Transcriptomic analysis of regulatory pathways involved in female reproductive physiology of Rhodnius prolixus under different nutritional states

The triatomine Rhodnius prolixus, a vector of the etiological agent of Chagas disease, has long been used as model to understand important aspects of insect physiology. Despite this history, the impact of the nutritional state on regulatory pathways associated with reproductive performance in triatomines has never been studied. The insulin-like peptide/target of rapamycin (ILP/ToR) signaling pathway is typically responsible for detecting and interpreting nutrient levels. Here, we analyzed transcriptomes from the central nervous system, fat bodies and ovaries of adult females in unfed and fed conditions, with a focus on the ILP/ToR signaling. The results show an up-regulation of transcripts involved in ILP/ToR signaling in unfed insects. However, we demonstrate that this signaling is only activated in tissues from fed insects. Moreover, we report that FoxO (forkhead box O) factor, which regulates longevity via ILP signaling, is responsible for the up-regulation of transcripts related with ILP/ToR signaling in unfed insects. As a consequence, we reveal that unfed females are in a sensitized state to respond to an increase of ILP levels by rapidly activating ILP/ToR signaling. This is the first analysis that correlates gene expression and protein activation of molecules involved with ILP/ToR signaling in R. prolixus females in different nutritional states.

total gene expression levels and co-expression analysis. Violin plots were used to compare total gene expression levels of each tissue under different nutritional states ( Supplementary Fig. S1). The results show that the same tissues, even under different nutritional conditions, i.e. UFC and FC, have a gene population comparable in distribution and density. However, differences could be observed between CNS, FB and OV. Differentially expressed genes (DEG) were also screened and summarized in Venn diagrams (Fig. 1). The results reveal the existence of genes that are uniquely expressed within each tissue and each condition, along with genes with overlapping regions that are expressed in the same tissue in both nutritional states (Fig. 1a) or different tissues in the same nutritional state (Fig. 1b). It is important to keep in mind that the unique gene expression percentages in each tissue during a specific nutritional state are low relative to overall gene expression. These results suggest that the expression levels of the same genes (overlapping) could define the physiological/metabolic response of a tissue according to nutritional state and not the expression of unique genes.
To assess the relationship between gene expression profiles of the CNS, FB and OV in the different nutritional states, we performed unsupervised hierarchical clustering of our complete RNA-Seq transcriptome data (Supplementary Fig. S2). As expected, genes of the same tissue cluster together, even during different nutritional conditions, and genes of different tissues, i.e. CNS, FB and OV, cluster separately, consistent with the unique functions of each.
Differentially expressed genes (DEG) analysis. Biological replicates were performed to demonstrate that the experiments are repeatable and to reveal differences in gene expression between the two nutritional states. A comparison among the 3 biological replicates of each nutritional state had a correlation coefficient (R 2 ) close to 1 (most > 0.9) (Supplementary Fig. S3). With these data, we were confident to advance to the next analysis. First, DEG were screened and volcano plots were used to infer overall distribution (Fig. 2). Genes with p-adj < 0.05 were assigned as differentially expressed. The results show 30  iLp/toR signaling analysis on cnS, fat bodies and ovaries. In insects, the availability of nutrients influences multiple signaling pathways. Nutrient depletion favors activation of processes involved in energy production, stress resistance and survival. On the other hand, in some species of insects, when the nutrients are abundant, ILP/ToR signaling is activated and cooperatively works with other pathways leading to successful reproduction 6 . For R. prolixus, specific genes related to ILP/ToR signaling were selected and analysed. As can Scientific RepoRtS | (2020) 10:11431 | https://doi.org/10.1038/s41598-020-67932-4 www.nature.com/scientificreports/ be seen, the CNS is metabolically stable under both nutritional conditions, i.e. non-DEG were found (Table 2). Previously we reported for fifth instars of R. prolixus that no significant changes in ILP transcript expression was observed relative to feeding 10 . In D. melanogaster, ILP2 transcript levels were also insensitive to nutrient deprivation but the protein secretion and signaling activity depends on metabolic needs; thus ILP2 is present in a basal level in fed insects and accumulates upon starvation 13 . ILPs are mainly synthesized in the CNS, but in some insects several ILPs have been reported to be produced and released by the FB as well as by other tissues 6 . In adults of R. prolixus, ILP is exclusively expressed in the CNS with higher expression compared with other transcripts related to this signaling pathway (Table 2). Also, in fifth instars of R. prolixus, ILP transcript is expressed around 25,000 times higher in CNS than in other tissues 10 .
