Figure 6. tweedledee and tweedledum are required for reproductive resilience during drought.

(A) Schematic of Δdeedum mutant that deletes both tweedledee and tweedledum. (B) Attraction of wild type and Δdeedum mutant females to a human forearm. Data are plotted as violin plots with median and 1st/3rd quartiles and showing all data points. Each point represents a single trial with ~20 females, n=5 trials/group. Significantly different groups are indicated by different letters (one-way ANOVA, Holm-Šídák’s multiple comparisons test, p<0.0001). (C) Averaged weights of 5 females of the indicated genotype not fed or blood fed, n=14 groups of 5 females per group. Data are plotted as violin plots with median and 1st/3rd quartiles and showing all data points. Significantly different groups are indicated by different letters (one-way ANOVA, Tukey’s multiple comparisons test, p<0.0001). (D, G) Photographs of ovaries (left, scale bars: 20 µm) and spermathecae with filled sperm (right) from wild type and Δdeedum females 6 days (D) or 12 days (G) post blood-meal with eggs retained. (E, H) Number of melanized eggs laid by (top) and larvae hatched from (bottom) single wild type and Δdeedum mutant females 6 days (E) and 12 days (H) post blood-meal, depicting moderate and extended egg retention, respectively. Females laying no melanized eggs are depicted by open circles. Lines connect melanized eggs laid by and larvae hatched from the same individual. Larger circles and bold lines represent medians. Numbers at right show hatching rate (mean ± S.E.M) from each egg retention group, n=48–50 females/ group. (F, I) Distribution of melanized eggs laid (left) and larvae hatched (right) after egg retention in wild type and Δdeedum mutant females 6 days (F) or 12 days (I) post-blood-meal. 0 values are binned separately for each group. All other bins are [1,10], [11,20] … [101,110], [111,120] where “[,]” denote closed/inclusive intervals. (F, I) The groups between each genotype for melanized eggs laid and larvae hatched respectively were compared at each of the time points to determine significant difference (Mann-Whitney tests, *** p<0.001; **** p<0.0001). Distributions in (F) are analyzed from data in (E) and distributions in (I) are analyzed from data in (H). (J) Summary of tweedledee and tweedledum function in drought resilience of female Aedes aegypti mosquitoes.

Figure 6—figure supplement 1. Δdeedum double mutant genotyping strategy and additional egg-laying data.

(A) Genotyping strategy for Δdeedum double mutants. Primer sequences are at https://doi.org/10.5281/zenodo.5945524. See Figure 6—figure supplement 1—source data 1 . (B–D) Melanized eggs laid by females of the indicated genotype 6 (B), 12 (C), or 13 (D) days post-blood-meal. The genotype of the males to which they were mated is indicated below the female genotypes. Data are plotted as violin plots with median and 1st/3rd quartiles and showing all data points. Each point represents the melanized eggs laid by a single female (B: n=50/genotype; C: n=48–50/genotype; D: n=38–39/genotype). In B,C, data labeled with different letters are significantly different (Kruskal-Wallis, Dunn’s multiple comparisons test, p<0.05). In D significance established by Mann-Whitney test, p<0.0001.

Figure 6—figure supplement 2. tweedledum mutants show defects in reproductive resilience during drought.

(A) Schematic of Δdum mutant that deletes tweedledum, but not tweedledee. (B) In silico predicted translation of Δdum. (C) Averaged weights of 5 females of the indicated genotype not fed or blood fed, n=11–16 groups of 5 females per group. Data are plotted as violin plots with median and 1st/3rd quartiles and showing all data points. Significantly different groups are indicated by different letters (one-way ANOVA, Tukey’s multiple comparisons test, p<0.05). (D, G) Photographs of ovaries (left, scale bars: 10 µm) and spermathecae with filled sperm (right) from wild type and Δdum females 6 days (D) or 12 days (G) post blood-meal with eggs retained. (E) Melanzied eggs laid by females of the indicated genotype 6 days post-blood-meal. Data are plotted as violin plots with median and 1st/3rd quartiles and showing all data points. Each point represents the melanized eggs laid by a single female (n=47–49/genotype). ns, not significant (Mann-Whitney test). (F, I, J) Distribution of melanized eggs laid by (F, I) or larvae hatched from (J) wild type and Δdum mutant females 6 days (F) and 12 days (I, J) post-blood-meal. 0 values are binned separately for each group. All other bins are [1,10], [11,20] … [141,150], [151,160] where “[,]” denote closed/inclusive intervals. (F, I, J) The groups between each genotype for melanized eggs laid and larvae hatched respectively were compared at each of the time points to determine significant difference (Mann-Whitney tests, **** p<0.0001). Distributions in (F) are analyzed from data in (E) and distributions in (I, J) are analyzed from data in (H). (H) Number of melanized eggs laid by (top) and larvae hatched from (bottom) single wild type and Δdum mutant females 12 days post blood-meal, depicting extended egg retention. Females laying no melanized eggs are depicted by open circles. Lines connect melanized eggs laid by and larvae hatched from the same individual. Larger circles and bold lines represent medians. Numbers at right show hatching rate (mean ± S.E.M) from each egg retention group, n=40–45 females/ group.