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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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Related in: MedlinePlus

Selective effects of diapause conditions on expression of innate immune genes.The relative expression of four immune genes was determined in six groups of female Canton S flies: virgin 3–6 h old flies (C0), one week old uninfected and non-diapausing flies (C1), uninfected flies kept for 3 weeks either at 11°C and 10L:14D (diapause, D3) or at 25°C and 12L:12D, (normal conditions, N3) and infected flies (cross hatched bars) kept under diapause (D3) or non-diapause conditions (N3) for 3 weeks. Infected flies were injected with a suspension of Micrococcus luteus and Escherichia coli and kept for an additional 3 hours before freezing and RNA extraction. Data are presented as means ± S.E.M, n = 3–4 replicates with 10–15 flies in each. Significance compared to the newly hatched control (C1) which was set at one, or as indicated by connectors: * p<0.05, ** p<0.01, *** p<0.001, N.S. – not significant (ANOVA followed with Tukey test) or #p<0.05, ##p<0.01 (Kruskal–Wallis test followed by pairwise comparisons using Wilcoxon rank sum test). A The Drosomycin expression was significantly upregulated in flies diapausing for 3 weeks (D3) in both infected and uninfected flies compared to non-diapausing 1-week old (C1) and 3-week old flies (N3). Infection further increased transcripts in both N3 and D3 flies. BCecropin A1 was significantly upregulated during diapause (D3) versus normal conditions (C1), but not N3, in uninfected flies only if comparing C1 to D3. Infection drastically increased transcripts in both diapausing and nondiapausing flies. C The peptidoglycan recognition protein SB1 (PGRP-SB1) transcript level are not affected by diapause, only infection increased it. DDiptericin also increased only due to infection. For the investigated immune genes we did not find a differences in expression levels between 1-week old (C1) and newly eclosed (C0) flies. Similar results were obtained with flies that were reared on food supplemented with antibiotics (see Fig. S2 in File S1).
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pone-0113051-g007: Selective effects of diapause conditions on expression of innate immune genes.The relative expression of four immune genes was determined in six groups of female Canton S flies: virgin 3–6 h old flies (C0), one week old uninfected and non-diapausing flies (C1), uninfected flies kept for 3 weeks either at 11°C and 10L:14D (diapause, D3) or at 25°C and 12L:12D, (normal conditions, N3) and infected flies (cross hatched bars) kept under diapause (D3) or non-diapause conditions (N3) for 3 weeks. Infected flies were injected with a suspension of Micrococcus luteus and Escherichia coli and kept for an additional 3 hours before freezing and RNA extraction. Data are presented as means ± S.E.M, n = 3–4 replicates with 10–15 flies in each. Significance compared to the newly hatched control (C1) which was set at one, or as indicated by connectors: * p<0.05, ** p<0.01, *** p<0.001, N.S. – not significant (ANOVA followed with Tukey test) or #p<0.05, ##p<0.01 (Kruskal–Wallis test followed by pairwise comparisons using Wilcoxon rank sum test). A The Drosomycin expression was significantly upregulated in flies diapausing for 3 weeks (D3) in both infected and uninfected flies compared to non-diapausing 1-week old (C1) and 3-week old flies (N3). Infection further increased transcripts in both N3 and D3 flies. BCecropin A1 was significantly upregulated during diapause (D3) versus normal conditions (C1), but not N3, in uninfected flies only if comparing C1 to D3. Infection drastically increased transcripts in both diapausing and nondiapausing flies. C The peptidoglycan recognition protein SB1 (PGRP-SB1) transcript level are not affected by diapause, only infection increased it. DDiptericin also increased only due to infection. For the investigated immune genes we did not find a differences in expression levels between 1-week old (C1) and newly eclosed (C0) flies. Similar results were obtained with flies that were reared on food supplemented with antibiotics (see Fig. S2 in File S1).

Mentions: Innate immunity is likely to play a critical role during insect diapause and genes of the Toll/Imd pathways are known to display strong allelic variation in a North American cline of D. melanogaster that show variations in important life history traits [77]. We set out to analyze expression of a selection of key immune genes in diapausing flies. Both infected and uninfected flies were analyzed. Uninfected flies were kept for 3 weeks in diapause or non-diapause conditions, and one week of normal conditions. For infected specimens we injected flies previously kept for 3 weeks at 11°C and 10L:14D, and non-diapausing control flies of the same age, with a mix of Gram-negative (Escherichia coli) and Gram-positive (Micrococcus luteus) bacteria. Three hours after bacterial injection flies were sampled, along with non-infected flies, for analysis of gene expression. We examined the transcriptional activation of three antimicrobial peptide (AMP) genes and one peptidoglycan recognition protein (PGRP-SB1) gene regulated by the Toll and Imd (immune deficiency) pathways (Fig. 7).


