Question

Design a meaningful study in a well-equipped lab that will determine the impact of pH change on your favourite food in the year 2049.

Answer 1

Background: Despite the prevalent use of Drosophila as a model in studies of nutrition, the effects of fundamental food properties, such as pH, on animal health and behavior are not well known.

Objectives: We examined the effect of food pH on adult Drosophilalifespan, feeding behavior, and microbiota composition and tested the hypothesis that pH-mediated changes in palatability and total consumption are required for modulating longevity.

Methods: We measured the effect of buffered food (pH 5, 7, or 9) on male gustatory responses (proboscis extension), total food intake, and male and female lifespan. The effect of food pH on germfree male lifespan was also assessed. Changes in fly-associated microbial composition as a result of food pH were determined by 16S ribosomal RNA gene sequencing. Male gustatory responses, total consumption, and male and female longevity were additionally measured in the taste-defective Pox neuro (Poxn) mutant and its transgenic rescue control.

Results: An acidic diet increased Drosophila gustatory responses (40–230%) and food intake (5–50%) and extended survival (10–160% longer median lifespan) compared with flies on either neutral or alkaline pH food. Alkaline food pH shifted the composition of fly-associated bacteria and resulted in greater lifespan extension (260% longer median survival) after microbes were eliminated compared with flies on an acidic (50%) or neutral (130%) diet. However, germfree flies lived longer on an acidic diet (5–20% longer median lifespan) compared with those on either neutral or alkaline pH food. Gustatory responses, total consumption, and longevity were unaffected by food pH in Poxn mutant flies.

Conclusions: Food pH can directly influence palatability and feeding behavior and affect parameters such as microbial growth to ultimately affect Drosophila lifespan. Fundamental food properties altered by dietary or drug interventions may therefore contribute to changes in animal physiology, metabolism, and survival.

Food composition strongly affects Drosophila behavior, metabolism, and survival (5, 26, 29–32). Nonetheless, fundamental properties that might be affected by food composition, such as acidity and buffering capacity, are not routinely considered for their impact on fly health and physiology. YE and Brewer’s yeast have greater capacity to buffer media than the other common fly food ingredients that we tested. Because yeast is the main source of protein in fly food and its concentration is typically manipulated in dietary restriction studies (27, 33, 34), our results suggest that, in addition to modulating nutrition, changing dietary yeast concentration might substantially affect buffering capacity and food pH.

Flies lived longer on acidic food compared with neutral or alkaline diets, and this effect was conserved in different genetic backgrounds and diets and in both sexes. Our findings differ from previous studies that reported that neutral pH medium resulted in longer lives compared with acidic or alkaline food (10, 11). These studies were possibly confounded by the comparison of different buffer salts (10), which we ruled out as an explanation by using only phosphate buffer, or the poor solubility of casein in acid (11, 35), which we ruled out by testing a sucrose-only diet that did not contain any protein. Different fly lines might also show different sensitivity to food pH, although we showed a consistent effect on lifespan in 3 distinct genetic backgrounds. Although dietary protein is not necessary for the longevity mediated by acid, some nutrition is required to observe a pH effect on survival because starvation resistance—measured in buffered agar with no other nutrients—was insensitive to pH. These results suggest that acid does not directly affect fly metabolism or physiology in extending life.

We thus hypothesized that food pH might affect palatability, which we assessed quantitatively by measuring PER. Although low pH buffer alone did not elicit a response, suggesting that acid is not a positive tastant on its own, acid-buffered sucrose induced higher PER than either neutral- or alkaline-buffered sucrose. Salt (sodium chloride) content was ruled out as a confound in our studies because salt concentration equivalent to that of phosphate buffer had no effect on PER to unbuffered sucrose. Using sodium acetate to raise the pH of unbuffered sucrose also significantly reduced PER. Collectively, our results suggest that acidic pH enhances or maintains the palatability of food, whereas alkaline pH reduces it. These findings are consistent with, and extend upon, previous studies that showed that strong acids (pH <3) inhibit sweet sensing (16), whereas milder conditions (pH 3–6) seem to have no effect unless bitter compounds are present (17). Given the abundance of acids in the natural food substrate for D. melanogaster(36, 37) and the potential benefit of acid-producing microbes to fly health and development (21, 38, 39), it is perhaps not surprising to find that flies prefer a mildly acidic diet. Toxic alkaloids, phenols, and terpenoids—often alkaline and/or bitter tasting—may also drive avoidance and reduced feeding. Further studies will be needed to fully delineate the effect of broad pH ranges, buffer capacity, and different buffer salts on fly chemosensation.

To our knowledge, palatability and its relation to actual food intake have never been studied in Drosophila. To determine whether food pH-mediated differences in gustatory responses affect total consumption, we used 2 independent methods to measure fly food intake over 24 h (18) and observed a significant systematic increase in consumption as food pH was reduced on both sucrose-only and YE diets. Pairwise comparisons between the ingestion of pH 5, 7, and 9 food also revealed significantly greater feeding (P < 0.05) or the tendency for greater feeding (P < 0.10) on acidic food compared with pH 7 and/or 9 diets. Although flies seemed to overcome differences in palatability to consume food to nearly meet nutritional requirements, careful measurements of total consumption revealed consistent, small changes in intake that correlated with gustatory responses.

