Fig 1.
Experimental design of feeding assay.
On the 2nd day PE mosquitoes were grouped into each of the light training cycles, LD and DL and continued for three days then an additional day in the same or continuous darkness depending on groups LDLD or LDDD before starting the feeding assay LD - Typical 12 hrs. light and dark cycle, DL - Reversed light dark cycle, White - Light on period, Black - Light off period, Grey - Light off period under subjective light period. The corresponding zeitgeber timepoints are; 08.00AM - ZT0, 12.00PM - ZT4, 04.00PM - ZT8, 08.00PM - ZT12, 12.00AM - ZT16, 04.00AM - ZT20. Fig 1 was drawn using Biorender (https://www.biorender.com).
Fig 2.
A- Feeding percentages measured under conventional 12h, light and dark cycle (LDLD). B- Feeding percentages under constant darkness after conventional light training (LDDD). C- Feeding percentages under reversed lighting conditions (DLDL). D- Feeding percentages under constant darkness after reversed light conditions (DLDD). Letters indicate statistical significance between each group (P < 0.005) (n = 20, per group). E- Total fed percentage comparison between daytime and subjective daytime and night and subjective nighttime under LDLD and LDDD conditions. F- Total fed percentage comparison between day and subjective day time and night and subjective night time under reversed DLDL and DLDD conditions. G- Fed comparison between LDLD and LDDD conditions under day and nighttime. H- Fed comparison between DLDL and DLDD conditions under day and nighttime. Readings from transition timepoints (ZT0 and ZT12) were excluded when calculating total fed percentages (n = 40, per group). Background represents the lighting conditions provided. White -Lights on period, Dark grey - Lights off period, Light grey - Lights off period correlates to subjective lights on period under LDLD or DLDL conditions. Statistical analysis was performed through a generalized linear mixed model followed by ANOVA with Sidak adjustment with two replicates, * P < 0.05, **** P < 0.0001. Data is represented as mean ± SEM.
Fig 3.
Endogenous expression of downstream neurotransmitter and olfactory response between LDLD and LDDD conditions.
A -sNPF expression profile comparison between LDLD and LDDD. B - sNPF expression between LDLD and LDDD at individual time points. C - OR114 expression profile comparisons between LDLD and LDDD. Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001. D - OR5 expression profile comparison between LDLD and LDDD. ANOVA single-factor, post hoc Dunnett’s test, *P < 0.05. The color bars at the top represent the lighting conditions: White - Lights on, Dark grey - Lights off, Light grey - Lights off period under LDDD conditions. E - Schematic representation of the three-cage olfactory assay. F - Olfactory response of mosquitoes under LDLD conditions. G - Olfactory response of mosquitoes under LDDD conditions. Chi-square of independence, **P < 0.01, ****P < 0.000, (n = 35-40, per group). Background color represents light conditions: White - Lights on, Dark grey - Lights off, Light grey - Lights off period under LDDD conditions. All data are represented as Mean±SEM. Fig 3E was drawn using Biorender (www.biorender.com).
Fig 4.
Core clock gene CYC is responsible for blood feeding propensity regulation.
A - Experimental design for core clock gene expressions under different feeding conditions. B - CYC expression under different feeding conditions. C - PER expression under different feeding conditions. D - TIM expression under different feeding conditions. E - CLK expression under different feeding conditions. GC - Glucose control, RF - Reverse feeding conditions, BF - 12 hrs. post blood meal, BF EXT - 24 hrs. post blood meal, S24 - 24 hrs. starvation (with access to water), F - Experimental design for CYC expression vs different food sources. G - CYC expression under different food sources. GC - Glucose control, Star - 12 hrs. complete starvation (no access to water), BM - Immediately after blood meal, Wat - 12 hrs. water only diet. H - CYC expression in daytime between LDLD and LDDD conditions. I - CYC expression at night time between LDLD and LDDD conditions. Student’s t-test, *P < 0.05, **P < 0.01. All data are represented as Mean±SEM. Fig 4A and 4F were drawn using Biorender (www.biorender.com).
Fig 5.
Mistimed blood feeding alters normal blood digestion and reproductive health.
