Transcriptomics Applied to the Study of Chagas Disease Vectors

ABSTRACT.

Chagas disease is a neglected disease caused by the protozoan Trypanosoma cruzi, and is transmitted mainly by the feces of contaminated triatomines. Knowledge of the biological, ecological, behavioral, genetic, taxonomic, and systematic aspects of these vectors can contribute to the planning of vector control programs, because all species are considered to be potential vectors of Chagas disease. Transcriptomic studies, in general, provided a new view of the physiology of triatomines (aiding in the knowledge of reproductive aspects of the hematophagy process and even the immune system and the sensory apparatus) and even contributed, as a new tool, to the taxonomy and systematics of these insects. Thus, we conducted a review of the transcriptomic studies on Chagas disease vectors.

INTRODUCTION

Chagas disease is a neglected disease caused by the protozoan Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae), and is transmitted mainly by the feces of contaminated triatomines (Hemiptera: Triatominae) (hematophagous insects that have a habit of defecating/urinating during or after the blood meal, which can release blood parasites in excreta if they are infected). ¹ Currently, there are about 8 million people infected and other 25 million at risk of infection, with vector control being the main measure to prevent new incidences of Chagas disease. ¹

Although the first species of triatomine (Triatoma rubrofasciata) was described in 1773, Chagas disease was only discovered after 136 years. ² Currently, there are 157 species of triatomines described. ^{3 – 5} Knowledge of the biological, ecological, behavioral, genetic, taxonomic, and systematic aspects of these vectors can contribute to the direction of vector control programs, because all species are considered to be potential vectors of Chagas disease. ^{3, 6}

One of the milestones of the application of omic tools in the study of these insect vectors was the characterization of the genome of Rhodnius prolixus. ⁷ However, before knowledge of the genome, omic studies had already been applied to triatomines, contributing the knowledge of taxonomy, systematics, as well as vector biology. ⁸

Transcriptomic studies, in general, provided a new view of the physiology of triatomines (aiding in the knowledge of reproductive aspects, ^{9, 10} of the hematophagy process, ^{11, 12} and even the immune system ¹³ and the sensory apparatus ¹⁴), and also contributed to the taxonomy and systematics of these insects. ^{15, 16} Thus, we conducted a review of the transcriptomic studies on Chagas disease vectors.

METHODS

The review was based on scientific articles published from 2004 to 2021, with 2004 being the year for the first publication of transcriptomic data in triatomines. ^{17, 18} Based on the keywords “transcriptome,” transcripts,” and “transcriptomics”, which were combined with the keywords “triatominae/triatomines,” searches were performed in the Scientific Electronic Library Online (SciELO), Medical Literature Analysis and Retrieval System Online (MEDLINE/PubMed), SCOPUS, Web of Science, and Web BIBliografía de TRIatominos (BibTri) V3.0 databases, which resulted in 70 articles. Of the total scientific articles retrieved, 62 articles were used to write the review article.

TRANSCRIPTOMICS

Saliva/salivary gland transcriptome.

The transcriptome of a cell or tissue is the collection of RNAs transcribed in it (composed of messenger RNA (mRNA) or coding RNAs and a variety of non-coding RNAs). ¹⁷ For triatomines, the first work published with transcriptome analysis was that of Ribeiro et al., ¹⁸ who analyzed the transcriptome of the R. prolixus salivary gland (sialotranscriptome or sialoma) to gain further insight into the biochemistry and potent pharmacological substances of its saliva.

