Figure 1. Caper is expressed throughout the female germline. Single z-plane confocal slices indicate that caper−/− ovarioles (A–H) show normal patterning as compared to yw ovarioles (I–K). Nuclei are marked with DAPI in blue (A,E,I). The Caper:GFP fusion protein (B,F) is shown in green and marks nurse cell nuclei, the oocyte nucleus and follicle cell nuclei. An anti-Caper antibody shown in red (C,G,J) marks nurse cell nuclei, the oocyte nucleus, follicle cell nuclei and is also found in the oocyte cytoplasm. Overlays of all channels are shown in (D,H,K). White arrows mark the oocyte nucleus. Yellow arrows show cytoplasmic expression of Caper marked with an anti-Caper antibody. A 50 micron scale bar is shown in (K).

Figure 1. Caper is expressed throughout the female germline. Single z-plane confocal slices indicate that caper−/− ovarioles (A–H) show normal patterning as compared to yw ovarioles (I–K). Nuclei are marked with DAPI in blue (A,E,I). The Caper:GFP fusion protein (B,F) is shown in green and marks nurse cell nuclei, the oocyte nucleus and follicle cell nuclei. An anti-Caper antibody shown in red (C,G,J) marks nurse cell nuclei, the oocyte nucleus, follicle cell nuclei and is also found in the oocyte cytoplasm. Overlays of all channels are shown in (D,H,K). White arrows mark the oocyte nucleus. Yellow arrows show cytoplasmic expression of Caper marked with an anti-Caper antibody. A 50 micron scale bar is shown in (K).

Figure 2. Reproductive output is decreased with caper dysfunction. (A) Reproductive output is affected in caper−/− females regardless of the genotype of the males they are mated with, and these deficits are increased with age. However, caper−/− females mated with caper−/− males show the greatest reduction in reproductive output. In each of the four genotypic pairings, 25 vials of 5 females each were scored. Statistical analysis of reproductive output revealed a significant female × male × day interaction (GLMM: χ2 = 35.4, p = 2.70 × 10−9). (B) Knockdown of caper specifically within the germline using nanosGal4 also results in a decreased reproductive output compared to nanosGal4 female controls and this phenotype was exacerbated by age (GLMM: genotype × day interaction, χ2 = 240.0, p = 2.2 × 10−16). In both genotypic pairings, 25 vials of 5 females each were scored. The number of eggs laid is plotted on the y-axis, with the age of the female in days plotted on the x-axis. Genotypes are indicated in the legend for each panel using color coding.

Figure 2. Reproductive output is decreased with caper dysfunction. (A) Reproductive output is affected in caper−/− females regardless of the genotype of the males they are mated with, and these deficits are increased with age. However, caper−/− females mated with caper−/− males show the greatest reduction in reproductive output. In each of the four genotypic pairings, 25 vials of 5 females each were scored. Statistical analysis of reproductive output revealed a significant female × male × day interaction (GLMM: χ2 = 35.4, p = 2.70 × 10−9). (B) Knockdown of caper specifically within the germline using nanosGal4 also results in a decreased reproductive output compared to nanosGal4 female controls and this phenotype was exacerbated by age (GLMM: genotype × day interaction, χ2 = 240.0, p = 2.2 × 10−16). In both genotypic pairings, 25 vials of 5 females each were scored. The number of eggs laid is plotted on the y-axis, with the age of the female in days plotted on the x-axis. Genotypes are indicated in the legend for each panel using color coding.

Figure 3. Stages during which embryos arrest development due to caper dysfunction. (A) The number of embryos that did not complete development and the stage at which they arrested is shown. Number of embryos is plotted on the y-axis; parental genotypes are indicated on the x-axis. caper−/− embryos die significantly more than yw embryos (caper−/− n = 696, 7.47% dead; yw n = 759, 1.71% dead; Tukey’s test: z-ratio = −4.9, p = 6.58 × 10−6) and caper +/− embryos derived from caper mutant females outcrossed to yw males (n = 793, 3.53% dead; Tukey’s test: z-ratio = −3.3, p = 0.0055) or yw females outcrossed to caper mutant males (Tukey’s test: z-ratio = −5.4, p = 3.45 × 10−7). More caper +/− embryos derived from caper mutant females outcrossed to yw males are embryonically arrested than embryos derived from yw females outcrossed to caper mutant males (n = 834, 1.20% dead; Tukey’s test: z-ratio = −3.0, p = 0.016). (B) Knockdown of caper specifically within the female germline results in a significant increase in the arrest of embryonic development compared to controls (GLM: χ2 = 9.7, p = 0.00186).

Figure 3. Stages during which embryos arrest development due to caper dysfunction. (A) The number of embryos that did not complete development and the stage at which they arrested is shown. Number of embryos is plotted on the y-axis; parental genotypes are indicated on the x-axis. caper−/− embryos die significantly more than yw embryos (caper−/− n = 696, 7.47% dead; yw n = 759, 1.71% dead; Tukey’s test: z-ratio = −4.9, p = 6.58 × 10−6) and caper +/− embryos derived from caper mutant females outcrossed to yw males (n = 793, 3.53% dead; Tukey’s test: z-ratio = −3.3, p = 0.0055) or yw females outcrossed to caper mutant males (Tukey’s test: z-ratio = −5.4, p = 3.45 × 10−7). More caper +/− embryos derived from caper mutant females outcrossed to yw males are embryonically arrested than embryos derived from yw females outcrossed to caper mutant males (n = 834, 1.20% dead; Tukey’s test: z-ratio = −3.0, p = 0.016). (B) Knockdown of caper specifically within the female germline results in a significant increase in the arrest of embryonic development compared to controls (GLM: χ2 = 9.7, p = 0.00186).