A - Comparison of the blood digestion state in the midgut between daytime and nighttime fed mosquitoes. B - Comparison of early trypsin (eTRP) expression in midgut between daytime and nighttime fed mosquitoes. C - Comparison of late trypsin (ITRP) expression in midgut between daytime and nighttime fed mosquitoes. D - Comparison of chymotrypsin (cTRP) expression in mosquito midguts between daytime and nighttime fed mosquitoes. E - Ovarian development comparison between daytime-fed and nighttime-fed mosquitoes. F - Ovary length comparison between daytime and nighttime-fed mosquitoes (n = 15-20, per group). G - Follicle count comparison between daytime and nighttime fed mosquitoes (n = 21, per group). H - Vitellogenin (Vg) expression comparison in mosquito fat body between day-time and night-time fed mosquitoes in PBM conditions. I - Egg raft comparison between daytime and nighttime-fed mosquitoes (n = 7-12, per group). J - Egg count per raft comparison between daytime and nighttime fed mosquitoes. Student’s t-test, *P < 0.05, **P < 0.01, ****P < 0.0001. K - Linear regression analysis results for the constructed hatch rate. The hatch rate was constructed using a mean egg count for each feeding time, and an arcsine-square root transformation to compensate. PBM - Post Blood Meal. NF - Non-blood-fed. All the scale bars represent 500μM. All data are represented as Mean±SEM.
Fig 6.
CYC-mediated shutdown of olfactory under metabolic stress.
A - CYC response to extended starvation. B - sNPF response to extended starvation. C - OR114 response to extended starvation. GC - Glucose control, S24 - 24 hrs. water diet, S48 - 48 hrs. water diet. Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001. D - Olfactory assay results for starvation conditions. S12 - 12 hrs. water diet. Chi. Square of indipendence (n = 32-40, per group), *P < 0.05. E - Periodic acid staining (PAS) of the fat body for glycogen deposits and areas positive for PAS in tissue. All scale bars represent 100uM. Statistical analysis was done through the Kruskal-Wallis test (n = 4, per group), followed by Mann-Whitney pairwise comparisons with Bonferroni correction. Letters indicate the significance between groups. All data are represented as Mean±SEM.
Fig 7.
Loss of function of CYC disrupts the day-night differences in core clock expression and alters sNPF expression.
A - siRNA efficiency against CYC. siCYC2 showed around 83% efficiency and will be referred to as siCYC from here on. B - CYC expression day vs night between WT and siCYC. C - TIM expression day vs night between WT and siCYC. D - PER expression day vs night between WT and siCYC. E - CLK expression day vs night between WT and siCYC. F - sNPF expression day vs night between WT and siCYC. All the expression was measured at two anchor points ZT4 and ZT16. Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001. All data are represented as Mean±SEM. NC - Negative control, WT - Wild type, siCYC1 - RNA interference group 1, siCYC2 - RNA interference group 2.
Fig 8.
Silence of CYC alters Olfactory-related gene day-night expression contrasts and alters blood feeding propensity.
A - Odorant binding protein day-night expression contrasts in wild-type mosquitoes. B - Odorant binding protein day-night expression contrasts in the siCYC group. C - Odorant receptor day-night expression contrasts in wild-type mosquitoes. D - Odorant receptor day-night expression contrasts in the siCYC group. All expression data was measured at two anchor points (ZT4 and ZT16). Student’s t-test, *P < 0.05, **P < 0.01. E - Blood feeding propensity in daytime between the wild type and siCYC group. F - Blood feeding propensity at night time between wildtype and siCYC group. Feeding propensity in E-F was assayed at the same non-transition time points used in Fig 2. G - Compiled blood feeding propensity differences between day and nighttime between wild-type and siCYC groups. Mosquito whole heads with the antenna was utilized as the tissue site, where the siRNA efficiency was already validated. Statistical analysis through generalized linear mixed model followed by ANOVA with Sidak adjustment (n = 13-25, per group), **P < 0.01, ***P < 0.001. All data are represented as Mean±SEM.
Fig 9.
Loss of CYC alters the expression of trypsin transcription.
A - Late trypsin (1TRP) expression. B - Late trypsin expression at night. C - Early trypsin (eTRP) expression in daytime. D - Early trypsin expression at night. E - Chymotrypsin (cTRP) expression in daytime. F - Chymotrypsin expression at night. Student’s t-test, *P < 0.05. All data are represented as Mean±SEM.