•We analyzed the spreading of kdr mutations in T. infestans populations from the Gran Chaco.•L1014F mutation is present in the resistance focus from the Argentinean-Bolivian border.•L925I mutation is ...present in the resistance focus from the Argentinean Chaco.•Mutations in the sodium channel is a major cause of pyrethroid resistance in T. infestans.•PASA and REA assays are sensitive and efficient tools for resistance monitoring in T. infestans.
Point mutations in the voltage-gated sodium channel, the primary target of pyrethroid insecticides, have been associated with the resistance in Triatoma infestans, an important vector of Chagas’ disease. Hence, the sustainability of vector control programs requires the implementation of resistance management strategies.
We determined the sensitivity of the molecular assays previously designed for early resistance detection to be used in pooled samples from a wide area of the endemic region, and validated them for their routine use in control campaigns for the monitoring of insecticide resistance in T. infestans. Consequently, we used these methods to examine the distribution of resistance-associated mutations in the sodium channel gene in populations of T. infestans from the Argentinean and Bolivian Gran Chaco.
The PASA and REA assays tested proved sensitive enough to detect kdr SNPs in pooled samples, indicating these assays are suitable for routine screening in insecticide resistance surveillance. Two geographically differentiated foci were detected in T. infestans populations from the Argentinean and Bolivian Gran Chaco, with populations on the Bolivian-Argentinean border carrying L1014F mutation, and those from the Argentinean Chaco carrying L925I mutation. In all highly resistant populations analyzed, one of both kdr mutations was present, and toxicological assays determined that all pyrethroid resistant populations analyzed herein were sensitive to fenitrothion.
The principal cause of pyrethroid resistance in T. infestans from the Gran Chaco ecoregion is kdr mutations in the sodium channel. Different levels of resistance occur in different populations carrying identical mutation, suggesting the existence of contributory mechanisms.
The importance of Chagas disease motivated the scientific effort to obtain the complete genomic sequence of the vector species Rhodnius prolixus, this information is also relevant to the ...understanding of triatomine biology in general. The central nervous system is the key regulator of insect physiology and behavior. Neurohormones (neuropeptides and biogenic amines) are the chemical messengers involved in the regulation and integration of neuroendocrine signals. In insects, this signaling is mainly mediated by the interaction of neurohormone ligands with G protein coupled receptors (GPCRs). The recently sequenced R. prolixus genome provides us with the opportunity to analyze this important family of genes in triatomines, supplying relevant information for further functional studies. Next-generation sequencing methods offer an excellent opportunity for transcriptomic exploration in key organs and tissues in the presence of a reference genome as well as when a reference genome is not available. We undertook a genomic analysis to obtain a genome-wide inventory of opsines and the GPCRs for neurohormones in R. prolixus. Furthermore, we performed a transcriptomic analysis of R. prolixus central nervous system, focusing on neuropeptide precursor genes and neurohormone and opsines GPCRs. In addition, we mined the whole transcriptomes of Triatoma dimidiata, Triatoma infestans and Triatoma pallidipennis – three sanitary relevant triatomine species – to identify neuropeptide precursors and GPCRs genes. Our study reveals a high degree of sequence conservation in the molecular components of the neuroendocrine system of triatomines.
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•We identified GPCRs for neurohormones and opsins in Rhodnius prolixus genome sequence.•Thirty-seven of the identified proteins had matching EST evidence in R. prolixus CNS.•ESTs from GPCRs were identified in Triatoma spp.•We describe the transcriptome of R. prolixus central nervous system.•The study reveals a high degree of conservation in the neuroendocrine system in triatomines.
In hematophagous insects, blood intake triggers a prompt response mediated by neuropeptides, which regulates a variety of physiological processes. Here we report a quantitative proteomic analysis of ...the postfeeding response in the central nervous system of Rhodnius prolixus, a vector of Chagas disease. The concentration of neuropeptides NVP-like, ITG-like, kinin-precursor peptide, and neuropeptide-like precursor 1 (NPLP1) significantly changes in response to blood intake. We also performed a neuropeptidomic analysis of other feeding-related organs, namely salivary glands and gut. We identified NPLP1 in salivary glands and myosuppressin in midgut. This is the first report suggesting a role for NPLP1, involving the peptides processed from this precursor in the hormonal control of the production and/or release of saliva. Our results contribute to the understanding of the postprandial neuroendocrine response in hematophagous and provide important information for physiological and pharmacological studies aimed to the design of next-generation insecticides such as peptidomimetics.
