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Knockout mutations of insulin-like peptide genes enhance sexual receptivity in Drosophila virgin females
2015 年 90 巻 4 号 p. 237-241
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2015 年 90 巻 4 号 p. 237-241
In the fruitfly Drosophila melanogaster, females take the initiative to mate successfully because they decide whether to mate or not. However, little is known about the molecular and neuronal mechanisms regulating sexual receptivity in virgin females. Genetic tools available in Drosophila are useful for identifying molecules and neural circuits involved in the regulation of sexual receptivity. We previously demonstrated that insulin-producing cells (IPCs) in the female brain are critical to the regulation of female sexual receptivity. Ablation and inactivation of IPCs enhance female sexual receptivity, suggesting that neurosecretion from IPCs inhibits female sexual receptivity. IPCs produce and release insulin-like peptides (Ilps) that modulate various biological processes such as metabolism, growth, lifespan and behaviors. Here, we report a novel role of the Ilps in sexual behavior in Drosophila virgin females. Compared with wild-type females, females with knockout mutations of Ilps showed a high mating success rate toward wild-type males, whereas wild-type males courted wild-type and Ilp-knockout females to the same extent. Wild-type receptive females retard their movement during male courtship and this reduced female mobility allows males to copulate. Thus, it was anticipated that knockout mutations of Ilps would reduce general locomotion. However, the locomotor activity in Ilp-knockout females was significantly higher than that in wild-type females. Thus, our findings indicate that the high mating success rate in Ilp-knockout females is caused by their enhanced sexual receptivity, but not by improvement of their sex appeal or by general sluggishness.
In the fruitfly Drosophila melanogaster, a virgin female’s decision to accept or reject a courting male is one of the important factors for mating success. Thus, to understand the genetic and neural bases of the Drosophila mating system, it is essential to identify neural circuits and signaling pathways involved in the regulation of female sexual receptivity. Behavioral genetic studies of Drosophila have identified critical brain neurons that regulate female sexual behavior (Ferveur, 2010). Electrical silencing of SAG neurons of the abdominal ganglion inhibits female sexual receptivity (Feng et al., 2014). Similarly, inactivation of subsets of doublesex-expressing brain neurons (pCd and pC1) also inhibits female sexual receptivity (Zhou et al., 2014). In addition, we have reported that insulin-producing cells (IPCs) in the adult brain regulate female sexual receptivity (Sakai et al., 2014). In contrast to SAG, pCd and pC1 neurons, IPCs downregulate female sexual receptivity. When IPCs are ablated by expressing the pro-apoptotic gene reaper (rpr) in IPCs, female sexual receptivity is dramatically enhanced (Sakai et al., 2014). As was observed in IPC-ablated females, conditional inactivation of IPCs also enhances female sexual receptivity (Sakai et al., 2014). Thus, it is likely that insulin secretion from IPCs modulates female sexual receptivity.
IPCs produce and release three types of insulin-like peptide (Ilp2, Ilp3 and Ilp5) (Broughton et al., 2005). In this study, we examined whether knockout mutations of Ilp2, Ilp3 and Ilp5 affect female sexual receptivity using Ilp21 (Bloomington Stock Center #30881), Ilp31 (Bloomington Stock Center #30882), and Ilp51 (Bloomington Stock Center #30884) females (Fig. 1A). These three knockout mutant flies were generated by ends-out homologous recombination, and each locus is replaced by the marker gene white (Grönke et al., 2010). These knockout lines were outcrossed for six generations to white flies with the wild-type Canton-S (CS) genetic background. Mating behavior was observed as described previously (Sakai et al., 2009). A pair of virgin male and female flies was introduced into an acrylic plastic observation chamber (15 mm diameter × 3 mm depth). Using hard disk video recorders (HDR-CX590V, Sony, Japan), the mating behaviors were recorded for 20–25 min in a temperature- and humidity-controlled cabinet (temperature, 24.5 ± 0.5℃; humidity, 50 ± 10%; illuminance, 180 ± 50 lux). After recording, we observed mating behaviors for 20 min by video playback, and then calculated mating success rate, courtship latency (the period between the moment the flies were placed in the chamber and the first courtship) and courtship index (the percentage of time spent courting during 10 min or until the moment of copulation after courtship initiation). Wild-type CS males were used in all the observations. All flies used in the experiment were 4 to 6 days old. The mating success rates of females homozygous for Ilp21, Ilp31 and Ilp51 were significantly higher than those of control wild-type females (Fig. 1B). However, no significant difference was observed in courtship latency or courtship index between wild-type and Ilp-knockout females (Fig. 1, C and D). In addition, females heterozygous for Ilp21 (Ilp21/+) also showed a high mating success rate, although neither their courtship latency nor courtship index was affected (Fig. 1, E–G). These results suggest that virgin females homozygous or heterozygous for Ilp21, Ilp31 and Ilp51 show enhanced sexual receptivity without improvement of their sex appeal.
