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The sleeping beauty: how reproductive diapause affects hormone signaling, metabolism, immune response and somatic maintenance in Drosophila melanogaster.

Kubrak OI, Kučerová L, Theopold U, Nässel DR - PLoS ONE (2014)

Bottom Line: Drosophila melanogaster females can enter a shallow reproductive diapause in the adult stage, which drastically reduces organismal senescence, but little is known about the physiology and endocrinology associated with this dormancy, and the genes involved in its regulation.We induced diapause in D. melanogaster and monitored effects over 12 weeks on dynamics of ovary development, carbohydrate and lipid metabolism, as well as expression of genes involved in endocrine signaling, metabolism and innate immunity.Diapausing flies display very low mortality, extended lifespan and decreased aging of the intestinal epithelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Stockholm University, S-106 91 Stockholm, Sweden.

ABSTRACT
Some organisms can adapt to seasonal and other environmental challenges by entering a state of dormancy, diapause. Thus, insects exposed to decreased temperature and short photoperiod enter a state of arrested development, lowered metabolism, and increased stress resistance. Drosophila melanogaster females can enter a shallow reproductive diapause in the adult stage, which drastically reduces organismal senescence, but little is known about the physiology and endocrinology associated with this dormancy, and the genes involved in its regulation. We induced diapause in D. melanogaster and monitored effects over 12 weeks on dynamics of ovary development, carbohydrate and lipid metabolism, as well as expression of genes involved in endocrine signaling, metabolism and innate immunity. During diapause food intake diminishes drastically, but circulating and stored carbohydrates and lipids are elevated. Gene transcripts of glucagon- and insulin-like peptides increase, and expression of several target genes of these peptides also change. Four key genes in innate immunity can be induced by infection in diapausing flies, and two of these, drosomycin and cecropin A1, are upregulated by diapause independently of infection. Diapausing flies display very low mortality, extended lifespan and decreased aging of the intestinal epithelium. Many phenotypes induced by diapause are reversed after one week of recovery from diapause conditions. Furthermore, mutant flies lacking specific insulin-like peptides (dilp5 and dilp2-3) display increased diapause incidence. Our study provides a first comprehensive characterization of reproductive diapause in D. melanogaster, and evidence that glucagon- and insulin-like signaling are among the key regulators of the altered physiology during this dormancy.

