Transcriptomic analysis and molecular docking reveal genes involved in the response of Aedes aegypti larvae to an essential oil extracted from Eucalyptus.

BACKGROUND: Aedes aegypti (L.) is an urban mosquito, vector of several arboviruses that cause severe diseases in hundreds of million people each year. The resistance to synthetic insecticides developed by Ae. aegypti populations worldwide has contributed to failures in vector control campaigns, increasing the impact of arbovirus diseases. In this context, plant-derived essential oils with larvicidal activity could be an attractive alternative for vector control. However, the mode of action and the detoxificant response of mosquitoes to plant derived compounds have not been established, impairing the optimization of their use. METHODS AND FINDINGS: Here we compare gene expression in Ae. aegypti larvae after 14 hrs of exposure to Eucalyptus camaldulensis essential oil with a control group exposed to vehicle (acetone) for the same lapse, by using RNA-Seq. We found differentially expressed genes encoding for cuticle proteins, fatty-acid synthesis, membrane transporters and detoxificant related gene families (i.e. heat shock proteins, cytochromes P450, glutathione transferases, UDP-glycosyltransferases and ABC transporters). Finally, our RNA-Seq and molecular docking results provide evidence pointing to a central involvement of chemosensory proteins in the detoxificant response in mosquitoes. CONCLUSIONS AND SIGNIFICANCE: Our work contributes to the understanding of the physiological response of Ae. aegypti larvae to an intoxication with a natural toxic distilled from Eucalyptus leafs. The results suggest an involvement of most of the gene families associated to detoxification of xenobiotics in insects. Noteworthy, this work provides important information regarding the implication of chemosensory proteins in the detoxification of a natural larvicide. Understanding the mode of detoxification of Eucalyptus distilled compounds could contribute to their implementation as a tool in mosquito control.

Insecticide Resistance Mechanisms in Triatoma infestans (Reduviidae: Triatominae): The Putative Role of Enhanced Detoxification and Knockdown Resistance (kdr) Allele in a Resistant Hotspot From the Argentine Chaco.

Chagas disease affects around 6 million people in the world, and in Latin America, it is mainly transmitted by the kissing bug. Chemical control of the vector with pyrethroid insecticides has been the most frequently used tool to reduce the disease incidence. Failures of field control have been detected in areas of the Argentinian Gran Chaco that correlate with high levels of insecticide resistance. Here, we provide evidence of the mechanisms involved in the resistance to insecticides of field populations of T. infestans from General Güemes Department (Chaco Province, Argentina). The biochemical analysis suggests the increase in the activity of the degradative enzymes P450 oxidases and esterases as a minor contributive mechanism in low-resistance populations. The molecular study revealed high frequencies of the kdr L925I mutation at the voltage-gated sodium channel as responsible for the high resistance ratios detected. This knowledge contributes to the generation of comprehensive vector control strategies that reduce the incidence of the disease.

Comparative and functional triatomine genomics reveals reductions and expansions in insecticide resistance-related gene families.

BACKGROUND: Triatomine insects are vectors of Trypanosoma cruzi, a protozoan parasite that is the causative agent of Chagas' disease. This is a neglected disease affecting approximately 8 million people in Latin America. The existence of diverse pyrethroid resistant populations of at least two species demonstrates the potential of triatomines to develop high levels of insecticide resistance. Therefore, the incorporation of strategies for resistance management is a main concern for vector control programs. Three enzymatic superfamilies are thought to mediate xenobiotic detoxification and resistance: Glutathione Transferases (GSTs), Cytochromes P450 (CYPs) and Carboxyl/Cholinesterases (CCEs). Improving our knowledge of key triatomine detoxification enzymes will strengthen our understanding of insecticide resistance processes in vectors of Chagas' disease. METHODS AND FINDINGS: The discovery and description of detoxification gene superfamilies in normalized transcriptomes of three triatomine species: Triatoma dimidiata, Triatoma infestans and Triatoma pallidipennis is presented. Furthermore, a comparative analysis of these superfamilies among the triatomine transcriptomes and the genome of Rhodnius prolixus, also a triatomine vector of Chagas' disease, and other well-studied insect genomes was performed. The expression pattern of detoxification genes in R. prolixus transcriptomes from key organs was analyzed. The comparisons reveal gene expansions in Sigma class GSTs, CYP3 in CYP superfamily and clade E in CCE superfamily. Moreover, several CYP families identified in these triatomines have not yet been described in other insects. Conversely, several groups of insecticide resistance related enzymes within each enzyme superfamily are reduced or lacking in triatomines. Furthermore, our qRT-PCR results showed an increase in the expression of a CYP4 gene in a T. infestans population resistant to pyrethroids. These results could point to an involvement of metabolic detoxification mechanisms on the high levels of pyrethroid resistance detected in triatomines from the Gran Chaco ecoregion. CONCLUSIONS AND SIGNIFICANCE: Our results help to elucidate the potential insecticide resistance mechanisms in vectors of Chagas' disease and provide new relevant information for this field. This study shows that metabolic resistance might be a contributing cause of the high pyrethroid resistance observed in wild T. infestans populations from the Gran Chaco ecoregion, area in which although subjected to intense pyrethroid treatments, vector control has failed. This study opens new avenues for further functional studies on triatomine detoxification mechanisms.