The use of chemical insecticides is the main control method for Blattella germanica worldwide. The prolonged and frequent use of insecticides produced the selection of insecticide-resistant ...individuals. The German cockroach is one of the most widespread urban pests in Argentina. In the last decades, resistance monitoring studies in this country demonstrated that there is a high prevalence of pyrethroid-resistant populations of B. germanica in the field. In this work, we studied the resistance mechanisms of a field-collected strain of B. germanica at toxicological, enzymatic, and molecular levels. A resistance ratio of 100 was obtained for the resistant strain when it was exposed to β-cypermethrin. The pretreatment with specific synergists (piperonyl butoxide and triphenyl phosphate) led to a significant increase in the toxicity of the pyrethroid, suggesting an involvement of oxidases and esterases in the detoxification of this insecticide. Moreover, esterase and oxidase activities in the resistant strain were 1.5-fold and 2-fold higher respectively, compared to the susceptible individuals. On the other hand, the voltage-gated sodium channel gene of the resistant cockroaches did not show nucleotidic substitutions in the domain II which are associated to knockdown resistance in this species. These results suggest that the main mechanism of resistance of the studied cockroaches' strain is metabolic, mainly due to an increase in the activity of oxidase and esterase enzymes. The results of this work in addition to other reports found in literature show that the extended use of a single active principle for cockroach control promotes the development of resistance leading to control failure in the field. In contrast, integrated pest management strategies include the use of different control tools in addition to chemical insecticides, which delay the appearance of resistance increasing the efficacy of pest control.
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•B. germanica OK gene codes for two mRNAs, BgOKA and BgOKB.•BgOKA and BgOKB encode mainly OKA and OKB peptides, respectively.•BgOKA mRNA is localized in brain, while BgOKB mRNA is ...localized in brain and midgut.•BgOKs RNAi reduces oocyte growth and Vg expression, independently of JH.•Results suggest that BgOK expression contributes to the regulation of vitellogenesis.
Orcokinins (OKs) are neuropeptides that were first identified in crustacean through their myotropic activity. In insects, the OK gene gives rise to two mRNAs coding for two different families of conserved mature neuropeptides: OKA and OKB. Although OKs are conserved in many insect species, its physiological role in this animal class is not fully understood. Until now prothoracicotropic, regulatory of light entrainment to the circadian clock and “awakening” activities have been reported for these peptides in different insect species. Here we report the identification of OKA and OKB precursors in the cockroach Blattella germanica. OKA mRNA was detected in brain, whereas OKB mRNA was detected both in brain and midgut. In vivo silencing of OK precursors suggests the involvement of OK gene products in the regulation of vitellogenin expression in the fat body, an action that appears to be independent of juvenile hormone. This is the first time that a function of this kind has been reported for OKs.
Abstract
The use of insecticides has been a central approach to control disease-transmitting mosquitoes for the last century. The high prevalence of pyrethroid use as public health insecticides has ...resulted in the evolution of pyrethroid resistance in many populations of Aedes aegypti (Linnaeus) (Diptera: Culicidae), throughout its global distribution range. Insecticide resistance is often correlated with an associated fitness cost. In this project, we studied the phenotypes of hybrid mosquitoes derived from crossing a pyrethroid-resistant strain of Ae. aegypti (Puerto Rico PR) with a more susceptible one (Rockefeller ROCK). We first sequenced and compared the para gene of both original strains. We then crossed males from one strain with females of the other, creating two hybrids (Puertofeller, Rockorico). We used a Y-tube choice assay to measure the attraction of these strains towards a human host. We then compared the levels of pyrethroid resistance in the different strains. We found three known resistance mutations in the para gene sequence of the PR strain. In our attraction assays, PR females showed lower attraction to humans, than the ROCK females. Both hybrid strains showed strong attraction to a human host. In the insecticide resistance bottle assays, both hybrid strains showed marginal increases in resistance to permethrin compared to the more susceptible ROCK strain. These results suggest that hybrids of sensitive and permethrin-resistant mosquitoes have an incremental advantage compared to more susceptible mosquitoes when challenged with permethrin. This explains the rapid spread of permethrin resistance that was observed many times in the field.
► We predicted the sequence of the region IIS4–IIS6 from the para-type sodium channel gene in the triatomine Rhodnius prolixus. ► We sequenced the IIS4–IIS6 region in the triatomine Triatoma ...infestans. ► We identified a point mutation in the IIS4–IIS6 region that is associated with pyrethroid resistance in T. infestans. ► We designed and developed a method based in PCR for the detection of the kdr mutation in T. infestans from the field.
