Identification of chironomid species as natural reservoirs of toxigenic Vibrio cholerae strains with pandemic potential

Abstract

Vibrio cholerae causes the fatal cholera diarrhea. Chironomids (Diptera; Chironomidae) are abundant in freshwater aquatic habitats and estuaries and are natural reservoirs of V. cholerae. Until now, only the non-O1/O139 serogroups of V. cholerae were identified in chironomids. Here, we explored whether chironomids are natural reservoirs of V. cholerae O1/O139 serogroups, which are associated with cholera endemics and pandemics. All four life stages of chironomids were sampled from two rivers, and a laboratory culture in Pune, India, and from a pond in Israel. In total, we analyzed 223 chironomid samples. The presence of V. cholerae O1/O139 serogroups was verified using molecular tools. Nine chironomid species were identified; of them, Chironomus circumdatus was the most abundant. The presence of V. cholerae serogroup O1 and the cholera toxin genes were detected in samples from all chironomid species. However, serogroup O139 was detected in only two chironomid species. Besides PCR to detect specific genes, a metagenomic analysis that was performed in three selected C. ramosus larvae, identified a list of virulence genes associated with V. cholerae. The findings provide evidence that chironomids are natural reservoirs of toxigenic V. cholerae O1/O139. Chironomid populations and V. cholerae show biannual peak patterns. A similar pattern is found for cholera epidemics in the Bengal Delta region. Thus, we hypothesize that monitoring chironomids in endemic areas of the disease may provide a novel tool for predicting and preventing cholera epidemics. Moreover, serogroup O139 was detected only in two chironomid species that have a restricted distribution in the Indian subcontinent, possibly explaining why the distribution of the O139 serogroup is limited.

Fig 1. Chironomid species relative abundance (%) in Mula and Mutha Rivers, India.

Fig 2. Detection rates of virulence genes and toxigenic V. cholerae serogroups at all sampling sites.

To increase clarity, specimens of species with two or fewer individuals were not included in the figures and calculations. n, the total number of individuals.

Fig 3. The presence of V. cholerae (ompW), the toxigenic strains (O1, O139), and cholera toxin subunit A (ctxA) in chironomid environmental samples vs. laboratory samples.

The figure presents the prevalence of these genes in the two dominant Chironomus species (C. circumdatus and C. ramosus), identified in India.