Dietary Stimuli, Intestinal Bacteria and Peptide Hormones Regulate Female Drosophila Defecation Rate
<p>Ingestion of virulent bacteria and specific gut microbiota increase gut motility in <span class="html-italic">Drosophila</span>. (<b>A</b>) Defecation assay customized to examine the impact of PA14 on the defecation rate. At 15 h of feeding on PA14 or vehicle (4% sucrose) female flies were starved in empty fly vials for 5 h and then allowed to feed again for 5 h in fly vials containing 4% sucrose and 0.5% bromophenol blue (BPB) which colored fly food blue (at pH = 7). Conditioned flies were then transferred to plates feeding on 4% sucrose and 0.5% BPB for another day. Defecation rate was calculated as fecal spots per fly per day excreted by groups of 10 flies in 6 independent replicates. (<b>B</b>) Fecal spots per fly per day upon PA14 infection or without infection, of five populations of Oregon-R (OR) flies, reared in parallel in the lab for one year. Statistical analysis was performed using two-way ANOVA test with Tukey’s post-hoc correction. Statistical significance was observed in all cases (*** <span class="html-italic">p</span> < 0.001). (<b>C</b>) CFUs of intestinal bacteria per fly, shaped on LB or BHI media, in the absence or presence of rifampicin. Statistical analysis was performed using two-way ANOVA test with Tukey’s post-hoc correction. Statistical significance was observed in all cases (*** <span class="html-italic">p</span> < 0.001). (<b>D</b>) Bacterial species found in adult female flies of the OR9 and OR3 populations feeding on LB or BHI media, with and without rifampicin. Extraction of genomic DNA from single colonies and sequencing followed by BLAST search of the sequences, revealed five rifampicin-sensitive bacterial species, common to both OR9 and OR3 and two rifampicin-resistant strains (<span class="html-italic">E. haemoperoxidus</span> and <span class="html-italic">S. arlettae</span>), exclusive to OR9. (<b>E</b>) Fecal spots per day per female of the OR9 and OR3 populations, following ingestion of either PA14 or rifampicin-resistant strains <span class="html-italic">E. haemoperoxidus</span> or <span class="html-italic">S. arlettae</span>, alone or in combinations, in six independent replicates. Statistical analysis was performed using the Mann–Whitney U test. Statistical significance is indicated as * <span class="html-italic">p</span> < 0.05. All error bars represent standard deviation of the mean.</p> "> Figure 2
<p>Starvation and dietary ethanol reduce <span class="html-italic">Drosophila</span> gut motility and lifespan. (<b>A</b>) Kaplan–Meyer survival curves of Oregon-R female flies, fed with either a 4% sucrose control diet (grey line) or 4% sucrose plus 25% ethanol (black line) (<b>B</b>) Survival curves of Oregon-R female flies, fed with either 4% sucrose (grey line) or starved, given only water (black line). For all experiments, <span class="html-italic">n</span> = 120 flies per condition. Statistical analysis was performed using the log-rank test. Statistical significance was observed in all cases (*** <span class="html-italic">p</span> < 0.001). (<b>C</b>) Fecal spots per fly per day upon feeding on 4% sucrose with and without 25% ethanol or upon water only starvation. Each column represents the mean value of six biological replicates. Error bars represent standard deviation of the mean. Statistical analysis was performed using the Mann–Whitney U test. Statistical significance indicated as * <span class="html-italic">p</span> < 0.05 or ** <span class="html-italic">p</span> < 0.01.</p> "> Figure 3