Transcriptomic analyses made it possible to observe a large expansion of lipocaine family proteins, as well as members of the nitrophorine families (with vasodilatory properties), ¹⁹ and triabine (a potent and selective thrombin inhibitor) ^{20, 21} and palidipine (an inhibitor of platelet aggregation)—both also identified from the T. pallidipennis saliva transcriptome. ^{21 – 23} The most abundant transcripts and proteins in T. pallidipennis saliva were anti-hemostatic triabines, although others involving antimicrobial and thrombolytic polypeptides were also found in addition to unique proteins such as angiotensin-converting enzyme. ²³

In Panstrongylus megistus, P. chinai, R. neglectus, T. matogrossensis, T. brasiliensis, T. infestans, T. dimidiata, and T. rubida sialoma, high numbers and a variety of lipocalins were found, as observed in R. prolixus saliva: palidipine, triabine, and procaline (a salivary allergen), in addition to a large number of Kazal-type proteins (serine protease inhibitors). ^{13, 18, 21, 24 – 35} In P. megistus and T. infestans, relatively well-expressed transcripts for the enzymes apyrase, inositol phosphatase, serine proteases, peptides containing the Kazal domain, family of antigen 5, family of hemolysin and trialisin, and some families of desorphanized proteins have also been identified. ^{31, 32}

In P. lignarius salivary transcripts lipocalins, small-molecule binding proteins, two apyrases-like proteins of the hydrolysate adenosine triphosphate and adenosine diphosphate, and proteins of the Kazal family have been found. ¹³ In Dipetalogaster maxima, a salivary transcriptome was observed that includes lipocalins like salivary proteins of other triatomines species, such as palidipine and procaline. ³⁶ However, when comparing sets of the D. maxima salivary transcriptome with other triatomines, it was possible to see that Dipetalogaster has a less complex sialome compared with species of the Triatoma genus, which presents a set of extra proteins such as trialysin, triatox, Kazal, pheromone binding, inositol phosphatase, and serine protease. ^{21, 23, 37 – 39} It was suggested that these proteins may be related to the adaptation of Triatoma to feeding on mammalian blood, whereas Dipetalogaster feeds mainly on lizards. ³⁶

Metabolism transcriptome.

Martínez-Barnetche et al. ⁴⁰ analyzed transcripts involved in the metabolism of three species of triatomines (T. pallidipennis, T. dimidiata, and T. infestans). They identified a numerical enrichment of transcripts that encode the protease inhibitor domains of the pacifastin (T. dimidiata and T. pallidipennis) and cystatin (the three species) families. Pacifastins are a family of protease inhibitors belonging to the MEROPS family of inhibitors, involved in the regulation of different proteolytic cascades, including phenoloxidase-dependent melanization. ⁴¹ The representation of pacifastine was most notable in T. pallidipennis, with 17 unique transcripts compared with nine genes in the R. prolixus genome. ⁴⁰ Cystatins belong to a large and widespread family of cysteine ​​inhibitors in plants, prokaryotes, and animals. ⁴² Cystatins also play an anti-hemostatic role during the blood meal in ticks, ^{43, 44} but they appear to be absent in the Triatoma sialotranscriptomes described so far. ^{21, 26, 29, 30, 36} There is only one protease inhibitor gene with cystatin domain in R. prolixus (IPR000010), and cystatins were more abundant in T. dimidiata (n = 9), T. pallidipennis (n = 6), and T. infestans (n ​​= 4). ⁴⁰ However, the authors point out that the cystatin expansion documented in T. pallidipennis and T. dimidiata deserves further studies to elucidate its biological significance. ⁴⁰

Martínez-Barnetche et al. ⁴⁰ found an important difference between the three Triatoma species. The largest number of transcriptionally active transposable elements is in T. infestans, particularly non-LTR retrotransposons (increased between 8 and 12 times), ⁴⁰ which may be related in part to the size of the T. infestans genome, which is one of the largest among all analyzed reduvids. ⁴⁵ Although most enzymes in the glycolytic pathway have been highly conserved in the three species of Triatoma, a sequence divergence has been identified in the enzymes involved in anabolism (gluconeogenesis, glycogen synthesis, and pentose phosphate pathway), and an enzyme that participates in glycolysis. ⁴⁰ Greater evolution rates in critical enzymes that control the metabolic flow may represent genetic adaptations favoring characteristics of lifestyles of triatomines. Glycogen storage serves to deal with prolonged hunger and reproductive success; antioxidant mechanisms can be used to deal with oxidative stress, which favors longevity; and optimization of aerobic metabolism meets the energy demands of hematophagy. ⁴⁰