In the FB, 28 transcripts involved with ILP/ToR signaling were analyzed, and 9 are up-regulated in the UFC (Table 3), including InR, venus kinase receptor (VKR), insulin receptor substrate 1 (IRS1), phosphatase and tensin homolog (PTEN), ribosomal protein S6 kinase (S6) and IGF. In the OV, and in agreement with the findings from the FB, 13 of 28 transcripts related with ILP/ToR signaling are up-regulated in the UFC, including InR, VKR, IRS1, ToR, ribosomal protein S6 kinase (S6K) and IGF (Table 4). To validate this data, different Table 1. Summary of RNA-seq metrics from R. prolixus transcriptomes for CNS, ovaries and fat bodies under both fed and unfed conditions. Raw Reads, the original sequencing reads counts; Clean Reads, number of reads after filtering; Raw Bases, raw reads number multiply read length, saved in G unit; Clean Bases, clean reads number multiply read length, saved in G unit; Error Rate: average sequencing error rate; Q20: percentages of bases whose correct base recognition rates are greater than 99% in total bases; Q30: percentages of bases whose correct base recognition rates are greater than 99.9% in total bases; GC content: percentages of G and C in total bases. Total mapped, total number of reads that can be mapped to the reference genome; Multiple mapped, number of reads that can be mapped to multiple sites in the reference genome; Uniquely mapped, number of reads that can be uniquely mapped to the reference genome.   www.nature.com/scientificreports/ mRNAs involved with this signaling were chosen and their relative transcripts abundance in FB and OV in both nutritional states monitored by RT-qPCR ( Fig. 3a and b, respectively). The results are in agreement with the DEG analysis, suggesting that the transcriptome data is accurate. However, it is important to note that an increase in transcript levels does not necessarily mean that the protein is translated and active 14 . Insulin signaling involves phosphorylation and protein-protein interactions to promote a response 8 . In light of this, we performed western blot studies to understand if the ILP/ToR signaling pathway was activated depending on nutritional condition. The results show that phosphorylated proteins involved with signaling, such as p-Akt, p-GSK, p-FoxO, p-p70S6K, p-ToR and p-4E-BP1, are only expressed in both FB and OV during the FC (Fig. 4). The ribosomal protein S6 was the first identified substrate of S6K, an important regulator of cell growth and cell size, and the translation initiation factor eIF4E was the second well-characterized ToR target 9 . By transcriptome analysis our results show that both mRNA are up-regulated in the OV in fed insects (Table 4), which also suggests that ToR signaling is active. In our experimental conditions, following feeding, the oocytes begin to grow rapidly, and 5-6 days after the blood meal female R. prolixus begin egg laying ( Supplementary Fig. S4). In some insect species, ILP/ToR signaling plays a key role in nutritional signal transduction activating egg development. With respect to the OV, in D. melanogaster the ILP/ToR signaling is involved in germinal stem cell proliferation and maintenance, germline growth and in the control of follicle growth 15 . In the desert locust, Schistocerca gregaria, ILP/ToR signaling disruption results in the development of small oocytes 16 as well as in Tribolium castaneum loss-of-function studies of several components of the ILP/ToR signaling results in impairment in the maturation of the primary oocyte and defective oocyte growth 17 . Moreover, in Aedes aegypti the ILP/ToR signaling is involved in the regulation of lipid droplet accumulation in oocytes 18 . With respect to the FB, in some species there is a stimulatory ILP/ToR signaling effect on vitellogenin (Vg) synthesis, an essential event for egg growth 6,17,[19][20] . IGF is highly expressed in the FB (Table 3) of adult female R. prolixus but it remains to be demonstrated whether this peptide could acts on Vg synthesis stimulation. Overall, although we demonstrate an increase in the transcript levels of molecules involved in ILP/ToR signaling in unfed insects (Tables 3 and 4), we did not detect phosphorylated proteins in either FB or OV (Fig. 4). In general, the UFC might favour the up-regulation of hormones involved in catabolism, while those involved in anabolism, such as ILPs, would be down-regulated. In agreement with