<p>Phenotypic ranking and additive genetic basis of defecation rate of 150 DGRP strains. (<b>A</b>) Z-score analysis of the defecation rate of 150 DGRP strains, upon oral ingestion of P. aeruginosa. (<b>B</b>) Validation of defecation rate of 27 DGRP strains deviating by ≥1.25 SDs above or below the mean of the Z-score analysis, upon and without PA14 infection. Vertical red and blue arrows indicate a ≥2-fold change in the defecation rate, upon PA14 ingestion, in 6 out of 16 strains exhibiting extremely enhanced defecation rates and in 3 out of 11 strains exhibiting extremely reduced defecation rates, respectively. Each column represents the mean value of six biological replicates. Error bars represent standard deviation of the mean. Statistical analysis was performed using the Mann–Whitney U test. Statistical significance indicated as * <span class="html-italic">p</span> < 0.05 or ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001. (<b>C</b>) Narrow sense heritability (<math display="inline"><semantics> <mrow> <msup> <mi>h</mi> <mn>2</mn> </msup> </mrow> </semantics></math>) of the defecation rate was calculated based on crossing schemes between DGRP strains displaying high defecation rates (25201, 28171 and 25208) or low defecation rates (28182, 28150 and 28153). The standard error for the calculation of <math display="inline"><semantics> <mrow> <msup> <mi>h</mi> <mn>2</mn> </msup> </mrow> </semantics></math> was 0.11.</p> "> Figure 4
<p>GWAS-identified and hormone gene downregulation either ubiquitously or specifically in EEs or neurons affects fly defecation rate. Fecal spots per fly per day upon (<b>A</b>) ubiquitous (via <span class="html-italic">act-Gal4 UAS-RNAi</span>), (<b>B</b>) EE-specific (via <span class="html-italic">prosV1-Gal4 UAS-RNAi</span>) and (<b>C</b>) pan-neuron-specific (via <span class="html-italic">elav-Gal4 UAS-RNAi</span>) downregulation of indicated GWAS-identified genes, divided by that of the uninfected progeny of Gal4 crossed to <span class="html-italic">w<sup>1118</sup></span>. (<b>D</b>) Fecal spots per uninfected fly per day upon EE-specific (via <span class="html-italic">prosV1-Gal4 UAS-RNAi</span>) downregulation of hormone genes, divided by that of the progeny of Gal4 crossed to <span class="html-italic">w<sup>1118</sup></span> flies. Each column represents the mean value of six biological replicates. Error bars represent standard deviation of the mean. Statistical significance using the Mann–Whitney U test indicated as * <span class="html-italic">p</span> < 0.05 or ** <span class="html-italic">p</span> < 0.01. (<b>E</b>) Schematic of the <span class="html-italic">Drosophila</span> midgut, indicating the anterior and posterior regions and the expression of <span class="html-italic">Dh31-Gal4 UAS-mCherry-NLS</span> reporter (red) that colocalizes with anti-Prospero antibody staining of EEs (green), indicated by white arrows.</p> "> Figure 5
<p>High defecation rate DGRP lines exhibit higher midgut expression of <span class="html-italic">NPF</span> and <span class="html-italic">CG11307</span>, indicated by horizontal red arrows. Midgut expression of hormone encoding genes (<span class="html-italic">AstA</span>, <span class="html-italic">AstC</span>, <span class="html-italic">Dh31</span>, <span class="html-italic">Ilp3</span>, <span class="html-italic">Ms</span>, <span class="html-italic">Tk</span>, <span class="html-italic">Dar-2</span>) and GWAS-identified genes considered either positive regulators of the defecation rate, (<span class="html-italic">Pmp70</span>, <span class="html-italic">mub</span>, <span class="html-italic">meso18E</span>) or negative regulators (<span class="html-italic">Bin3</span>, <span class="html-italic">ckn</span> and <span class="html-italic">jumu</span>), in eight DGRP lines displaying high (25208, 28128, 25201, 28180, 28171, 28123, 29658, 28244) versus eight DGRP lines displaying low (28206, 28182, 28153, 28246, 25745, 25189, 28242, 28150) defecation rates. For each of the 16 DGRP lines the mean expression value of six biological replicates per gene was calculated and normalized to that of the strain OR6, and then used to find the relative average expression per group of eight high and eight low defecation rate lines. Statistical analysis was performed using the Mann–Whitney U test. Statistical significance (<span class="html-italic">p</span> < 0.05) is indicated with asterisks and red font for <span class="html-italic">NPF</span> and <span class="html-italic">CG11307</span>.</p> "> Figure 6
