Feeding behaviours of An. stephensi

A. Reproductive cycle of An. stephensi. Upon emergence [1, D0-D5] females have a dual appetite for protein-rich blood and carbohydrate-rich sources of sugar [2,3]. After a blood-meal [3,4,5, D1B-D4B], the eggs mature and are laid in water [6]. The next reproductive cycle can now begin. B. Blood-feeding assay. 0-7h old mosquitoes were collected in cups and aged appropriately on sugar. Prior to the test, mosquitoes were ‘activated’ for 5 minutes by presenting a human hand, followed by 3 exhalations. Blood-meals were presented through a Hemotek perfumed with human skin odours. Number of blood-fed mosquitoes were visually scored, irrespective of the quantity fed. C. Blood-feeding behaviour of Ae. aegypti. 8-days old virgin and mated females were assessed for their first blood-meal. Fully fed mated females were assessed for second blood-meal (D1B-D4B). Each dot represents a single trial with 18-21 females, n=10-12trials/group. Kruskal-Wallis with Dunn’s multiple comparisons test, p<0.05. Data labelled with different letters are significantly different. D. Blood-feeding behaviour of An. stephensi. Co-housed and virgin females were tested on the day of emergence (D0) and each day for the next 5 days (D1-D5). D1B-D4B represents their blood-feeding 1-4 days after their first blood-meal and D1○-D2○ represents their blood-feeding 1-2 days after oviposition (see methods for assay details). 18-21 females/trial, n=9-20trials/group. Generalized Linear Mixed Model with post-hoc pairwise comparisons using estimated marginal means and Bonferroni-corrected p-values; *p<0.05; **p<0.01; ****p<0.0001. E. Sugar-feeding behaviour of An. stephensi. Females of similar conditions as in (D) were tested for sugar-feeding. 18-21females/trial, n=9-20trials/group. Generalized Linear Mixed Model with posthoc pairwise comparisons using estimated marginal means and Bonferroni-corrected p-values; **p<0.01; ***p<0.001; ****p<0.0001. F. Choice assay for blood and sugar. Sugar-sated females or sugar-starved females, co-housed with males (indicated by the dashed abdomens) were given a choice between blood and sugar and assessed for their choice of food (bottom): blood and sugar; blood only; sugar only; none. While most sugar-sated females took blood (left of dashed line), most sugar-starved females fed on both (right of dashed line). As an internal control, co-housed males were accessed for sugar-feeding only and most were found to be fed in both conditions. 17-24 mosquitoes/trial; n=11-12 trials/condition. One-way ANOVA with Holm-Šídák multiple comparisons test, p<0.05. Data labelled with different letters are significantly different. G. Mating and blood-feeding. The mating status of D0-D5 co-housed females assayed in (D) were determined post-hoc by dissecting their spermathecae. n=232-239 females were analysed for each day. Many blood-fed females were unmated. H. Mating after blood-feeding. Blood-fed virgins were allowed to mate and then offered a second blood-meal. A suppression in blood-appetite was observed. 15-20females/trial, n=10 trials. Unpaired t-test; ****p<0.0001.

Host-seeking is modulated like blood-feeding is in An. stephensi.

A. Schematic of the Y-maze used to assess host-seeking behaviour of female An. stephensi. Females were allowed to acclimatise in the chamber for 5 minutes while being exposed to host kairomones that were presented in a test arm. A fan sucked the air from both host and control arms at 0.3-0.6m/s. Mosquitoes were released after acclimatisation and allowed to choose between the two arms. B. Percent females attracted to either host-cues or control arm at the indicated time points in the Y-maze. 17-21 females/trial, n=10 trials/group. Unpaired t-test; n.s.: not significant, p>0.05.; * p<0.05; ****p<0.0001. (D0: day of emergence; D5M: 5days post-emergence, mated; D5: 5days post-emergence, virgin; D3BM: 3days post-blood-meal, mated; D3B: 3days post-blood-meal, virgin; D2○: 2days post-oviposition)

