Aerobic Metabolism Alterations as an Evidence of Underlying Deltamethrin Resistance Mechanisms in Triatoma infestans (Hemiptera: Reduviidae).

Triatoma infestans (Klug, 1834), the main vector of Chagas disease in Latin America, is regularly controlled by spraying the pyrethroid deltamethrin, to which some populations have developed resistance. The three main mechanisms of resistance are 1) metabolic resistance by overexpression or increased activity of detoxifying enzymes, 2) target site mutations, and 3) cuticle thickening/modification. We use open-flow respirometry to measure real-time H2O loss rate (V˙H2O) and CO2 production rate (V˙CO2), on nymphs from susceptible and resistant populations before and after exposure to the insecticide to understand the underlying mechanisms of resistance in live insects. Lack of differences in V˙H2O between populations suggested that cuticular thickness/composition is not acting as a relevant resistance mechanism. Similarly, there was no difference in resting V˙CO2, suggesting a trade-off between resistance mechanisms and other physiological processes. The increment in V˙CO2 after application of deltamethrin was similar in both populations, which suggested that while enhanced enzymatic detoxification may play a role in resistance expression in this population, the main mechanism involved should be a passive one such as target site mutations. Open-flow respirometry provided useful evidence for evaluating the mechanisms involved in deltamethrin resistance. Using this technique could improve efficiency of scientific research in the area of insecticide resistance management, leading to a faster decision making and hence improved control results.

Silencing of ATG6 and ATG8 promotes increased levels of triacylglycerol (TAG) in the fat body during prolonged starvation periods in the Chagas disease vector Rhodnius prolixus.

Rhodnius prolixus is an obligatorily hematophagous insect known as an important vector of Chagas disease. Autophagy is a conserved cellular mechanism that acts in response to nutrient starvation, where components of the cytoplasm are sequestered by a double membrane organelle, named autophagosome, which is targeted to fuse with the lysosome for degradation. Lipophagy is the process of lipid degradation by selective autophagy, where autophagosomes sequester lipid droplets and degrade triacylglycerol (TAG) generating free fatty acids for ß-oxidation. Here, two essential genes of the autophagic pathway, Atg6/Beclin1 (RpAtg6) and Atg8/LC3 (RpAtg8), were silenced and the storage of lipids during starvation in Rhodnius prolixus was monitored. We found that RNAi knockdown of both RpAtg6 and RpAtg8 resulted in higher levels of TAG in the fat body and the flight muscle, 24 days after the blood meal, as well as a larger average diameter of the lipid droplets in the fat body, as seen by Nile Red staining under the confocal fluorescence microscope. Silenced starved insects had lower survival rates when compared to control insects. Accordingly, when examined during the starvation period for monitored activity, silenced insects had lower spontaneous locomotor activity and lower forced flight rates. Furthermore, we found that some genes involved in lipid metabolism had their expression levels altered in silenced insects, such as the Brummer lipase (down regulated) and the adipokinetic hormone receptor (up regulated), suggesting that, as previously observed in mammalian models, the autophagy and neutral lipolysis machineries are interconnected at the transcriptional level. Altogether, our data indicate that autophagy in the fat body is important to allow insects to mobilize energy from lipid stores.

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.

Glycosylation on proteins of the intestine and perimicrovillar membrane of Triatoma (Meccus) pallidipennis, under different feeding conditions.

Trypanosoma cruzi, the causative agent of Chagas disease, interacts with molecules in the midgut of its insect vector to multiply and reach the infective stage. Many studies suggest that the parasite binds to midgut-specific glycans. We identified several glycoproteins expressed in the intestine and perimicrovillar membrane (PMM) of Triatoma (Meccus) pallidipennis under different feeding conditions. In order to assess changes in protein-linked glycans, we performed lectin and immunoblot analyses on glycoprotein extracts from these intestinal tissues using well-characterized lectins, and an antibody, which collectively recognize a wide range of different glycans epitopes. We observed that the amount and composition of proteins and glycoproteins associated with different glycans structures changed over time in the intestines and PMM under different physiological conditions. PMM extracts contained a wide variety of glycoproteins with different sugar residues, including abundant high-mannose and complex sialylated glycans. We propose that these molecules could be involved in the process of parasite-vector interactions.

Developmental changes in the levels and redox potentials of main hemolymph thiols/disulfides in the Jamaican field cricket Gryllus assimilis.

