Genotype and environmental effects shape the house fly microbiome (Musca domestica)

This study demonstrates that while environmental temperature exerts a stronger influence than host genotype on the house fly microbiome, the specific cool and warm temperature ranges tested do not induce dysbiosis.

The Gist

Imagine a house fly not just as a buzzing nuisance, but as a tiny, walking ecosystem. Inside and on the surface of every fly lives a bustling city of bacteria, known as the microbiome. Think of this microbiome like a personal garden that the fly carries with it. Some of these bacteria are helpful gardeners, while others might be weeds.

This study asks a simple question: What determines what grows in this garden? Is it the fly's own DNA (its "blueprint"), or is it the environment where the fly lives (the "soil and weather")?

Here is the story of what the researchers found, broken down into everyday concepts:

1. The Experiment: A Temperature Test

The scientists set up a controlled experiment with two types of house flies.

• The "Genotype" (The Blueprint): They used two different strains of flies. One strain (let's call them the "Sun-Lovers") naturally prefers hot weather. The other strain (the "Cool-Seekers") is built for colder climates.
• The "Environment" (The Weather): They raised both types of flies in two different temperatures: a cozy warm room (29°C / 84°F) and a chilly cool room (18°C / 64°F).

They wanted to see if the flies' DNA or the temperature they lived in had a bigger impact on their bacterial gardens.

2. The Big Discovery: Weather Wins

The results were clear: The temperature mattered way more than the fly's DNA.

• The Analogy: Imagine you have two different types of seeds (the fly genotypes). If you plant them in a desert (hot) versus a tundra (cold), the plants will look completely different because of the weather, even if the seeds are different.
• The Finding: When the flies were in the warm room, their bacterial gardens changed dramatically compared to when they were in the cool room. The flies' genetic background (whether they were "Sun-Lovers" or "Cool-Seekers") barely made a difference in the grand scheme of things. The environment was the boss.

3. What Happened in the Heat?

When the flies were kept warm, their bacterial gardens became more diverse and more chaotic.

• The Analogy: Think of the cool room as a quiet, orderly library where everyone sits in assigned seats. The warm room, however, turned into a bustling, noisy music festival. There were more types of bacteria, and the mix of bacteria varied wildly from fly to fly.
• The Surprise: Usually, scientists worry that extreme heat "breaks" a system (a state called dysbiosis, like a garden being overrun by weeds). But here, the heat didn't break the garden; it just made it wilder and more varied. The flies were actually thriving, not suffering.

4. The Wild Fly Comparison: Location, Location, Location

To double-check their lab results, the researchers went out into the wild in Texas. They caught flies from two different farms:

• Farm A (Bastrop): Had chickens, goats, and donkeys.
• Farm B (Washington): Had only horses.

Even though all the wild flies had the same genetic background (they were all "Sun-Lovers"), their bacterial gardens were totally different depending on which farm they came from.

• The Analogy: It's like two people living in different neighborhoods. One lives near a bakery and smells like bread; the other lives near a fish market and smells like the ocean. The flies picked up bacteria from the animal poop and manure around them. The flies from the diverse livestock farm had a much richer, more complex bacterial garden than the ones from the horse farm.

5. The Bottom Line

This paper teaches us three main things:

1. Environment is King: For house flies, where they live and the temperature they experience shape their internal bacterial world much more than their own genes do.
2. Heat isn't Always Bad: Moving a warm-adapted animal to a cooler temperature didn't "break" their microbiome. In fact, the warm temperature made their bacterial communities more diverse and interesting.
3. Flies are Messengers: Because flies pick up bacteria from their surroundings so easily, looking at what's inside a fly can tell you a lot about the animals and environment it has been visiting.

In short: If you want to know what's living inside a house fly, don't look at its family tree; look at the temperature of the room and the farm it's been visiting. The environment writes the story, and the fly's genes just provide the paper.

Technical Summary

1. Problem Statement and Research Context

Animal-associated microbiomes are critical determinants of host phenotypes, fitness, and local adaptation. While it is established that both host genotype and environment influence microbiome composition, the relative contributions of these factors vary significantly across taxa.

• The Gap: Most existing research on thermal stress and microbiomes focuses on heat stress in warm-adapted animals. There is a lack of understanding regarding how low temperatures affect the microbiomes of warm-adapted species and whether such shifts cause dysbiosis (microbiome disruption).
• The Specific Question: How do host genotype and temperature interact to shape the bacterial communities of the house fly (Musca domestica), a warm-adapted insect with known genetic variation in thermal tolerance? Does shifting a warm-adapted host to a cooler temperature induce dysbiosis?

2. Methodology

The study employed a dual approach: controlled laboratory experiments and wild population sampling.

A. Experimental Design (Laboratory)

• Subjects: Two distinct house fly genotypes were used, differing in their male-determining chromosomes:
  • $III_M$ (CSkab): Associated with heat tolerance and preference for warmer temperatures (common in lower latitudes).
  • $Y_M$ (IsoCS): Associated with cold tolerance and preference for cooler temperatures (common in higher latitudes).
• Conditions: Both genotypes were reared for one complete generation (egg-to-adult) at two temperatures:
  • Cool: 18°C
  • Warm: 29°C
• Sampling: 26 unmated males per Genotype $\times$ Temperature (G$\times$T) combination were collected within 24 hours of emergence.
• Wild Sampling: 87 wild male house flies were collected from two sites in Texas (Bastrop and Washington Counties) with different livestock environments (diverse livestock vs. horse stable). All wild flies were genotyped (all found to be $III_M$).

