**Background:** Host-associated bacterial communities can influence host nutrition, development, and stress tolerance, but the extent to which host genetics versus environmental factors shape these communities varies across animal groups. In Daphnia, a well-established model in ecology and evolutionary biology, previous studies have reported conflicting results regarding the role of host genotype in shaping bacterial communities. This study aimed to determine whether Daphnia galeata clonal lineages harbor genotype-specific bacteria after long-term common garden rearing, and whether bacterial community composition correlates with host genetic distance.
**Methods:** Eight D. galeata genotypes were hatched from resting eggs collected from two sediment layers (1989 and 2009) of a single sediment core from Lake Greifensee, Switzerland. These genotypes represent distinct genetic clusters based on whole-genome sequencing data (41,771 SNPs). After hatching, genotypes were maintained as clonal lineages under identical laboratory conditions for 5 years. For the experiment, four genotypes from 1989 (GR_020, GR_023, GR_024, GR_025) and four from 2009 (GR_052, GR_053, GR_054, GR_055) were moved to 20°C, split into three replicate lines, and maintained simultaneously. Twenty adult Daphnia per replicate were dissected to separate gut and remaining body tissue. DNA was extracted using the Qiagen Blood & Tissue kit, and a nested PCR approach was used to amplify the 16S rRNA gene. Sequencing produced approximately 8.3 million reads (range: 55,804–137,685 per sample). After quality filtering and denoising, 432 ZOTUs (zero-radius OTUs) were retained. Beta diversity was assessed using Weighted Unifrac distance with PERMANOVA (genotype nested within sediment layer, 9999 permutations). Mantel tests tested correlation between bacterial community dissimilarity and host genetic distance. Alpha diversity (ZOTU richness and Shannon Index) was analyzed by ANOVA.
**Key Results:** Beta diversity of both gut and body bacterial communities varied significantly by genotype (gut: F=2.127, R²=0.438, p=0.0079; body: F=1.898, R²=0.41, p=0.006) but not by sediment layer (gut: R²=0.013, p=0.9155; body: R²=0.015, p=0.916). Medium bacterial communities varied by neither genotype nor sediment layer. The PERMANOVA models explained 45.5% of variation for gut and 43.4% for body bacterial communities. There was no significant correlation between host genetic distance and bacterial community dissimilarity (gut: r=−0.21, p=0.889; body: r=−0.26, p=0.935). Hierarchical clustering showed that Daphnia genotypes clustered by sediment layer based on genetic distance, but bacterial communities did not. At the class level, Betaproteobacteria dominated Daphnia guts (65.9±15.6%) and bodies (56.6±7.8%), while Flavobacteriia was more abundant in the medium (22.4±7.3%) than in guts (9.8±4.2%). Alphaproteobacteria was significantly more abundant in guts of 2009 genotypes (9.01±9.8%) compared to 1989 (3.89±2.35%), but this was driven by a single genotype (GR055). ZOTU richness varied significantly by genotype (p=0.00045) and sample type (p=0.03049) but not by sediment layer (p=0.706). The Shannon Index varied only by sample type (p<0.00001), being higher in medium than in gut or body.
**Clinical Implications:** While this study does not have direct clinical applications, it contributes to the fundamental understanding of host-microbe interactions in a well-established ecological model system. The finding that host genotype influences bacterial community composition but is not correlated with genetic distance suggests that stochastic processes, interspecies microbial interactions, or specific dispersal abilities may be more important than host genetic divergence in shaping these communities. This has implications for understanding how host-associated microbiomes evolve and are maintained in natural populations, and highlights the need for functional studies to determine whether genotype-specific bacterial diversity provides adaptive benefits to hosts. The study also demonstrates the importance of long-term common garden experiments to disentangle genetic from environmental effects on microbiome composition.