Kazal inhibitors have also been found and characterized in the midgut of some triatomines, including D. maxima (dipetalogastin), R. prolixus (rhodinin), T. infestans (infestin), and T. brasiliensis (brasiliensina). ²³ Among the gastrointestinal tract transcripts of R. prolixus, digestive enzymes, protease inhibitors, lipocalin family proteins, carrier proteins, cytoskeleton-related genes, immunity-related transcripts (such as PAMP recognition molecules and signal transducers), effector proteins, as well as genes that belong to the TOLL pathway were found. ⁴⁶ Furthermore, lipid metabolism-related transcripts showed genes involved in β-oxidation, suggesting that the Rhodnius midgut uses fatty acid oxidation as the main energy source whereas amino acid metabolism-related transcripts showed a marked predominance of β-oxidation enzymes and amino acid degradation. ⁴⁶

For R. prolixus, an insulin receptor has also been identified that is present in 10 tissues studied, and it is expressed in different ways in each tissue, suggesting that this receptor is probably involved in a variety of physiological processes. ⁴⁷ For P. lignarius fatty body transcripts, the predominant protein subclass was classified as secreted proteins related to peptide synthesis with distinct functions, many of which were intended for the hemolymph compartment, and the other highly expressed subclass was also associated with storage of the secreted proteins intended for hemolymph. ¹³ In addition, transferrin (which is synthesized and stored in the adipose body for later hemolymph secretion and may function in insect defense mechanisms) has also been identified in the fatty body. ¹³

Reproductive system transcriptome.

Analyses of ovarian follicular tissue and testis transcripts of R. prolixus have been performed. ^{48, 49} In the study of ovarian follicular tissue, the most abundant transcript was a small proline-rich secreted protein (S) that may play a role in oocyte maturation. ⁴⁸ A protein called Rp45 was also identified and described as a component of Rhodnius eggshell. Furthermore, VASA-type transcripts that may play a role in oocyte development have also been found. The overall analysis of the transcript classification showed that almost two thirds of all expressed proteins were functional type and somehow related to transcription, translation, protein turnover, and vesicular traffic, reflecting the immense metabolic effort for egg maturation. ⁴⁸ In testis, the most abundant families of transcripts were related to spermatogenesis and fertility, such as myosins, actins, tubulins, and dyneins. ^{49, 50} Myosins may be related to acrosome development, vesicle transport, gene transcription, and nuclear modeling, whereas actins may be related to correct nuclear positioning, germinal vesicle breakdown, spindle migration and rotation, and chromosome segregation in spermatogenesis. ^{49, 50} Dyneins and tubulins are involved with flagella structure and motility. ^{49, 51} Also, transcripts of lipocalin family proteins and serine protease inhibitors were found, showing an important role in sperm maturation and the role of serine protease inhibitors in spermatogenesis, respectively. ⁴⁹ In addition, immunity-related transcripts have also been identified, and most of them are lysozymes. ⁴⁹

Brito et al., ⁹ investigated the molecular mechanisms that coordinate and direct oogenesis in Rhodnius using transcriptomic analyses in ovaries of females fed with blood. The study focused on the pre-vitellogenic phase of oogenesis and used the transcriptome profile to determine the extent of evolutionary and functional conservation of the piwi-interacting RNA (piRNA) pathway in R. prolixus. ⁹ They found a helicase, critical to producing piRNAs in Drosophila, as well as the piwi orthologist Rp-piwi1, which appears to be expressed at very modest levels or not expressed in Rhodnius ovaries. Furthermore, analysis of normalized RNAseq readings showed that Rp-piwi2, Rp-piwi3, and Rp-ago3 are expressed at intermediate or low levels during Rhodnius oogenesis. ⁹

