AgTRPA1 mediates larval responses within the upper temperature range.

<p><b>a</b>) Arithmetic means ± S.E.M of total distance travelled in 300s for injected larvae reared at 27° C were plotted. Asterisks indicate <i>p</i><0.05 comparing AgTRPA1 and Non-specific siRNA-treatment using Mann–Whitney <i>U</i> test. Black rectangle labels the temperature range at which larval mobility was significantly modified following the knockdown of AgTRPA1. <b>b</b>) A stack of larval trajectories (n≥10) recorded in 28-38°C gradient for buffer-alone and AgTRPA1, Non-specific siRNA-injected treatments were shown. Thermotactic indices ± S.E.M were plotted for injected larvae. Asterisks indicate <i>p</i><0.05 comparing AgTRAP1 and Non-specific siRNA treatment (Mann–Whitney <i>U</i> test).</p

Thermal preferences of WT 4^th instar <i>An</i>.<i>gambiae</i>

<div><p><b>larvae</b>.</p> <p><b>a</b>) Individual <i>An. gambiae</i> larva was introduced into the center of the behavioral arena and recording started following a 15s acclimation period. Swimming trajectories from a minimum of 10 individual larvae reared at 27° C were superimposed. Each color represents a separate trial. <b>b</b>) Arithmetic means ± S.E.M (n≥10) of thermotactic indices in 7 different thermal gradients were plotted. Mann–Whitney <i>U</i> test was used to compare thermotactic indices at 27 and 33°C with a <i>p</i> value > 0.05.</p></div

Expression of AgTRPA1 in larval tissues.

<p>cDNA libraries from larval antennae, heads and bodies were generated by extracting mRNA followed by <i>in intro</i> reverse transcription. <i>rps7</i> and <i>agtrpa1</i> were amplified using gene-specific primers and run on a 2% agarose gel. “+” or “-“ indicates the presence or absence of reverse transcriptase, respectively.</p

Thermal preferences of WT 4^th instar An.

<div><p><b><i>gambiae</i> larvae following the shift of cultivation</b>. </p> <p><b>a</b>) A stack of larval trajectories (n≥10) recorded in 7 different thermal gradients were shown for larvae reared at 30° C. <b>b</b>) Larval thermotactic indices ± S.E.M were plotted for larvae reared at 30° C. Mann-Whitney <i>U</i> test was used to compare thermotactic indices at 30 and 36°C with a <i>p</i> value > 0.05.</p></div

AgTRPA1 mediates larval behavior within the shifted hot range.

<p><b>a</b>) Arithmetic means ± S.E.M of total distance travelled in 300s for injected larvae reared at 30° C were plotted. Asterisks indicate <i>p</i><0.05 comparing AgTRAP1 and Non-specific siRNA-injected larvae (Mann–Whitney <i>U</i> test). <b>b</b>) Stack of larval trajectories (n≥10) recorded in 31-41°C gradient for buffer and AgTRPA1, Non-specific siRNA treatments were shown. Thermotactic indices ± S.E.M were shown for injected larvae reared at 30° C. Asterisks indicate <i>p</i><0.05 comparing AgTRPA1 and Non-specific siRNA-injected larvae (Mann–Whitney <i>U</i> test).</p

Thermal-induced larval mobility following the shift of cultivation.

<p>Arithmetic means ± S.E.M recorded from larvae reared at both 27 and 30°C of total distance travelled in 300s were plotted (n≥12). White arrow shows the shift of cultivation temperature from 27 to 30°C. Black circle shows the temperature at which larval mortality was evident for both 27 and 30°C-reared colony. This figure indicates the change of larval mobility pattern matches the shift of rearing temperature.</p

Thermal-induced mobility in WT 4^th instar <i>An</i>.

<div><p><b><i>gambiae</i> larvae</b>. </p> <p>Arithmetic means ± standard error of the mean (S.E.M) of total distance travelled by individual larva in 300s were plotted (n≥15). Red circle indicates the two individual temperatures that generated lowest larval mobility in the neighboring temperature ranges (27 and 33°C) while black circle shows the temperature at which larvae experienced morbidity/death after 2-3 mins of assaying (41° C), thus the total distance was calculated based on the time frame before larval mortality.</p></div

Larval antenna is a peripheral thermosensory organ.

<p><b>a</b>) Arithmetic means ± S.E.M of total distance travelled in 300s for individual larva recorded from larvae lacking either antennae or maxillary palp were plotted (n≥12). Asterisks suggest <i>p</i><0.05 using Mann–Whitney <i>U</i> test to compare antennal ablation to sham ablation treatment. Kruskal-Wallis one-way analysis of variance was also utilized to compare larval mobility at all 5 temperatures following antennal ablation with <i>p</i>>0.05, indicating larvae without antennae were not capable of eliciting differential mobility at varying ambient temperatures comparing to sham treatment. <b>b</b>–<b>k</b>) Localization of AgTRPA1 mRNA was detected by fluorescent in situ hybridization (FISH). White arrow indicates localization of AgTRPA1 mRNA while green labels neuronal axons and dendrites. <b>l</b>–<b>o</b>) Red fluorescence indicates AgTRPA1 mRNA while green indicates the localization of AgOrco protein that is expressed in all ORNs. White arrow indicates AgTRPA1-expressing neuronal cell bodies while hollow arrow shows cluster of ORNs (Scale bar, 25µm).</p

Knockdown of AgTRPA1 mRNA via RNAi.

<p>Means of cycle threshold (CT) values for amplification of <i>agtrpa1</i> and <i>rps7</i> were shown (n=2). Quantitative RT-PCR was performed on cDNA isolated from whole larvae receiving AgTRPA1, non-specific siRNA and buffer injection. Relative mRNA abundance + S.E.M was plotted with data normalized to non-specific siRNA treatment using PFAFFL method.</p

Oviposition behavior of <i>An</i>. <i>coluzzii</i> gravid females is negatively affected by DMDS, DMTS and sulcatone.

An. coluzzii gravid females were allowed oviposit between control water and DMDS, DMTS and sulcatone with serial dilutions. Asterisks represent significant OI value different from zero (**, p p < 0.05; Wilcoxon signed-rank test, two-sided). Error bar = s.e.m. (n = 14 ~ 35).</p