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)

Selective effects of diapause conditions on expression of innate immune genes.The relative expression of four immune genes was determined in six groups of female Canton S flies: virgin 3–6 h old flies (C0), one week old uninfected and non-diapausing flies (C1), uninfected flies kept for 3 weeks either at 11°C and 10L:14D (diapause, D3) or at 25°C and 12L:12D, (normal conditions, N3) and infected flies (cross hatched bars) kept under diapause (D3) or non-diapause conditions (N3) for 3 weeks. Infected flies were injected with a suspension of Micrococcus luteus and Escherichia coli and kept for an additional 3 hours before freezing and RNA extraction. Data are presented as means ± S.E.M, n = 3–4 replicates with 10–15 flies in each. Significance compared to the newly hatched control (C1) which was set at one, or as indicated by connectors: * p<0.05, ** p<0.01, *** p<0.001, N.S. – not significant (ANOVA followed with Tukey test) or #p<0.05, ##p<0.01 (Kruskal–Wallis test followed by pairwise comparisons using Wilcoxon rank sum test). A The Drosomycin expression was significantly upregulated in flies diapausing for 3 weeks (D3) in both infected and uninfected flies compared to non-diapausing 1-week old (C1) and 3-week old flies (N3). Infection further increased transcripts in both N3 and D3 flies. BCecropin A1 was significantly upregulated during diapause (D3) versus normal conditions (C1), but not N3, in uninfected flies only if comparing C1 to D3. Infection drastically increased transcripts in both diapausing and nondiapausing flies. C The peptidoglycan recognition protein SB1 (PGRP-SB1) transcript level are not affected by diapause, only infection increased it. DDiptericin also increased only due to infection. For the investigated immune genes we did not find a differences in expression levels between 1-week old (C1) and newly eclosed (C0) flies. Similar results were obtained with flies that were reared on food supplemented with antibiotics (see Fig. S2 in File S1).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0113051-g007: Selective effects of diapause conditions on expression of innate immune genes.The relative expression of four immune genes was determined in six groups of female Canton S flies: virgin 3–6 h old flies (C0), one week old uninfected and non-diapausing flies (C1), uninfected flies kept for 3 weeks either at 11°C and 10L:14D (diapause, D3) or at 25°C and 12L:12D, (normal conditions, N3) and infected flies (cross hatched bars) kept under diapause (D3) or non-diapause conditions (N3) for 3 weeks. Infected flies were injected with a suspension of Micrococcus luteus and Escherichia coli and kept for an additional 3 hours before freezing and RNA extraction. Data are presented as means ± S.E.M, n = 3–4 replicates with 10–15 flies in each. Significance compared to the newly hatched control (C1) which was set at one, or as indicated by connectors: * p<0.05, ** p<0.01, *** p<0.001, N.S. – not significant (ANOVA followed with Tukey test) or #p<0.05, ##p<0.01 (Kruskal–Wallis test followed by pairwise comparisons using Wilcoxon rank sum test). A The Drosomycin expression was significantly upregulated in flies diapausing for 3 weeks (D3) in both infected and uninfected flies compared to non-diapausing 1-week old (C1) and 3-week old flies (N3). Infection further increased transcripts in both N3 and D3 flies. BCecropin A1 was significantly upregulated during diapause (D3) versus normal conditions (C1), but not N3, in uninfected flies only if comparing C1 to D3. Infection drastically increased transcripts in both diapausing and nondiapausing flies. C The peptidoglycan recognition protein SB1 (PGRP-SB1) transcript level are not affected by diapause, only infection increased it. DDiptericin also increased only due to infection. For the investigated immune genes we did not find a differences in expression levels between 1-week old (C1) and newly eclosed (C0) flies. Similar results were obtained with flies that were reared on food supplemented with antibiotics (see Fig. S2 in File S1).
Mentions: Innate immunity is likely to play a critical role during insect diapause and genes of the Toll/Imd pathways are known to display strong allelic variation in a North American cline of D. melanogaster that show variations in important life history traits [77]. We set out to analyze expression of a selection of key immune genes in diapausing flies. Both infected and uninfected flies were analyzed. Uninfected flies were kept for 3 weeks in diapause or non-diapause conditions, and one week of normal conditions. For infected specimens we injected flies previously kept for 3 weeks at 11°C and 10L:14D, and non-diapausing control flies of the same age, with a mix of Gram-negative (Escherichia coli) and Gram-positive (Micrococcus luteus) bacteria. Three hours after bacterial injection flies were sampled, along with non-infected flies, for analysis of gene expression. We examined the transcriptional activation of three antimicrobial peptide (AMP) genes and one peptidoglycan recognition protein (PGRP-SB1) gene regulated by the Toll and Imd (immune deficiency) pathways (Fig. 7).

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