<p>Midgut expression levels of hormone-encoding and GWAS genes upon ubiquitous downregulation of GWAS-identified positive regulator genes of the defecation rate. Relative expression levels of (<b>A</b>) hormones and (<b>B</b>) GWAS-identified genes associated with defecation rate, in the midgut of flies upon ubiquitous downregulation (via act-Gal4-UAS-RNAi) of positive regulators of the defecation rate, <span class="html-italic">Pmp70</span>, or <span class="html-italic">mub</span>, or <span class="html-italic">meso18E</span>, or <span class="html-italic">CG11307</span>, normalized to flies prepared from Gal4 lines crossed to <span class="html-italic">w<sup>1118</sup></span>. Ubiquitous downregulation of either <span class="html-italic">jumu</span> or <span class="html-italic">CG7166</span> was used as a control. Values indicated in red represent a ≥0.3 decrease (red arrow pointing down), whereas values in blue represent a ≥0.3 increase (blue arrow pointing up) from the baseline value of 1. Each number represents the average of six values.</p> "> Figure 7
<p>Downregulation of GWAS genes, <span class="html-italic">mub</span>, <span class="html-italic">CG11307</span> and <span class="html-italic">Pmp70</span>, in EEs or neurons consistently reduces <span class="html-italic">Dh31</span> and increases <span class="html-italic">AstC</span> and <span class="html-italic">AstA</span> expression in the midgut, while <span class="html-italic">Dh31</span>, <span class="html-italic">AstC</span> and <span class="html-italic">AstA</span> expression are all increased in the head. Relative expression levels of (<b>A</b>) hormone-encoding genes <span class="html-italic">Dh31</span>, <span class="html-italic">AstC</span> and <span class="html-italic">AstA</span> and (<b>B</b>) GWAS-identified genes <span class="html-italic">ckn</span>, NPF and <span class="html-italic">meso18E</span> in the midgut and head of flies upon EE-specific (via prosV1-Gal4-UAS-RNAi) or pan-neuronal (via elav-Gal4-UAS-RNAi) downregulation of <span class="html-italic">Pmp70</span>, <span class="html-italic">mub</span> or <span class="html-italic">CG11307</span>, normalized to flies prepared from Gal4 lines crossed to <span class="html-italic">w<sup>1118</sup></span>. Values in red font accompanied by a red arrow pointing down represent statistically significant decrease. Values in blue font accompanied by a blue arrow pointing up represent statistically significant increase. Each number represents the average of 12 values. Statistical analysis was performed using the <span class="html-italic">t</span>-test. Statistical significance indicated as * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01 or *** <span class="html-italic">p</span> < 0.001.</p> "> Figure 8
<p>Starvation reduces expression of hormonal genes, <span class="html-italic">Dh31</span>, <span class="html-italic">AstC</span> and <span class="html-italic">AstA</span>, and positive regulators, <span class="html-italic">Pmp70</span> in the midgut, and <span class="html-italic">meso18E</span> in the head. Relative expression of (<b>A</b>) hormone-encoding genes <span class="html-italic">Dh31</span>, <span class="html-italic">AstA</span>, <span class="html-italic">AstC</span> and <span class="html-italic">NPF</span> and (<b>B</b>) GWAS-identified positive (<span class="html-italic">Pmp70</span>, <span class="html-italic">CG11307</span>, <span class="html-italic">meso18E</span> and <span class="html-italic">mub</span>) and negative (<span class="html-italic">ckn</span>) regulators of the defecation rate, in midgut and head, upon starvation vs. feeding on 4% sucrose of female OR6 adults. Values indicated in red accompanied by a red arrow pointing down represent statistically significant decrease in the relative hormone gene expression. Each number represents the average of twelve values. Statistical significance using the <span class="html-italic">t</span>-test is indicated as * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01 or *** <span class="html-italic">p</span> < 0.001.</p> "> Figure 9
