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

Chemosensory appendages of gravid <i>An</i>. <i>coluzzii</i> electrophysiologically respond to DMDS, DMTS and sulcatone.

<p>(A) EAG, (B) EPG and (C) ELG responses (each chemosensory organ is highlighted in red in a schematic diagram of mosquito head) are expressed as response difference to solvent control (oil) of <i>An</i>. <i>coluzzii</i> females to DMDS, DMTS and sulcatone. Y axis represents response amplitude subtracted by control values and X axis represents log transformed molar concentration. Asterisks represent significant response amplitude different from zero (***, <i>p</i> < 0.001; **, <i>p</i> < 0.01; *, <i>p</i> < 0.05; one sample <i>t</i>-test, one-sided). Error bar = s.e.m. (n = 7).</p

Neurons in capitate peg sensillum of maxillary palp are activated by DMDS, DMTS and sulcatone.

<p>Electrophysiological activities of (A) cpA, (B) cpB and (C) cpC neurons in capitate peg sensillum (highlighted in a red box; picture modified from a previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149800#pone.0149800.ref035" target="_blank">35</a>]) of maxillary palp in gravid <i>An</i>. <i>coluzzii</i> females are identified by spike amplitudes, and changes in spike frequency are quantified to characterize individual neuronal response to varying concentrations of DMDS, DMTS and sulcatone. Y axis represents response spike number subtracted by control response values (DMSO) and X axis represents log transformed molar concentration of DMDS, DMTS and sulcatone. Asterisks represent significant response amplitude different from zero (***, <i>p</i> < 0.001; **, <i>p</i> < 0.01; *, <i>p</i> < 0.05; one sample <i>t</i>-test, one-sided). Error bar = s.e.m. (n = 7~10).</p

Volatiles from overcrowded/starved larval water habitats affect oviposition behavior of <i>An</i>. <i>coluzzii</i> gravid females.

<p>(A) Schematic of oviposition dual choice behavioral assay designed to examine olfactory-driven responses (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149800#sec002" target="_blank">Methods</a> for details). Oviposition preference of gravid females to larval water samples with varied treatment by (B) incubating different number of late instars for 72 h, (C) incubating 300 late instars for different time period, and (D) diluting LW sample obtained by incubating 300 late instars for 72 h. Asterisks represent significant OI values different from zero (***, <i>p</i> < 0.001; **, <i>p</i> < 0.01; Wilcoxon signed-rank test, two-sided). Error bar = s.e.m. (n = 17 ~ 36).</p