The R. prolixus colony was maintained under 50% humidity at 25°C, and insects were fed on defibrinated rabbit blood (Cedarlane Laboratories Inc., Burlington, ON, Canada) once per instar.

Seven Drosophila ILPs (accession numbers NP_648361.1, NP_524012.1, NP_996037.1, NP_648360.2, NP_648359.1, NP_570070.1, NP_570000.1), the computationally predicted R. prolixus ILPs (Ons et al., 2011; Mesquita et al., 2015), and the recently described R. prolixus ILP, Rhopr-ILP (Defferrari et al., 2016), were used for screening the predicted R. prolixus peptidome (available at https://www.vectorbase.org) using the program Geneious 8.1.7 (Kearse et al., 2012). The putative amino acid sequence was identified and its corresponding mRNA sequence was used as a template for designing primers and for subsequently cloning the transcript.

Total RNA was extracted using a PureLink® RNA mini kit (Life Technologies Corporation, Carlsbad, CA, USA) from the CNS and the fat body of fifth instar R. prolixus. For the amplification of the 5′ and 3′ ends of the Rhopr-IGF transcript, cDNA from each of the tissues was synthesized using a RACE PCR kit (Roche Applied Science, Mannheim, Germany) in combination with gene-specific primers (GSPs) (Supplementary Table 1). The final PCR products were cloned using the pGEM-T Easy Vector system (Promega, Madison, WI, USA) and the clones expressing the desired products were sequenced at the Center for Applied Genomics, at the Hospital for Sick Children in Toronto (MaRS Centre, Toronto, ON, Canada).

The complete coding sequence of Rhopr-IGF was isolated as follows: total RNA was extracted from the CNS and the fat body of fifth instars and cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied-Biosystems, Fisher Scientific, Toronto, ON, Canada). PCR was performed combining each of the cDNAs with GSPs (Supplementary Table 1) and the products were cloned using the pGEM-T Easy Vector system (Promega, Madison, WI, USA). The clones expressing the desired products were sequenced at the Center for Applied Genomics, at the Hospital for Sick Children (Toronto, ON, Canada). Sequences were confirmed from at least three independent clones from each tissue to ensure base accuracy.

The exon-exon boundaries within the Rhopr-IGF transcript were first predicted with a local BLASTN search against the R. prolixus genome (Mesquita et al., 2015), using the program Geneious 8.1.7 (Kearse et al., 2012), where the cDNA sequence of Rhopr-IGF was compared to the genomic sequence in order to predict location and size of exons. The splice regions were confirmed on the platform Berkeley Drosophila Genome Project (http://www.fruitfly.org/seq_tools/splice.html) by using the online tool Splice Site Prediction. The sequence was also analyzed for a potential signal peptide and for subcellular localization, using SignalP 4.0 (http://www.cbs.dtu.dk/services/SignalP) and TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP), respectively. The predicted protein sequence of Rhopr-IGF (KX185519) was aligned with 12 ILP and IGF precursor peptides from different insects and three human insulin and IGF precursor peptides using the program MUSCLE 3.8 (Multiple Sequence Comparison by Log-Expectation—http://www.ebi.ac.uk/Tools/msa/muscle).

The spatial expression profile of Rhopr-IGF was quantified using different tissues from unfed fifth instars. RNA was isolated using a PureLink® RNA mini kit (Life Technologies Corporation, Carlsbad, CA, USA) and cDNA was synthesized using 250 ng of RNA from each tissue using the High Capacity cDNA Reverse Transcription Kit (Applied-Biosystems, Fisher Scientific, Toronto, ON, Canada). The cDNAs were individually diluted to the appropriate concentrations and 5 ng of template was used per well for the real time qPCR reactions. Primers for the amplification of Rhopr-IGF (Supplementary Table 1) were designed to amplify fragments of similar size across all experimental and reference genes (rp49, β-actin, and α-tubulin) (Paluzzi and O'Donnell, 2012; Defferrari et al., 2014). The qPCR reactions were carried out on a CFX384 Touch™ Real-Time PCR Detection System (Bio-Rad, Mississauga, ON, Canada) and the expression levels were quantified using the ΔΔCt method (Pfaffl, 2001). Experiments were repeated for a total of three biological replicates with three technical replicates each.