<p>Downregulation of <span class="html-italic">Dh31</span> in EEs or neurons reduces <span class="html-italic">Dh31</span> expression in both the midgut and the head, while downregulation of <span class="html-italic">Dh31</span> in EEs increases <span class="html-italic">AstC</span> and <span class="html-italic">AstA</span> expression in the midgut. Relative expression levels of hormone-encoding genes <span class="html-italic">Dh31</span>, <span class="html-italic">AstA</span> and <span class="html-italic">AstC</span> in the midgut and the head upon EE-specific (via prosV1-Gal4-UAS-RNAi) or pan-neuron-specific (via elav-Gal4-UAS-RNAi) downregulation of <span class="html-italic">Dh31</span> or VM-specific (via 24B-Gal4-UAS-RNAi) downregulation of <span class="html-italic">Dh31-R</span>, normalized to flies prepared from Gal4 lines crossed to <span class="html-italic">w<sup>1118</sup></span>. Values indicated in red and blue represent statistically significant decrease (red arrow pointing down) and increase (blue arrow pointing up) respectively, of the relative hormone gene expression. Each number represents the average of 12 values. Statistical analysis was performed using the <span class="html-italic">t</span>-test. Statistical significance indicated as * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01 or *** <span class="html-italic">p</span> < 0.001.</p> "> Figure 10
<p><span class="html-italic">Pmp70</span>, <span class="html-italic">mub</span> and <span class="html-italic">CG11307</span>, control <span class="html-italic">Dh31</span> positively in the midgut and negatively in the head, irrespective of the tissue being targeted for downregulation (midgut EEs or neurons). Proposed mechanism depicting the communication between GWAS-identified genes and <span class="html-italic">Dh31</span> along the gut-brain axis. While a positive regulator of defecation rate, <span class="html-italic">Dh31</span> is induced in the midgut via <span class="html-italic">Pmp70</span>, <span class="html-italic">mub</span>, <span class="html-italic">CG11307</span> expression in EEs or neurons. Interestingly, <span class="html-italic">Dh31</span> is inhibited in the head via <span class="html-italic">Pmp70</span>, <span class="html-italic">mub</span>, <span class="html-italic">CG11307</span> expression in EEs or neurons. However, <span class="html-italic">Dh31</span> positively regulates itself, locally and remotely, in the midgut and head tissues, when expressed in either midgut EEs or neurons. Red lines on the diagram represent negative regulation while the black arrows represent positive regulation of defecation rate.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Preparation of LB and BHI Agar Plates with or without Rifampicin
2.2. Bacteria Strains, Infection and Determination of Bacterial Load
2.2.1. Bacteria Strains
2.2.2. Bacterial Infection
2.2.3. Bacterial Load
2.2.4. Isolation of Bacterial Species from Drosophila
2.2.5. Single Colony PCR and Analysis of 16S rRNA Genes
2.2.6. DNA Extraction from Bacterial Species for Sequencing
2.3. Drosophila Melanogaster Diet, Maintenance, Strains and Experiments
2.3.1. Drosophila Diet and Maintenance
2.3.2. Germ-Free Flies
2.3.3. Drosophila Strains
2.3.4. Defecation Assay
2.3.5. Survival Assay
2.3.6. Determination of Narrow-Sense Heritability (h2)
2.3.7. Fly Midgut and Head Dissections
2.4. RNA Extraction, cDNA Synthesis and RT-qPCR
2.5. Immunohistochemistry
2.6. Image Acquisition and Analysis
2.7. Statistical Analysis
3. Results
3.1. Specific Gut Microbiota Composition and Virulent Bacteria Ingestion Increase Defecation Rate in Drosophila Females
3.2. Water-Only and Ethanol-Containing Food Reduce Lifespan and Defecation Rate in Female Adults
3.3. Genome Wide Analysis Reveals the Genetic Basis of Female Drosophila Defecation
3.4. Defecation Rate Is Controlled in Female Midgut EEs and Neurons by GWAS-Identified and Hormone-Encoding Genes