Figure 4. caper dysfunction results in smaller ovaries. (A–F) Images of ovaries dissected from yw and caper−/− females at days 3, 5 and 14 post-eclosion using a Brightfield microscope. (G) Quantification of ovary area shows that ovaries from caper−/− females are significantly smaller at day 3 (caper−/− n = 50, average = 0.406 mm2, yw n = 46, average = 0.508 mm2, Tukey’s test: t-ratio = −2.128, p = 0.0352) and significantly larger at day 14 (caper−/− n = 44, average = 0.593 mm2, yw n = 68, average = 0.451 mm2, Tukey’s test: t-ratio = 3.147, p = 0.002) than ovaries from yw females. There is no significant difference in ovary size at day 5 (caper−/− n = 36, average = 0.759 mm2, yw n = 40, average = 0.782 mm2, Tukey’s test: t-ratio = −0.427, p = 0.6697). (H–M) Images of ovaries dissected from nosGal4 ctl and nosGal4, UASCaperRNAiGLC01382 females at days 3, 5 and 14 post-eclosion using a Brightfield microscope. (N) Quantification of ovary area shows that ovaries from nosGal4, UASCaperRNAiGLC01382 females are significantly smaller at day 3 (nosGal4, UASCaperRNAiGLC01382 n = 54, average = 0.666 mm2, nosGal4 ctl n = 30, average = 0.835 mm2, Tukey’s test: t-ratio = −4.188, p = 4.92e × 10−5) and day 5 (nosGal4:UASCaperRNAiGLC01382 n = 56, average = 0.552 mm2, nosGal4 ctl n = 50, average = 0.848 mm2, Tukey’s test: t-ratio = −8.619, p = 1.19 × 10−14) than ovaries from nosGal4 ctl. At day 14 there is not a significant difference in ovary size (nosGal4:UASCaperRNAiGLC01382 n = 48, average = 0.536 mm2, nosGal4 ctl n = 58, average = 0.491 mm2, Tukey’s test: t-ratio = 1.298, p = 0.1964). Scale bar on panel A represents 500 μm.Lines within each graph represent the mean and 95% confidence interval and significance is indicated by * p ≤ 0.05, **, p ≤ 0.01, *** p ≤ 0.001 or ns (not significant).

Figure 4. caper dysfunction results in smaller ovaries. (A–F) Images of ovaries dissected from yw and caper−/− females at days 3, 5 and 14 post-eclosion using a Brightfield microscope. (G) Quantification of ovary area shows that ovaries from caper−/− females are significantly smaller at day 3 (caper−/− n = 50, average = 0.406 mm2, yw n = 46, average = 0.508 mm2, Tukey’s test: t-ratio = −2.128, p = 0.0352) and significantly larger at day 14 (caper−/− n = 44, average = 0.593 mm2, yw n = 68, average = 0.451 mm2, Tukey’s test: t-ratio = 3.147, p = 0.002) than ovaries from yw females. There is no significant difference in ovary size at day 5 (caper−/− n = 36, average = 0.759 mm2, yw n = 40, average = 0.782 mm2, Tukey’s test: t-ratio = −0.427, p = 0.6697). (H–M) Images of ovaries dissected from nosGal4 ctl and nosGal4, UASCaperRNAiGLC01382 females at days 3, 5 and 14 post-eclosion using a Brightfield microscope. (N) Quantification of ovary area shows that ovaries from nosGal4, UASCaperRNAiGLC01382 females are significantly smaller at day 3 (nosGal4, UASCaperRNAiGLC01382 n = 54, average = 0.666 mm2, nosGal4 ctl n = 30, average = 0.835 mm2, Tukey’s test: t-ratio = −4.188, p = 4.92e × 10−5) and day 5 (nosGal4:UASCaperRNAiGLC01382 n = 56, average = 0.552 mm2, nosGal4 ctl n = 50, average = 0.848 mm2, Tukey’s test: t-ratio = −8.619, p = 1.19 × 10−14) than ovaries from nosGal4 ctl. At day 14 there is not a significant difference in ovary size (nosGal4:UASCaperRNAiGLC01382 n = 48, average = 0.536 mm2, nosGal4 ctl n = 58, average = 0.491 mm2, Tukey’s test: t-ratio = 1.298, p = 0.1964). Scale bar on panel A represents 500 μm.Lines within each graph represent the mean and 95% confidence interval and significance is indicated by * p ≤ 0.05, **, p ≤ 0.01, *** p ≤ 0.001 or ns (not significant).

Table 1. caper is required for viability during larval stages but is dispensable during pupariation.

Table 1. caper is required for viability during larval stages but is dispensable during pupariation.

MalesFemales ywcaper−/−RNAi Controlcaper RNAiywcaper−/−RNAi Controlcaper RNAiLarval Death3(63)5(46)0(46)3(43)0(59)9(50)2(53)2(49)Pupal Death6(60)8(41)2(46)1(40)8(59)3(41)4(51)1(47)

Table 2. caper dysfunction results in decreased mating rates for females but not males.

Table 2. caper dysfunction results in decreased mating rates for females but not males.

Female Genotype ywcaper−/−yw; nosGal4nosGal4; UAScaperRNAiTotalsMale Genotypeyw6024270 caper−/−60393829270 Totals150150120120