Vibrio cholerae Invasion Dynamics of the Chironomid Host Are Strongly Influenced by Aquatic Cell Density and Can Vary by Strain

Abstract

Cholera has been a human scourge since the early 1800s and remains a global public health challenge, caused by the toxigenic strains of the bacterium Vibrio cholerae. In its aquatic reservoirs, V. cholerae has been shown to live in association with various arthropod hosts, including the chironomids, a diverse insect family commonly found in wet and semiwet habitats. The association between V. cholerae and chironomids may shield the bacterium from environmental stressors and amplify its dissemination. However, the interaction dynamics between V. cholerae and chironomids remain largely unknown. In this study, we developed freshwater microcosms with chironomid larvae to test the effects of cell density and strain on V. cholerae-chironomid interactions. Our results show that chironomid larvae can be exposed to V. cholerae up to a high inoculation dose (10⁹ cells/mL) without observable detrimental effects. Meanwhile, interstrain variability in host invasion, including prevalence, bacterial load, and effects on host survival, was highly cell density-dependent. Microbiome analysis of the chironomid samples by 16S rRNA gene amplicon sequencing revealed a general effect of V. cholerae exposure on microbiome species evenness. Taken together, our results provide novel insights into V. cholerae invasion dynamics of the chironomid larvae with respect to various doses and strains. The findings suggest that aquatic cell density is a crucial driver of V. cholerae invasion success in chironomid larvae and pave the way for future work examining the effects of a broader dose range and environmental variables (e.g., temperature) on V. cholerae-chironomid interactions. IMPORTANCE Vibrio cholerae is the causative agent of cholera, a significant diarrheal disease affecting millions of people worldwide. Increasing evidence suggests that the environmental facets of the V. cholerae life cycle involve symbiotic associations with aquatic arthropods, which may facilitate its environmental persistence and dissemination. However, the dynamics of interactions between V. cholerae and aquatic arthropods remain unexplored. This study capitalized on using freshwater microcosms with chironomid larvae to investigate the effects of bacterial cell density and strain on V. cholerae-chironomid interactions. Our results suggest that aquatic cell density is the primary determinant of V. cholerae invasion success in chironomid larvae, while interstrain variability in invasion outcomes can be observed under specific cell density conditions. We also determined that V. cholerae exposure generally reduces species evenness of the chironomid-associated microbiome. Collectively, these findings provide novel insights into V. cholerae-arthropod interactions using a newly developed experimental host system.

Keywords: Vibrio cholerae; chironomids; host-microbe interactions; microbiome; microcosm.

FIG 1

Survival ratio of chironomid larvae exposed to different V. cholerae strains at different inoculation doses. (A) Illustration of the experiments using microcosms. The chironomid larvae were exposed to V. cholerae in freshwater microcosms at four different cell densities (10⁶, 10⁷, 10⁸, and 10⁹ cells/mL). (B) Fractional survival was monitored and plotted every 12 h for 7 days (n = 20 microcosm wells of single larva per treatment). (C) Hazard ratio (HR) of larvae exposed to individual V. cholerae strains against the control (unexposed larvae). The error bars indicate 95% confidence intervals. HR values were calculated using Cox PH model with Firth’s penalized likelihood, which estimates the effect of treatment relative to the baseline (control). HR ranges from 0 to infinity. An HR of 1 indicates no effect, while an HR of >1 indicates a higher risk of death compared to the control. An HR of <1 means a lower risk, which suggests a survival benefit compared to the control. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 2

Invasion and prevalence of V. cholerae in the chironomid larvae. The proportion of V. cholerae-positive chironomid larvae (prevalence) after exposure to six different strains at different cell densities (10⁶, 10⁷, 10⁸, and 10⁹ cells/mL) was assayed every 24 h for 72 h (n = 16 microcosm wells of single larvae per treatment; the assay was repeated for three times).

FIG 3

V. cholerae CFU in chironomid larvae after exposure. Chironomid larvae were exposed to V. cholerae in freshwater microcosms at four different cell densities (10⁶, 10⁷, 10⁸, and 10⁹ cells/mL). The CFU numbers per larva (log transformed) were assayed every 24 h for 72 h, and those with CFU number larger than 0 were included in the following analysis (n = 20 to 32 microcosm wells of single larvae per treatment).

FIG 4

Temporal dynamics of V. cholerae CFU in the water. Freshwater microcosms containing chironomid larvae and without larvae were inoculated with single V. cholerae strains at four different cell densities (10⁶, 10⁷, 10⁸, and 10⁹ cells/mL). V. cholerae CFU in the water were sampled every 24 h for 72 h. A linear model was fitted for the whole data set. The regression lines generated from the model (red, microcosms with larvae; blue, without larvae) were plotted. The statistical significance of difference between CFU per milliliter in microcosms with and without larvae is shown (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 5

Microbiome diversity and composition in V. cholerae-exposed and control (unexposed) chironomid larvae at 24 h and 48 h, revealed by 16S rRNA gene amplicon sequencing. (A) Simpson index; (B) observed taxa. The estimated marginal mean of each index and its corresponding 95% confidence intervals are shown for each treatment group. The pairwise comparison between alpha diversity index of V. cholerae exposed larvae and control (unexposed) larvae is indicated (*, P < 0.05). Information for other alpha diversity indices can be found in Fig. S2. (C) Relative abundances of bacterial components detected by 16S rRNA gene amplicon sequencing. Each bar represents a biological replicate sample. Genera that account for less than 1% of the total abundance are grouped together and summarized as “Genera < 1% abundance.”