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

Chironomids are aquatic insects that undergo a complete metamorphosis of four life stages. Here we studied, for the first time, the microbiota composition of Chironomus circumdatus, a tropical midge species, both from the Mula and Mutha Rivers in Pune, India and as a laboratory-reared culture. We generated a comparative microbial profile of the eggs, larvae and pupae, the three aquatic life stages of C. circumdatus. Non-metric multidimensional scaling analysis (NMDS) demonstrated that the developmental stage had a more prominent effect on the microbiota composition compared to the sampling location. Notably, the microbiota composition of the egg masses from the different sampling points clustered together and differed from laboratory culture larvae. Proteobacteria was the dominant phylum in all the environmental and laboratory-reared egg masses and pupal samples, and in the laboratory-reared larvae, while Fusobacteria was the dominant phylum in the larvae collected from the field environment. The most abundant genera were Cetobacterium, Aeromonas, Dysgonomonas, Vibrio, and Flavobacterium. The ten amplicon sequence variants (ASVs) that most significantly contributed to differences in microbiota composition between the three sampled locations were: Burkholderiaceae (ASVs 04 and 37), C39 (Rhodocyclaceae, ASV 14), Vibrio (ASV 07), Arcobacter (ASV 21), Sphaerotilus (ASV 22), Bacteroidia (ASVs 12 and 28), Flavobacterium (ASV 29), and Gottschalkia (ASV 10). No significant differences were found in the microbial richness (Chao1) or diversity (Shannon H') of the three sampled locations. In contrast, significant differences were found between the microbial richness of the three life stages. Studying the microbiota of this Chironomus species may contribute to a better understanding of the association of C. circumdatus and its microbial inhabitants.

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

Chironomids (Diptera; Chironomidae) are aquatic insects that are abundant in freshwater. We aimed to study the endogenous microbiota composition of Chironomus ramosus larvae that were sampled from the Mutha River and a laboratory culture in India. Furthermore, we performed a metagenomic analysis of the larval microbiome, sampled from the Mutha River. Significant differences were found between the bacterial community composition of C. ramosus larvae that were sampled from the Mutha River and the laboratory culture. A total of 54.7% of the amplicon sequence variants (ASVs) that were identified in the larvae from the Mutha River were unique, compared to only 12.9% of unique ASVs that were identified from the laboratory-reared larvae. The four most abundant phyla across all samples were: Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes, while the nine most abundant genera were: Aeromonas, Alkanindiges, Breznakia, Cetobacterium, Chryseobacterium, Desulfovibrio, Dysgonomonas, Thiothrix, and Vibrio. Moreover, in the metagenomic analysis, we detected bacterial genes and bacterial pathways that demonstrated the ability to degrade different toxic compounds, detoxify metal, and confer resistance to antibiotics and UV radiation, amongst other functions. The results illuminate the fact that there are detoxifying enzymes in the C. ramosus larval microbiome that possibly play a role in protecting the insect in polluted environments.

Copper and chromium exposure affect chironomid larval microbiota composition.

Chironomids are aquatic insects that are known to be pollution tolerant. We have recently demonstrated that endogenous chironomid microbiota protects its host from toxic metals. Following these findings, we hypothesized that under different environmental conditions, a different bacterial consortium will evolve. Our aim was to explore the change in chironomid larval microbiota composition triggered by exposure to toxic copper and hexavalent chromium. Chironomid larvae were collected from the environment and treated in the laboratory with copper, hexavalent chromium, and no metal (control). After six days, the microbial composition of the surviving larvae was examined. We found a significant change in larval microbiota composition between the three treatments and for different copper concentrations. The abundance of specific taxa varied significantly between the treatments. At the genus level, the abundance of some genera (e.g. Yersinia, Acinetobacter) increased in the presence of copper, and some genera (e.g. Yersinia, Dysgonomonas, Delftia, Enterococcus) increased in the presence of hexavalent chromium, compared to the control. The change in the larval microbiota composition was rapid and metal-specific. We suggest that each larva hosts a consortium of bacterial species that can proliferate under a specific environmental change and thus, protect the insect under unstable environmental conditions.

Quorum-sensing signaling by chironomid egg masses' microbiota, affects haemagglutinin/protease (HAP) production by Vibrio cholerae.

Vibrio cholerae, the causative agent of cholera, is commonly isolated, along with other bacterial species, from chironomid insects (Diptera: Chironomidae). Nevertheless, its prevalence in the chironomid egg masses' microbiota is less than 0.5%. V. cholerae secretes haemagglutinin/protease (HAP) that degrades the gelatinous matrix of chironomid egg masses and prevents hatching. Quorum sensing (QS) activates HAP production in response to accumulation of bacterial autoinducers (AIs). Our aim was to define the impact of chironomid microbiota on HAP production by V. cholerae. To study QS signaling, we used V. cholerae bioluminescence reporter strains (QS-proficient O1 El-Tor wild-type and QS-deficient mutants) and different bacterial species that we isolated from chironomid egg masses. These egg mass isolates, as well as a synthetic AI-2, caused an enhancement in lux expression by a V. cholerae QS-deficient mutant. The addition of the egg mass bacterial isolate supernatant to the QS-deficient mutant also enhanced HAP production and egg mass degradation activities. Moreover, the V. cholerae wild-type strain was able to proliferate using egg masses as their sole carbon source, while the QS-deficient was not. The results demonstrate that members of the chironomid bacterial consortium produce external chemical cues that, like AI-2, induce expression of the hapA gene in V. cholerae. Understanding the interactions between V. cholerae and the insects' microbiota may help uncover the interactions between this pathogen and the human gut microbiota.

