The role of neuropeptides in regulating ecdysis and reproduction in the hemimetabolous insect Rhodnius prolixus.

In ecdysozoan animals, moulting entails the production of a new exoskeleton and shedding of the old one during ecdysis. It is induced by a pulse of ecdysone that regulates the expression of different hormonal receptors and activates a peptide-mediated signalling cascade. In Holometabola, the peptidergic cascade regulating ecdysis has been well described. However, very little functional information regarding the neuroendocrine regulation of ecdysis is available for Hemimetabola, which display an incomplete metamorphosis. We use Rhodnius prolixus as a convenient experimental model to test two hypotheses: (1) the role of neuropeptides that regulate ecdysis in Holometabola is conserved in hemimetabolous insects; and (2) the neuropeptides regulating ecdysis play a role in the regulation of female reproduction during the adult stage. The RNA interference-mediated reduction of ecdysis triggering hormone (ETH) mRNA levels in fourth-instar nymphs resulted in lethality at the expected time of ecdysis. Unlike in holometabolous insects, knockdown of eth and orcokinin isoform A (oka) did not affect oviposition in adult females, pointing to a different endocrine regulation of ovary maturation. However, eth knockdown prevented egg hatching. The blockage of egg hatching appears to be a consequence of embryonic ecdysis failure. Most of the first-instar nymphs hatched from the eggs laid by females injected with dsRNA for eclosion hormone (dsEH), crustacean cardioactive peptide (dsCCAP) and dsOKA died at the expected time of ecdysis, indicating the crucial involvement of these genes in post-embryonic development. No phenotypes were observed upon corazonin (cz) knockdown in nymphs or adult females. The results are relevant for evolutionary entomology and could reveal targets for neuropeptide-based pest control tools.

Transcriptome analysis of Anastrepha fraterculus sp. 1 males, females, and embryos: insights into development, courtship, and reproduction.

BACKGROUND: Anastrepha fraterculus sp. 1 is considered a quarantine pest in several American countries. Since chemical control applied in an integrated pest management program is the only strategy utilized against this pest, the development of pesticide-free methods, such as the Sterile Insect Technique, is being considered. The search for genes involved in sex-determination and differentiation, and in metabolic pathways associated with communication and mating behaviour, contributes with key information to the development of genetic control strategies. The aims of this work were to perform a comprehensive analysis of A. fraterculus sp. 1 transcriptome and to obtain an initial evaluation of genes associated with main metabolic pathways by the expression analysis of specific transcripts identified in embryos and adults. RESULTS: Sexually mature adults of both sexes and 72 h embryos were considered for transcriptome analysis. The de novo transcriptome assembly was fairly complete (62.9% complete BUSCO orthologs detected) with a total of 86,925 transcripts assembled and 28,756 GO annotated sequences. Paired-comparisons between libraries showed 319 transcripts differently expressed between embryos and females, 1242 between embryos and males, and 464 between sexes. Using this information and genes searches based on published studies from other tephritid species, we evaluated a set of transcripts involved in development, courtship and metabolic pathways. The qPCR analysis evidenced that the early genes serendipity alpha and transformer-2 displayed similar expression levels in the analyzed stages, while heat shock protein 27 is over-expressed in embryos and females in comparison to males. The expression of genes associated with courtship (takeout-like, odorant-binding protein 50a1) differed between males and females, independently of their reproductive status (virgin vs mated individuals). Genes associated with metabolic pathways (maltase 2-like, androgen-induced gene 1) showed differential expression between embryos and adults. Furthermore, 14,262 microsatellite motifs were identified, with 11,208 transcripts containing at least one simple sequence repeat, including 48% of di/trinucleotide motifs. CONCLUSION: Our results significantly expand the available gene space of A. fraterculus sp. 1, contributing with a fairly complete transcript database of embryos and adults. The expression analysis of the selected candidate genes, along with a set of microsatellite markers, provides a valuable resource for further genetic characterization of A. fraterculus sp. 1 and supports the development of specific genetic control strategies.

Orcokinins regulate the expression of neuropeptide precursor genes related to ecdysis in the hemimetabolous insect Rhodnius prolixus.

Ecdysis is a vital process for insects, during which they shed the old cuticle in order to emerge as the following developmental stage. Given its relevance for survival and reproduction, ecdysis is tightly regulated by peptidic hormones that conform an interrelated neuromodulatory network. This network was studied in species that undergo a complete metamorphosis, but not in hemimetabola. In a recent work, we demonstrated that orcokinin neuropeptides are essential for ecdysis to occur in the kissing bug Rhodnius prolixus. Here we performed gene silencing, quantitative PCR and in vitro treatments in order to study the interrelationships between RhoprOKs and hormones such as ecdysis triggering hormone, corazonin, eclosion hormone, crustacean cardioactive peptide and ecdysone. Our results suggest that RhoprOKs directly or indirectly regulate the expression of other genes. Whereas RhoprOKA is centrally involved in the regulation of gene expression, RhoprOKB is implicated in processes related to midgut physiology. Therefore, we propose that the different transcripts encoded in RhoprOK gene could integrate signaling cues, in order to coordinate the nutritional state with development and ecdysis. Given the emerging data that point to OKs as important factors for survival and reproduction, they could be candidates in the search for new insect management strategies based on neuroendocrine targets.

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.

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.

Cardiac contractility structure-activity relationship and ligand-receptor interactions; the discovery of unique and novel molecular switches in myosuppressin signaling.

Peptidergic signaling regulates cardiac contractility; thus, identifying molecular switches, ligand-receptor contacts, and antagonists aids in exploring the underlying mechanisms to influence health. Myosuppressin (MS), a decapeptide, diminishes cardiac contractility and gut motility. Myosuppressin binds to G protein-coupled receptor (GPCR) proteins. Two Drosophila melanogaster myosuppressin receptors (DrmMS-Rs) exist; however, no mechanism underlying MS-R activation is reported. We predicted DrmMS-Rs contained molecular switches that resembled those of Rhodopsin. Additionally, we believed DrmMS-DrmMS-R1 and DrmMS-DrmMS-R2 interactions would reflect our structure-activity relationship (SAR) data. We hypothesized agonist- and antagonist-receptor contacts would differ from one another depending on activity. Lastly, we expected our study to apply to other species; we tested this hypothesis in Rhodnius prolixus, the Chagas disease vector. Searching DrmMS-Rs for molecular switches led to the discovery of a unique ionic lock and a novel 3-6 lock, as well as transmission and tyrosine toggle switches. The DrmMS-DrmMS-R1 and DrmMS-DrmMS-R2 contacts suggested tissue-specific signaling existed, which was in line with our SAR data. We identified R. prolixus (Rhp)MS-R and discovered it, too, contained the unique myosuppressin ionic lock and novel 3-6 lock found in DrmMS-Rs as well as transmission and tyrosine toggle switches. Further, these motifs were present in red flour beetle, common water flea, honey bee, domestic silkworm, and termite MS-Rs. RhpMS and DrmMS decreased R. prolixus cardiac contractility dose dependently with EC50 values of 140 nM and 50 nM. Based on ligand-receptor contacts, we designed RhpMS analogs believed to be an active core and antagonist; testing on heart confirmed these predictions. The active core docking mimicked RhpMS, however, the antagonist did not. Together, these data were consistent with the unique ionic lock, novel 3-6 lock, transmission switch, and tyrosine toggle switch being involved in mechanisms underlying TM movement and MS-R activation, and the ability of MS agonists and antagonists to influence physiology.