Table 2. Expression profiles of genes involved in ILP/ToR signaling in CNS under fed and unfed conditions. Gray boxes, genes which are not differentially expressed (non-DEG) between unfed and fed insects; Data shown as log 2 fold change of FC versus UFC. Gene ID: VectorBase code (the official gene number in the RproC3 genome assembly); annotations: the protein name we are assigning. In A. aegypti it was demonstrated that starvation significantly increased the mRNA expression of the InR in corpora allata, the gland that synthesizes and releases JH 22 . Also, in Bombyx mori, expression of InR, IRS, PI3K, and phosphoinositide dependent protein kinase (PDK) is elevated in the FB when animals cease feeding 23 . Thus, the cells of these tissues could accumulate higher levels of various factors involved in the ILP/ToR signaling, thereby establishing a sensitized state to respond quickly to changes in ILP levels. In this way, when nutrient conditions become favorable, the cells are able to respond rapidly by turning on the mechanisms that stimulate growth. With this in mind, we perform ILP/ToR signaling activation tests by in vivo assays, injecting porcine insulin into unfed insects. By western blot, the results show that phosphorylated Akt and mToR proteins (p-Akt and p-mToR, respectively) are up-regulated by insulin, demonstrating that unfed insects are able to respond quickly to insulin levels (Fig. 4d). In fifth instar R. prolixus, it has been shown that treatment with porcine insulin leads to an increase of p-Akt expression in the FB after a half-hour incubation 12 . Also, it is interesting to note that insulin injection into unfed insects decreases the transcriptional expression of InR in both FB and OV (Fig. 4e). Thus, injection of insulin could be simulating a scenario following a blood meal, where the transcripts involved with the ILP/ToR signaling are down-regulated with respect to the UFC.
VKR belongs to the large tyrosine kinase receptor family, as does the InR. This receptor possesses a Venus Fly Trap extracellular module, a bilobate structure that binds small ligands to induce receptor kinase activity 24 . In Table 3. Expression profiles of genes involved in ILP/ToR signaling in fat body under fed and unfed conditions. Gray boxes, genes which are not differentially expressed (non-DEG) between unfed and fed insects; Green boxes, DEG up-regulated in unfed insects (UFC). Data shown as log 2 fold change of FC versus UFC. Gene ID: VectorBase code (the official gene number in the RproC3 genome assembly); annotations: the protein name we are assigning. www.nature.com/scientificreports/ mosquitoes, ovary ecdysteroidogenic hormone (OEH), a neuropeptide belonging to a family of small cysteinerich proteins referred to as neuroparsins, triggers phosphorylation of downstream components associated with the insulin signaling pathway via VKR 25 . Here, we show a higher expression in FB and OV of VKR transcripts in unfed female R. prolixus (Tables 3 and 4). In addition, the neuroparsins display clear sequence similarities with the N-terminal hormone-binding module of IGF binding proteins (IGFBPs). Based on studies in locusts, it is suggested that neuroparsins may act in vivo by controlling ILP availability 26 . In our experimental conditions, neuroparsin-1 transcript is exclusively present in the CNS of R. prolixus, with a comparable level of expression in both nutritional states. Also, for the first time, we report an IGFBP-related protein in R. prolixus with high homology with IGFBP-7. It is interesting to note that IGFBP-7 transcript is only found in the CNS, as is the neuroparsin ( Table 2). On the other hand, imaginal morphogenesis protein-Late 2 (Imp-L2), the first functionally characterized insulin-binding protein in invertebrates, counteracts insulin signaling in D. melanogaster and was reported as an essential factor for starvation resistance 27 . It was suggested to be a putative homolog of vertebrate IGFBP-7, but in R. prolixus it seems to be present as two different transcripts, maybe with a common precursor, but with different pattern of expression. In R. prolixus, we show that Imp-L2-like is expresses in CNS, FB and OV in both UFC and FC but only in the OV V_FC the expression is up-regulated (Tables 2, 3, 4). These results suggests that VKR, neuroparsin, IGFBP-7 and Imp-L2-likes could be working on the regulation of ILP/IGF availability, each