Neurotranscriptome of An. stephensi

A. Central brains were sampled from males and females at different ages and feeding preferences for bulk RNA-seq. Uninterested in blood: D0 males, D0 females, D5 males. Blood-hungry: D5, sugar-fed virgin females, D1B virgin females, and D1○ females. Blood-sated: D1B mated females. n=100 central brains per replicate; 3 biological replicates per sample, 7 samples. B. Principal Component Analysis (PCA) of the normalised counts-per-million from different brain RNA-seq samples. Replicates cluster closely, and age and sex contribute most to the differences between the samples. C. Genes (listed on the right) with known expression patterns plotted across different samples. These known expression patterns are reproduced in our data. Average of the triplicate data are represented as z-score normalised log10 (TPM+1) values. Grey cells represent no data. D. Identification of candidates potentially involved in promoting blood-feeding behaviour. Two types of comparisons were made: (left) genes commonly up-regulated in the three blood-hungry conditions (see figure 3A) when compared against D0 females and excluded in males, and (right) genes up-regulated in D5 sugar-fed virgin females when compared against D1B mated females (blood-sated), and excluded in males. Number of candidate genes identified from two different sets of analyses are boxed in red. E. Nine candidate genes (listed on the right), shortlisted for further validation. RNA-seq data from central brain samples in triplicates are represented as z-score normalised log10 (TPM+1) values. (D0: day of emergence; D5: 5days post-emergence, virgin; D1B: 1day post-blood-meal, virgin; D1BM: 1day post-blood-meal, mated; D1○: 1day post-oviposition)

short Neuropeptide F (sNPF) and RYamide (RYa) together promote blood-feeding in An. stephensi

A. Schematic representation of the pipeline used for functional validation. dsRNAs against candidate genes were injected in adult virgins for targeted RNAi. After recovery these were tested for blood-feeding. Knockdown efficiency was determined via qPCR in whole heads and abdomens of fed and/or unfed females after the behaviour. B. Comparison of uninjected females and dsGFP-injected females for blood-feeding behaviour revealed no difference between the two, indicating that the animals recovered from the injection procedure. 19-25 females/ replicate, n= 8-9 replicates/group. Unpaired t-test; n.s.: not significant, p>0.05. C,D. Blood-feeding and sugar-feeding behaviour of females when both RYa and sNPF are knocked down in the head (C). Relative mRNA expressions of RYa and sNPF in the heads of dsRYa+dssNPF - injected blood-fed and unfed females, as compared to that in uninjected females, analysed via qPCR (D). Simultaneous knockdown of both neuropeptides suppressed the blood-feeding behaviour of females, without affecting the sugar-feeding behaviour. Note the more efficient knockdown in the heads of unfed females, as compared to blood-fed females. 14-25 females/replicate, n=8-9 replicates/group. Unpaired t-test; n.s.: not significant, p>0.05; ****p<0.0001 (C). One-way ANOVA with Holm-Šídák multiple comparisons test, p<0.05 (D). Data labelled with different letters are significantly different. E,F,G: Blood-feeding and sugar-feeding behaviour of females when both RYa and sNPF are knocked down only in the abdomen (E). Relative mRNA expressions of RYa and sNPF in the heads (F) and abdomens (G) of dsRYa+dssNPF - injected fed females, as compared to that in dsGFP-injected females, analysed via qPCR. No change in the blood-feeding or sugar-feeding behaviour was observed, despite the strong knockdown of both the genes in the abdomens, while the levels in the heads remain unaltered. 17-21 females/replicate, n=3 replicates/group.Unpaired t-test; n.s.: not significant, p>0.05. (E). One-way ANOVA with Holm-Šídák multiple comparisons test, p<0.05 (F,G). Data labelled with different letters are significantly different. H, I. Whole mount mRNA in situ hybridisation of central brain of 5days old sugar-fed virgin female, with RYa and sNPF probes (H). Both neuropeptides are expressed in multiple clusters (cyan: RYa, magenta: sNPF, blue: nc82). Co-expression was observed only in the Kenyon cells of the mushroom body (I). Maximum intensity projections of the confocal stacks are shown. Scale bar, 50µm. J. sNPF expression in the SEZ region of the central brains of females uninterested in blood (D0), blood-hungry females (D5) and blood-sated females (D2BM). Note the appearance of a novel cluster only in the blood-hungry (D5) condition. Maximum intensity projections of selected confocal stacks are shown. Scale bar, 50µm. K. Whole mount mRNA in situ hybridisation of gut of 5days old sugar-fed virgin female with sNPF probes. sNPF expression was observed in the enteroendocrine cells (EECs) in the posterior midgut, shown in higher magnification in K’. Scale bar, 50µm. L,M. sNPF expression in the posterior midguts of females uninterested in blood (D0), blood-hungry females (D5) and blood-sated females (D2BM). In addition to the EECs in the posterior midgut, two cells in the anterior midgut also show strong sNPF expression (L, inset). Higher number of cells express sNPF in the blood-hungry condition (M). n=5-7 guts per condition. One-way ANOVA with Holm-Šídák multiple comparisons test, p<0.05. N. Whole mount mRNA in situ hybridisation of central brain (left) and midgut (right) of 5days old sugar-fed virgin female, with sNPF receptor (sNPFR) and RYa receptor(RYaR) probes. While both receptors are expressed in the brain, only sNPFR is expressed in the gut. Scale bar, 50µm. O. Summary schematic of sNPF and RYa function in modulating blood-appetite in An. stephensi. RYa and RYaR (cyan) are expressed only in the brain, while sNPF and sNPFR (magenta) are expressed both in the brain and the gut. Increased sNPF levels in both the tissues (dashed circles) promote a state of blood-hunger, which may drive feeding behaviour either by sNPF’s action in the two tissues independently or via a communication between them. This happens in the context of RYa signalling in the brain. (D0: day of emergence; D5: 5days post-emergence, virgin; D2BM: 2days post-blood-meal, mated)