In order to evaluate any changes in the expression of Rhopr-IGF transcript throughout the fifth instar, insects were dissected at different time points under nuclease-free PBS and RNA was extracted as described above. Insects were dissected immediately and 1 week after ecdysis into fifth instars. Two weeks after ecdysis, a group of 30 insects was fed on defibrinated rabbit blood and tissues (CNS, dorsal vessel and fat body) were dissected at 3 h, 1, 3, 7, 14, and 21 days after feeding. Insects dissected at 21 days after feeding had already started ecdysis into adults but had not yet ecdysed. The expression was quantified by real time qPCR as described above. Five insects were dissected at each time point and the experiment was repeated for a total of three biological replicates.

A double-stranded RNA fragment consisting of 560 base pairs from the coding region of Rhopr-IGF transcript (dsIGF) was synthesized using the T7 Ribomax Express RNAi System (Promega, Madison, WI, USA), following the manufacturer instructions, combined with GSPs conjugated with the T7 RNA polymerase promoter (Supplementary Table 1). A dsRNA fragment of the ampicillin resistance gene (dsARG) from the pGEM-T Easy Vector system (Promega, Madison, WI, USA) was used as negative control throughout the experiments (Lee et al., 2013; Defferrari et al., 2014). Rhopr-ILP dsRNA was produced as previously described (Defferrari et al., 2016).

Injections of dsIGF and dsARG were performed using with a Hamilton syringe of 10 μL volume capacity (Hamilton Company, Reno, NV, USA) into the hemocoel of fifth instar R. prolixus 1 week after the insects had been fed on a blood meal. Insects were divided into two groups of 20–25 individuals: (1) dsIGF-injected and (2) control dsARG-injected. For the quantification of Rhopr-IGF expression knockdown, the CNS, the fat body and the dorsal vessel from each of the two groups were dissected on day 2 and 7 post-injection. The knockdown was analyzed through quantitative PCR, as described above, and Rhopr-IGF transcript expression in dsIGF-injected insects was quantified relative to the expression of the transcript in dsARG-injected insects.

In order to investigate possible changes in Rhopr-IGF expression after Rhopr-ILP knockdown, and in Rhopr-ILP expression after Rhopr-IGF knockdown, a third group of 10 insects was injected with the dsRNA targeting the Rhopr-ILP transcript. The CNS and the fat body of such insects were dissected on day 7-post-injection and the expression of Rhopr-IGF was analyzed. The expression of Rhopr-ILP was quantified in the CNS of dsIGF-injected insects that were dissected on day 7-post-injection. As described above, the expression of the transcripts was analyzed by quantitative PCR, relative to the expression of dsARG-injected insects.

One week after the injection of the dsRNA, the fed fifth instars were immobilized and 5 μL of hemolymph were collected from the cut end of a leg, using a 10 μL capacity micro-pipette. The hemolymph was placed in 50 μL of 10% cold TCA and the samples were centrifuged for 10 min at 20°C and 8000 g. The supernatants were transfered to new tubes and both pellets and supernatants were kept for subsequent measurements of lipids (as lipoproteins) and carbohydrates (including both glucose and trehalose), respectively. The pellets were resuspended in 200 μL of isopropanol for the lipid measurements.

One week after the dsRNAs were injected into the insects, the fat body sheet lining the ventral abdominal segments was removed from fed fifth instars under R. prolixus physiological saline (NaCl 150 mM, KCl 8.6 mM, CaCl₂ 2.0 mM, MgCl₂ 8.5 mM, NaHCO₃ 4.0 mM, glucose 34.0 mM, HEPES 5.0 mM, pH 7.0). The fat body was placed in either 200 μL isopropanol or 200 μL 10% cold TCA, sonicated for two cycles of 3 s each and centrifuged for 10 min at 20°C and 8000 g. Forty microliter of the supernatants were transfered to new tubes and kept for the measurements of total lipid (isopropanol) or carbohydrate content (10% TCA) in the fat body.

The lipids associated with lipoproteins in the hemolymph (pellets of TCA precipitated samples), and the lipid content in the fat body (isopropanol extracted fat body) of Rhopr-IGF knockdowns and dsARG-injected insects, were measured as previously described (Patel et al., 2014).

The hemolymph and fat body carbohydrates were measured as previously described (Defferrari et al., 2016). For the measurement of carbohydrates, 40 μL of the supernatant (after TCA precipitation of protein) was used from either hemolymph or fat body samples.

The fed fifth instars that were injected with dsIGF or dsARG were allowed to ecdyse into adults and 2–3 days after ecdysing the following measurements were taken: (1) body length, (2) body width, at the widest part of the abdomen, (3) wing length, (4) wing width, at the widest part of each wing, and (5) body weight. The experiment was repeated three times with 4–6 insects per group each time.