one by specific signaling but possible modulating the binding to its receptors. The tumor suppressor PTEN was originally identified as a negative regulator of PI3K signaling, the main sensor of cell growth, metabolism and survival 28 . In R. prolixus, PTEN mRNA is up-regulated in FB and OV during the UFC, possibly to prevent PI3K Table 4. Expression profiles of genes involved in ILP/ToR signaling in ovaries under fed and unfed conditions. Orange boxes, DEG up-regulated in fed insects (FC); Green boxes, DEG up-regulated in unfed insects (UFC). Gray boxes, genes which are not differentially expressed (non-DEG) between FC and UFC. Data shown as log 2 fold change of FC versus UFC. Gene ID: VectorBase code (the official gene number in the RproC3 genome assembly); annotations: the protein name we are assigning. www.nature.com/scientificreports/ activation. GSK3β is described as a key enzyme involved in glycogen metabolism in mammals. GSK3β transcript expression is increased in the FB of R. prolixus during vitellogenesis respect to the UFC (Table 3). In Rhipicephalus (Boophilus) microplus, it was proposed that the synthesis of GSK3β is up-regulated in the vitellogenic FB to be released and stored by developing oocytes to promote successful oviposition and hatching 29 . Autophagy, a process which requires the activation of different peptidases, is inherently related to ILP/ToR signaling. For example, in the FB of D. melanogaster, the loss of mToR activity induces autophagy which has a  www.nature.com/scientificreports/ protective role as a growth suppressor 30 . Our results show that two transcripts which encode to serine-type peptidase (RPRC004789 and RPRC000107) are up-regulated in the FB_UFC (Supplementary Table S1). These type of enzymes are involved in the proteolytic degradation of cellular macromolecules during autophagy 31 . In addition, cathepsin F, a less studied member of the papain-protease family, is ubiquitously expressed in most tissues. Here, we find an up-regulation of transcripts which encodes to a cathepsin F-like peptidase in the FB_UFC (Table 3). The involvement of this peptidase in apoptosis has been reported 32 . In D. melanogaster and the silkworm, Bombyx mori, two forms of programmed cell death (PCD) in the remodeling of FB are regulated by hormonal and nutritional signals. In both insects, autophagy gradually increases in larval fat body cells during metamorphosis and is followed by apoptotic events 33 . The regulation between PCD and the FB of R. prolixus during different nutritional states is unknown, but it is clear that cathepsin F could be working on the development and/or progress of PCD in this tissue. Therefore, the FB could release nutrients during the UFC to help maintain a stable metabolic state when the animal is deprived of nutrient. In addition, LKB1 mRNA expression is up-regulated in the OV_UFC (Table 4). This transcript encodes a serine/threonine kinase that directly phosphorylates and activates AMPK, a central metabolic sensor 34 . Upon activation, AMPK phosphorylates and activates TSC1/2. In our experiments, the transcripts for TSC1 and TSC2 are increased in the OV_UFC (Table 4); the TSC1/2 complex typically results in downstream inhibition of ToR signaling 9 . In R. prolixus, mToR signaling inhibition in ovaries could limit pro-growth signals and also induce autophagy, which in turn provides ATP through the recycling of degradation products. Indeed, 4 transcripts which encode to serine-type peptidases are up-regulated in the OV_UFC (RPRC009729, RPRC009383, RPRC004789, RPRC000107; Supplementary Table S1). It is important to highlight that autophagy in OVs of triatomines during starvation has previously been reported 35 . In addition, our results also show an increase of cathepsin D-like peptidase mRNA levels in the FB and an up-regulation in OVs of fed insects (Tables 3 and 4, respectively). In R. prolixus it was reported that cathepsin D is stored in the eggs during vitellogenesis 36 and then takes part in yolk mobilization during embryogenesis 37 . Supporting this finding, in the triatomine Dipetalogaster maxima, it was demonstrated that cathepsin D peptidase is synthesized during vitellogenesis by FB and OV as a yolk protein precursor contributing to the total of cathepsin D stored in the oocytes. However, the activity of this peptidase is higher during the unfed condition in both tissues 38 .