Orcokinin neuropeptides regulate ecdysis in the hemimetabolous insect Rhodnius prolixus.

To grow and develop insects must undergo ecdysis. During this process, the individual sheds the old cuticle to emerge as the following developmental stage. During ecdysis, different programed behaviors are regulated by neuropeptidergic pathways. In general, components of these pathways are better characterized in crustacean and holometabolous insects than in hemimetabola. In insects, the orkoninin gene produces two different neuropeptide precursors by alternative splicing: orcokinin A and orcokinin B. Although orcokinins are well conserved in insect species, their physiological role remains elusive. Here we describe a new splicing variant of the orcokinin gene in the hemimetabolous triatomine Rhodnius prolixus. We further analyze the expression pattern and the function of the alternatively spliced RhoprOK transcripts by means of immunohistochemistry and RNAi-mediated gene silencing. Our results indicate that orkoninis play an essential role in the peptidergic signaling pathway regulating ecdysis in the hemimetabolous insect Rhodnius prolixus.

Neuropeptidomics in Triatoma infestans. Comparative transcriptomic analysis among triatomines.

Chagas' disease, affecting up to 6-7 million people worldwide, is transmitted to humans through the feces of triatomine kissing bugs. From these, Rhodnius prolixus, Triatoma dimidiata, Triatoma infestans and Triatoma pallidipennis are important vectors distributed throughout the Latin American subcontinent. Resistance to pyrethroids has been developed by some triatomine populations, especially T. infestans, obstructing their control. Given their role in the regulation of physiological processes, neuroendocrine-derived factors have been proposed as a source of molecular targets for new-generation insecticides. However, the involvement of neuropeptides in insecticide metabolism and resistance in insects has been poorly studied. In the present work, the sequences of 20 neuropeptide precursor genes in T. infestans, 16 in T. dimidiata, and 13 in T. pallidipennis detected in transcriptomic databases are reported, and a comparative analysis in triatomines is presented. A total of 59 neuropeptides were validated by liquid chromatography-tandem mass spectrometry in brain and nervous ganglia from T. infestans, revealing the existence of differential post-translational modifications, extended and truncated forms. The results suggest a high sequence conservation in some neuropeptide systems in triatomines, whereas remarkable differences occur in several others within the core domains. Comparisons of the basal expression levels for several neuropeptide precursor genes between pyrethroid sensitive and resistant population of T. infestans are also presented here, in order to introduce a proof of concept to test the involvement of neuropeptides in insecticide resistance. From the precursors tested, NVP and ITG peptides are significantly higher expressed in the resistant population. To our knowledge, this is the first report to associate differential neuropeptide expression with insecticide resistance. The information provided here contributes to creating conditions to widely extend functional and genetic studies involving neuropeptides in triatomines.

Kdr mutations in Triatoma infestans from the Gran Chaco are distributed in two differentiated foci: Implications for pyrethroid resistance management.

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.

Identification of G protein coupled receptors for opsines and neurohormones in Rhodnius prolixus. Genomic and transcriptomic analysis.

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.