Since empty-spiracles (ems) was identified and characterized in Drosophila melanogaster as a head-gap gene, several studies have been carried out in other insect orders to confirm its evolutionary ...conserved function. Using the blood-sucking bug Rhodnius prolixus as biological model, we found an ems transcript with three highly conserved regions: Box-A, Box-B, and the homeodomain. R. prolixus embryos silenced by parental RNAi for two of these ems conserved regions showed both maternal and zygotic defects. Rp-emsB fragment results in early lethal embryogenesis, with eggs without any embryonic structure inside. Rp-emsB expression pattern is only maternally expressed and localized in the ovary tropharium, follicular cells, and in the unfertilized female pronucleus. Rp-emsA fragment is zygotically expressed during early blastoderm formation until late developmental stages in two main patterns: anterior in the antennal segment, and in a segmentary in the neuroblast and tracheal pits. R. prolixus knockdown embryos for Rp-emsA showed an incomplete larval hatching, reduced heads, and severe neuromotor defects. Furthermore, in situ hybridization revealed a spatial and temporal expression pattern that highly correlates with Rp-ems observed function. Here,Rp-ems function in R. prolixus development was validated, showing that empty-spiracles does not act as a true head-gap gene, but it is necessary for proper head development and crucial for early embryo determination and neurodevelopment.
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•We functionally describe the empty-spiracles gene in Rhodnius prolixus•Rp-ems is maternally expressed in ovary tropharium, follicular cells, and oocyte nucleus.•Zygotic expression is located in the head region and in a body segmented pattern.•Maternal RNAi phenotype shows eggs without any embryological structure.•Zygotic Rp-ems knockdown results in embryos with cephalic and neuromotor defects.
Insects operate complex humoral and cellular immune strategies to fend against invading microorganisms. The majority of these have been characterized in Drosophila and other dipterans. Information on ...hemipterans, including Triatominae vectors of Chagas disease remains incomplete and fractionated.
We identified putative immune-related homologs of three Triatominae vectors of Chagas disease, Triatoma pallidipennis, T. dimidiata and T. infestans (TTTs), using comparative transcriptomics based on established immune response gene references, in conjunction with the predicted proteomes of Rhodnius prolixus, Cimex lecticularis and Acyrthosiphon pisum hemimetabolous. We present a compressive description of the humoral and cellular innate immune components of these TTTs and extend the immune information of other related hemipterans. Key homologs of the constitutive and induced immunity genes were identified in all the studied hemipterans.
Our results in the TTTs extend previous observations in other hemipterans lacking several components of the Imd signaling pathway. Comparison with other hexapods, using published data, revealed that the absence of various Imd canonical components is common in several hemimetabolous species.
Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people ...worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome (∼ 702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immune-deficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods.
Stinkbugs (Hemiptera: Pentatomidae) are of major economic importance as pest of crops. Among the species composing the stinkbug complex, Nezara viridula is one of the most abundant in Brazil, ...Argentina and the Southern USA. However, this species has been poorly characterized at the genetic and physiological level. Here we sequenced and analyzed the complete transcriptome of N. viridula male and female adults. We identified neuropeptide precursor genes and G-protein coupled receptors for neuropeptides in this transcriptome. Mature neuropeptides were identified in N. viridula brain extracts by liquid chromatography-tandem mass spectrometry. We also analyzed the neuropeptide precursor complement in the genome sequence of Halyomorpha halys, another pentatomid of economic relevance. We compared the results in both pentatomids with the well-characterized neuropeptide repertoire from the kissing bug Rhodnius prolixus (Hemiptera: Reduviidae). We identified both group-specific features (which could be related to the different feeding habits) and similarities that could be characteristic of Heteroptera. This work contributes to a deeper knowledge of the genetic information of these pests, with a focus on neuroendocrine system characterization.
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.
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.
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.
Chagas disease is a parasitic infection caused by Trypanosoma cruzi. It is an important public health problem affecting around seven to eight million people in the Americas. A large number of ...hematophagous triatomine insect species, occupying diverse natural and human-modified ecological niches transmit this disease. Triatomines are long-living hemipterans that have evolved to explode different habitats to associate with their vertebrate hosts. Understanding the molecular basis of the extreme physiological conditions including starvation tolerance and longevity could provide insights for developing novel control strategies. We describe the normalized cDNA, full body transcriptome analysis of three main vectors in North, Central and South America, Triatoma pallidipennis, T. dimidiata and T. infestans.