Overall, these studies indicate the importance of a cathepsin D peptidase in oocytes of triatomines and suggests that this peptidase could be a yolk protein precursor produced by the OV itself in addition to the FB. Also, it has been reported that the specific temporal pattern of cathepsin F expression in fish indicates a specific role for this peptidase in yolk protein processing events occurring during oocyte maturation and/or early embryogenesis 39 . This finding supports our results (cathepsin F-like peptidase expression up-regulated in the OV_FC), thereby identifying a potential new regulator of reproductive processes in R. prolixus. ILP/ToR signaling regulates organ size by stimulating cell growth, and thereby increasing cell size but also acts via Akt to inhibit Hippo pathway signaling, which controls organ size by restricting cell number via inhibition of proliferation and induction of apoptosis 40 . KEGG analysis reveals up-regulation of Hippo signaling in both FB and OV during the UFC in R. prolixus (Tables 5, 6), reinforcing the hypothesis that ILP/ToR signaling is not active in this state. In R. prolixus, this signaling may drive the "organ size checkpoint" controlling the volume of both tissues under low nutritional conditions. Also, by KEGG enrichment, we see an increase in Longevity regulating pathway during the UFC (Tables 5, 6). One target of this pathway is FoxO signaling, which has been described to have pleiotropic effects including those related to stress resistance, metabolism, cellular differentiation and apoptosis 41 . Here, KEGG enrichment show that FoxO signaling is present in FB and OV during the UFC. It was reported in Blattella germanica that FoxO transcript expression is not nutritionally regulated 42 and in accordance, our results indicate that FoxO transcript levels remain constant under the different nutritional states in both FB and OV (Tables 3, 4). It is widely known that Akt phosphorylates FoxO (p-FoxO) in the cytoplasm, inhibiting its nuclear translocation 41 . For that, FoxO signaling could represent a pathway that indirectly indicates that insulin signaling is absent. By western blot, ours results show that in unfed insects, p-FoxO expression is not detected (Fig. 4), promoting the hypothesis that non-phosphorylated FoxO is translocated to the nucleus during the UFC. Using immunofluorescence we found that FoxO staining (red signal) is co-localized with DAPI staining (blue signal) in the nucleus of numerous cells of the FB_UFC but not of the FB_FC (Fig. 5). Control experiments were carried out by omitting one of the antibodies (primary or secondary) and no fluorescence signal was detected ( Supplementary Fig. S5). In this sense, FoxO could activate or repress the transcription of a wide array of genes, probably in a tissue-specific manner. In D. melanogaster, transcriptional activation of genes by FoxO is a critical step in maintaining survival during amino acid withdrawal 43 . In this scenario, one of the events that depends on FoxO signaling during starvation is the promotion of InR expression 44 . To check if FoxO is responsible for increasing transcript levels involved in ILP/ToR signaling during the UFC in R. prolixus, we interfered FoxO signaling in unfed insects by RNAi treatment. The transcripts related to nutritional signaling decrease in unfed R. prolixus treated with dsFoxO with respect to the control (dsARG) (Fig. 6). Also, in the same insects, Vg levels in the FB tend to increase with the same treatment. These results suggest that effectively, during the UFC, FoxO is involved in both the insulin sensitivity pathway and the repression of Vg expression. In B. germanica, RNAi against FoxO produced a large induction of Vg transcripts expression, which was also reflected in an increase of Vg protein in the haemolymph 42 . In addition, in this cockroach, it has been suggested that the starvation-induced activation of FoxO stimulates the transcription of different genes related to catabolic processes, basically genes involved in lipolysis, glycogenolysis and gluconeogenesis 45 . Furthermore, in the beetle T. castaneum, expression of Vg genes is under the control of FoxO regulated by the InR/PI3K pathway 18 . Overall, these results confirm that FoxO signaling is involved in the regulation of different pathways aimed at maintaining the survival of the insect in a specific metabolic state allowing them to respond quickly to the next food supply using ILP/ToR signaling activation. www.nature.com/scientificreports/ Overall, we propose a model for the role of ILP/ToR signaling pathway involved in nutritional states that have effects on reproductive performance (Fig. 7). This research is an important foundation to understanding physiological processes that orchestrate overall reproductive success, as well as the mechanisms involved in maintaining an optimal metabolic state during starvation. It is also important to keep in mind that this work identifies several starting points for new investigations, not only to answer fundamental biological questions but also to advance the development of tools for bio-rational insect control and/or methods for species conservation.

