Opsin1 regulates light-evoked avoidance behavior in Aedes albopictus

Abstract

Background: Mosquitoes locate a human host by integrating various sensory cues including odor, thermo, and vision. However, their innate light preference and its genetic basis that may predict the spatial distribution of mosquitoes, a prerequisite to encounter a potential host and initiate host-seeking behaviors, remains elusive.

Results: Here, we first studied mosquito visual features and surprisingly uncovered that both diurnal (Aedes aegypti and Aedes albopictus) and nocturnal (Culex quinquefasciatus) mosquitoes significantly avoided stronger light when given choices. With consistent results from multiple assays, we found that such negative phototaxis maintained throughout development to adult stages. Notably, female mosquitoes significantly preferred to bite hosts in a shaded versus illuminated area. Furthermore, silencing Opsin1, a G protein-coupled receptor that is most enriched in compound eyes, abolished light-evoked avoidance behavior of Aedes albopictus and attenuated photonegative behavior in Aedes aegypti. Finally, we found that field-collected Aedes albopictus also prefers darker area in an Opsin1-dependent manner.

Conclusions: This study reveals that mosquitoes consistently prefer darker environment and identifies the first example of a visual molecule that modulates mosquito photobehavior.

Keywords: Aedes aegypti; Aedes albopictus; Biting behavior; Blood feeding; Compound eyes; Culex quinquefasciatus; Light preference; Opsin1; Photobehavior; Vision.

Fig. 1

Photobehavior of adult female mosquitoes. a, b Y-maze assay. a Assay schematic. b Preference index between illuminated and shaded environment (n = 400, 350, 250 females). c, d Tube assay. c Assay schematic and d preference index between illuminated and shaded environment (n = 250, 250, 150 females). e, f Shading assay. e Assay schematic. f Photobehavior of Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus upon shading (n = 209, 325, 246 females). g–i Photobehavior assay with blacklight. g Assay schematic. h Photobehavior assay with UV light of 345 nm performed at ZT17-ZT23 (n = 250 females per species). i Photobehavior assay with UV light of 395 nm performed at ZT17-ZT23 (n = 250 females per species). j Photobehavior of adult female mosquitoes across zeitgeber time. Photobehavior of Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus across zeitgeber time (n = 450 females per species). Mosquitoes were maintained on 12 h light/12 h dark cycles, with dark periods highlighted in black. Kruskal-Wallis test, followed by Dunn’s multiple comparisons test was performed for testing among different groups for Ae. aegypti. One-way ANOVA, followed by Tukey’s multiple comparisons test was performed for testing among different groups for Ae. albopictus and Cx. quinquefasciatus. Data labelled with different lowercase letters are significantly different from each other. Experimental groups denoted by “ab” are not significantly different from either “a” or “b” groups. Data includes over three biological repeats, each with four technical replicates. b, d, h–i Dots represent PI of individual repeats, which includes over three biological repeats, each with two technical replicates. b, d, f, h–j Data are presented as mean ± SEM. Total number of mosquitoes released are indicated. Photobehavior was analyzed using one sample t test or Wilcoxon signed-rank test for tests against chance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant

Fig. 2

Mosquito preference index over different illumination intensities. a–c Binary photopreference. a Photopreference between 0 lux and 15 lux (n = 200 females per species). b Photopreference between 15 lux and 150 lux (n = 200 females per species) and c photopreference between 150 lux and 1500 lux (n = 200 females per species). d, e Trinary photopreference. d Photopreference over 0 lux, 15 lux and 150 lux (n = 150 females per species) and e photopreference over 15 lux, 150 lux, and 1500 lux (n = 150 females per species). f Quaternary photopreference. Histogram showing mosquito photopreference over 0 lux, 15 lux, 150 lux, and 1500 lux (n = 200 females per species). a–c Dots represent PI of individual repeats, which includes over three biological repeats, with two technical replicates for each biological repeat. Photobehavior were analyzed using one sample t test or Wilcoxon signed-rank test for tests against chance. **P < 0.01, ***P < 0.001, ****P < 0.0001. a–f Data are presented as mean ± SEM. Total number of mosquitoes used are indicated