Main thiols and disulfides were determined in the hemolymph of the Jamaican field cricket Gryllus assimilis at various developmental stages. On the basis of these data, redox potentials of the glutathione, cysteine and homocysteine redox systems were calculated. The concentrations of all thiols studied decreased during development (at a stage of 6 molts) with respect to young crickets, and increased again in adult insects. Redox potentials of the glutathione and cysteine systems increased from values of -131.0±5.6 mV and -86.9±17.1 mV, respectively in young crickets to -58.0±3.6 mV and -36.1±4.2 mV, respectively, at the stage of 6 molts and decreased to values of -110.4±24.8 mV and -66.3±12.2 mV, respectively, in adult insects. Redox potentials of the glutathione and cysteine systems in the hemolymph of young and adult insects were similar to those reported for human plasma.

Oral delivery of double-stranded RNAs induces mortality in nymphs and adults of the Asian citrus psyllid, Diaphorina citri.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is one of the most important citrus pests. ACP is the vector of the phloem-limited bacteria Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus, the causal agents of the devastating citrus disease huanglongbing (HLB). The management of HLB is based on the use of healthy young plants, eradication of infected plants and chemical control of the vector. RNA interference (RNAi) has proven to be a promising tool to control pests and explore gene functions. Recently, studies have reported that target mRNA knockdown in many insects can be induced through feeding with double-stranded RNA (dsRNA). In the current study, we targeted the cathepsin D, chitin synthase and inhibitor of apoptosis genes of adult and nymph ACP by feeding artificial diets mixed with dsRNAs and Murraya paniculata leaves placed in dsRNAs solutions, respectively. Adult ACP mortality was positively correlated with the amount of dsRNA used. Both nymphs and adult ACP fed dsRNAs exhibited significantly increased mortality over time compared with that of the controls. Moreover, qRT-PCR analysis confirmed the dsRNA-mediated RNAi effects on target mRNAs. These results showed that RNAi can be a powerful tool for gene function studies in ACP and perhaps for HLB control.

Saliva from nymph and adult females of Haemaphysalis longicornis: a proteomic study.

BACKGROUND: Haemaphysalis longicornis is a major vector of Theileria spp., Anaplasma phagocytophilum, Babesia spp. and Coxiella burnetti in East Asian countries. All life stages of ixodid ticks have a destructive pool-feeding style in which they create a pool-feeding site by lacerating host tissue and secreting a variety of biologically active compounds that allows the tick to evade host responses, enabling the uptake of a blood meal. The identification and functional characterization of tick saliva proteins can be useful to elucidate the molecular mechanisms involved in tick development and to conceive new anti-tick control methods. METHODS: H. longicornis tick saliva was collected from fully engorged nymphs and fully engorged adults induced by dopamine or pilocarpine, respectively. Saliva was digested with trypsin for LC-MS/MS sequencing and peptides were searched against tick and rabbit sequences. RESULTS: A total of 275 proteins were identified, of which 135 were tick and 100 were rabbit proteins. Of the tick proteins, 30 proteins were identified exclusively in fully engorged nymph saliva, 74 in fully engorged adult females, and 31 were detected in both stages. The identified tick proteins include heme/iron metabolism-related proteins, oxidation/detoxification proteins, enzymes, proteinase inhibitors, tick-specific protein families, and cytoskeletal proteins. Proteins involved in signal transduction, transport and metabolism of carbohydrate, energy, nucleotide, amino acids and lipids were also detected. Of the rabbit proteins, 13 were present in nymph saliva, 48 in adult saliva, and 30 were present in both. The host proteins include immunoglobulins, complement system proteins, antimicrobial proteins, serum albumin, peroxiredoxin, serotransferrin, apolipoprotein, hemopexin, proteinase inhibitors, and hemoglobin/red blood cells-related products. CONCLUSIONS: This study allows the identification of H. longicornis saliva proteins. In spontaneously detached tick saliva various proteins were identified, although results obtained with saliva of fully engorged ticks need to be carefully interpreted. However, it is interesting to note that proteins identified in this study were also described in other tick saliva proteomes using partially engorged tick saliva, including hemelipoprotein, proteases, protease inhibitors, proteins related to structural functions, transporter activity, metabolic processes, and others. In conclusion, these data can provide a deeper understanding to the biology of H. longicornis.

Chitin deposition on the embryonic cuticle of Rhodnius prolixus: the reduction of CHS transcripts by CHS-dsRNA injection in females affects chitin deposition and eclosion of the first instar nymph.