Materials and methods
insects. Adult insects of R. prolixus were obtained from an established colony at the University of Toronto Mississauga. Insects were reared in incubators at 25 °C under high humidity (~ 50%). The insects were fed through an artificial feeding membrane as described previously 46 on defibrinated rabbit blood (Cedarlane Laboratories Inc., Burlington, ON, Canada). For all experiments, males and females, during the last nymphal instar (fifth stage), were separated and fed 30 days post-ecdysis from fourth instars. Insects that gorged at least nine times their own initial body weight (a typical blood meal for fifth instars) were chosen and allowed to molt into the adult stage. Newly-emerged adult females were segregated individually and placed together with a recently www.nature.com/scientificreports/ fed male. Mating was verified by examining the cubicle for the deposited spermatophore. After copulation, females were fed with a blood meal 10 days post-ecdysis to promote egg growth. Only insects that fed 2.5 to 3 times their initial body weight (a typical blood meal for adults) were used to experiments. CNS, fat body (FB) and ovaries (OV) were sampled from adult females on representative days of the unfed condition (UFC) and fed condition (FC): (a) 10 days post-ecdysis as UFC and (b) 3 days post-feeding as FC (vitellogenesis). In supplementary Fig. S4, the FB and OV morphologies in both nutritional states can be observed. Briefly, triatomine ovarioles are composed of a tropharium containing nurse cells and a vitellarium containing oocytes or follicles in different development stages 4 . The nurse cells transport nutrient to immature oocytes (β and ϒ oocytes or pre-vitellogenic oocytes). After an appropriate nutritional stimulus, the follicular cells surrounding the mature oocytes (α oocytes or vitellogenic oocytes) shrink, a phenomenon called patency, generating an enlargement of interfollicular channels to allow for macromolecules from the hemolymph to be taken up by oocytes 4 . The ovarioles from insects during the FC were further separated according to Brito et al. 47 : (a) pre-vitellogenic ovariole (OV PV_FC), which include the tropharium and immature oocytes, and (b) vitellogenic ovariole (OV V_FC), which are the follicles containing mature oocytes. Ovarioles during UFC were used in their entirety (OV_UFC) (Supplementary Fig. S4).
Indirect immunofluorescence, DAPI and phalloidin-TRITC staining. The tissues for indirect immunofluorescence, DAPI or phalloidin-TRITC staining were dissected under R. prolixus saline (NaCl 150 mM, KCl   Bioinformatic analyses. The present work was analyzed using gene annotation from the RproC1.3 gene set (ftp://ftp.ensem blgen omes.org/pub/metaz oa/relea se-42/gff3/rhodn ius_proli xus/Rhodn ius_proli xus.RproC 3.42.gff3.gz), and Rhodnius prolixus alternative annotation-gene set 48 . FASTX-Toolkit (https ://hanno nlab.cshl. edu/fastx _toolk it/) was used to filter and trim sequences based on quality. HISAT2 was selected to map the filtered sequenced reads to the reference genome. In order to analyze gene expression levels, Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM) was used. HTSeq v0.6.1 software was performed to analyze the gene expression levels. To compare total gene expression levels in CNS, FB and OV during different nutritional conditions, violin plots were used. Venn diagrams were performed to analyse the number of genes that were uniquely expressed within each sample with the number of genes that were expressed in two or more samples. To infer the differentially expressed genes (DEG) with good statistical power, DESeq R package software (1.18.0) was used to normalize. DESeq provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg's approach for controlling the false discovery rate. Genes with p-adj (p-value after normalization) < 0.05 were assigned as differentially expressed. The results are shown as log 2 fold change: log 2 (FC/UFC). Volcano plots were performed to infer the overall distribution of DEG. We used KOBAS software to test the statistical enrichment of differential expression genes in KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. KEGG enrichment with corrected p-values < 0.05 were significantly enriched in DEGs.

Quantitative real-time pcR (Rt-qpcR).