Fig. 3

Photobehavior of larvae, pupae and adult male mosquitoes. a–d Plate assay (a, b) and tray assay (c, d) to test photobehavior of larvae and pupae. a, c Assay schematic. Fifty larvae or pupae were released in the center of the testing plate/tray in each individual test. b, d Preference index of Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus between illuminated and shaded environment (n = 150 larvae or pupae per group for Ae. aegypti and Ae. albopictus, n = 300 larvae or pupae per group for Cx. quinquefasciatus). e, f Y-maze assay (n = 250, 300, 250 males) (e) and tube assay (n = 200 males per species) (f) to test photobehavior of male mosquitoes. b–d Data are presented as mean ± SEM. Total number of larvae or pupae tested are indicated. e, f Dots represent PI of individual repeats, which includes over three biological repeats, with two technical replicates for each biological repeat. Total number of mosquitoes tested are indicated. b–f Photobehavior were analyzed using one sample t test or Wilcoxon signed-rank test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

Biting behavior of female mosquitoes under different light intensities. a–d Biting preference between hosts from illuminated and shaded environment. a Assay schematic and b–d percent of Ae. aegypti (b), Ae. albopictus (c), and Cx. quinquefasciatus (d) that bitted hosts in illuminated and shaded environment (n = 400, 400, 450 females). e–h Biting preference for hosts in environment with different illumination intensities. e Assay schematic and f–h percent of Ae. aegypti (f), Ae. albopictus (g), and Cx. quinquefasciatus (h) that bitted hosts in environment with indicated illumination intensities (n = 800, 800, 1200 females). b–d, f–h Data are presented as mean ± SEM. Each dot represents the blood feeding rate of each biological repeat. Total number of mosquitoes used are indicated. The data are presented as the number of fully engorged mosquitoes that bitted host in environment with indicated illumination intensities in relative to the total mosquitoes released. b–d Mann-Whitney test (b, d) or unpaired t test with Welch’s correction (c) was performed for testing between two groups. **P < 0.01, ***P < 0.001, ****P < 0.0001. f, h Kruskal-Wallis test, followed by Dunn’s multiple comparisons test was performed for testing among different groups. g One-way ANOVA, followed by Tukey’s multiple comparisons test was performed for testing among different groups. f–h Different lowercase letters indicate significantly different. Experimental groups denoted by “ab” are not significantly different from either “a” or “b” groups

Fig. 5

Role of Opsins in mosquito photobehavior. a Effect of eye occlusion on mosquito photobehavior. b Effect of eye occlusion on Ae. albopictus biting preference (Mock, n = 104 females; Blindfolded, n = 65 females). Data is presented as the number of fully engorged mosquitoes that bitted host in illuminated or shaded environment relative to the total number of mosquitoes released. Two-way ANOVA, followed by Tukey’s multiple comparisons test was performed for testing among different groups. Different lowercase letters are significantly different. Experimental groups denoted by “ab” are not significantly different from either “a” or “b” groups. c Heatmap showing expression of opsin genes in Ae. albopictus across developmental stages. Expression levels of opsin genes in mixed-gender first instar larvae (1st), mixed-gender second instar larvae (2nd), mixed-gender third instar larvae (3rd), mixed-gender forth instar larvae (4th), mixed-gender pupae, adult females, and adult males. n = 50 for first and second instar larvae; n = 30 for third instar larvae; n = 20 for forth instar larvae and pupae; n = 3 for adult female and male. d Opsin gene expression of adult females at 1 day and 7 days after eclosion. Expression levels of opsin genes were normalized against Ae. albopictus actin (AALF010408). n = 9 females per group. e, f Effects of knocking down opsin genes on mosquito photobehavior. e Preference index for shaded area upon knocking down indicated opsin genes in Ae. albopictus (dsGFP, n = 328 females; dsAalbOpsin1, n = 103 females; dsAalbOpsin2, n = 166 females; dsAalbOpsin8, n = 88 females; dsAalbOpsin9, n = 95 females). f Effect of knocking down Opsin1 on Ae. aegypti photobehavior (dsGFP, n = 126 females; dsAaegOpsin1, n = 72 females). a–f Data are presented as mean ± SEM. Total number of female mosquitoes tested for each group was indicated by n in the figure. Photobehavior was analyzed using one sample t test. a, d, f Unpaired t test was performed for testing among different groups. e One-way ANOVA, followed by Tukey’s multiple comparisons test was performed for testing among different groups. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant

