Standard ingredient of <i>Drosophila</i> medium reduces transmission and virulence of the gut pathogen <i>Pseudomonas entomophila</i>

In the last 20 years, <i>Pseudomonas entomophila</i> (Pe) has emerged as a model to explore insect immunity to bacterial intestinal pathogens. Laboratory studies have characterized multiple detrimental effects of Pe on <i>Drosophila melanogaster</i>. However, these effects require that the bacteria are ingested in extremely high concentrations of 10¹⁰-10¹¹ CFU per mL (OD₆₀₀ 20-200), questioning the relevance of this pathogen in nature. Here, we tested whether the need for such high doses may be due to protective effects of the antifungal agent methylparaben (Nipagin), a standard ingredient of laboratory <i>Drosophila</i> diets. While significant mortality of flies fed diet containing methylparaben required pathogen concentrations of >10¹⁰ CFU per mL, we could induce mortality with 500,000-fold lower dose when methylparaben was absent. Here, even this small infection dose (10⁵ CFU per mL) led to high bacterial loads (10⁶ CFU per fly) after several days, indicating the ability of Pe to grow and overcome the flies' defenses in the absence of methylparaben. Consistent with these results, we show strong bactericidal properties of methylparaben on Pe <i>in vitro</i>. We also demonstrate that, in the absence of methylparaben, infected flies can easily transmit the pathogen to other adults and to offspring, resulting in high mortality and thus highlighting the potential of Pe as a pathogen of <i>Drosophila</i> in nature. For those reasons, careful consideration should be given to food additives used in standard diets in laboratory research on host-pathogen interactions.IMPORTANCEAccurate characterization of pathogen infections requires appropriate experimental methodologies. Infections of insects with Pe are frequently studied using fruit flies as a model organism, with laboratory cultures typically maintained on artificial media containing various food preservatives. In this study, we show that one commonly used preservative, methylparaben, significantly influences the outcome of oral infections with Pe. We found that minimal infection doses, far below the standards of the field, could still be lethal to flies raised on media without methylparaben. This increased virulence was also associated with increased transmission of the pathogen, both from infected adult flies to their offspring and to uninfected adults. Our findings show how subtle variations in experimental conditions can profoundly affect how we perceive pathogenic threats.