Standardization of various behavioural assays to study feeding behaviours in An. stephensi

A. Detailed schematic of the assay designed to assess the blood-feeding behaviour of virgins and co-housed females across the reproductive cycle. 0-7h old mosquitoes were collected and about 20 females (with or without 20 males) were separated into cups. Each cup was aged appropriately and tested on the relevant day (see methods for more details). Number of blood-fed mosquitoes were visually scored, irrespective of the quantity fed. Representative images of differently fed females are shown in A’. Females were tested for first blood-meal from the day of emergence (D0) to five days of age, every 24h. Spermathecae of co-housed females were dissected post-hoc to determine the mating status. To assay for the second blood-meal, fully fed females were collected and aged appropriately in groups of 20. To assess feeding 1day post-first blood-meal (D1B), Rhodamine B was spiked in the second blood-meal offered to the females. The subsequent days (D2B-D4B) were tested without any such spiking (grey box; see methods for more details). To assay for blood-feeding behaviour in females after oviposition (D1○-D2○), freshly oviposited females were collected and aged in groups of 20 for the appropriate time in cups (blue box; see methods for more details). In all cases, females were presented with host-cues prior to the assay and testing was performed as shown in figure 1B (see methods for more details). B. Standardisation of spiking Rhodamine B dye in blood to assess feeding 24h post-first blood-meal (D1B). To assay for the second blood-meal, blood-fed females were collected, aged for 24h and offered Rhodamine B-spiked blood in a Hemotek. All other assay parameters remained the same as for the first blood-meal (see methods for more details). Widefield fluorescent images of Ae. aegypti and An. stephensi fed on second blood-meal spiked with Rhodamine B are shown. Images on the left (black box) show mosquitoes not fed and fed on un-spiked blood (without Rhodamine B), as controls for autofluorescence from body tissues and blood itself, respectively. Fluorescence from Rhodamine B added to the second blood-meal enabled identification of fresh blood intake in previously fed mosquitoes reliably. C,D. Comparison of feeding behaviours of Ae. aegypti females (C) and An. stephensi females (D) fed on blood with and without Rhodamine B at the indicated age. Females fed similarly on both indicating that Rhodamine B itself does not affect feeding behaviour. Unpaired t-test; n.s.: not significant, p>0.05. 16-20 females/trial. n= 5 trials/group. D5M: 5days post-emergence, mated; D2BM: 2days post-blood-meal, mated; D8M: 8days post-emergence, mated). E. Determining optimal oviposition timing in An. stephensi. Females were tested in groups of 10 and average number of eggs laid per group are shown at the indicated day after the first blood-meal. Dashed lines connect the eggs laid by each group on the indicated day. Majority of the eggs were laid 2days after blood-meal. 10 females/group, n=10 replicates/group. D0B-D5BM: 0-5days post-blood-meal, mated. F. Standardisation of the dual choice assay, to determine the choice between blood and sugar in sugar-sated and sugar-starved An. stephensi females, co-housed with males. Widefield fluorescent images of females fed on either blood, sugar or both are shown. White arrowhead marks the fluorescence from sugar (spiked with Rhodamine B) in the abdomens of females engorged on blood, indicating that the female has fed on both. This is not observed in the females fed on blood alone. Females fed on sugar alone can be visualised by the fluorescent lean bellies. Unfed females exhibit no fluorescence or engorgement. G. Widefield fluorescent images of female An. stephensi fed on sugar spiked with Rhodamine B. This setup was used to study the sugar-feeding behaviour of virgins and co-housed females across the reproductive cycle. Addition of Rhodamine B to standard sucrose solution enabled visualisation of even the smallest amounts ingested, as indicated by the white arrowheads, under the fluorescent microscope. H. Mating status of sugar-fed and sugar-unfed co-housed females tested in figure 1E, 24-120h post-emergence (D0-D5). n=175-179 females per time-point. I. Percent participation of virgin and co-housed females tested for host-seeking behaviour in the Y-maze olfactometer (figure 2B), at different stages of the reproductive cycle. All females tested participated well. 17-21 females/trial, n=10 trials/group. Kruskal-Wallis, with Dunn’s multiple correction test, p<0.05. Data labelled with different letters are significantly different.D0: day of emergence; D5M: 5days post-emergence, mated; D5: 5days post-emergence, virgin; D3BM: 3days post-blood-meal, mated; D3B: 3days post-blood-meal, virgin; D2○: 2days post-oviposition.