Brito et al. ⁹ proposed that Rp-piwi1 is a pseudogene, resulting from the absence of intronic sequences, and that it may not be necessary for the development of Rhodnius. Transcriptomic data supported this hypothesis, although the authors have not ruled out that Rp-piwi1 can be expressed in tissues other than the ovary. ⁹ With this work, it was demonstrated that the central components of the piRNA pathway, first described in Drosophila, are conserved in R. prolixus and that this insect houses four putative piwi genes. In addition, it was possible to observe that Rp-piwi2, Rp-piwi3, and Rp-ago3, but not Rp-piwi1, are expressed in ovaries. ⁹ The experiments carried out using piRNA against the Rp-piwi2 gene resulted in the interruption of oogenesis and complete sterility of the adult female. Data also revealed that a variety of transposable elements are expressed in the early stages of oogenesis, and their transcripts accumulate in the developing oocyte. ¹⁰ Thus, the authors suggest that the piRNA pathway decreases the transposable and repetitive sequences efficiently in R. prolixus oogenesis, and proves that piwi genes are essential for adult Rhodnius oogenesis and fertility, and probably have similar functions in other species of triatomines. ^{9, 10}

Analyzing the ovary transcriptome of R. prolixus, Coelho et al. ¹⁰ were able to observe many genes that probably encode critical factors in the development of this insect, as they include histone-modifying enzymes, chromatin remodeling factors, homeotic genes, signaling molecules, kinases, and metabolic enzymes. Moreover, they were able to determine transcription variants generated by alternative splicing events for more than 6,000 genes. Through data related to the production of oocytes, they observed that the production of oocytes in R. prolixus seems to be restricted to the stages of nymph, and that the expression of several orthologous genes occurs in pre-vitellogenic stages and their transcripts are deposited in mature eggs. It may represent a consequence of the structure of the egg chambers and ovarioles of R. prolixus. ¹⁰

The data presented also showed that the transcripts encoding a variety of ribosomal proteins are among the top 25 genes expressed at the beginning of oogenesis; however, they are underrepresented in unfertilized mature eggs. The authors then suggested that it is possible that in R. prolixus, ribosomal proteins, instead of the corresponding transcripts, are deposited in the egg and used in the early stages of embryogenesis, and this can ensure that ribosomal proteins are readily available to assemble ribosomes quickly. ¹⁰ In addition, a differentially expressed gene (Yellow-g2 [RPRC009244]) has been identified that has been associated with the resistance of the insect’s eggs to desiccation and the production of cuticle pigments, and at least two genes that can be activated in oogenesis to control oxidative stress caused by blood ingestion. ^{10, 52}

Detoxification system transcriptome.

Traverso et al. ⁵³ carried out a study of superfamily genes of triatomines related to detoxification by analyzing the transcriptomes of three species of triatomines T. infestans, T. dimidiata, and T. pallidipennis, and also used data extracted from genomic sequences of R. prolixus. They reported that P450 cytochromes are actively involved in the detoxification response of synthetic insecticides. ⁵³ The insect CYP genes fall into four main classes: CYP2, CYP3, CYP4, and mitochondrial clade, with each class being subdivided into several families and subfamilies. ⁵⁴ The authors noted that CYP3 is the most abundant class in insects, ⁵³ and its members are generally the genes that belong to the CYP6 and CYP9 families that are involved with insecticide resistance. ⁵⁴ The CYP9 family was not found in triatomines ⁵⁵; thus, CYP6 is the only family in the CYP3 clade of triatomines that is involved with insecticide resistance. ⁵⁵