B. Molecular and Bioinformatic Analysis

• Sequencing: 16S rRNA gene amplicon sequencing (V3-V4 region) was performed on all samples.
• Processing: Data were processed using QIIME 2 with the DADA2 plugin to generate Amplicon Sequence Variants (ASVs).
• Normalization: Samples were rarefied to standardize sequencing depth (1,628 reads for lab flies; 975 reads for wild flies).
• Statistical Analysis:
  • Alpha Diversity: Faith's Phylogenetic Diversity, Shannon, Pielou's Evenness, Observed Features.
  • Beta Diversity: Unweighted/Weighted UniFrac, Bray-Curtis, Jaccard distances.
  • Tests: ANOVA, PERMANOVA (Adonis), PERMDISP (dispersion), and ANCOM for differential abundance.

3. Key Results

A. Temperature Dominates Genotype in Lab-Raised Flies

• Alpha Diversity: Temperature had a significant effect on Faith's Phylogenetic Diversity ($p < 0.0001$), with flies at 29°C exhibiting higher diversity than those at 18°C. Most other alpha metrics showed no significant difference, though a Genotype $\times$ Temperature (G$\times$T) interaction was detected for Pielou's evenness.
• Beta Diversity: Temperature was the primary driver of community composition differences across all metrics. While genotype had a minor effect on weighted UniFrac and Bray-Curtis, temperature explained significantly more variation than genotype.
• Dispersion: Flies reared at 29°C showed significantly higher inter-individual variability (dispersion) in microbiome composition compared to those at 18°C.
• Taxonomic Shifts:
  • 18°C: Enriched in Gammaproteobacteria, Clostridia, and Bacilli. Specific genera like Leucobacter, Myroides, and Haloplasma were more abundant.
  • 29°C: Enriched in Proteobacteria (specifically Gammaproteobacteria like Ochrobactrum, Pseudomonas, Stenotrophomonas) and Enterococcus.
  • Genotype Specificity: While some genera (e.g., Chryseobacterium, Stenotrophomonas) were consistently more abundant in $Y_M$ flies, the overall higher-order taxonomic response to temperature was genotype-dependent.

B. Environmental Effects in Wild Populations

• Wild flies from Bastrop County (diverse livestock: chickens, goats, donkeys) had significantly higher alpha diversity than those from Washington County (horses only).
• Beta diversity analysis confirmed distinct community structures between the two sites, driven by the presence of livestock-associated taxa (e.g., Fusobacteriota, Desulfobacterota, Bacteroidaceae, Ruminococcaceae) in Bastrop.
• All wild flies possessed the $III_M$ genotype, isolating the environmental effect from genetic variation.

C. Dysbiosis Assessment

• The study tested the hypothesis that low temperature (18°C) causes dysbiosis in warm-adapted flies.
• Finding: Dysbiosis was not detected. Dysbiosis is typically characterized by reduced alpha diversity and increased beta diversity (instability).
• Observation: The 18°C group actually had lower alpha diversity but lower beta diversity (more uniform communities). The 29°C group had higher alpha and beta diversity.
• Conclusion: The shift to 18°C did not disrupt the microbiome; rather, the 29°C environment promoted a more diverse and variable community, suggesting 29°C is within the optimal range and not a stressor causing dysbiosis.

4. Key Contributions

1. Quantification of G vs. E: Demonstrated that in Musca domestica, environmental temperature exerts a stronger influence on microbiome composition than host genotype, contrasting with some findings in Drosophila melanogaster where genotype filtering is stronger.
2. Thermal Stress Mechanism: Provided evidence that shifting a warm-adapted host to a cooler temperature does not induce dysbiosis. Instead, warmer temperatures (within the tested range) increase both the diversity and variability of the microbiome.
3. Genotype-Environment Interaction: Identified that while temperature is the dominant factor, the specific bacterial taxa responding to temperature changes are often genotype-specific (G$\times$E interaction), particularly at the genus level.
4. Ecological Insight: Validated that local agricultural environments (livestock diversity) significantly shape wild house fly microbiomes, with specific taxa serving as markers for animal husbandry practices.

5. Significance

• Evolutionary Biology: The findings suggest that for warm-adapted ectotherms like house flies, environmental plasticity in the microbiome is a dominant feature, potentially facilitating rapid adaptation to varying thermal niches without requiring specific host genetic filtering.
• Vector Control & Public Health: House flies are vectors for human and animal pathogens. Understanding that temperature and local livestock environments drive microbiome composition helps predict the carriage of specific pathogens (e.g., Staphylococcus, Acinetobacter, Escherichia-Shigella) in different agricultural settings.
• Climate Change Resilience: The study indicates that moderate cooling (18°C) does not destabilize the microbiome of warm-adapted insects, whereas warming (29°C) increases diversity. This challenges the assumption that any deviation from a "thermal optimum" causes dysbiosis, suggesting a more nuanced relationship between thermal stress and microbial health.

In summary, this paper establishes that while host genotype plays a role, temperature and local environmental context are the primary architects of the house fly microbiome, and that within the tested range, these environmental shifts do not result in pathological dysbiosis.