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.

Chironomus ramosus Larval Microbiome Composition Provides Evidence for the Presence of Detoxifying Enzymes

“…Recently, Vibrio and Aeromonas were suggested to serve as C. transvaalensis symbionts [ 15 ]. Moreover, toxigenic V. choerae O1 and O139 were previously detected in C. ramosus larvae [ 16 ]. In the current study, we confirmed these findings by identifying Vibrio and Aeromonas in larvae that were sampled both from the laboratory culture and the Mutha River, using both 16S rRNA gene sequencing and metagenomics methods.…”

“…Chironomid larvae were sampled in November 2018 from (i) the Mutha River, Pune, India (18.2901° N, 73.4956° E), and (ii) a laboratory culture which was initially collected from the Mula River, Pune, India (18.5551° N, 73.8618° E), in April 2018, and maintained under laboratory conditions for eight months until it was sampled for our current study. The sampling procedure and the conditions for rearing the laboratory culture were previously described, in detail, by Laviad-Shitrit et al [ 16 ]. In total, 31 larvae that were identified as C. ramosus were selected for further analyses in the current study ( Supplementary Table S1 ).…”

Comparative Microbiota Composition Across Developmental Stages of Natural and Laboratory-Reared Chironomus circumdatus Populations From India

“…C. circumdatus egg masses, larvae, and pupae were collected in November 2018 from plants (Eichhornia crassipes) and riverbank sediments from urban settings surrounding two rivers in Pune, India: (i) the Mula River (18.5551 • N, 73.8618 • E) and (ii) the Mutha River (18.2901 • N, 73.4956 • E) (Laviad-Shitrit et al, 2020). Samples were also collected from (iii) laboratory cultures being reared in the Animal House Facility at the Department of Zoology, SPPU, Pune, India.…”

“…All the samples that were investigated in the current study were taxonomically identified as C. circumdatus by Laviad-Shitrit et al (2020). Each sample was treated individually after collection by washing it five times in 1 ml sterile water to remove the microorganisms that were not tightly connected to the samples.…”

“…Sela et al (2020) compared the bacterial composition of Chironomus transvaalensis egg masses, larvae, pupae, and adults. Recently, Laviad-Shitrit et al (2020) sampled nine chironomid species from the Mula and Mutha Rivers in India and from a waste stabilization pond in Israel and found evidence for the presence of toxigenic O1 and O139 V. cholerae serogroups in these chironomid species.…”