Carboxyl/cholinesterases hydrolyze carboxylic esters to alcohols and acids. ⁵⁶ They are subdivided into three main classes, according to their function: 1) the food class, 2) the degradation class of hormones and pheromones, and 3) the neuro group. ⁵⁶ The authors noted that, of the observed SCCs, those belonging to the food class were absent in the four species of triatomines analyzed, and suggest an absence or extremely low representation of this class in triatomines, and that almost all detected SCCs are included in the class of hormones that degrade hormones and pheromones. ⁵³ The absence of the food class may be a result of a blood-only diet, with the absence of other types of metabolites. In addition, the deficiency of an entire class of SCC may be associated with a lesser potential for the detoxification of chemical insecticides compared with other species. ⁵³

Glutathione transferases (GSTs) are a family of enzymes that can use glutathione in reactions, contributing to the biodegradation and elimination of xenobiotics and reactive oxygen species generated during aerobic metabolism. ⁵⁷ Of the existing GST classes, the Delta and Epsilon classes are exclusive to insects and are the main classes associated with resistance to insecticides. ⁵³ However, they were observed with little abundance in the insects studied (T. infestans, T. dimidiata, and T. pallidipennis). ⁵³ The levels of three T. infestans genes, related to insecticide resistance, were compared between sensitive and pyrethroid-resistant populations (CYP4, carboxyl/cholinesterases Clade ii, and GST Delta). ⁵³ The authors realized that the analyzed CYP4 gene was overexpressed in insects in the resistant population, suggesting that this could be an expanded mechanism of resistance to pyrethroids in this species. The expression of the other studied enzymes did not differ significantly between sensitive and resistant populations. ⁵³

Immune system transcriptome.

Some work on triatomines using the transcriptomic technique has already identified components of the insect’s immune system. ^{13, 49} Recently, Zumaya-Estrada et al. ⁵⁸ performed a transcriptomic analysis of the reproductive and digestive tracts, Malpighian tubules, brain, adipose body, and salivary glands to describe innate immune response genes to triatomines. Through the analyses, the authors were able to provide evidence of the presence of important immune components in four important Chagas disease vectors (T. pallidipennis, T. dimidiata, T. infestans, and R. prolixus), as well as identify molecules related to the main immune categories (microbial recognition and activation, signaling, effectors, regulation, antioxidant system, RNA interference, and coagulation). ⁵⁸

Proteins involved in microbial recognition, immune activation, and various antimicrobial peptides have been documented in most species analyzed; lysozymes related to digestive and immune defense functions and defensins were detected. ⁵⁸ Most canonical components of the TOLL and Jak-STAT signaling cascades are conserved in the studied insects; however, the major components of the immunodeficiency pathway were absent. ⁵⁸

Sensory system transcriptome.

Aspects of the sensory apparatus of triatomines, such as the use of thermal and humidity signals, as well as vibratory signals, were also studied. As in other insects, antennae are the main chemosensory structures. ¹⁴ In addition, the expression of a variety of modulating process components, such as neuropeptides, GPCRs, and nuclear receptors, has already been observed in the R. prolixus antenna. ⁵⁹ Latorre-Estivalis et al. ^{14, 59} developed works related to the sensory apparatus of triatomines using transcriptomics. The study involving the R. prolixus antenna showed that these insects seem to increase the expression of several chemosensory genes after reaching adulthood. This can serve to improve the most necessary chemosensory skills in adulthood, as activities that require sensory action from the device, such as sexual behavior, oviposition, and escape, are activities performed exclusively by adult insects. ¹⁴

A large number of mechanoreceptors have also been observed, and this seems to correspond to the known ability of triatomines to detect vibratory signals. ⁶⁰ Several genes belonging to this category seem to have a more intense expression in male insect antennae, known in triatomines to detect vibratory sexual signals. ^{14, 60} It has also been reported that R. prolixus antennae produce a diversity of neuropeptide coding transcripts, with significantly lower expression of some neuropeptides in male antennae, which may suggest a specific role for the sex antenna. ¹⁴ In addition, some nuclear receptors are expressed on the antennae of this insect, suggesting that these organs have a wide capacity to respond to endocrine signals. ¹⁴

