDL, and delipidated plasma) in agglutination assays. The results confirmed the identity of the PA as a high density lipoprotein (HDL) in the plasma of the vertebrate blood meal which agglutinates parasites in the R. prolixus gut. In addition, the use of single or double labeled HDL in fluorescence and confocal microscopy showed the interaction of the labeled HDL with the parasite surface and its internalization at later times. Finally, results of T. cruzi parasitization of R. prolixus, incorporating various vertebrate blood components, resulted in highly significant increases in infectivity in the presence of HDL from the 2nd day of infection, thus confirming the important role of this molecule in T. cruzi infection of R. prolixus.

Heme crystallization in a Chagas disease vector acts as a redox-protective mechanism to allow insect reproduction and parasite infection.

Heme crystallization as hemozoin represents the dominant mechanism of heme disposal in blood feeding triatomine insect vectors of the Chagas disease. The absence of drugs or vaccine for the Chagas disease causative agent, the parasite Trypanosoma cruzi, makes the control of vector population the best available strategy to limit disease spread. Although heme and redox homeostasis regulation is critical for both triatomine insects and T. cruzi, the physiological relevance of hemozoin for these organisms remains unknown. Here, we demonstrate that selective blockage of heme crystallization in vivo by the antimalarial drug quinidine, caused systemic heme overload and redox imbalance in distinct insect tissues, assessed by spectrophotometry and fluorescence microscopy. Quinidine treatment activated compensatory defensive heme-scavenging mechanisms to cope with excessive heme, as revealed by biochemical hemolymph analyses, and fat body gene expression. Importantly, egg production, oviposition, and total T. cruzi parasite counts in R. prolixus were significantly reduced by quinidine treatment. These effects were reverted by oral supplementation with the major insect antioxidant urate. Altogether, these data underscore the importance of heme crystallization as the main redox regulator for triatomine vectors, indicating the dual role of hemozoin as a protective mechanism to allow insect fertility, and T. cruzi life-cycle. Thus, targeting heme crystallization in insect vectors represents an innovative way for Chagas disease control, by reducing simultaneously triatomine reproduction and T. cruzi transmission.

In vitro Trypanocidal Activity, Genomic Analysis of Isolates, and in vivo Transcription of Type VI Secretion System of Serratia marcescens Belonging to the Microbiota of Rhodnius prolixus Digestive Tract.

Serratia marcescens is a bacterium with the ability to colonize several niches, including some eukaryotic hosts. S. marcescens have been recently found in the gut of hematophagous insects that act as parasite vectors, such as Anopheles, Rhodnius, and Triatoma. While some S. marcescens strains have been reported as symbiotic or pathogenic to other insects, the role of S. marcescens populations from the gut microbiota of Rhodnius prolixus, a vector of Chagas' disease, remains unknown. Bacterial colonies from R. prolixus gut were isolated on BHI agar. After BOX-PCR fingerprinting, the genomic sequences of two isolates RPA1 and RPH1 were compared to others S. marcescens from the NCBI database in other to estimate their evolutionary divergence. The in vitro trypanolytic activity of these two bacterial isolates against Trypanosoma cruzi (DM28c clone and Y strain) was assessed by microscopy. In addition, the gene expression of type VI secretion system (T6SS) was detected in vivo by RT-PCR. Comparative genomics of RPA1 and RPH1 revealed, besides plasmid presence and genomic islands, genes related to motility, attachment, and quorum sensing in both genomes while genes for urea hydrolysis and type II secretion system (T2SS) were found only in the RPA1 genome. The in vitro trypanolytic activity of both S. marcescens strains was stronger in their stationary phases of growth than in their exponential ones, with 65-70 and 85-90% of epimastigotes (Dm28c clone and Y strain, respectively) being lysed after incubation with RPA1 or RPH1 in stationary phase. Although T6SS transcripts were detected in guts up to 40 days after feeding (DAF), R. prolixus morbidity or mortality did not appear to be affected. In this report, we made available two trypanolytic S. marcescens strains from R. prolixus gut to the scientific community together with their genomic sequences. Here, we describe their genomic features with the purpose of bringing new insights into the S. marcescens adaptations for colonization of the specific niche of triatomine guts. This study provides the basis for a better understanding of the role of S. marcescens in the microbiota of R. prolixus gut as a potential antagonist of T. cruzi in this complex system.

A Metagenomic Analysis of Bacterial Microbiota in the Digestive Tract of Triatomines.

The digestive tract of triatomines (DTT) is an ecological niche favored by microbiota whose enzymatic profile is adapted to the specific substrate availability in this medium. This report describes the molecular enzymatic properties that promote bacterial prominence in the DTT. The microbiota composition was assessed previously based on 16S ribosomal DNA, and whole sequenced genomes of bacteria from the same genera were used to calculate the GC level of rare and prominent bacterial species in the DTT. The enzymatic reactions encoded by coding sequences of both rare and common bacterial species were then compared and revealed key functions explaining why some genera outcompete others in the DTT. Representativeness of DTT microbiota was investigated by shotgun sequencing of DNA extracted from bacteria grown in liquid Luria-Bertani broth (LB) medium. Results showed that GC-rich bacteria outcompete GC-poor bacteria and are the dominant components of the DTT microbiota. In addition, oxidoreductases are the main enzymatic components of these bacteria. In particular, nitrate reductases (anaerobic respiration), oxygenases (catabolism of complex substrates), acetate-CoA ligase (tricarboxylic acid cycle and energy metabolism), and kinase (signaling pathway) were the major enzymatic determinants present together with a large group of minor enzymes including hydrogenases involved in energy and amino acid metabolism. In conclusion, despite their slower growth in liquid LB medium, bacteria from GC-rich genera outcompete the GC-poor bacteria because their specific enzymatic abilities impart a selective advantage in the DTT.

Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels.

Glycoside Hydrolases (GHs) are enzymes able to recognize and cleave glycosidic bonds. Insect GHs play decisive roles in digestion, in plant-herbivore, and host-pathogen interactions. GH activity is normally measured by the detection of a release from the substrate of products as sugars units, colored, or fluorescent groups. In most cases, the conditions for product release and detection differ, resulting in discontinuous assays. The current protocols result in using large amounts of reaction mixtures for the obtainment of time points in each experimental replica. These procedures restrain the analysis of biological materials with limited amounts of protein and, in the case of studies regarding small insects, implies in the pooling of samples from several individuals. In this respect, most studies do not assess the variability of GH activities across the population of individuals from the same species. The aim of this work is to approach this technical problem and have a deeper understanding of the variation of GH activities in insect populations, using as models the disease vectors Rhodnius prolixus (Hemiptera: Triatominae) and Lutzomyia longipalpis (Diptera: Phlebotominae). Here we standardized continuous assays using 4-methylumbelliferyl derived substrates for the detection of α-Glucosidase, ß-Glucosidase, α-Mannosidase, N-acetyl-hexosaminidase, ß-Galactosidase, and α-Fucosidase in the midgut of R. prolixus and L. longipalpis with results similar to the traditional discontinuous protocol. The continuous assays allowed us to measure GH activities using minimal sample amounts with a higher number of measurements, resulting in data that are more reliable and less time and reagent consumption. The continuous assay also allows the high-throughput screening of GH activities in small insect samples, which would be not applicable to the previous discontinuous protocol. We applied continuous GH measurements to 90 individual samples of R. prolixus anterior midgut homogenates using a high-throughput protocol. α-Glucosidase and α-Mannosidase activities showed the normal distribution in the population. ß-Glucosidase, ß-Galactosidase, N-acetyl-hexosaminidase, and α-Fucosidase activities showed non-normal distributions. These results indicate that GHs fluorescent-based high-throughput assays apply to insect samples and that the frequency distribution of digestive activities should be considered in data analysis, especially if a small number of samples is used.

Lethal and sublethal effects of essential oil of Lippia sidoides (Verbenaceae) and monoterpenes on Chagas´ disease vector Rhodnius prolixus

The aim of this study was to identify the composition of the essential oil from leaves of Lippia sidoides (EOLS), a typical shrub commonly found in the dry northeast of Brazil, popularly known as “alecrim-pimenta”. Additionally, we investigated the nymphicidal, ovicidal, phagoinhibitory and excretion effects of EOLS, its major constituent thymol and its isomer carvacrol, on fourth instar nymphs and eggs of Rhodnius prolixus, the Chagas’ disease vector. The nymphicidal and ovicidal activity of thymol, carvacrol, and EOLS was assessed by tests using impregnated Petri dishes. The lethal concentration values (LC50) for EOLS, carvacrol, and thymol were 54.48, 32.98, and 9.38 mg/cm2, respectively. The ovicidal test showed that both carvacrol and thymol (50 mg/cm2) inhibited hatching (50% and 23.3%, respectively), while treatments with 10 mg/cm2 or 50 mg/cm2 EOLS did not affect the hatching rate at all (80% and 90%, respectively). We observed an anti-feeding effect in insects fed with blood containing natural products at the higher concentrations (100 µg/mL). Finally, excretion rate was affected by EOLS and carvacrol, but not by thymol. These findings offer novel insights into basic physiological processes that make the tested natural compounds interesting candidates for new types of insecticides.

Lethal and sublethal effects of essential oil of Lippia sidoides (Verbenaceae) and monoterpenes on Chagas' disease vector Rhodnius prolixus.

The aim of this study was to identify the composition of the essential oil from leaves of Lippia sidoides (EOLS), a typical shrub commonly found in the dry northeast of Brazil, popularly known as "alecrim-pimenta". Additionally, we investigated the nymphicidal, ovicidal, phagoinhibitory and excretion effects of EOLS, its major constituent thymol and its isomer carvacrol, on fourth instar nymphs and eggs of Rhodnius prolixus, the Chagas' disease vector. The nymphicidal and ovicidal activity of thymol, carvacrol, and EOLS was assessed by tests using impregnated Petri dishes. The lethal concentration values (LC50) for EOLS, carvacrol, and thymol were 54.48, 32.98, and 9.38 mg/cm2, respectively. The ovicidal test showed that both carvacrol and thymol (50 mg/cm2) inhibited hatching (50% and 23.3%, respectively), while treatments with 10 mg/cm2 or 50 mg/cm2 EOLS did not affect the hatching rate at all (80% and 90%, respectively). We observed an anti-feeding effect in insects fed with blood containing natural products at the higher concentrations (100 µg/mL). Finally, excretion rate was affected by EOLS and carvacrol, but not by thymol. These findings offer novel insights into basic physiological processes that make the tested natural compounds interesting candidates for new types of insecticides.