Figures from Portwood et al 2026

Fig. 1: Tralopyril exposure prior to P. berghei infection negatively impacts parasite development in the mosquito. A Schematic of potential chlorfenapyr interaction with Plasmodium in the mosquito. Following uptake of a gametocyte-containing blood meal, male and female gametes fuse to form a zygote, which develops into a motile ookinete and subsequently an oocyst. Mature oocysts release sporozoites that move to- and invade- the mosquito’s salivary glands, completing development. Female mosquitoes feed approximately every 48-72 hours, and may encounter a chlorfenapyr net once or multiple times. Depending on the stage of the life cycle harboured during feeding, chlorfenapyr may target multiple stages of the Plasmodium life cycle. B Mean concentration of tralopyril per mosquito (x-axis) in mosquitoes topically exposed to 31 µM (LC30 dose, solubilised in acetone) and maintained over a period of 10 days (y-axis) at 21oC (light purple) and 28oC (dark purple), quantified by LC-MS. Points represent mean and error bars indicate standard deviation of three replicates at each timepoint. C Schematic of experimental design to assess the impact of tralopyril exposure on P. berghei development in An. stephensi. Mosquitoes were topically exposed to 31 µM (LC30 dose) of tralopyril, 48 h prior to receiving an infectious blood meal. On day 0, mosquitoes fed on a P. berghei-infected blood-meal. Midguts were dissected at six, nine, and 12-days post-infection (dpi) to assess oocyst prevalence, intensity and GFP signal prevalence. Salivary glands were dissected on 18 and 20 dpi for sporozoite presence via GFP signal prevalence. D Pie charts display infection prevalence, defined as the proportion of mosquitoes with at least one detectable oocyst. No significant differences were observed between control (Con) and tralopyril-exposed (Tralo) mosquitoes (Fisher’s exact test). Dot plots show oocyst counts per midguts across 6, 9 and 12 dpi pooled (x-axis) for control and tralopyril-exposed mosquitoes (y-axis). Vertical lines indicate median values; error bars indicate 95% confidence intervals. A significant reduction in oocyst number per midgut was observed in the tralopyril group compared to the controls (binomial GLM). Data represents 3-4 independent biological replicates; n = number of total mosquito midguts counted. E Motility (y-axis) was assessed at one and two-hours in control (Con, black) and tralopyril-exposed (Tralo, purple) groups (x-axis). A significant reduction was observed at two hours (Fisher’s exact test). Data represents 4-5 biological replicates; n = number of individual ookinetes tracked. F Ookinete speed (y-axis) for control (Con, black) and tralopyril-treated (Tralo, purple) groups (x-axis). At one-hour, tralopyril-treated ookinetes exhibited significantly increased speed compared to controls (unpaired t-Test with Welch’s correction). No significant difference in speed was observed at two-hours (Mann-Whitney test; data not normally distributed). Horizonal lines indicate median; error bars indicated 95% confidence intervals. Data represents 4-5 biological replicates; n = number of individual ookinetes tracked. G Average oocyst area per midgut (y-axis) was measured in mosquitoes at six, nine and 12 dpi for control (Con, black) and tralopyril-exposed (Tralo, purple) mosquitoes (x-axis). At six dpi, oocysts were significantly smaller in the tralopyril-exposed group (Mann-Whitney test). No significant difference was detected at nine dpi (unpaired t-Test with Welch’s correction). By 12 dpi, oocysts in the tralopyril group were significantly larger than controls (Mann-Whitney test on transformed data). Horizontal lines indicate mean; error bars indicate standard deviation. Data represents three-four biological replicates; n = number of mosquito midguts counted. Figures A and C were created using BioRender. Fig. 2. Tralopyril induces early arrest of P. berghei oocyst development and mitochondrial disruption. A-B Pie charts display GFP fluorescence in mosquito midguts on nine, 12, 18 and 20 dpi (A) and salivary glands 18 and 20 dpi (B) for control (Con, black) and tralopyril exposure (Tralo, purple) respectively. Representative GFP channel images of mosquito midguts and salivary glands are shown. Scale bars, 200 µm. Data represents three-four independent biological replicates. Statistical significance was assessed by Fisher’s exact test; **** denotes p < 0.0001. C Ultrastructure expansion microscopy images of oocysts from midguts of control (Con, black) or tralopyril-exposed (Tralo, purple) mosquitoes on 12 dpi, stained with NHS ester (total protein), anti-CSP antibody (sporozoite surface marker), and Hoechst (DNA). Individual channels are shown in grey scale (left panels), and merged images are shown in colour (right) display NHS ester in grey, CSP in green, and DNA in blue. Scale bars, 50 µm. D Transmission electron microscopy images of 12 dpi oocysts. Left: overview images showing sporulated oocysts in control (Con) mosquitoes and arrested development in tralopyril-exposed (Tralo) mosquitoes. Scale bars, 5 µM. Middle: magnified view of mitochondria. Scale bars, 500 nm. Right: magnified view of mitochondrial cristae highlighting ultrastructure. Scale bars, 50 nm. E Mitochondrial cristae circularity was assessed from transmission electron microscopy (TEM) images of oocysts on day 12 dpi. Kernel density plot of the long-to-short axis ratio of mitochondrial cristae measured from TEM images (n = 77 cristae control, grey, n= 153 cristae tralopyril, purple). Statistical significance was evaluated with a Kolmogorov-Smirnov test. Fig. 3 – Tralopyril irreversibly halts P. berghei oocyst maturation and disrupts sporozoite motility. A Schematic of the recovery experimental design to assess the impact of tralopyril exposure on P. berghei development on An. stephensi. Mosquitoes were topically exposed to 31 µM tralopyril (solubilised in Acetone) 48 h prior to receiving an infectious blood meal. Mosquitoes received non-infectious blood meals on three and six dpi. Midguts were dissected on 12 and 20 dpi to assess GFP signal prevalence and 20 dpi to assess sporozoite GFP prevalence. B Pie charts display GFP signal prevalence assessed in midguts (12 and 20 dpi) in control (black) and tralopyril (purple) groups following two additional non-infectious blood meals (three and six dpi). Data represent four biological replicates. Statistical significance relative to control was assessed by Fisher’s exact test; **** denotes p < 0.0001. C Pie chart displays GFP signal prevalence in salivary glands assessed at day 20 post-infection in control (black) and treatment (purple) groups. Data represent three biological replicates. Statistical significance was assessed by Fisher’s exact test; **** denotes P < 0.0001. D Representative midgut images reveal reduced fluorescence in tralopyril (Tralo, right, purple) exposure compared to acetone-only (Con, black, left). Scale bars, 200 µm. E Motility (y-axis) of sporozoites was assessed at 15 min (left) and one hour (1 h, right) in control (black) and tralopyril groups (purple) (solubilised in 2% DMSO in RPMI) at 4 concentrations (0.8-15 µM). Filled bars represent the mean, and error bars represent standard deviation. Data represent three-12 biological replicates. Control n = 1061-1289 and tralopyril n= 133-635 sporozoites per condition. Two-way ANOVA and multiple comparison. *P< 0.05, **P< 0.01, ***P