In addition to nuclear receptors, one study by Latorre-Estivalis et al. ⁵⁹ showed that R. prolixus antennae produce a diversity of neuropeptide coding transcripts, including high levels of allatostatin-CC peptide transcripts and ITG, in addition to orcokinin and IDLSRF-type peptide, which showed increased antennal expression after imaginal change, suggesting that these peptides can modulate sensory processes specific to adults underlying dispersion by flight and mating. However, the significantly lower expression of allatostatin-CC in male antennae may suggest a sex-specific antennal role. ⁵⁹ With the data analyzed, the authors were able to show that most nuclear insect receptors are expressed in an insect’s antennae, suggesting that these organs have a wide capacity to respond to endocrine signals. ⁵⁹

Transcriptome applied in taxonomic and systematic studies.

Transcriptomics has also been shown to be useful for triatomine systematics. ¹⁵ The identification of triatomines has been largely based on the observation of morphological characteristics. ⁶ As a result of the existence of cryptic or closely related species, many authors resorted to molecular data to differentiate these species. ⁶¹ In 2017, Carvalho et al. ¹⁵ used transcriptomics to look for differences in cephalic transcripts of R. montenegrensis and R. robustus. They were able to identify several sequences with a high level of differentiation between these species—in addition to the substantial number of fixed polymorphisms suggesting a clear separation of lineages—and these results support the hypothesis that these species are valid and genetically distinct. ¹⁵

A subsequent study using a transcriptome from members of the R. prolixus–R. robustus cryptic species complex found evidence confirming that R. montenegrensis and R. robustus genotype II are genetically identical and therefore are representative of the same species. ¹⁶ However, the authors point out that their findings do not invalidate R. montenegrensis; they only show that “R. montenegrensis” is the specific name to the so-called “R. robustus genotype II.” ¹⁶ In addition, the researchers found genetic evidence that the reference stocks for “R. robustus” used in the description of R. montenegrensis and transcriptome-based studies are from R. prolixus, probably mixed with R. robustus of type II genotype (known as also as R. montenegrensis). ¹⁶

Multiple-organ transcriptome.

Leyria et al. ^{11, 12} analyzed how a blood meal influences the expression of mRNA in the central nervous system (CNS), adipose body, and ovaries of R. prolixus and did not observe major genetic differences in the CNS between the non-fed condition and the fed condition. ^{11, 12} Vitellogenins, the main precursors of yolk protein, are large molecules synthesized predominantly by the adipose body. ¹² The results of the study by Leyria et al. ¹² showed that the levels of mRNA for vitellogenins are considerably greater in the adipose body in relation to the ovaries, and there was no significant difference between the fed and unfed conditions. The authors showed that in the adipose body, the levels of Rhodnius heme binding protein mRNA (which functions as an antioxidant in hemolymph) were regulated positively in females after feeding. In addition, the authors identified that the metabolism of arginine and proline is regulated positively in the adipose body after a meal of blood, and that proline metabolism can also contribute to the energy required by the ovary during vitellogenesis. ¹²

In R. prolixus, the data showed that Hsp70 (considered a good marker for an inducible stress response ^{62, 63}) is regulated positively in the adipose body of unfed females, a condition inherently associated with a stress condition. A member of the Hsp70 family, Grp78 has already been reported as a regulatory factor for vitellogenin synthesis and homeostasis in the adipose body. ⁶⁴ In the studied insect, regulation of a protein like Grp78 was observed in the adipose body and the ovaries of fed insects, which led the authors to suggest a new regulatory mechanism involved in the vitellogenic process of R. prolixus. ¹²