Our collective results are consistent with the idea that pH is an important modulator of the gustatory response to food and that longevity on acidic diets results from increased palatability and food intake. To further support this idea, we measured PER, food intake, and lifespan of taste-defective Poxn mutant flies (19), which we hypothesized would be insensitive to food pH. Whereas control flies showed higher PER and greater food intake on acidic medium compared with those on pH 7 or 9 food, the Poxn mutant showed gustatory responses and consumption that were insensitive to food pH. As with Canton-S and Dahomey flies, the Poxn rescue control lived longer on acid-buffered sucrose than on pH 7 or 9 food, and this was consistent in both sexes. In stark contrast, the lifespan of Poxn mutant males was insensitive to food pH. Female Poxn mutants showed statistically significant differences in survival, but these results were clearly different from the effects observed in control flies. Overall, our studies strongly support the idea that food pH affects palatability and total consumption to influence survival because the elimination of external taste sensation abolishes these phenotypes. In addition to the tarsi and labellum, pharyngeal sweet sensing, which lies at the interface of external and internal nutrient-sensing mechanisms, is required for the sustained feeding of sugar (40). Interestingly, the Poxn mutant has intact pharyngeal sweet taste (40), suggesting that the effect of food pH on total consumption is driven only by labellar and/or tarsal chemosensation. Given the limited number of Drosophila studies that incorporate food intake measurements, much is still unknown about how information from the various sense organs is integrated to both initiate and sustain feeding.

Another consideration of changing food properties is their effect on microbial growth. Previous studies on the impact of microbes on fly lifespan have been inconsistent (21, 22, 41, 42), suggesting that microbial influences on fly health and physiology are highly dependent on environmental factors. Although we observed minimal microbial growth on sucrose-only food surfaces, microbes were clearly associated with flies on a YE diet. Removing fly-associated microbes extended lifespan on the YE diet regardless of pH, which is consistent with studies that have reported that infection might be a primary cause of mortality in older flies (22) and that germfree flies show enhanced intestinal homeostasis during aging (43–45)—a critical determinant of fly lifespan (46, 47). Regardless, axenic and antibiotic-treated flies continued to show greater longevity on the acidic YE diet compared with pH 7 or 9 food. We also observed an increase in the ratio of Firmicutes to Proteobacteria, the predominant phylum present under standard conditions in our laboratory flies, with increased food pH. At the genus level, Bacillus was present only on neutral or alkaline food, whereas more common fly-associated microorganisms such as Acetobacter,Enterococcus, and Lactobacillus (48, 49) were prevalent under acidic conditions. We hypothesize that high food pH might contribute to dysbiosis, increasing the presence of pathogenic compared with commensal bacteria, which ultimately alters intestinal homeostasis and impairs health (50). The shift toward Firmicutes on high pH food is reminiscent of the shift in the dominant phyla or ratio of phyla seen in aging mammals. In mammals, these changes can be associated with detrimental effects on health (51, 52). Future work using more comprehensive approaches to assess microbial species diversity might better reveal how changes in microbiota composition and microbial load are associated with survival in different environments (50, 53, 54).

Our results demonstrate that although acidic food can independently affect fly feeding behavior and extend life, the presence of microbes is detrimental and a shift in the microbes present in alkaline pH diets might exacerbate this deleterious effect. In addition, many microbes are known to acidify the substrates upon which they grow (55). Consistent with this finding, we observed that flies were exposed to a lifelong cycling of food pH as a result of medium acidification from microbial growth and subsequent, periodic transfers to fresh food. How the additional complexity of a food pH-microbiota interaction affects fly health and physiology will require further study. Although the Poxnmutants we used in this study were on a sucrose-only diet, which minimizes microbial growth, they may be valuable in future work for independently assessing the impact of different microbes on health and survival while eliminating differences in nutrient intake as a potential confound.

Our results emphasize not only the importance of considering fundamental food properties in Drosophila studies but also the value of food intake measurements. We hypothesize that small differences in nutrient intake over a lifetime can profoundly affect health and survival. This is evident not only in our studies on sucrose-only diets where flies were short-lived but also on low-protein diets typically associated with dietary restriction-mediated longevity (5, 26, 27, 33), where reduced feeding might lead to undernutrition. Food additives, including acids or drugs, are often supplemented to fly media without considering the effect they have on pH. In particular, many of these studies use diets containing only sucrose, which has negligible buffering capacity, perhaps confounding results. Given the fundamental nature of feeding behavior to animal health, we suggest that studies of Drosophilaphysiology and metabolism should include rigorous assessments of nutrient ingestion that are comparable to the total consumption and body weight measurements in mammalian model studies.