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Altered gene expression in diapausing flies assessed by qPCR suggest endocrine diapause phenotypes.Relative steady state expression of genes encoding DILPs, InR and AKH in virgin female flies (Canton S), kept for 1–9 weeks under diapause conditions (11°C and 10L:14D), and for one week recovery (R1′) under non-diapause conditions (25°C and 12L:12D) after 3 weeks of diapause. Virgin flies kept for one week at normal conditions (C1) were used as comparison. The expression values were calculated with the 2−ΔΔCt method relative to that of the 3–6-h old virgin flies (C0) in each assay. Data are presented as means ± S.E.M, n = 4 independent replicates with 10–15 flies in each replicate. Significance of differences from 1 week non-diapause control (1) is indicated as well between groups indicated by connectors, p<0.05, ** p<0.01, *** p<0.001, N.S. not significantly different (ANOVA followed with Tukey test) or alternatively #p<0.05, ##p<0.01 or ####p<0.01 (Kruskal–Wallis test followed by pairwise comparisons using Wilcoxon rank sum test). A Compared to 1-week old non-diapausing flies (C1) the dilp2 expression increased significantly after one week of diapause (D1) and remained significantly higher over diapause (D3–D9). After one week of recovery from diapause (R1′) the dilp2 expression decreased significantly back to the C1 level. Interestingly the recently hatched control flies (C0) display a significantly higher dilp2 expression compared to 1-week non-diapausing flies (C1). B Compared to C1 controls dilp3 increased after one week of diapause and remained higher during 9 weeks (D1–D9). After one week of recovery (R1′) from diapause dilp3 decreased back to the control level (C1). The dilp3 expression is significantly higher in recently eclosed flies (C0) than in one week normal controls (C1). C The dilp5 expression showed a profile similar to that of dilp3. D The dilp6 expression was significantly higher throughout diapause than in C1 controls, but with a slight decrease after 6 weeks (D6). After one week of recovery from diapause dilp6 decreased significantly. Recently hatched flies (C0) display higher dilp6 expression than the one week old ones (C1). E The insulin receptor (InR) transcript displayed no significant differences between the treatments. F The Akh mRNA increased drastically after one week in diapause (D1) and peaked after three weeks (D3). The transcript level returned to the one week control (C1) level after recovery from diapause. The Akh expression is not significantly different in recently eclosed flies (C0) and one week normal controls (C1).
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pone-0113051-g004: Altered gene expression in diapausing flies assessed by qPCR suggest endocrine diapause phenotypes.Relative steady state expression of genes encoding DILPs, InR and AKH in virgin female flies (Canton S), kept for 1–9 weeks under diapause conditions (11°C and 10L:14D), and for one week recovery (R1′) under non-diapause conditions (25°C and 12L:12D) after 3 weeks of diapause. Virgin flies kept for one week at normal conditions (C1) were used as comparison. The expression values were calculated with the 2−ΔΔCt method relative to that of the 3–6-h old virgin flies (C0) in each assay. Data are presented as means ± S.E.M, n = 4 independent replicates with 10–15 flies in each replicate. Significance of differences from 1 week non-diapause control (1) is indicated as well between groups indicated by connectors, p<0.05, ** p<0.01, *** p<0.001, N.S. not significantly different (ANOVA followed with Tukey test) or alternatively #p<0.05, ##p<0.01 or ####p<0.01 (Kruskal–Wallis test followed by pairwise comparisons using Wilcoxon rank sum test). A Compared to 1-week old non-diapausing flies (C1) the dilp2 expression increased significantly after one week of diapause (D1) and remained significantly higher over diapause (D3–D9). After one week of recovery from diapause (R1′) the dilp2 expression decreased significantly back to the C1 level. Interestingly the recently hatched control flies (C0) display a significantly higher dilp2 expression compared to 1-week non-diapausing flies (C1). B Compared to C1 controls dilp3 increased after one week of diapause and remained higher during 9 weeks (D1–D9). After one week of recovery (R1′) from diapause dilp3 decreased back to the control level (C1). The dilp3 expression is significantly higher in recently eclosed flies (C0) than in one week normal controls (C1). C The dilp5 expression showed a profile similar to that of dilp3. D The dilp6 expression was significantly higher throughout diapause than in C1 controls, but with a slight decrease after 6 weeks (D6). After one week of recovery from diapause dilp6 decreased significantly. Recently hatched flies (C0) display higher dilp6 expression than the one week old ones (C1). E The insulin receptor (InR) transcript displayed no significant differences between the treatments. F The Akh mRNA increased drastically after one week in diapause (D1) and peaked after three weeks (D3). The transcript level returned to the one week control (C1) level after recovery from diapause. The Akh expression is not significantly different in recently eclosed flies (C0) and one week normal controls (C1).

Mentions: All four investigated dilps (dilp2, 3, 5 and 6) display similar expression profiles in Canton S flies under the experimental conditions (Fig. 4A–D). Thus, in newly eclosed flies (C0) dilp mRNA levels are higher than in flies kept for 1 week at normal conditions (C1) (Fig. 4A–D). The largest differences are observed for dilp3 and dilp5 transcripts, where C1 flies show only 14% of the values seen in C0 flies (Fig. 4B–C). In flies subjected to diapause conditions, we observed by 2.2–4.5-fold elevated levels of all four dilps already after one week of diapause (D1) compared to non-diapausing flies (C1) (Fig. 4A–D). High relative expressions were also registered in 3-week diapausing flies (D3). After 6–9 weeks of diapause (D6–D9) dilp levels decrease, but are still higher than in the C1 non-diapausing flies (Fig. 4A–D). Flies that had recovered for one week from three weeks of diapause (R1′) display a decrease in all four dilp transcripts to the control (C1) levels (Fig. 4A–C). In contrast to alterations in expression of dilps, the insulin receptor, InR mRNA does not change significantly over 9 weeks of diapause or after recovery compared to non-diapausing flies (C1) and is similar to that of the newly eclosed flies (Fig. 4E).