Knockout mutations of Ilps enhance female sexual receptivity. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant. (A) Genomic structures of Ilp2, Ilp3 and Ilp5 in wild-type (WT), Ilp21, Ilp31 and Ilp51. Black and white boxes represent the coding and noncoding regions, respectively. An Ilp2, Ilp3 or Ilp5 locus is replaced by the marker gene white (gray boxes). (B) Cumulative mating success rate (%) in Ilp21 (black circles), Ilp31 (black triangles), Ilp51 (black squares) and wild-type (open circles) females. The observation period was 20 min. N = 119 for WT, N = 96 for Ilp21, N = 88 for Ilp31, N = 94 for Ilp51. The G test with Williams’s correction (Sokal and Rohlf, 1995) was used for comparison of mating success rates. (C) Courtship latency (sec). Females homozygous for Ilp21, Ilp31 and Ilp51, and wild-type females, were used. N = 119 for WT, N = 96 for Ilp21, N = 88 for Ilp31, N = 94 for Ilp51. Non-parametric ANOVA (Kruskal-Wallis test) was used for statistical analysis. (D) Courtship index (%). Females homozygous for Ilp21, Ilp31 and Ilp51, and wild-type females, were used. N = 30 for each genotype. Non-parametric ANOVA (Kruskal-Wallis test) was used for statistical analysis. (E) Cumulative mating success rate (%) in Ilp21 (black circles), Ilp21/+ (gray circles) and wild-type (open circles) females. For each genotype, 69–72 pairs were observed. (F) Courtship latency (sec). Ilp21, Ilp21/+ and wild-type females were used. N = 72 for WT, N = 70 for Ilp21/+, N = 72 for Ilp21. (G) Courtship index (%). Ilp21, Ilp21/+ and wild-type females were used. N = 30 for each genotype. (C, D, F, G) In each box plot, the box encompasses the interquartile range, a line is drawn at the median, and error bars correspond to the 10th and 90th percentiles. Each black square is the mean.
In Drosophila, receptive females retard their movement before mating and this receptivity-dependent reduction of female movement allows males to copulate (Hall, 1994; Bussell et al., 2014). One possible explanation for the enhanced female sexual receptivity due to Ilp21, Ilp31 and Ilp51 could simply be sluggishness, regardless of whether males court them or not. Thus, we measured locomotor activity using a video-tracking system as described previously (Sakai et al., 2009, 2014). Single virgin flies were used for the quantification of locomotor activity, for which total walking distance (mm) in 10 min was used as an index. The total distance was calculated using Move-tr/2D 7.0 (Library Co., Japan). The locomotor activity of females homozygous for Ilp21, Ilp31 or Ilp51 was significantly higher than that of wild-type females (Fig. 2A). The hyperlocomotion phenotype was also detected in males homozygous for Ilp21, Ilp31 or Ilp51, indicating that this phenotype is not female-specific (Fig. 2B). To further confirm that flies lacking Ilps show the hyperlocomotion phenotype, we next used IPC-ablated females. Ilp2-GAL4 drives the expression of UAS-linked genes specifically in the IPCs. We generated F1 flies of crosses between Ilp2-GAL4-III and UAS-rpr. In Ilp2-GAL4/UAS-rpr flies, the IPCs are ablated by expressing the pro-apoptotic gene rpr in IPCs (Broughton et al., 2005). As was observed in Ilp21, Ilp31 and Ilp51 females, IPC-ablated females also showed an increased locomotor activity (Fig. 2C). Although the regulatory mechanisms of locomotor activity through insulin signaling remain largely unknown, our findings indicate that the enhanced sexual receptivity in females lacking IPCs or Ilps does not simply result from general sluggishness.