Two-thirds of the de novo assembled transcriptomes map to the Rhodnius prolixus genome and proteome. A Triatoma expansion of the calycin family and two types of protease inhibitors, pacifastins and cystatins were identified. A high number of transcriptionally active class I transposable elements was documented in T. infestans, compared with T. dimidiata and T. pallidipennis. Sequence identity in Triatoma-R. prolixus 1:1 orthologs revealed high sequence divergence in four enzymes participating in gluconeogenesis, glycogen synthesis and the pentose phosphate pathway, indicating high evolutionary rates of these genes. Also, molecular evidence suggesting positive selection was found for several genes of the oxidative phosphorylation I, III and V complexes.
Protease inhibitors and calycin-coding gene expansions provide insights into rapidly evolving processes of protease regulation and haematophagy. Higher evolutionary rates in enzymes that exert metabolic flux control towards anabolism and evidence for positive selection in oxidative phosphorylation complexes might represent genetic adaptations, possibly related to prolonged starvation, oxidative stress tolerance, longevity, and hematophagy and flight reduction. Overall, this work generated novel hypothesis related to biological adaptations to extreme physiological conditions and diverse ecological niches that sustain Chagas disease transmission.
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.
The segmentation process in insects depends on a hierarchical cascade of gene activity. The first effectors downstream of the maternal activation are the gap genes, which divide the embryo in broad ...fields. We discovered a sequence corresponding to the leucine-zipper domain of the orthologue of the gene
giant (
Rp-
gt) in traces from the genome of
Rhodnius prolixus, a hemipteran with intermediate germ-band development. We cloned the
Rp-
gt gene from a normalized cDNA library and characterized its expression and function. Bioinformatic analysis of 12.5
kbp of genomic sequence containing the
Rp-
gt transcriptional unit shows a cluster of
bona fide regulatory binding sites, which is similar in location and structure to the predicted posterior expression domain of the
Drosophila orthologue.
Rp-gt is expressed in ovaries and maternally supplied in the early embryo. The maternal contribution forms a gradient of scattered patches of mRNA in the preblastoderm embryo. Zygotic
Rp-gt is expressed in two domains that after germ band extension are restricted to the head and the posterior growth zone. Parental RNAi shows that
Rp-gt is required for proper head and abdomen formation. The head lacks mandibulary and maxillary appendages and shows reduced clypeus-labrum, while the abdomen lacks anterior segments. We conclude that
Rp-gt is a gap gene on the head and abdomen and, in addition, has a function in patterning the anterior head capsule suggesting that the function of
gt in hemipterans is more similar to dipterans than expected.
► We studied the gap gene
giant in
Rhodnius prolixus. ► We predict regulatory regions by comparative genomics. ► We show
the Rp-gt expression pattern and its correlation with the phenotype in the head and abdomen. ► We show that
giant is involved in the formation of the anterior most head capsule.
The establishment of the anterior–posterior segmentation in insects requires the concerted action of a hierarchical gene network. Here, we study the orthologue of Krüppel gap gene in the hemipteran ...Rhodnius prolixus (Rp-Kr). We characterized its structure, expression pattern and function. The genomic sequence upstream of the Rp-Kr transcriptional unit shows a putative regulatory region conserved in the orthologue genes from Drosophila melanogaster and Tribolium castaneum. Rp-Kr expression is zygotic and it is expressed in the anterior half of the embryo (the posterior half of the egg) during the blastoderm stage and germ band formation; later, during germ band extension, it is expressed in a central domain, from T2 to A3. The Rp-Kr loss of function phenotypes shows disrupted thoracic and abdominal segmentation. Embryos with weak segmentation phenotypes show homeotic transformations, in which an ectopic tibial comb, typical of T1 leg, appears in T2, which correlates with the ectopic expression of Rp-sex-comb reduced in this leg.
•We identify the Krüppel ortholog gene in R. prolixus.•A Rp-Kr conserved regulatory cluster corresponds to Drosophila CD1.•RNAi experiments evidence a classical gap phenotype.•Rp-Kr-embryos show a homeotic phenotype likely due to derepression of Scr.
Rhodnius prolixusnot only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people ...worldwide. We sequenced the genome ofR. prolixus,generated assembled sequences covering 95% of the genome (∼702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immunedeficiency (IMD)-mediated immune responses were observed,R. prolixusputatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affectedTrypanosoma cruzi,the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms.R. prolixushas experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes fromWolbachiasp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences inR. prolixus,including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods.
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