The sleeping beauty: how reproductive diapause affects hormone signaling, metabolism, immune response and somatic maintenance in Drosophila melanogaster.

Kubrak OI, Kučerová L, Theopold U, Nässel DR - PLoS ONE (2014)

Altered gene expression in diapausing flies assessed by qPCR suggest endocrine diapause phenotypes.Relative steady state expression of genes encoding DILPs, InR and AKH in virgin female flies (Canton S), kept for 1–9 weeks under diapause conditions (11°C and 10L:14D), and for one week recovery (R1′) under non-diapause conditions (25°C and 12L:12D) after 3 weeks of diapause. Virgin flies kept for one week at normal conditions (C1) were used as comparison. The expression values were calculated with the 2−ΔΔCt method relative to that of the 3–6-h old virgin flies (C0) in each assay. Data are presented as means ± S.E.M, n = 4 independent replicates with 10–15 flies in each replicate. Significance of differences from 1 week non-diapause control (1) is indicated as well between groups indicated by connectors, p<0.05, ** p<0.01, *** p<0.001, N.S. not significantly different (ANOVA followed with Tukey test) or alternatively #p<0.05, ##p<0.01 or ####p<0.01 (Kruskal–Wallis test followed by pairwise comparisons using Wilcoxon rank sum test). A Compared to 1-week old non-diapausing flies (C1) the dilp2 expression increased significantly after one week of diapause (D1) and remained significantly higher over diapause (D3–D9). After one week of recovery from diapause (R1′) the dilp2 expression decreased significantly back to the C1 level. Interestingly the recently hatched control flies (C0) display a significantly higher dilp2 expression compared to 1-week non-diapausing flies (C1). B Compared to C1 controls dilp3 increased after one week of diapause and remained higher during 9 weeks (D1–D9). After one week of recovery (R1′) from diapause dilp3 decreased back to the control level (C1). The dilp3 expression is significantly higher in recently eclosed flies (C0) than in one week normal controls (C1). C The dilp5 expression showed a profile similar to that of dilp3. D The dilp6 expression was significantly higher throughout diapause than in C1 controls, but with a slight decrease after 6 weeks (D6). After one week of recovery from diapause dilp6 decreased significantly. Recently hatched flies (C0) display higher dilp6 expression than the one week old ones (C1). E The insulin receptor (InR) transcript displayed no significant differences between the treatments. F The Akh mRNA increased drastically after one week in diapause (D1) and peaked after three weeks (D3). The transcript level returned to the one week control (C1) level after recovery from diapause. The Akh expression is not significantly different in recently eclosed flies (C0) and one week normal controls (C1).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231144&req=5

