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. 2016 Jun 2:6:27242.
doi: 10.1038/srep27242.

Molecular mechanism and functional significance of acid generation in the Drosophila midgut

Gayle Overend  1 , Yuan Luo  2 , Louise Henderson  1 , Angela E Douglas  2 , Shireen A Davies  1 , Julian A T Dow  1
Affiliations

Molecular mechanism and functional significance of acid generation in the Drosophila midgut

Gayle Overend et al. Sci Rep. .

Abstract

The gut of Drosophila melanogaster includes a proximal acidic region (~pH 2), however the genome lacks the H(+)/K(+) ATPase characteristic of the mammalian gastric parietal cell, and the molecular mechanisms of acid generation are poorly understood. Here, we show that maintenance of the low pH of the acidic region is dependent on H(+) V-ATPase, together with carbonic anhydrase and five further transporters or channels that mediate K(+), Cl(-) and HCO3(-) transport. Abrogation of the low pH did not influence larval survival under standard laboratory conditions, but was deleterious for insects subjected to high Na(+) or K(+) load. Insects with elevated pH in the acidic region displayed increased susceptibility to Pseudomonas pathogens and increased abundance of key members of the gut microbiota (Acetobacter and Lactobacillus), suggesting that the acidic region has bacteriostatic or bacteriocidal activity. Conversely, the pH of the acidic region was significantly reduced in germ-free Drosophila, indicative of a role of the gut bacteria in shaping the pH conditions of the gut. These results demonstrate that the acidic gut region protects the insect and gut microbiome from pathological disruption, and shed light on the mechanisms by which low pH can be maintained in the absence of H(+), K(+) ATPase.

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Figures

Figure 1
Figure 1. The acidic region of the larval Drosophila midgut.
(A) Midgut of an intact Canton S larva fed m-Cresol purple pH dye (red pH <2.4 yellow pH 2.5–8 purple pH >8); (B) Excised midgut of a Canton S larvae maintained on m-Cresol purple pH dye showing five regions of pH. The anterior acidic region is spatially distant from the posterior alkaline region after midgut dissection (B), but lies parallel in the intact larva (A).
Figure 2
Figure 2. The H+ V-ATPase complex is highly expressed in the acidic region of the midgut.
Transcript abundance of three representative subunits of the H+ V-ATPase complex–vha55 (A), vha68-2 (B) and vhaPPA1-1 (C) -in the five regions of larval gut pH, showing enrichment in the acidic region. Expression of three genes which transcribe the H+ V-ATPase V0 ‘a’ subunit–vha100-2 (D), vha100-4 (E) and vha100-5 (F)–which are differentially expressed along the length of the midgut. Transcript abundance is determined by RNAseq, and expressed as FPKM (mean ± s.e.m., N = 3).
Figure 3
Figure 3. The H+ V-ATPase complex is required for acidic pH generation.
Midgut pH was assessed using Thymol blue pH dye (red pH <2.4, yellow pH 2.5–8, blue pH >8). Canton S and parental controls (A–C,E,G) all maintain a region of pH 2 (orange/red staining) in the acidic region (annotated by white arrows), whereas acidity is reduced in the vha100-2 (D) and vha100-4 (F) knockdown lines (yellow staining), but not the vha100-5 knockdown line (H).
Figure 4
Figure 4
Transcript abundance of additional genes enriched in the acid pH region, as determined by RNAseq; (A) CAH1; (B) Kcc; (C) Slowpoke; (D) CG8177; (E) CG11340; and (F) Labial. Transcript abundance is expressed as FPKM (mean ± s.e.m., N = 3).
Figure 5
Figure 5. Multiple genes are required for pH generation.
Midgut pH was assessed using Thymol blue pH dye (red pH <2.4, yellow pH 2.5–8, blue pH >8). (A) Control parental RNAi line (acidic region ∼pH 2). Knockdown of CAH1 (B), Kcc (C), Slowpoke (D), the SLC4A anion exchanger CG8177 (E) or the ligand-gated chloride channel CG11340 (F) reduced acidity in comparison to parental controls. (G) There was no effect of the H+, K+-ATPase inhibitor omeprazole (1 mM), but inhibition of carbonic anhydrase activity using the inhibitor compound acetazolamide (100 μM) reduced acidity (H).
Figure 6
Figure 6. vha100-4 knockdown increases susceptibility to ion loading and Pseudomonas infection.
(A) Knockdown of vha100-4 does not impede larval survival to pupation under standard lab conditions, (B) but does result in a significant decrease in adult lifespan. (C) Pseudomonas infection significantly increases larval development time and decreases survival to pupation, and (D) Tsp42Ec-Gal4>vha100-4-RNAi flies have an increased midgut bacterial load after Pseudomonas infection. (E) Knockdown of vha100-4 also compromises larval survival to pupation when maintained on a 2.5% NaCl or 5% KCl diet. Data are expressed as percent pupation (n = ~120 larvae) or percent survival (n = ~120 flies). Statistically significant differences were assessed by Kaplan-Meier testing with the logrank test (A–C) or one-way ANOVA analysis (D,E), critical level P = 0.05.
Figure 7
Figure 7. Bacterial abundance is increased in vha100-4 knockdown larvae.
(A) The abundance of Acetobacter is significantly elevated in the neutral, transition and alkaline regions of the midgut in the Tsp42Ec-Gal4>vha100-4-RNAi strain (pink squares), in comparison to the Tsp42Ec-Gal4/+ (grey circles) and vha100-4-RNAi/+ (black triangles) parental controls. (B) The abundance of Lactobacillus is significantly increased in the acidic and neutral regions of Tsp42Ec-Gal4> vha100-4-RNAi strain, in comparison to both parental controls. Each treatment has 10 replicates, each comprising the gut segment dissected from 10 larvae. In the lower panels, significantly different median values between groups (Dunn’s multiple comparison post hoc test) are indicated by different letters (a,b).
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