Notch is a receptor that directly translates information from cell–cell contact to gene expression in the nucleus. ⁶⁵ By analyzing the KEGG database, Leyria et al. ^{11, 12} showed that Notch signaling is regulated positively in the ovaries of fed females, ¹¹ and that the transcripts involved in Notch developmental functions are regulated upward in ovaries in the fed condition. ¹² By KEGG analysis, two pathways related to the juvenile hormone—insect hormone biosynthesis and terpenoid backbone biosynthesis—are regulated positively in the adipose body and ovaries during the fed condition. ¹¹ The authors also found five other related enzymes that are involved in the mevalonate pathway that are regulated positively in ovaries after a blood meal, enzymes that convert inactive juvenile hormone precursors into active juvenile hormones in the final stage of the juvenile hormone biosynthesis pathway in insects. ^{12, 66} Some precursors of the juvenile hormone have been identified, and all are present in the adipose body and ovaries. The authors suggest that the majority of vitellogenic ovaries and the adipose body may have the potential to synthesize juvenile hormone in R. prolixus. ¹²

The data showed a differentiated pattern of expression of takeout (To) genes that changes according to the tissue analyzed and the feeding condition. ¹² The mRNA expression of To1, To2, To4, and To7 is highly expressed in the CNS of unfed insects, and the authors suggest that hunger may induce the expression of these genes. The mRNA expression of To9, To11, To12, and To15 is increased in the adipose body of females after a blood meal, whereas the transcripts of To5, To12, and To13 show a significantly increased expression in ovaries in the fed condition. ¹² Thus, Leyria et al. ¹² presented the first report of an analysis of To genes in different tissues involved in reproduction in R. prolixus, providing new information on the mechanisms involved in the formation of eggs.

Leyria et al. ¹² found a trehalose transporter (the main blood sugar of insects) regulated more than six times in fed-insect ovaries and, with this finding, they support the hypothesis that a direct capture of hemolymph trehalose through this transporter can be an important process for the storage of carbohydrates in the ovaries. Because the vitellogenic process is an event with a high energy demand, trehalose uptake is necessary for the development of oocytes. ^{12, 67}

The analyses made it possible to observe seven enzymes involved in the processing of neuropeptides, and all of them are expressed in the CNS, adipose body, and ovaries in both nutritional conditions. The results support the contribution of adipose body and ovaries to the production of neuropeptides in both nutritional conditions. ¹² The authors found a positive regulation of NPLP1 transcript expression (NPLP1 peptides are involved in the food response ⁶⁸) in ovaries in the unfed condition, and the physiological role of NLPL1 signaling in reproduction is still unknown. In addition, they found a positive regulation of the corticotropin releasing factor receptor mRNA CRF/DH in ovaries and adipose body of unfed insects, where vitellogenesis is inhibited. This result supports its effects as a negative reproduction regulator, as it has been reported by Mollayeva et al. ⁶⁹ that the adult female of R. prolixus, injected with CRF/DH, produces and lays significantly fewer eggs.

The signaling pathway of the insulin-like peptide/rapamycin target (ILP/ToR) is responsible for nutritional detection. ⁷⁰ Leyria et al. ¹¹ analyzed the transcripts involved in ILP/ToR signaling. The data showed a positive regulation of ILP/ToR transcripts in unfed insects and demonstrated that this signaling is only activated in fed-insect tissues. The authors also noted that the forkhead box O, or FoxO, transcription factor, which regulates longevity through ILP signaling, is responsible for the positive regulation of transcripts related to ILP/ToR signaling in unfed insects. Given this, they found that unfed females quickly activate ILP/ToR signaling because they are in a sensitive state to respond to changes in ILP levels. ¹¹

CONCLUSION AND PROSPECTS

We reviewed all studies related to transcriptomics of Chagas disease vectors, contributing to the direction of further research with these insects, because it was evident that most studies focus on the physiological knowledge of the triatomines, contributing to the knowledge of saliva/salivary gland and metabolism, as well as the immune, reproductive, sensory, and detoxification systems of Chagas disease vectors. Given the importance of transcriptomics for taxonomy and systematics of Rhodnius, the need to apply this technique in other genera and species of the subfamily Triatominae (especially in those with taxonomic problems) is evident.