In a previous study, we found that the embryonic cuticle of Rhodnius prolixus is a chitin-based structure that helps the first instar nymph to hatch from the chorion. Here, we investigated how the reduction of transcripts induced by CHS dsRNA injection affects R. prolixus embryogenesis and eclosion. Deposition of chitin in the embryonic cuticle begins later at embryogenesis, around day 8, and ends approximately at day 15, when the insects are ready for eclosion. In R. prolixus, chitin deposition follows pari passu with the synthesis of the chitin synthase mRNA, indicating a regulation at the transcriptional level. The reduction of the chitin synthase gene transcripts by the injection of CHS dRNA prevented chitin deposition during embryonic cuticle formation, being lethal to hatching nymphs, which end up dying while stuck in the chorionic border trying to leave the chorion. The successful eclosion rates were reduced by 60% in animals treated with CHS dsRNA when compared to animals injected with a control (dsRNA no related gene or water). We found that the harmful effects on oviposition and eclosion are possibly due to changes in the structure of the embryonic cuticle, as observed by directly comparing the morphology of control and chitin-deficient embryonic cuticles under the transmission electron microscope. The lack of chitin and changes in its morphological characteristics appears to alter the embryonic cuticle physiology and functionality. Additionally, we observed that the effects of CHS dRNA treatment on R. prolixus females lasted up to 3 egg-laying cycles (∼100 days), pointing to R. prolixus as a useful model for developmental studies.

Defensive compounds and male-produced sex pheromone of the stink bug, Agroecus griseus.

Agroecus griseus is a serious corn pest in Brazil. Contents of the dorsal abdominal glands (DAGs) in nymphs, and the metathoracic gland (MTG) in adults of this species were characterized and quantified. Compounds found were similar to those of other Pentatomidae species and included aliphatic hydrocarbons, aldehydes, oxo-alkenals, and esters. However, two compounds were found in the MTG that have not been described previously for this family. Mass spectrometry, infrared spectroscopy, and gas chromatographic analysis using coinjection with authentic standards confirmed the identities of the compounds as enantiopure (S)-2-methylbutyl acetate and 3-methyl-2-butenyl acetate. The five nymphal instars showed significantly different ratios of components, mainly between those of the first and later instars. No significant differences were detected in MTG contents between sexes. Gas chromatography (GC) analysis of aeration extracts of males and females showed the presence of a compound released exclusively by males. Gas chromatography - electroantennographic detection (GC-EAD) assays indicated that the male-specific compound is bioactive in females, suggesting the presence of an attractant pheromone. The mass spectrum and infrared data for this compound matched with methyl 2,6,10-trimethyltridecanoate, a sex pheromone component previously detected in the stink bugs, Euschistus heros and E. obscurus. The synthetic standard coeluted with the natural pheromone on two different GC stationary phases, confirming the proposed structure. Y-tube olfactometer assays showed that the synthetic standard was strongly attractive to females, and GC-EAD tests produced responses with antennae from females similar to those of the natural pheromone.

Effects of shape variations on the energy metabolism of the sand cricket Gryllus firmus: a geometric morphometric analysis.

Respiration and energy metabolism are key processes in animals, which are severely constrained by the design of physical structures, such as respiratory structures. Insects have very particular respiratory systems, based on gas diffusion across tracheae. Since the efficiency of the tracheal respiratory system is highly dependent on body shape, the pattern of morphological variation during ontogeny could have important metabolic consequences. We studied this problem combining through-flow respirometry and geometric morphometrics in 88 nymphs of the sand cricket, Gryllus firmus. After measuring production in each individual, we took digital photographs and defined eight landmarks for geometric morphometric analysis. The analysis suggested that ontogenic deformations were mostly related to enlargement of the abdomen, compared to thorax and head. We found that (controlling for body size) metabolic variables and especially resting metabolism are positively correlated with a shape-component associated to an elongation of the abdomen. Our results are in agreement with the mechanics of tracheal ventilation in orthopterans, as gas circulation occurs by changes in abdominal pressures due to abdominal contractions and expansions along the longitudinal axis.

Lipid accumulation and utilization by oocytes and eggs of Rhodnius prolixus.

Insect eggs must contain the necessary nutrients for embryonic growth. In this article, we investigated the accumulation of triacylglycerol (TAG) in growing oocytes and its utilization during embryonic development. TAG makes up about 60% of the neutral lipids in oocytes and accumulates as oocytes grow, from 2.2 ± 0.1 µg in follicles containing 1.0 mm length oocytes to 10.2 ± 0.8 µg in 2.0 mm length oocytes. Lipophorin (Lp), the hemolymphatic lipoprotein, radioactively labeled in free fatty acid (FFA) or diacylglycerol (DAG), was used to follow the transport of these lipids to the ovary. Radioactivity from both lipid classes accumulated in the oocytes, which was abolished at 4°C. The capacity of the ovary to receive FFA or DAG from Lp varied according to time after a blood meal and reached a maximum around the second day. (3) H-DAG supplied by Lp to the ovaries was used in the synthesis of TAG as, 48 hr after injection, most of the radioactivity was found in TAG (85.7% of labeling in neutral lipids). During embryogenesis, lipid stores were mobilized, and the TAG content decreased from 16.4 ± 2.1 µg/egg on the first day to 10.0 ± 1.3 µg on day 15, just before hatching. Of these, 7.4 ± 0.9 µg were found in the newly emerged nymphs. In unfertilized eggs, the TAG content did not change. Although the TAG content decreased during embryogenesis, the relative lipid composition of the egg did not change. The amount of TAG in the nymph slowly decreased during the days after hatching.