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

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.

Changes in Microbiota Composition Along the Metamorphosis Developmental Stages of Chironomus transvaalensis.

Chironomids (Diptera; Chironomidae), also known as non-biting midges, are one of the most abundant insects in freshwater habitats. Our aim was to understand whether the metamorphosis developmental stages affect the endogenous microbiota composition of Chironomus transvaalensis. Toward our objective, we analyzed the endogenous microbiota composition of C. transvaalensis' four life stages: egg masses, larvae, pupae, and adults. Significant differences were found between the microbiota compositions of the different developmental stages of this Chironomus species. We observed a decline in bacterial diversity as the insect evolved from egg mass to adult, while the highest richness was observed in the pupal stage. Although there were significant differences between the microbiota compositions of each life stage, a bacterial core, which included 27 Amplicon Sequence Variants (ASVs), was found in all the developmental life stages (in ≥75% of samples). Chironomids are natural reservoirs of Vibrio cholerae and Aeromonas species, and the Vibrio and Aeromonas ASVs were part of the core bacteria. The presence of the ompW gene, which is specific to V. cholerae, confirmed the presence of this species in all the chironomid's life stages. Thus, the results provide important insights about the host-microbe interactions in chironomids with a specific understanding of chironomids-Vibrio-Aeromonas-microbiota interactions.

Seasonal dynamics of Chironomus transvaalensis populations and the microbial community composition of their egg masses.

Chironomids (Diptera; Chironomidae) are the most abundant insects in freshwater environments and are considered natural reservoirs of Vibrio cholerae. We monitored the annual dynamics of chironomid populations along with their microbiota in order to better understand host-microbiota interactions. Chironomus transvaalensis populations peaked biannually in August and May-June. The composition of the endogenous bacterial communities of their egg masses clustered in two groups according to the sampling periods August-November and May-July. Nevertheless, a core bacterial community (43%) was present in all egg-mass samples. The most abundant phyla were: Proteobacteria, Firmicutes, Cyanobacteria and Bacteroidetes. The abundance of several genera (e.g. Rheinheimera and Pseudomonas) was positively correlated with C. transvaalensis population dynamics, while a predator-prey interaction was observed between the relative abundance of Vibrio OTUs and C. transvaalensis population size. Chironomids are known to tolerate toxic and stress conditions, and our results demonstrated that bacterial genera that may protect the insect under these conditions are present in the egg masses. After hatching, the first larval meal is the gelatinous matrix that surrounds the eggs. This meal contains a probiotic consortium that may protect the larva during its metamorphosis. The results provide important insights into the host-microbe interactions of chironomids.

Virulence Traits of Environmental and Clinical Legionella pneumophila Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Genotypes.

Legionella pneumophila causes water-based infections resulting in severe pneumonia. Recently, we showed that different MLVA-8 (multilocus variable-number tandem-repeat analysis using 8 loci) genotypes dominated different sites of a drinking-water distribution system. Each genotype displayed a unique temperature-dependent growth behavior. Here we compared the pathogenicity potentials of different MLVA-8 genotypes of environmental and clinical strains. The virulence traits studied were hemolytic activity and cytotoxicity toward amoebae and macrophages. Clinical strains were significantly more hemolytic than environmental strains, while their cytotoxicity toward amoebae was significantly lower at 30°C. No significant differences were detected between clinical and environmental strains in cytotoxicity toward macrophages. Significant differences in virulence were observed between the environmental genotypes (Gt). Gt15 strains showed a significantly higher hemolytic activity. In contrast, Gt4 and Gt6 strains were more infective toward Acanthamoeba castellanii Moreover, Gt4 strains exhibited increased cytotoxicity toward macrophages and demonstrated a broader temperature range of amoebal lysis than Gt6 and Gt15 strains. Understanding the virulence traits of Legionella genotypes may improve the assessment of public health risks of Legionella in drinking water.IMPORTANCELegionella pneumophila is the causative agent of a severe form of pneumonia. Here we demonstrated that clinical strains were significantly more cytotoxic toward red blood cells than environmental strains, while their cytotoxicity toward macrophages was similar. Genotype 4 (Gt4) strains were highly cytotoxic toward amoebae and macrophages and lysed amoebae in a broader temperature range than to the other studied genotypes. The results can explain the relatively high success of Gt4 in the environment and in clinical samples; thus, Gt4 strains should be considered a main factor for the assessment of public health risks of Legionella in drinking water. Our findings shed light on the ecology, virulence, and pathogenicity potential of different L. pneumophila genotypes, which can be a valuable parameter for future modeling and quantitative microbial risk assessment of Legionella in drinking-water systems.

Release of neurotransmitter induced by Ca2+-uncaging: reexamination of the ca-voltage hypothesis for release.

The primacy of Ca2+ in controlling the amount of released neurotransmitter is well established. However, it is not yet clear what controls the time-course (initiation and termination) of release. Various experiments indicated that the time-course is controlled by membrane potential per se. Consequently the phenomenological Ca-Voltage-Hypothesis (CVH) was formulated. The CVH was later embodied in a molecular level mathematical model, whose key predictions were affirmed experimentally. Nonetheless, the single most important basis for the CVH, namely that depolarization per se is needed to induce physiological phasic release, was challenged by two major experimental findings. (i) Release was induced by Ca2+ alone by means of Ca2+-uncaging. (ii) There was at most a small additional effect when depolarization was applied after release was induced by Ca2+-uncaging. Point (i) was dealt with previously, but additional conclusions are drawn here. Here we concentrate on (ii) and show that the experimental results can be fully accounted for by the molecular level CVH model, with essentially the same parameters.