pone-0113051-g004: Altered gene expression in diapausing flies assessed by qPCR suggest endocrine diapause phenotypes.Relative steady state expression of genes encoding DILPs, InR and AKH in virgin female flies (Canton S), kept for 1–9 weeks under diapause conditions (11°C and 10L:14D), and for one week recovery (R1′) under non-diapause conditions (25°C and 12L:12D) after 3 weeks of diapause. Virgin flies kept for one week at normal conditions (C1) were used as comparison. The expression values were calculated with the 2−ΔΔCt method relative to that of the 3–6-h old virgin flies (C0) in each assay. Data are presented as means ± S.E.M, n = 4 independent replicates with 10–15 flies in each replicate. Significance of differences from 1 week non-diapause control (1) is indicated as well between groups indicated by connectors, p<0.05, ** p<0.01, *** p<0.001, N.S. not significantly different (ANOVA followed with Tukey test) or alternatively #p<0.05, ##p<0.01 or ####p<0.01 (Kruskal–Wallis test followed by pairwise comparisons using Wilcoxon rank sum test). A Compared to 1-week old non-diapausing flies (C1) the dilp2 expression increased significantly after one week of diapause (D1) and remained significantly higher over diapause (D3–D9). After one week of recovery from diapause (R1′) the dilp2 expression decreased significantly back to the C1 level. Interestingly the recently hatched control flies (C0) display a significantly higher dilp2 expression compared to 1-week non-diapausing flies (C1). B Compared to C1 controls dilp3 increased after one week of diapause and remained higher during 9 weeks (D1–D9). After one week of recovery (R1′) from diapause dilp3 decreased back to the control level (C1). The dilp3 expression is significantly higher in recently eclosed flies (C0) than in one week normal controls (C1). C The dilp5 expression showed a profile similar to that of dilp3. D The dilp6 expression was significantly higher throughout diapause than in C1 controls, but with a slight decrease after 6 weeks (D6). After one week of recovery from diapause dilp6 decreased significantly. Recently hatched flies (C0) display higher dilp6 expression than the one week old ones (C1). E The insulin receptor (InR) transcript displayed no significant differences between the treatments. F The Akh mRNA increased drastically after one week in diapause (D1) and peaked after three weeks (D3). The transcript level returned to the one week control (C1) level after recovery from diapause. The Akh expression is not significantly different in recently eclosed flies (C0) and one week normal controls (C1).
Mentions: All four investigated dilps (dilp2, 3, 5 and 6) display similar expression profiles in Canton S flies under the experimental conditions (Fig. 4A–D). Thus, in newly eclosed flies (C0) dilp mRNA levels are higher than in flies kept for 1 week at normal conditions (C1) (Fig. 4A–D). The largest differences are observed for dilp3 and dilp5 transcripts, where C1 flies show only 14% of the values seen in C0 flies (Fig. 4B–C). In flies subjected to diapause conditions, we observed by 2.2–4.5-fold elevated levels of all four dilps already after one week of diapause (D1) compared to non-diapausing flies (C1) (Fig. 4A–D). High relative expressions were also registered in 3-week diapausing flies (D3). After 6–9 weeks of diapause (D6–D9) dilp levels decrease, but are still higher than in the C1 non-diapausing flies (Fig. 4A–D). Flies that had recovered for one week from three weeks of diapause (R1′) display a decrease in all four dilp transcripts to the control (C1) levels (Fig. 4A–C). In contrast to alterations in expression of dilps, the insulin receptor, InR mRNA does not change significantly over 9 weeks of diapause or after recovery compared to non-diapausing flies (C1) and is similar to that of the newly eclosed flies (Fig. 4E).

Bottom Line: Drosophila melanogaster females can enter a shallow reproductive diapause in the adult stage, which drastically reduces organismal senescence, but little is known about the physiology and endocrinology associated with this dormancy, and the genes involved in its regulation.We induced diapause in D. melanogaster and monitored effects over 12 weeks on dynamics of ovary development, carbohydrate and lipid metabolism, as well as expression of genes involved in endocrine signaling, metabolism and innate immunity.Diapausing flies display very low mortality, extended lifespan and decreased aging of the intestinal epithelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Stockholm University, S-106 91 Stockholm, Sweden.

ABSTRACT
Some organisms can adapt to seasonal and other environmental challenges by entering a state of dormancy, diapause. Thus, insects exposed to decreased temperature and short photoperiod enter a state of arrested development, lowered metabolism, and increased stress resistance. Drosophila melanogaster females can enter a shallow reproductive diapause in the adult stage, which drastically reduces organismal senescence, but little is known about the physiology and endocrinology associated with this dormancy, and the genes involved in its regulation. We induced diapause in D. melanogaster and monitored effects over 12 weeks on dynamics of ovary development, carbohydrate and lipid metabolism, as well as expression of genes involved in endocrine signaling, metabolism and innate immunity. During diapause food intake diminishes drastically, but circulating and stored carbohydrates and lipids are elevated. Gene transcripts of glucagon- and insulin-like peptides increase, and expression of several target genes of these peptides also change. Four key genes in innate immunity can be induced by infection in diapausing flies, and two of these, drosomycin and cecropin A1, are upregulated by diapause independently of infection. Diapausing flies display very low mortality, extended lifespan and decreased aging of the intestinal epithelium. Many phenotypes induced by diapause are reversed after one week of recovery from diapause conditions. Furthermore, mutant flies lacking specific insulin-like peptides (dilp5 and dilp2-3) display increased diapause incidence. Our study provides a first comprehensive characterization of reproductive diapause in D. melanogaster, and evidence that glucagon- and insulin-like signaling are among the key regulators of the altered physiology during this dormancy.

Show MeSH
Related in: MedlinePlus