Systematic analysis of nutrient-microbiome interactions and their effects on host phenotypes in <i>Drosophila</i>

Diet and the gut microbiome are interconnected forces shaping host physiology and behavior. However, disentangling their independent and combined effects on phenotypic variation remains a major challenge. Here, we systematically assessed 120 combinations of nutrient and microbiome conditions to evaluate their effects on <i>Drosophila melanogaster</i> phenotypes. By varying dietary yeast-to-sucrose ratios (Y:S) and concentrations, coupled with five defined microbiome configurations, we disentangled the complex diet-microbiome interplays on metabolic, reproductive, and behavioral traits. Our analyses show that abundances of the major gut bacteria <i>Acetobacter pasteurianus</i> and <i>Levilactobacillus brevis</i> are differentially shaped by macronutrient components. <i>A. pasteurianus</i> exhibits a linear positive association with dietary yeast, whereas <i>L. brevis</i> displays a non-linear response driven more strongly by the yeast-sucrose ratio. In the host phenotypes, body protein and triglyceride levels are primarily determined by the nutrient environment, while the microbiome exerts a more notable influence on glucose metabolism. Microbial species exhibited context-dependent effects: <i>A. pasteurianus</i> facilitated protein assimilation and weight gain under high-yeast conditions, whereas co-colonization with <i>L. brevis</i> synergistically amplified fecundity. Critically, microbiome influences on locomotion and sleep varied by dietary Y:S, which helps explain discrepancies in prior studies that did not account for dietary variability when assessing microbiome-behavior relationships. By conceptualizing diet as a multidimensional nutrient environment, our study introduces a framework to better predict how dietary and microbial variation shape host phenotypes. These findings highlight that microbial contributions to host phenotypes are not fixed but emerge from interactions with the nutritional environment, thus advancing a more ecologically realistic framework for studying diet-microbiome relationships.IMPORTANCEThe interplay between diet and the gut microbiome is fundamental to shaping host physiology and behavior; however, their interactions remain poorly understood. Most studies treat diet as a single-dimensional variable (e.g., high-fat or high-sugar), overlooking the complexity of nutrient balance and density. This oversimplification neglects how diet and microbes function as an integrated system. This study addresses this gap by testing 120 different nutrient-microbiome combinations in <i>Drosophila melanogaster</i>, systematically varying yeast and carbohydrate levels and microbiome configurations. Our results show that dietary nutrient composition drives body protein and fat storage, whereas the microbiome plays a notable role in glucose metabolism and buffers against excess fat accumulation. Microbial effects on reproduction, locomotion, and sleep depend on nutrient composition, and our model reveals specific diet-microbiome patterns driving these outcomes. By treating diet as a dynamic, multidimensional factor, we provide a novel, ecologically relevant framework for understanding how diet and microbiome shape host.