Results are shown as means plus standard errors. The significance of differences between means of dsIGF-injected and dsARG-injected groups was determined using Student's t-test. The difference between each time point and the time point used as reference in the temporal analysis of Rhopr-IGF expression was also determined using Student's t-test. Results were considered statistically different when P < 0.05. All analyses were carried out using the program SigmaPlot (Systat Software, San Jose, California, USA).

We identified a sequence within the R. prolixus genome that showed similarity to Rhopr-ILP and other insect ILPs, but also to human and insect IGFs. We have cloned and sequenced 994 base pairs of Rhopr-IGF cDNA, which has six exons, with the coding sequence starting at base 319 and ending at base 972, yielding a protein of 218 amino acids (Figure 1). The predicted preproprotein contains a signal peptide with cleavage site between residues 22 and 23, according to SignalP v4.1 and to TargetP 1.1, which also suggested that the protein is secreted to the extracellular environment. The putative Rhopr-IGF protein has six conserved cysteines as seen in other members of the insulin/IGF family that form two interchain disulfide bonds, between chains/domains A and B, and one intrachain disulfide bond, within chain/domain A (Cohick and Clemmons, 1993; Davidson, 2004), located in positions 31, 43, 70, 71, 75, and 84 (Figures 1, 2). Additionally, the predicted structure of the mature hormone resembles that of other IGFs, consisting of a single polypeptide chain stabilized by the three disulfide bonds, without the release of an internal peptide (Cohick and Clemmons, 1993). The same sequence was partially identified in a previous study, named R. prolixus ILP2 (Ons et al., 2011).

We have also constructed an alignment where the predicted protein sequence of Rhopr-IGF was combined with 12 ILP and IGF precursor peptides from different insects and three human insulin and IGF precursor peptides. The alignment highlights conserved residues such as the six cysteines within the A and B chains/domains, but also shows the reduced amino acid sequence between the two chains/domains in IGF sequences. Like human IGFs, Rhopr-IGF displays a longer sequence when compared to other insect ILPs, except for the partial sequence of R. neglectus ILP2 that shows 100% identity to Rhopr-IGF except for being shorter by eight amino acids (Figure 2).

Rhopr-IGF transcript expression is most abundant in the fat body, followed by the dorsal vessel, the CNS, and the hindgut, where expression ranges from 50 to 20 times higher than in the anterior midgut (AMG). The AMG was used as the reference tissue and shows a similar expression level to the other tissues tested (Figure 3).

Changes in Rhopr-IGF transcript expression during development were analyzed in tissues where the transcript is most abundant: the CNS, the fat body and the dorsal vessel. In the CNS, we did not observe major variations in expression throughout the different times tested, whereas in the fat body expression peaks right after the insects ecdyse into the fifth instar and before they ecdyse into the adult (21 days post-feeding). Expression in the fat body is also high 1 week after the insects ecdyse into fifths, but drops after the blood meal and stays at lower levels in the post-feeding period. In the dorsal vessel, expression peaks at 1 week after the insects ecdyse into fifths and, similar to the fat body, it drops right after the blood meal, but remains at similar levels until the last time point tested (Figure 4).

Injection of dsIGF reduced the expression of the transcript by 65 and 50% in the CNS, 70 and 90% in the fat body, and 40 and 70% in the dorsal vessel, at 2 and 7 days post-injection, respectively (Supplementary Figure 1). Injection of dsIGF or dsILP on transcript expression of ILP or IGF, respectively was examined and found to induce a slight compensatory upregulation but this was not significant (Supplementary Figure 2).

At 1 week after the injection of dsIGF, hemolymph lipid, and carbohydrate levels were increased when compared to the control group (dsARG-injected) (Figures 5A,B). No significant changes in lipid or carbohydrate content of fat body were observed in Rhopr-IGF knockdown insects when compared to dsARG controls (Figures 5C,D).

When fifth instar R. prolixus injected with dsIGF 1 week after feeding were followed through to ecdyse into adults, their external appearance was different from that of insects injected with dsARG (Figure 6). In order to quantify and assess the differences between the two groups, we weighed the insects and measured the length and width of the adult insect body and wings. Insects that had reduced Rhopr-IGF transcript expression as fifth instars, developed into adults with shorter body length along with shorter and narrower wings when compared to the dsARG controls (Figures 7A,B). On the other hand, body width and body weight did not seem to be affected by the knockdown of Rhopr-IGF (Figures 7A, 8).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.