General locomotion of flies lacking Ilps or IPCs. Total walking distance (mm) was used as the index of general locomotion. A Mann-Whitney U test was used for statistical analysis. **, P < 0.01; ***, P < 0.001; NS, not significant. (A) Females homozygous for Ilp21, Ilp31 and Ilp51, and wild-type females, were used. N = 32 for WT, N = 32 for Ilp21, N = 39 for Ilp31, N = 39 for Ilp51. (B) Males homozygous for Ilp21, Ilp31 and Ilp51, and wild-type males, were used. N = 40 for WT, N = 40 for Ilp21, N = 39 for Ilp31, N = 40 for Ilp51. (C) Ilp2-GAL4/+, +/UAS-rpr and Ilp2-GAL4/UAS-rpr females were used. N = 64 for each genotype.
In this study, we found that Ilp21, Ilp31 and Ilp51 virgin females show high mating success rate. In Drosophila, inhibition of insulin signaling reduces body size (Goberdhan and Wilson, 2003; Mirth and Shingleton, 2012). Thus, as a different hypothesis, it is possible to explain this high mating success rate as follows. In wild-type flies, the body of females is larger than that of males, and males usually cannot forcibly copulate with unreceptive females of normal size. However, females with a small body size may find it difficult to detach themselves from courting males completely, and males could forcibly copulate with such females even though they are still unreceptive. Ilp21 females weigh less than wild-type females, but Ilp31 and Ilp51 females do not (Grönke et al., 2010). Thus, it seems unlikely that the high mating success rate induced by knockout mutations of Ilp2, Ilp3 and Ilp5 results from a reduced body size of females.
Previous studies have demonstrated that insulin signaling plays important roles in the regulation of several behaviors. Impairment of IPC function leads to increased adult ethanol sensitivity (Corl et al., 2005). Mutations of Ilps and conditional expression of a dominant-negative transgene of the insulin receptor (InR) affect feeding preference toward nutritive sugars (Stafford et al., 2012). Inhibition of insulin signaling inhibits learning and memory induced by olfactory conditioning (Naganos et al., 2012; Chambers et al., 2015) and reduces sleep (Cong et al., 2015). Considering the significance of IPCs and Ilp/InR signaling for the regulation of innate and learned behaviors, it is likely that Ilps also act as neuromodulators involved in the regulation of female sexual receptivity. Another hypothesis is that knockout mutations of Ilp2, Ilp3 and Ilp5 affect neurodevelopmental processes. Gu et al. (2014) have reported that inhibition of insulin signaling inhibits the growth of neuropeptide-expressing neurons during metamorphosis. Thus, the Ilp-knockout mutations may induce growth defects of brain neurons that downregulate female sexual receptivity, leading to the enhanced sexual receptivity observed in virgin females.
After mating, the physiology and behavior of females dramatically change and the mated females do not allow male copulation attempts for a certain period of time (Ferveur, 2010; Avila et al., 2011). Wigby et al. (2011) have reported that knockout mutations of Ilp2, Ilp3 and Ilp5 decrease the remating rate of mated females, suggesting that insulin signaling upregulates the sexual receptivity of mated females. In contrast to mated females, our results showed that insulin signaling downregulates the sexual receptivity of virgin females. Thus, it is possible that the properties of the nervous system regulating sexual receptivity are mating-dependently modified and that the role of insulin signaling in female sexual receptivity dramatically changes after mating.
We previously reported that IPC-ablated females copulate with wild-type males significantly earlier than control females (Sakai et al., 2014). More than 80% of IPC-ablated females copulate with wild-type males within 10 min. However, no such extremely rapid copulation was observed in Ilp21, Ilp31 or Ilp51 females (Fig. 1B). Thus, neurotransmitters other than Ilps released from IPCs may also be involved in the regulation of sexual receptivity in virgin females. Besides Ilps, IPCs also express a neuropeptide, drosulfakinin (DSK) (Söderberg et al., 2012). It is largely unknown whether DSK affects female sexual receptivity, but it would be interesting to identify the neurotransmitters released from IPCs and to examine whether such neurotransmitters also play a role in the regulation of female sexual receptivity in Drosophila.
We thank Minoru Saitoe for providing the Ilp2-GAL4 and UAS-rpr lines, and Yuki Suzuki for technical assistance. This work was supported by JSPS KAKENHI grant numbers 23370035 and 25650116 (to T.S.).
