Characterization of Reproductive Dormancy in Male Drosophila melanogaster

Abstract

Insects are known to respond to seasonal and adverse environmental changes by entering dormancy, also known as diapause. In some insect species, including Drosophila melanogaster, dormancy occurs in the adult organism and postpones reproduction. This adult dormancy has been studied in female flies where it is characterized by arrested development of ovaries, altered nutrient stores, lowered metabolism, increased stress and immune resistance and drastically extended lifespan. Male dormancy, however, has not been investigated in D. melanogaster, and its physiology is poorly known in most insects. Here we show that unmated 3-6 h old male flies placed at low temperature (11°C) and short photoperiod (10 Light:14 Dark) enter a state of dormancy with arrested spermatogenesis and development of testes and male accessory glands. Over 3 weeks of diapause we see a dynamic increase in stored carbohydrates and an initial increase and then a decrease in lipids. We also note an up-regulated expression of genes involved in metabolism, stress responses and innate immunity. Interestingly, we found that male flies that entered reproductive dormancy do not attempt to mate females kept under non-diapause conditions (25°C, 12L:12D), and conversely non-diapausing males do not mate females in dormancy. In summary, our study shows that male D. melanogaster can enter reproductive dormancy. However, our data suggest that dormant male flies deplete stored nutrients faster than females, studied earlier, and that males take longer to recover reproductive capacity after reintroduction to non-diapause conditions.

Keywords: Drosophila melanogaster; diapause; mating; metabolism; reproduction.

Figure 1

Diapausing flies display altered reproductive organs and spermatogenesis. (A) Unmated males (D. melanogaster) dormant for 3 weeks at 11°C and 10L:14D (D3) have significantly smaller seminal vesicles (SV) and accessory glands (AG) than the control flies kept for 1 or 3 weeks at 25°C and 12L:12D (C1 and C3). The size of the testis (T) does not change. The tissues were labeled with DAPI and phalloidin-rhodamine. The size of seminal vesicles (B) and accessory glands (C), sperm number (D) and sperm motility (E) in unmated male flies, 3–6 h old (C0), kept at non-diapause conditions for 1 or 3 weeks (C1 and C3), under diapause conditions for 3 weeks (D3) and after recovery for 1–3 weeks (R1–R3). Data are presented as means ± S.E.M, n = 18–20 flies. Data are significantly different from the control C1 flies as indicated with ^##p < 0.01, ^###p < 0.001 (Kruskal–Wallis test followed by pairwise comparisons using Wilcoxon rank sum test). N.S. not significant.

Figure 2

Dormancy leads to arrested spermatogenesis at the stage of sperm individualization. (A) Newly emerged flies (C0) and 1-week old control flies (C1) display visible early (blue arrows) and late (yellow arrows) individualization complexes in testes (T) and seminal vesicles (SV). In control (C1) flies there are many sperm nuclei (asterisks) in seminal vesicles. Seminal vesicles and testes of C3 flies are filled with sperm cells (inset shows details of sperm nuclei in testes at triple magnification). Dormant (D3) males have very few sperm in seminal vesicles, but early individualization complexes (blue arrows). (B) Details of early (blue arrows) and late (yellow arrows) individualization complexes in testis of control (C1) flies. Only DAPI staining is seen in late individualization complexes, whereas early ones display colocalized DAPI and phalloidin-rhodamine. (C). qPCR shows that expression of soti (encoding Scotti, a regulator of sperm individualization) is decreased in flies dormant for 3 weeks and increases after 1 week of recovery (R1), but decreases after 3 weeks of recovery (R3). (D) Expression of twe (encoding Twine, a meiosis marker protein) is decreased in 3 week dormant flies, D3 (but not D1) and increases after 1 week of recovery (R1), but again decreases after 3 weeks (R3). Data in (C,D) are presented as means ± S.E.M and represent 6 replicates with 10–15 flies in each, n = 60–90 flies. Data are significantly different from the C1 control flies or between indicated groups with ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 (ANOVA followed with Tukey test) or alternatively with ^#p < 0.05 (Kruskal–Wallis test followed by pairwise comparisons using Wilcoxon rank sum test). N.S. Not significant.

Figure 3

Dormant flies display strongly reduced mating and fecundity. (A) Number of copulation attempts of control (C1) males crossed with control (C1) diapausing (D3) or recovering (R1–R3) females. A drastic drop in copulations was observed when either male or female dormant flies were paired with controls. Recovering flies did not fully regain their rate of mating. (B) The number of eggs laid by a female fly after mating with dormant or recovered males (or vice versa) showed a similar reduction (compared to crosses between control partners). (C) The egg to adult viability is also reduced in crossings with diapausing flies, but increased for recovered flies crossed to controls, especially when the controls are females. Data are shown as mean ± S.E.M for six independent replicates with 5–7 fly couples in each replicate, n = 30–42 fly couples (male and female). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 (ANOVA followed by Tukey test) or alternatively ^#p < 0.05, ^##p < 0.01 (Kruskal96Wallis test followed by pairwise comparisons using Wilcoxon rank sum test).

Figure 4

Dormant flies display alterations in carbohydrate and lipid stores. (A) Hemolymph glucose is increased after 3 weeks of dormancy (D3) and 3 weeks of recovery from diapause (R3). (B) Hemolymph trehalose is not altered during dormancy. (C) Whole body (stored) glucose is low after 1 week of (D1) and then increases (D3) during dormancy. (D) Whole body trehalose is not significantly affected by dormancy, but elevated in recovering flies (R3). (E) Stored glycogen is lower in flies dormant for 1 week (D1). (F) Triacylglycerid (TAG) content first increases (D1) and then decreases (D3) during dormancy. After 1 (R1) and 3 (R3) weeks of recovery TAG levels increased back to control levels. Data significantly different from that in the C1 control flies or between groups indicated with connectors are shown: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 (ANOVA followed with Tukey test) or alternatively with ^#p < 0.05, ^##p < 0.01 (Kruskal–Wallis test followed by pairwise comparisons using Wilcoxon rank sum test). N.S. Not significant. Data represent 6 replicates with 10–15 flies in each replicate, n = 60–90 flies.

Figure 5

Dormancy affects expression of genes regulating metabolism, stress responses, and innate immunity. Using qPCR, we measured transcript levels of genes encoding (A) adipokinetic hormone (Akh), (B,C) the targets of IIS phosphoenolpyruvate carboxykinase (pepck) and Brummer lipase (bmm), stress-responsive genes, (D) Neural Lazarillo (NLaz) and (E) Turandot A (TotA), and (F) the immune gene Drosomycin (Drs). Of these bmm, pepck, NLaz, and TotA transcripts increased after 1 week of dormancy (D1) and Drs after 3 weeks (D3). After recovery for 1–3 weeks (R1–R3) from dormancy, transcription of all genes return to levels close to that in the control flies. Data significantly different from the C1 control flies, or between groups indicated with connectors: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 (ANOVA followed with Tukey test) or alternatively ^#p < 0.05 (Kruskal–Wallis test followed by pairwise comparisons using Wilcoxon rank sum test). N.S. Not significant. Data represent 6 replicates with 10–15 flies in each replicate, n = 60–90 flies. Results are analyzed with 2^−ΔΔCt method and shown as a fold of expression, normalized vs. expression in newly eclosed (C0) flies. rp49 was used as a housekeeping gene.