3.5. Higher Expression of NPF and CG11307 in DGRP Lines Displaying High Defecation Rates
3.6. GWAS-Identified and Hormonal Genes Participate in a Defecation Rate Signaling Network
3.7. GWAS-Identified Inducers of Defecation Induce Dh31 Expression in the Midgut and Reduce it in the Head
3.8. Water-Only Consumption Reduces Dh31, AstC, AstA and Pmp70 Expression in the Midgut and Reduced Expression of meso18E in the Head
3.9. Downregulation of Dh31 in the Midgut EEs or in Neurons Reduces Dh31 Expression in Both the Midgut and Head
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene Affected (FlyBase ID, Name) | Associated Variant ID (SNPs) | Variant Hits per Gene | Gene Function and Midgut Expression (Flybase and FlyGutSeq) |
---|---|---|---|
FBgn0016075, vkg | 2L_5025290_SNP 2L_5025252_SNP 2L_5025248_SNP | 3 | Subunit of Collagen IV, a major component of basement membranes. Anterior midgut visceral muscle expression and enteroendocrine cell induction by infection. |
FBgn0261388, CG42629 | X_12955420_SNP X_12955430_SNP | 2 | radish (rad) encodes a protein involved in anesthesia-resistant memory, heart contraction regulation, and small GTPase mediated signal transduction. |
FBgn0262222, mir-971 | X_12955420_SNP X_12955430_SNP | 2 | Micro-RNA gene (within the rad locus) of unknown function. Enterocyte and visceral muscle expression. |
FBgn0020306, dom | 2R_17221075_SNP 2R_17220640_SNP 2R_17220646_SNP | 3 | Tip60 acetyltransferase complex and functions in the exchange of histone His2Av. Uniform expression and stem cell induction upon infection. |
FBgn0031069, Pmp70 | X_19647084_SNP | 1 | ATPase-coupled peroxisomal transmembrane transporter of long-chain fatty acids. High enteroendocrine cell and visceral muscle expression. |
FBgn0037106, CG11307 | 3L_21612306_SNP 3L_21612307_SNP | 2 | Unknown function. Enteroendocrine cell expression. |
FBgn0037107, CG7166 | 3L_21612306_SNP 3L_21612307_SNP | 2 | Predicted to be involved in homophilic cell adhesion. Enteroendocrine cell expression. |
FBgn0262737, mub | 3L_21916473_SNP 3L_21853239_SNP | 2 | Regulation of alternative mRNA splicing. |
FBgn0033987, ckn | 2R_10853015_SNP | 1 | Cell contact and tyrosine phosphatase signaling pathway. Enteroendocrine cell expression. |
FBgn0024321, NK7.1 | 3R_10187071_SNP | 1 | Regulation of transcription by RNA polymerase II. Uniform expression. |
FBgn0040089, meso18E | X_19604135_SNP | 1 | Visceral muscle development. Uniform expression and progenitor cell bias. |
FBgn0004644, hh | 3R_18968168_SNP | 1 | Morphogen. Uniform expression and enteroblast bias. |
FBgn0016075, CG8065 | 3L_10208120_SNP | 1 | Unknown function. Enterocyte lineage expression. |
FBgn00263144, Bin3 | 2R_2126406_SNP | 1 | mRNA translation inhibition. Uniform expression and enterocyte bias. |
FBgn0052365, CG32365 | 3L_7873543_SNP | 1 | Unknown function. Uniform expression and progenitor cell bias. |
FBgn0001316, klar | 3L_461631_SNP | 1 | Organelle movement via microtubules. Uniform expression and enteroblast bias. |
FBgn0011828, pxn | 3L_2603619_SNP | 1 | Collagen IV cross-linking. Uniform expression, enteroendocrine cell induction by infection. |
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Kotronarou, K.; Charalambous, A.; Evangelou, A.; Georgiou, O.; Demetriou, A.; Apidianakis, Y. Dietary Stimuli, Intestinal Bacteria and Peptide Hormones Regulate Female Drosophila Defecation Rate. Metabolites 2023, 13, 264. https://doi.org/10.3390/metabo13020264
Kotronarou K, Charalambous A, Evangelou A, Georgiou O, Demetriou A, Apidianakis Y. Dietary Stimuli, Intestinal Bacteria and Peptide Hormones Regulate Female Drosophila Defecation Rate. Metabolites. 2023; 13(2):264. https://doi.org/10.3390/metabo13020264
Chicago/Turabian StyleKotronarou, Katerina, Anna Charalambous, Amalia Evangelou, Olympiada Georgiou, Andri Demetriou, and Yiorgos Apidianakis. 2023. "Dietary Stimuli, Intestinal Bacteria and Peptide Hormones Regulate Female Drosophila Defecation Rate" Metabolites 13, no. 2: 264. https://doi.org/10.3390/metabo13020264