The voltage-gated sodium channel is the target site of pyrethroid insecticides. Point mutations in the domain II region of the channel have been implicated in pyrethroid resistance of several insect species. We identified the sequence of domain II from the para sodium channel in Rhodnius prolixus, a vector of Chagas’ disease. With this information, we cloned and sequenced the domain II of the sodium channel from the other main Chagas’ disease vector: Triatoma infestans. We also identified the presence of a resistance-conferring mutation (L1014F) in a pyrethroid-resistant population of T. infestans from Argentina, and present a PCR-based method to detect this mutation in individuals from field populations. These findings have important implications for the implementation of strategies for resistance management, and for the rational design of campaigns for the control of Chagas’ disease transmission.
Stress exposure resulted in brain induction of immediate‐early genes (IEGs), considered as markers of neuronal activation. Upon repeated exposure to the same stressor, reduction of IEG response ...(adaptation) has been often observed, but there are important discrepancies in literature that may be in part related to the particular IEG and methodology used. We studied the differential pattern of adaptation of the IEGs c‐fos and arc (activity‐regulated cytoskeleton‐associated protein) after repeated exposure to a severe stressor: immobilization on wooden boards (IMO). Rats repeatedly exposed to IMO showed reduced c‐fos mRNA levels in response to acute IMO in most brain areas studied: the medial prefrontal cortex (mPFC), lateral septum (LS), medial amygdala (MeA), paraventricular nucleus of the hypothalamus (PVN) and locus coeruleus. In contrast, the number of neurons showing Fos‐like immunoreactivity was only reduced in the MeA and the various subregions of the PVN. IMO‐induced increases in arc gene expression were restricted to telencephalic regions and reduced by repeated IMO only in the mPFC. Double‐labelling in the LS of IMO‐exposed rats revealed that arc was expressed in only one‐third of Fos+ neurons, suggesting two populations of Fos+ neurons. These data suggest that c‐fos mRNA levels are more affected by repeated IMO than corresponding protein, and that arc gene expression does not reflect adaptation in most brain regions, which may be related to its constitutive expression. Therefore, the choice of a particular IEG and the method of measurement are important for proper interpretation of the impact of chronic repeated stress on brain activation.
Background Chagas' disease is an important public health concern in Latin America. Despite intensive vector control efforts using pyrethroid insecticides, the elimination of Triatoma infestans has ...failed in the Gran Chaco, an ecoregion that extends over Argentina, Paraguay, Bolivia and Brazil. The voltage-gated sodium channel is the target site of pyrethroid insecticides. Point mutations in domain II region of the channel have been implicated in pyrethroid resistance of several insect species. Methods and Findings In the present paper, we identify L925I, a new pyrethroid resistance-conferring mutation in T. infestans. This mutation has been found only in hemipterans. In T. infestans, L925I mutation occurs in a resistant population from the Gran Chaco region and is associated with inefficiency in the control campaigns. We also describe a method to detect L925I mutation in individuals from the field. Conclusions and Significance The findings have important implications in the implementation of strategies for resistance management and in the rational design of campaigns for the control of Chagas' disease transmission.
Metyrapone, a 11‐β steroid hydroxylase inhibitor that blocks stress‐induced glucocorticoid release, is extensively used to study the physiological and behavioural roles of glucocorticoids. However, ...there is circumstancial evidence suggesting that metyrapone could act as a pharmacological stressor. Thus, the effects of various doses of metyrapone on two well‐characterized stress markers (ACTH and glucose) were studied in male rats. Metyrapone administration, while exerting a modest effect on plasma corticosterone levels, dose‐dependently increased plasma ACTH and glucose levels. Using the highest doses previously tested (200 mg/kg) we further observed, as evaluated by fos‐like immunoreactivity (FLI), a strong activation of a wide range of brain areas, including the parvocellular region of the hypothalamic paraventricular nucleus (PVNp), the origin of the main ACTH secretagogues. Metyrapone‐induced FLI was observed in neocortical and allocortical areas, in several limbic, thalamic and hypothalamic nuclei and, to a lesser extent, in the brainstem. In a final experiment, a dose–response study of metyrapone‐induced FLI was carried out focusing on selected brain areas. The study revealed that the paraventricular thalamic nucleus and central amygdala were the areas most sensitive to metyrapone as they responded even to the lowest dose of the drug. Most areas, among them the PVNp, only showed enhanced FLI with the two highest doses, i.e. when it was associated with ACTH and glucose responses. These data suggest that some of the effects of metyrapone could be due to its stressful properties rather than its ability to inhibit glucocorticoid synthesis. The exact mechanisms involved remain to be established.
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