Effects of shape variations on the energy metabolism of the sand cricket Gryllus firmus: a geometric morphometric analysis

Respiration and energy metabolism are key processes in animals, which are severely constrained by the design of physical structures, such as respiratory structures. Insects have very particular respiratory systems, based on gas diffusion across tracheae. Since the efficiency of the tracheal respiratory system is highly dependent on body shape, the pattern of morphological variation during ontogeny could have important metabolic consequences. We studied this problem combining through-flow respirometry and geometric morphometrics in 88 nymphs of the sand cricket, Gryllus firmus. After measuring production in each individual, we took digital photographs and defined eight landmarks for geometric morphometric analysis. The analysis suggested that ontogenic deformations were mostly related to enlargement of the abdomen, compared to thorax and head. We found that (controlling for body size) metabolic variables and especially resting metabolism are positively correlated with a shape-component associated to an elongation of the abdomen. Our results are in agreement with the mechanics of tracheal ventilation in orthopterans, as gas circulation occurs by changes in abdominal pressures due to abdominal contractions and expansions along the longitudinal axis.

An insight into the sialome of the blood-sucking bug Triatoma infestans, a vector of Chagas' disease.

Triatoma infestans is a hemiptera, vector of Chagas' disease that feeds exclusively on vertebrate blood in all life stages. Hematophagous insects' salivary glands (SG) produce potent pharmacological compounds that counteract host hemostasis, including anticlotting, antiplatelet, and vasodilatory molecules. To obtain a further insight into the salivary biochemical and pharmacological complexity of this insect, a cDNA library from its SG was randomly sequenced. Also, salivary proteins were submitted to two-dimensional gel (2D-gel) electrophoresis followed by MS analysis. We present the analysis of a set of 1534 (SG) cDNA sequences, 645 of which coded for proteins of a putative secretory nature. Most salivary proteins described as lipocalins matched peptide sequences obtained from proteomic results.

Quantitative genetic variation of metabolism in the nymphs of the sand cricket, Gryllus firmus, inferred from an analysis of inbred-lines.

Compared with morphological and life history traits, quantitative genetic variation of metabolic and related traits in animals has been poorly studied. We used flow-through VCO(2) respirometry and simultaneous activity measurement on nymphs of the sand cricket (Gryllus firmus) from inbred lines to estimate broad-sense heritability of four metabolic variables. In addition, we measured a number of linear dimensions in the adults from the same inbred lines. There were significant multivariate effects of inbred lines for all traits and broad-sense heritability for physiological traits was 4.5%, 5.2%, 10.3% and 8.5% for average, resting, minimum and maximum CO(2) production in nymphs, respectively. Though the MANOVA indicated significant genetic variation among inbred lines in adult morphology, the broad-sense heritabilities were relatively low ranging from 0-18%. Our results indicate that the heritabilities of metabolic measures are large enough to potentially respond to selection.

Dissecting the variance-covariance structure in insect physiology: the multivariate association between metabolism and morphology in the nymphs of the sand cricket (Gryllus firmus).

Energy metabolism in animals has been largely studied in relation to exogenous sources of variation. However, because they give insight into the relationship between whole metabolism and lower organizational levels such as organs and tissues, examination of endogenous determinants of metabolism other than body mass is itself very important. We studied the multivariate association of body parts and several aspects of energy metabolism in an insect, the nymphs of the sand cricket, Gryllus firmus. By using a variety of both univariate and multivariate techniques, we explored the resultant variance-covariance matrix to build a path diagram with latent variables. After controlling for body mass, we found a significant canonical correlation between metabolism and morphology. According to the factor loadings and path coefficients, the most important contributions of morphology to the correlation were thorax and abdomen size measures, whereas the most important metabolic contribution was resting metabolism. Activity metabolism was mostly explained by body mass rather than body parts, which could be a result of resting rates being chronic consequences of the functioning of the metabolic machinery that the insect must maintain.