DOI: 10.6084/m9.figshare.19759863.v1
title: Additional file 2 of Opsin1 regulates light-evoked avoidance behavior in Aedes albopictus

description: Additional file 2: Fig. S1. Photonegative behavior of adult mosquitoes, related to Fig. 1. (a-b) Y-maze assay with host cues. (a) Assay schematic. (b) Preference between illuminated and shaded environment. n = 150 females per species. The data are presented as mean ± SEM. Photobehavior were analyzed using one sample t test. Rejection of the null hypothesis that the mean of the data set is chance: ****p &lt; 0.0001. (c-d) Mosquito activity post release. Upper panel: assay schematic. The assay was conducted with Y-maze or tube with the entire apparatus under 120 lux. Lower panels: mosquito activity in Y-maze assay (c) or tube assay (d) post release. Three sample activity patterns from a pool of 8-12 Ae. albopictus individuals is shown. n in the figure denotes the total number of mosquitoes tested. Fig. S2. Schematic presentation of photopreference assay, related to Fig. 2. (a) Schematic presentation of binary photopreference assay between 0 lux and 15 lux. (b) Schematic presentation of binary photopreference assay between 15 lux and 150 lux. (c) Schematic presentation of binary photopreference assay between 150 lux and 1500 lux. (d) Schematic presentation of trinary photopreference assay with 0 lux, 15 lux and 150 lux (e). Schematic presentation of trinary photopreference assay with 15 lux, 150 lux and 1500 lux. (f) Mosquitoes were allowed to make a choice from environment of 0 lux, 15 lux, 150 lux and 1500 lux. (a-f) Arrow head indicates where mosquitoes were released. Fig. S3. Photobehavior of forth instar larvae and pupae of Ae. albopictus mosquito, related to Fig. 3. (a-c) Plate assay. (a) Schematic indicating how the quadrants were annotated. (b-c) Percentage of forth instar larvae (b) or pupae (c) of Ae. albopictus that preferred the indicated region. n = 200 larvae or pupae. (d-f) Tray assay. (d) Schematic indicating how the regions were annotated. (e-f) Percentage of forth instar larvae (e) or pupae (f) of Ae. albopictus that preferred the indicated region. n = 200 larvae or pupae. (b-c, e-f) Dots represent the percentage of Ae. albopictus that preferred the indicated region in individual repeats. For each group, four biological replicates with four technical replicates were performed. Total number of animals tested are indicated. (b, e) One-way ANOVA, followed by Tukey's multiple comparisons test was performed for testing among different groups. (c, f) Kruskal-Wallis test, followed by Dunn's multiple comparisons test was performed for testing among different groups. Different lowercase letters indicate significantly different. Fig. S4. Biting behavior of female mosquitoes under indicated illumination intensities, related to Fig. 4. (a) Schematic of biting assay under indicated illumination intensities without preference between hosts. (b-d) Percent of Ae. aegypti (b), Ae. albopictus (c) and Cx. quinquefasciatus (d) that bitted hosts under environment with indicated illumination intensities. Data are presented as mean ± SEM. Each dot indicates the blood feeding rate of one biological replicate. n in the figure denotes the total number of mosquitoes tested. The total number of mosquitoes fully engorged relative to the total number of mosquitoes tested is shown at the top of each column. One-way ANOVA test performed for testing among different groups, which showed no significant difference. Fig. S5. Phylogenetic tree based on protein sequences of opsins from five species of Dipteran. generated with neighbor-joining tree, related to Fig. 5. Neighbor-joining tree constructed using MEGA5 with Poisson model+uniform rates. Aalb denotes Ae. albopictus, Aaeg denotes Ae. aegypti, Cqui denotes Cx. quinquefasciatus, Dmel denotes D. melanogaster. The bar indicates phylogenetic distance. Fig. S6. Knocking down efficiency and biting behavior of opsin1-silenced Ae. albopictus, related to Figs. 5 and 6. (a-d) Expression of AalbOpsins upon microinjection of dsRNA. AalbOpsin1 (a), AalbOpsin2 (b), AalbOpsin8 (c) and AalbOpsin9 (d) expression three days post microinjection of AalbOpsin1 dsRNA. n = 10 mosquitoes pre group. (e-h) Expression of AalbOpsins upon microinjection of dsRNA. AalbOpsin1 (e), AalbOpsin2 (f), AalbOpsin8 (g) and AalbOpsin9 (h) expression three days post microinjection of AalbOpsin2 dsRNA. n = 10 mosquitoes pre group. (i) Expression of Opsins upon microinjection of dsRNA. AalbOpsin8 expression three days post microinjection of AalbOpsin8 dsRNA. n = 10 mosquitoes pre group. (j) Expression of Opsins upon microinjection of dsRNA. AalbOpsin9 expression three days post microinjection of AalbOpsin9 dsRNA. n = 10 mosquitoes pre group. (k) Sequence identities between AalbOpsins in percentage. Pairwise comparison of amino acid sequences of opsins of Ae. albopictus. Sequence similarity was presented with heatmap. (l) Sequence identities between AalbOpsins and AaegOpsins in percentage. Pairwise comparison of amino acid sequences of AalbOpsins and AaegOpsins. Opsins of Ae. albopictus share 90.74% to 97.59% similarity with the corresponding opsins of Ae. aegypti. Sequence similarity was presented with heatmap. (m) AaegOpsin1 expression three days post microinjection of dsRNA. Expression levels of AaegOpsin1 were normalized against Ae. aegypti actin (AAEL011197). n = 10 females per group. (n) Photobehavior of opsin1-silenced Ae. albopictus. Three days post thoracic inoculation of dsRNA, biting preference between hosts in illuminated and shaded environment was assessed (dsGFP, n = 113 females; dsAalbOpisn1#1, n = 111 females). The data are presented as mean ± SEM. Two-way ANOVA, followed by Tukey's multiple comparisons test was performed for testing among different groups. Different lowercase letters indicate significantly different. (a-j, m) The data are presented as mean ± SEM, with each dot represents one mosquito. (a-j) Expression levels of AalbOpsin genes were normalized against Ae. albopictus actin (AALF010408). (a-c, f, h, j, m) Mann-Whitney test was performed for testing between two groups. (d-e, g, i) Unpaired t test was performed for testing among different groups. Rejection of the null hypothesis that two groups have the same mean: *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001, ns: not significant. Fig. S7. Field collection of Ae. albopictus in three districts and photobehavior of field-collected larval and pupae. (a) Ae. albopictus were collected from three sites in three districts. One site in district A (112°56′E, 28°11′N), one site in district B (112°1′E, 27°43′N) and one site in district C (111°56′E, 28°53′N). (b) Pictures of typical collection sites. (c) Tray assay to test photobehavior of field-collected larvae and pupae. Preference index of field-collected Ae. albopictus between illuminated and shaded environment (n = 150 larvae or pupae per group). Data are presented as mean ± SEM. Photobehavior were analyzed using one sample t test or Wilcoxon signed-rank test. *p &lt; 0.05, ***p &lt; 0.001, ****p &lt; 0.001.