Differential expression of genes across various states of blood-hunger and -satiety.

A. Number of genes identified to be differentially up- and down-regulated in the central brain of females across the reproductive cycle (indicated at the bottom), compared to either newly emerged males or females (D0), or blood-fed sated females (D2BM). Note that no genes were found to be differentially expressed between mated and virgin blood-fed females, despite their contrasting blood- appetites. B. Top 10 Gene Ontology terms for Biological Processes (GOBP), overrepresented in the samples indicated at the bottom, compared to newly emerged males or females (D0). Size of the dot indicates the p-value significance. Processes involved in protein metabolism (highlighted in red) were found to be highly enriched in blood-fed females. (D0: day of emergence; D5: 5days post-emergence, virgin; D1B: 1day post-blood-meal, virgin; D1BM: 1day post-blood-meal, mated; D1○: 1day post-oviposition)

Differential expression of genes involved in carbohydrate metabolism across different tissues and states of blood-hunger and -satiety in An. stephensi

Expression patterns of genes (listed on the right) involved in sugar metabolism via different pathways, across the reproductive cycle in the central brain and in the fat body, midgut and ovary of blood-hungry females, are represented as heatmaps. Green box shows downregulation of genes involved in glucose utilisation and the magenta box represents genes involved in glucose storage. Note that both are low in the brains of blood-fed females indicating that the brain goes into a state of ‘sugar rest’ after a blood-meal. Analysing publicly available data from the midgut, ovary, and fat body of sugar-fed females suggest that energy expenditure via the non-oxidative branch of the pentose phosphate pathway of glucose metabolism is high in these tissues, likely to prime them for the blood-meal. RNA-seq data from all central brain samples in triplicates and single sample data for fat body, midgut and ovary (taken from Prasad et al., 2017) are represented as z-score normalised log10 (TPM+1) values. TPM values of zero were excluded from z-score transformation and are displayed as grey cells. (D0: day of emergence; D2-D4: 2-4days post emergence; D5: 5days post-emergence, virgin; D1B: 1day post-blood-meal, virgin; D1BM: 1day post-blood-meal, mated; D1○: 1day post-oviposition)

Differential expression of genes involved in lipid metabolism across different tissues and states of blood-hunger and -satiety in An. stephensi

Expression patterns of genes (listed on the right) involved in lipid metabolism: lipid breakdown (top) and lipid synthesis (bottom), across the reproductive cycle in the central brain and in the fat body, midgut and ovary of blood-hungry females, are represented as heatmaps. No specific patterns were observed in the central brain (pre- and post-blood-meal), midgut or ovaries. As expected, fat body showed high expression of genes involved in both the catabolic and anabolic branches of fatty-acid metabolism. RNA-seq data from all central brain samples in triplicates and single sample data for fat body, midgut and ovary (taken from Prasad et al., 2017) are represented as z-score normalised log10 (TPM+1) values. TPM values of zero were excluded from z-score transformation and are displayed as grey cells. (D0: day of emergence; D2-D4: 2-4days post emergence; D5: 5days post-emergence, virgin; D1B: 1day post-blood-meal, virgin; D1BM: 1day post-blood-meal, mated; D1○: 1day post-oviposition)

Expression patterns of neuropeptides and neuropeptide-receptor across different tissues and states of blood-hunger and -satiety in An. stephensi

Expression patterns of neuropeptides (top) and neuropeptide-receptors (bottom), across the reproductive cycle in the central brain and in the fat body, midgut and ovary of blood-hungry females, are represented as heatmaps. While neuropeptides were observed to be downregulated in the central brain of newly emerged males and females (D0), neuropeptide-receptors showed no such patterns. Additionally, both neuropeptides and their receptors were much more abundant in the brain, as compared to fat body, midgut or ovaries. Transcripts for the receptors highlighted in red were visualised in situ both in the midgut and brain samples via HCR (figure S10, S11). RNA-seq data from all central brain samples in triplicates and single sample data for fat body, midgut and ovary (taken from Prasad et al., 2017) are represented as z-score normalised log10 (TPM+1) values. TPM values of zero were excluded from z-score transformation and are displayed as grey cells. (D0: day of emergence; D2-D4: 2-4days post emergence; D5: 5days post-emergence, virgin; D1B: 1day post-blood-meal, virgin; D1BM: 1day post-blood-meal, mated; D1○: 1day post-oviposition)

Expression patterns of neurotransmitter-related genes in the central brain of An. stephensi across different states of blood-hunger and-satiety

Expression patterns of genes (listed on the right) involved in neurotransmitter expression, processing and transport across the reproductive cycle in the central brain, are represented as heatmaps. Interestingly, many of these genes were observed to be up-regulated in the newly emerged (D0) mosquitoes as compared to the older and blood-fed mosquitoes. RNA-seq data from all central brain samples in triplicates are represented as z-score normalised log10 (TPM+1) values. (D0: day of emergence; D5: 5days post-emergence, virgin; D1B: 1day post-blood-meal, virgin; D1BM: 1day post-blood-meal, mated; D1○: 1day post-oviposition)

Candidates potentially involved in promoting blood-feeding behaviour in An. stephensi

71 candidate genes identified as potential promoters of blood-feeding behaviour are listed. These were identified to be commonly up-regulated in different conditions of blood-hunger (as compared to conditions of satiety or no-interest), and non-overlapping in males. Nine genes shortlisted for further validation are highlighted in red. RNA-seq data from all central brain samples in triplicates are represented as z-score normalised log10 (TPM+1) values. TPM values of zero were excluded from z-score transformation and are displayed as grey cells. (D0: day of emergence; D5: 5days post-emergence, virgin; D1B: 1day post-blood-meal, virgin; D1BM: 1day post-blood-meal, mated; D1○: 1day post-oviposition)

Screening of the shortlisted candidates, potentially involved in promoting blood-feeding behaviour in An. stephensi via dsRNAmediated gene knockdown

A-H. Blood-feeding behaviour of virgin females injected with dsRNA against Eiger (A), Juvenile hormone epoxide hydrolase-1 like (JHEH) (B), Orcokinin peptides type-A like (Ork) (C), Hairy (D), Prothoracicostatic peptides (Prp) (E), Na-dependent amino acid transporter-1 like 118504075 (F), Na-dependent amino acid transporter-1 like 118504077 (G), 118504075+118504077 (H). dsGFP was injected as a control in each case. No change in the behaviour was observed for any of the candidates tested. 18-25 females/replicate, n=2-6 replicates/group. Unpaired t-test; n.s.: not significant, p>0.05; *p<0.05 (A, D, F-H). A’-H’. Relative mRNA expression in the heads of virgin An. stephensi females injected with the indicated dsRNA, as compared to that in dsGFP injected females, analysed via qPCR. Efficient knockdown could not be achieved for Hairy and Prp. Unpaired t-test; ****p<0.0001 (A’, D’). One-way ANOVA with Holm-Šídák multiple comparisons test, p<0.05 (F’-H’). For candidates JHEH (B), Ork (C) and Prp (E), significance could not assessed, as statistical testing requires a minimum of three replicates, which were not available for these experiments.

Abdominal knockdown of RYa or sNPF alone does not contribute to the blood-feeding behaviour in An. stephensi

A. Widefield fluorescent images of females assayed for both sugar and blood appetite, post-dsRNA injections. Females were injected as described earlier (see methods for details) and offered Rhodamine B-spiked sugar 24h prior to the assay. Blood-feeding behaviour was tested as described earlier. Representative images of females fed on both blood and sugar, blood only, sugar only or none, are shown. B-H. Blood-feeding and sugar-feeding behaviour of virgin females injected with dsRYa (B) and dssNPF (E). dsGFP injected or uninjected females were used as controls. Relative mRNA expression in the heads and abdomens of the injected females were analysed via qPCR (C, F) to assay for the knockdown efficiency in both tissues. Percent females engorged on blood upon injection with dsRYa and dssNPF, as compared to control females are shown in D and G respectively. Representative widefield images of females considered to be engorged or not, are shown in H. While the mRNA levels in the abdomens were significantly downregulated, efficient knockdown could not be achieved in the heads. Under these conditions, no change in the blood- or sugar-feeding behaviour was observed for either of the targets. However, fewer females were able to fully engorge on blood in both cases and instead took smaller meals. 16-26 females/replicate, n=5-10 replicates/group. Unpaired t-test; n.s.: not significant, p>0.05; *p<0.05, ***p<0.001 (B,D,E,G). One-way ANOVA with Holm-Šídák multiple comparisons test, p<0.05 (C,F). I-L. Blood-feeding and sugar-feeding behaviour of females when RYa and sNPF are knocked down only in the abdomen. Virgin females were injected with dsRNA against RYa (H) and sNPF (J) and assayed for both sugar- and blood-appetite, as described before. dsGFP injected females were used as controls. Relative mRNA expression in the heads and abdomens of RYa (I) and sNPF (K) injected females were analysed via qPCR to assay for the knockdown efficiency in both tissues. No change in the blood- or sugar-feeding behaviour was observed when RYa or sNPF levels were significantly downregulated in the abdomens without altering the levels in the heads, suggesting that neuronal expression is necessary to promote the blood-feeding behaviour. 19-24 females/replicate, n=4-5 replicates/group. Unpaired t-test; n.s.: not significant, p>0.05 (I, K). One-way ANOVA with Holm-Šídák multiple comparisons test, p<0.05 (J,L).

Transcripts of neuropeptides sNPF, RYa and their receptors are expressed in An. stephensi central brain

A, B. RYa and sNPF mRNA expression in the central brain of females uninterested in blood (D0), blood-hungry females (D5) and blood-sated females (D2BM), analysed via HCR. Transcripts of both neuropeptides are expressed in several clusters across the central brain (A). While the clusters are largely non-overlapping, co-expression was observed in the Kenyon cells of the mushroom body, indicated by the marked area (B). Maximum intensity projections of confocal stacks are shown. Scale bar, 50µm. C. RYa receptor (RYaR) and sNPF receptor (sNPFR) mRNA expression in the central brain of females uninterested in blood (D0), blood-hungry females (D5) and blood-sated (D2BM), analysed via HCR. Transcripts of both receptors were found to be expressed in multiple cells. Maximum intensity projections of confocal stacks are shown. Scale bar, 50µm. D. sNPFR mRNA expression in the central complex. A magnified image is shown in D’. Maximum intensity projection of selected confocal stacks is shown. Scale bar, 50µm. E. central brain samples incubated with HCR-Amplifiers only, to serve as a control for background signal in the absence of HCR probes. No signal was observed for either of the amplifiers used (B1 labelled with 546 fluorophore (B1-546) and B2 labelled with 488 fluorophore (B2-488)). (D0: day of emergence; D5: 5days post-emergence, virgin; D2BM: 2days post-blood-meal, mated)

Transcripts of sNPF and sNPFR are expressed in An. stephensi posterior midgut

A-F. RYa (A), sNPF (B), RYaR (C) and sNPFR (D) mRNA expression in the guts of females uninterested in blood (D0), blood-hungry females (D5) and blood-sated females (D2BM), analysed via HCR. Transcripts of both sNPF and its receptor sNPFR are expressed in the posterior midgut, at all stages analysed. No expression of RYa or RYaR was observed. Magnified images of transcript expression of neuropeptides and their receptors are shown in E and F, respectively. Scale bar, 50µm. G,H. Gut samples incubated with HCR-Amplifiers only, to serve as a control for background signal in the absence of HCR probes. No signal was observed for either of the amplifiers used (B1 labelled with 546 fluorophore (B1-546) and B2 labelled with 488 fluorophore (B2-488)). Scale bar, 50µm. (D0: day of emergence; D5: 5days post-emergence, virgin; D2BM: 2days post-blood-meal, mated)

List of primers used in the study for dsRNA-mediated gene knockdown and quantitative PCR (qPCR).