Fig 1.
Reproductive physiology in fed virgin and mated females.
(A) Weight of insects (grams) measured before (unfed insects) and immediately post blood meal (fed insects) (n = 15–20 females). (B) Representative images showing the reproductive system of virgin (left panel) and mated females (right panel) at 6 (upper) and 12 (bottom) days post blood meal (d PBM) (representative image of n = 15–20 females). (C-D) Parameters indicating reproductive performance in fed virgin and mated females. The beginning of egg laying in virgin (highlight in blue) and mated (highlight in white) females is shown in (C), and the average cumulative eggs laid per female at 12 (left) and 28 d PBM (right) is shown in (D). *p < 0.05; ***p < 0.001; ****p < 0.0001 (Student’s t-test, n = 15 females). (E) Survival rate at 100 d PBM shown as a percentage (n = 15 females).
Fig 2.
Blood meal size, anterior midgut weight, lipid, protein, and vitellogenin quantification in fed virgin and mated females.
(A) Weight of insects (grams) was measured at 6 and 12 days post blood meal (d PBM); **p < 0.01 (Student’s t-test, n = 6–7 females). (B-C) Weight (B) and total protein content (C) in the anterior midgut (AMG) of virgin and mated females at 6 and 12 d PBM; *p < 0.05 (Student’s t-test, n = 6–7 independent biological replicates, where each n represents the AMG from 1 insect). (D-E) Total protein (D) and lipid content (E) in the fat body of virgin and mated females at 6 and 12 d PBM; *p < 0.05; **p < 0.01 (Student’s t-test, n = 6–7 females, where each n represents the fat body from 1 insect). (F-F’) Total protein (F) and SDS-PAGE analysis (F’) of the hemolymph (1 μL) of virgin and mated females at 6 and 12 d PBM; *p < 0.05 (Student’s t-test, n = 6–7 independent biological replicates, where each n represents hemolymph from 1 insect); in (F’) a representative image of 3 independent experiments is shown. (G) Total lipid circulating in the hemolymph of virgin and mated females at 6 and 12 d PBM; **p < 0.01 (Student’s t-test, n = 5 independent biological replicates, where each n represents hemolymph from 1 insect). (H-H’) Vg1 (H) and Vg2 (H’) mRNA expression in the fat body of virgin and mated females at 6 and 12 d PBM. Transcript levels were quantified using RT-qPCR and analyzed by the 2-ΔCt method. The y axes represent the relative expression obtained via geometric averaging using Rp49 and actin as reference genes. *p < 0.05; **p < 0.01; (Student’s t-test, n = 5–6 independent biological replicates, where each n represents an individual tissue from 1 insect). (I) Quantification of vitellogenin circulating in the hemolymph of virgin and mated females at 6 and 12 d PBM by ELISA. *p < 0.05 (Student’s t-test, n = 5–6 independent biological replicates, where each n represents hemolymph from 1 insect) (I’) Western blot image (1 μL of hemolymph in 1:20 dilution) showing Vg circulating in the hemolymph. A representative image of 3 independent experiments is shown.
Fig 3.
JHSB3 production in fed virgin and mated females of R. prolixus.
(A) Transcript levels of three JH biosynthetic enzymes from the JH-branch were quantified in the CNS-CC-CA complexes from fed virgin and mated females at 6 and 12 days post blood meal (PBM) by RT-qPCR. The analysis was performed using the 2−ΔCt method. The y axes represent the relative expression obtained via geometric averaging using Rp49 and 18S as reference genes. **p < 0.01; ****p < 0.0001 (Student’s t-test; n = 4 independent biological replicates, where each n represents a pool of CNS-CC-CA complexes from 3 insects). (B) Hemolymph was collected from fed virgin and mated females at 6 and 12 d PBM. The y axis represents JHSB3 titers (fmol) per μL of hemolymph. *p < 0.05; (Student’s t-test; n = 4–6 independent biological replicates, where each n is hemolymph from 5–7 insects). (C) Left: scheme of JHSB3-Met-Tai axis in the fat body modulating production of the downstream factor Kr-h1 and vitellogenin (Vg); right: transcript levels of Met, Tai and Kr-h1 were quantified in the fat body from fed virgin and mated females at 6 and 12 days post blood meal (PBM) using RT-qPCR. The analysis was performed using the 2−ΔCt method. The y axes represent the relative expression obtained via geometric averaging using Rp49, 18S and actin as reference genes. *p < 0.05; ***p < 0.001 (Student’s t-test, n = 5–6 independent biological replicates, where each n represents an individual tissue from 1 insect). FALDH, farnesal dehydrogenase; JHAMT, juvenile hormone acid methyltransferase; EPOX, methyl farneseoate epoxidase.
Fig 4.
Ecdysteroid production in fed virgin and mated females of R. prolixus.
(A) Transcript levels of ecdysteroid biosynthetic enzymes were quantified in the ovaries from fed virgin and mated females at 6 and 12 days post blood meal (PBM) by RT-qPCR. The analysis was performed using the 2−ΔCt method. The y axes represent the relative expression obtained via geometric averaging using Rp49 and actin as reference genes. *p < 0.05; **p < 0.01 (Student’s t-test; n = 4–5 independent biological replicates, where each n represents an individual tissue from 1 insect). (B) Ecdysteroid hemolymph titers of fed virgin and mated females at 6 and 12 PBM were quantified by ELISA. The y axis represents ecdysteroid titers (pg) per μL of hemolymph. **p < 0.001 (Student’s t-test, n = 4–5 independent biological replicates, where each n represents hemolymph from 1 insect). nvd, neverland; spo, spook; phm, phantom; dib, disembodied; sad, shadow; shd, shade.
Fig 5.
Locomotion of fed virgin and mated females of R. prolixus.
(A-B) Fed virgin (blue symbols) and mated females (gray symbols) were individually placed inside actometer units and their movements recorded for 24 hours (x axis) at day 6 (A) and day 12 (B) post blood meal (PBM). Total number of movements of virgin and mated females 6 d PBM and 12 d PBM is shown in (A’) and (B’), respectively. White and grey areas depict the photophase and scotophase, respectively. Data are presented as the square root of the mean locomotory activity. ****p < 0.0001 (Student’s t-test, n = 35 insects for each treatment).