Total RNA was extracted as described above, followed by cDNA synthesis using the High Capacity cDNA Reverse Transcription Kit (Applied-Biosystems, by Fisher Scientific, ON, Canada). RT-qPCR was performed using an advanced master mix with super green low rox reagent (Wisent Bioproducts Inc, QC, Canada). Three independent experiment were performed (n = 3) with each n composed of a pool of 5 tissues. Each reaction contained 3 technical replicates as well as a no template control and a no reverse transcriptase control. The reactions were performed using a CFX384 Touch Real-Time PCR Detection System (BioRad Laboratories Ltd., Mississauga, ON, Canada). Quantitative validation was analyzed by the 2 −ΔΔCt method 49 . The primers used (by Sigma-Aldrich, ON, Canada) for amplification are shown in Supplementary Table S2. β-actin, which was previously validated for transcript expression in FB and OV from R. prolixus at different nutritional conditions 11,50 , was used as reference genes. The stability test to confirm the use of actin as reference gene in this work is shown in Supplementary Fig. S9. The amplification efficiency for each pair of primers was calculated using standard curves generated by serial dilutions of cDNA. All amplification efficiencies ranged from 96 to 105% for the different pair of primers tested (Supplementary Table S2). For each pair of primers a dissociation curve with a single peak was seen, indicating that a single cDNA product was amplified. Specific target amplification was confirmed by automatic sequencing (Macrogen, NY, USA).   12 . Protein bands were separated under reducing conditions on 4-15% premade (Mini-Protean TGX Stain-Free Precast Gels, BioRad) 6.5% and 12% Tris-Glycine-SDS gels and loaded in equal amounts across all wells (40 μg each one). After electrophoresis, proteins were transferred to a low-fluorescence PVDF (LF-PVDF) membrane in transfer buffer over 3 min, using a Trans-Blot Turbo Transfer System (all reagents/materials: BioRad Laboratories Ltd., ON, Canada). Membranes were then blocked in PBS-T (PBS containing 0.1% Tween-20) and 5% non-fat milk for 1 h at room temperature. After blocking, the blots were incubated overnight at 4 °C, with primary antibody (1:1,000 dilution in PBS-T with 3% BSA): anti-phospho-Akt (ser473); anti-phospho-GSK3β (ser9); anti-phospho-FoxO (ser256); anti-phospho-ToR (ser2448); anti-phospho-p70S6K (ser434); anti-phospho-4E-BP1 (Thr37/46) (all rabbit antibodies from Cell Signaling Technology, MA, USA) and anti-tubulin (mouse monoclonal antibody from Life Technologies, ON, CA). The specificity of these antibodies has been previously reported 12 . Primary antibodies were washed-off with PBS-T followed by incubation in secondary antibody (1:5,000, horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit antibodies, from Cell Signaling Technology) for 1 h at room temperature with constant agitation. Blots were then washed with PBS-T and visualized using enhanced chemiluminescence (Clarity Western ECL Substrate, BioRad), imaged on a ChemiDoc XRS system and analyzed using Image Lab 5.0 (BioRad software and system). impact of the nutritional states. In R. prolixus, after a blood meal, ILP/IGF are release into the circulation and promote a phosphorylation cascade mediating the InR signaling activation. By western blot we showed that p-Akt, p-GSK, p-FoxO, p-mToR, p-p70S6K and p-4E-BP1 are up-regulated in both fat body and ovaries (green boxes) of fed females. The presence of other components of the ILP/ToR siganling pathway (gray boxes) were checked by transcriptome analysis or RT-qPCR. In the fed condition, we assume that all of them are actively participating in the signaling cascade, promoting lipogenesis, glycogenesis and translation regulation, along with other events. We suggest that the network of interactions and regulations informed by the nutritional state of the CNS, fat body and ovary, including those related with nutrient biosynthesis and storage, stimulate oocyte growth. In the unfed condition, we demonstrated that in the fat body and the ovaries there is an increase in gene expression of molecules related to the ILP/ToR signaling pathway. However, it is not possible to find these proteins activated (phosphorylated proteins). In unfed females, we suggest that nutrient stores decrease in both tissues and the oocyte growth is inhibited. In addition, by immunofluorescence and gene silencing assays, we show that FoxO factor is translocated to the nucleus in this state and assume that this player a role in increasing the insulin-sensitive pathway and modulating longevity signaling.

Double-stranded RnA design and synthesis.
A 161-base pair template was used to synthesize a double stranded RNA molecule (dsFoxO) using the T7 Ribomax Express RNAi System (Promega, WI, USA), according to the manufacturer protocol. Gene specific primers (GSP) were combined with GSPs containing the T7 RNA polymerase promoter sequence (Supplementary Table S2). As an experimental control, a dsRNA molecule based on the Ampicillin Resistance Gene (dsARG) from the pGEM-T Easy Vector system (Promega, WI, USA) was used throughout the study 12 .
Knockdown of foxo transcript expression using double stranded RnA. To knockdown the expression of FoxO, females 5 days post-ecdysis to adult stage, were injected into the hemocoel with 2 μg of dsARG or dsFoxO in 5 μL of ultrapure water using a Hamilton micro syringe (Hamilton Company, NV, USA). Insects were dissected 5 days post-injection and FoxO, IGF, InR, VkR, elF4E, ToR and Vg transcript expressions were measured in FB and OV by RT-qPCR assays, as described above.

Data availability
The raw sequence dataset of this project is registered with the National Center for Biotechnology Information (NCBI) under PRJNA624187 and PRJNA624904 bioprojects.