Fig. 6

Photobehavior of opsin1-silenced Ae. albopictus. a–c Photobehavior of Ae. albopictus injected with two additional dsRNA against opsin1. a Schematic depiction of the Opsin1 and the design of all the three dsRNAs used in this study (dsAalbOpsin1^#1, dsAalbOpsin1^#2, and dsAalbOpsin1^#3). b Knockdown efficiency of dsAalbOpsin1^#2 and dsAalbOpsin1^#3 assayed with qPCR (each dot denotes one mosquito; dsGFP, n = 20 females; dsAalbOpsin1^#2, n = 10 females; dsAalbOpsin1^#3, n = 10 females). c Photobehavior of Ae. albopictus injected with dsAalbOpsin1^#2 and AalbOpsin1^#3 (dsGFP, n = 105 females; dsAalbOpsin1^#2, n = 111 females; dsAalbOpsin1^#3, n = 106 females). Dots represent PI of individual repeats, which includes three biological repeats, with two technical replicates for each biological repeat. d–f Photobehavior of Ae. albopictus injected with dsAalbOpsin1^#1. d AalbOpsin1 expression at 3–5 days after thoracic inoculation of dsAalbOpsin1^#1 (each dot denotes one mosquito; n = 10 females per group). e Photobehavior of Opsin1-silenced Ae. albopictus with Y-maze assay. f Photobehavior of opsin1-silenced Ae. albopictus in the presence of host cues with Y-maze assay. b–f Data are presented as mean ± SEM. n in the figure denotes the total number of female mosquitoes tested for each group. Photobehavior was analyzed using one sample t test or Wilcoxon signed-rank test. b, d Expression levels of opsin were normalized against Ae. albopictus actin (AALF010408). b Kruskal-Wallis test, followed by Dunn’s multiple comparisons test was performed for testing among different groups. c One-way ANOVA, followed by Tukey’s multiple comparisons test was performed for testing among different groups. d–f Two-way ANOVA, followed by Sidak’s multiple comparisons test was performed for testing among different groups. *P < 0.05, **P < 0.01, ***P< 0.001, ****P < 0.0001, ns: not significant

Fig. 7

AalbOpsin1 mediates negative phototaxis behavior of field-collected Ae. albopictus. a, b Photobehavior of field-collected Ae. albopictus. a Photobehavior of female adult (n = 300 females for site A, n = 150 females for site B and site C). b Photobehavior of field-collected larvae and pupae (n = 150 larvae or pupae per group for site A and site C, n = 100 first and third instar larvae for site B, n = 150 pupae for site B). c, d Photobehavior of field-collected Ae. albopictus injected with dsRNA against AalbOpsin1. c Knockdown efficiency of dsAalbOpsin1 in field mosquitoes was measured with qPCR (each dot denotes one mosquito; n = 10 females per group). d Photobehavior of field-collected Ae. albopictus with AalbOpsin1-silenced. Starting from 3 days post-gene silencing, mosquito photobehavior was assessed with Y-maze assay. n in the figure denotes the total number of female mosquitoes tested for each group. a–d Data are presented as mean ± SEM. n denotes the total number of mosquitoes tested. Photobehavior was analyzed using one sample t test or Wilcoxon signed-rank test. c, d Two-way ANOVA, followed by Sidak’s multiple comparisons test was performed for testing among different groups. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant