Rafael D. Mesquita

Transcriptome and gene expression profile of ovarian follicle tissue of the triatomine bug Rhodnius prolixus.

Insect oocytes grow in close association with the ovarian follicular epithelium (OFE), which escorts the oocyte during oogenesis and is responsible for synthesis and secretion of the eggshell. We describe a transcriptome of OFE of the triatomine bug Rhodnius prolixus, a vector of Chagas disease, to increase our knowledge of the role of FE in egg development. Random clones were sequenced from a cDNA library of different stages of follicle development. The transcriptome showed high commitment to transcription, protein synthesis, and secretion. The most abundant cDNA was a secreted (S) small, proline-rich protein with maximal expression in the vitellogenic follicle, suggesting a role in oocyte maturation. We also found Rp45, a chorion protein already described, and a putative chitin-associated cuticle protein that was an eggshell component candidate. Six transcripts coding for proteins related to the unfolded-protein response (UPR) by were chosen and their expression analyzed. Surprisingly, transcripts related to UPR showed higher expression during early stages of development and downregulation during late stages, when transcripts coding for S proteins participating in chorion formation were highly expressed. Several transcripts with potential roles in oogenesis and embryo development are also discussed. We propose that intense protein synthesis at the FE results in reticulum stress (RS) and that lowering expression of a set of genes related to cell survival should lead to degeneration of follicular cells at oocyte maturation. This paradoxical suppression of UPR suggests that ovarian follicles may represent an interesting model for studying control of RS and cell survival in professional S cell types.

A nuclear single-nucleotide polymorphism (SNP) potentially useful for the separation of Rhodnius prolixus from members of the Rhodnius robustus cryptic species complex (Hemiptera: Reduviidae)

The design and application of rational strategies that rely on accurate species identification are pivotal for effective vector control. When morphological identification of the target vector species is impractical, the use of molecular markers is required. Here we describe a non-coding, single-copy nuclear DNA fragment that contains a single-nucleotide polymorphism (SNP) with the potential to distinguish the important domestic Chagas disease vector, Rhodnius prolixus, from members of the four sylvatic Rhodnius robustus cryptic species complex. A total of 96 primer pairs obtained from whole genome shotgun sequencing of the R. prolixus genome (12,626 random reads) were tested on 43 R. prolixus and R. robustus s.l. samples. One of the seven amplicons selected (AmpG) presented a SNP, potentially diagnostic for R. prolixus, on the 280th site. The diagnostic nature of this SNP was then confirmed based on the analysis of 154 R. prolixus and R. robustus s.l. samples representing the widest possible geographic coverage. The results of a 60% majority-rule Bayesian consensus tree and a median-joining network constructed based on the genetic variability observed reveal the paraphyletic nature of the R. robustus species complex, with respect to R. prolixus. The AmpG region is located in the fourth intron of the Transmembrane protein 165 gene, which seems to be in the R. prolixus X chromosome. Other possible chromosomal locations of the AmpG region in the R. prolixus genome are also presented and discussed.

Platelet-activating factor (PAF) activates casein kinase 2 in the protozoan parasite Herpetomonas muscarum muscarum

Herpetomonas muscarum muscarum is a flagellate parasite of the family Trypanosomatidae, whose cell differentiation can be triggered by the lipid mediator, PAF. In this study we demonstrate for the first time that PAF effect relies on the activation of casein kinase 2 (CK2). The classical antagonist of PAF receptor, WEB 2086, abrogated PAF-enhanced CK2 activity. CK2 activation by PAF was also inhibited when parasite extracts were assayed in the presence of modulators of PKC, MAPK, and both Ser/Thr and Tyr phosphatases. Finally, a cell permeable inhibitor of CK2 (DRB) suppressed PAF-induced cell differentiation in a dose-dependent manner.

Silencing of Iron and Heme-Related Genes Revealed a Paramount Role of Iron in the Physiology of the Hematophagous Vector Rhodnius prolixus

Iron is an essential element for most organisms However, free iron and heme, its complex with protoporphyrin IX, can be extremely cytotoxic, due to the production of reactive oxygen species, eventually leading to oxidative stress. Thus, eukaryotic cells control iron availability by regulating its transport, storage and excretion as well as the biosynthesis and degradation of heme. In the genome of Rhodnius prolixus, the vector of Chagas disease, we identified 36 genes related to iron and heme metabolism We performed a comprehensive analysis of these genes, including identification of homologous genes described in other insect genomes. We observed that blood-meal modulates the expression of ferritin, Iron Responsive protein (IRP), Heme Oxygenase (HO) and the heme exporter Feline Leukemia Virus C Receptor (FLVCR), components of major pathways involved in the regulation of iron and heme metabolism, particularly in the posterior midgut (PM), where an intense release of free heme occurs during the course of digestion. Knockdown of these genes impacted the survival of nymphs and adults, as well as molting, oogenesis and embryogenesis at different rates and time-courses. The silencing of FLVCR caused the highest levels of mortality in nymphs and adults and reduced nymph molting. The oogenesis was mildly affected by the diminished expression of all of the genes whereas embryogenesis was dramatically impaired by the knockdown of ferritin expression. Furthermore, an intense production of ROS in the midgut of blood-fed insects occurs when the expression of ferritin, but not HO, was inhibited. In this manner, the degradation of dietary heme inside the enterocytes may represent an oxidative challenge that is counteracted by ferritins, conferring to this protein a major antioxidant role. Taken together these results demonstrate that the regulation of iron and heme metabolism is of paramount importance for R. prolixus physiology and imbalances in the levels of these key proteins after a blood- meal can be extremely deleterious to the insects in their various stages of development.

Genome Wide Mapping of Peptidases in Rhodnius prolixus: Identification of Protease Gene Duplications, Horizontally Transferred Proteases and Analysis of Peptidase A1 Structures, with Considerations on Their Role in the Evolution of Hematophagy in Triatominae

Triatominae is a subfamily of the order Hemiptera whose species are able to feed in the vertebrate blood (i.e., hematophagy). This feeding behavior presents a great physiological challenge to insects, especially in Hemipteran species with a digestion performed by lysosomal-like cathepsins instead of the more common trypsin-like enzymes. With the aim of having a deeper understanding of protease involvement in the evolutionary adaptation for hematophagy in Hemipterans, we screened peptidases in the Rhodnius prolixus genome and characterized them using common blast (NCBI) and conserved domain analyses (HMMER/blast manager software, FAT, plus PFAM database). We compared the results with available sequences from other hemipteran species and with 18 arthropod genomes present in the MEROPS database. Rhodnius prolixus contains at least 433 protease coding genes, belonging to 71 protease families. Seven peptidase families in R. prolixus presented higher gene numbers when compared to other arthropod genomes. Further analysis indicated that a gene expansion of the protease family A1 (Eukaryotic aspartyl protease, PF00026) might have played a major role in the adaptation to hematophagy since most of these peptidase genes seem to be recently acquired, are expressed in the gut and present putative secretory pathway signal peptides. Besides that, most R. prolixus A1 peptidases showed high frequencies of basic residues at the protein surface, a typical structural signature of Cathepsin D-like proteins. Other peptidase families expanded in R. prolixus (i.e., C2 and M17) also presented significant differences between hematophagous (higher number of peptidases) and non-hematophagous species. This study also provides evidence for gene acquisition from microorganisms in some peptidase families in R. prolixus: (1) family M74 (murein endopeptidase), (2) family S29 (Hepatitis C virus NS3 protease), and (3) family S24 (repressor LexA). This study revealed new targets for studying the adaptation of these insects for digestion of blood meals and their competence as vectors of Chagas disease.