Chironomid egg masses as a natural reservoir of Vibrio cholerae non-O1 and non-O139 in freshwater habitats.

Cholera is a diarrheal disease caused by the gram-negative bacterium Vibrio cholerae, and an estimated 120,000 deaths from cholera occur globally every year. The natural reservoir of the bacterium is environmental. A recent report indicated an association between V. cholerae and chironomid egg masses. Chironomids, the "non-biting midges" (Diptera; Chironomidae), are the most widely distributed and frequently the most abundant insects in freshwater. Females attach egg masses, each containing hundreds of eggs encased in a layer of gelatin, to the water's edge where bacteria are abundant and may encounter the nutrient-rich substrate. Here we report the isolation of non-O1 and non-O139 V. cholerae from chironomid egg masses from different freshwater bodies in Israel, India, and Africa. In a yearly survey in Israel, chironomid populations were found to peak biannually, and it seemed that those peaks were followed by subsequent bacterial growth and disappearance during the winter in the Mediterranean region. The bacterial population rose as water temperature surpassed 25 degrees C. Thirty-five different serogroups of V. cholerae were identified among the bacteria isolated from chironomids, demonstrating population heterogeneity. Two strains of V. cholerae O37 and O201 that were isolated from chironomid egg masses in Zanzibar Island were NAG-ST positive. Our findings support the hypothesis that the association found between chironomids and the cholera bacteria is not a rare coincidence, indicating that chironomid egg masses may serve as yet another potential reservoir for V. cholerae.

Nuisance chironomids in waste water stabilization ponds: monitoring and action threshold assessment based on public complaints.

Large populations of non-biting midges (Chironomidae) that emerged from waste water stabilization ponds in central Israel created severe nuisance to nearby residents in 1998. A study was begun in summer 1998 to examine the dynamics and phenology of the population as a basis for a successful control strategy. The extensive waste pond area required the development of efficient, reliable and competent sampling methods. The efficiency of four sampling methods was tested: (1) egg-mass counts, (2) larval counts, (3) adult emergence traps, and (4) sampling adults with yellow sticky traps placed on the shoreline. The latter two methods were significantly correlated with and accurately detected midge outbreaks. Yellow sticky traps were safer, easier and more convenient to employ for large scale monitoring. An action threshold was determined based on public complaints that were correlated with the numbers of midges caught by yellow sticky traps.

The protective nature of Chironomus luridus larval tubes against copper sulfate.

The objective of this study was to determine whether the tubes in which Chironomus larvae dwell protect them against chemical toxicants. A laboratory culture of an Israeli benthic midge, Chironomus luridus, was exposed to copper sulfate. Two conditions were tested in bioassay experiments: larvae within silt tubes and larvae without tubes. The non toxic, anionic, fluorescent dye, fluorescein, was used to examine the effect of sub-lethal copper sulfate concentrations on the permeability of cuticular, gill and gut epithelia of the chironomids. Increased cell permeability, which is the cause of cell damage, was reflected by an increase in fluorescence intensity. Following exposure to copper sulfate, higher fluorescence was found in different body compartments: midgut, hindgut, tracheal gills, fat body and muscles, and the Malpighian tubules. The effect was significantly higher in tube-free larvae when compared to silt tube dwelling larvae. We conclude that in addition to its other functions in feeding, respiration, and anti-predation shelter, the Chironomus luridus tube protects its inhabitant from toxins such as copper sulfate.

Chloramine and copper sulfate as control agents of planktonic larvae of Chironomus luridus in water supply systems.

The latest approach to control of midge larvae in drinking-water supplies is suppression of the planktonic 1st-stage larvae, by using 2 disinfectants, chloramine and copper sulfate. The median lethal concentration for 24-h exposure of the 1st-stage larvae of Chironomus luridus to chloramine and copper sulfate individually was 0.51 and 0.38 mg/liter, respectively. The increase of copper sulfate to 0.5 mg of copper per liter to water containing chloramine (0.5 mg/liter) created a synergistic reaction that resulted in 96% (+/-8% SD) mortality of the planktonic larvae. This treatment may serve as an effective control of 1st-stage larvae in municipal drinking-water supplies.

Shock chloramination: potential treatment for Chironomidae (Diptera) larvae nuisance abatement in water supply systems.

In the early 1990s, infestations of midge larvae (Chironomidae, Chironomus sp.) were discovered in the potable water system of Tel Aviv, Israel. Control measures, such as draining and cleaning tanks, spraying water into the tank's air space, and electrocution traps of midge adults, were either inadequate or ineffective. In this system, monochloramine concentrations of up to 0.75 mg/liter are used routinely as a secondary disinfectant. This chemical was tested in the laboratory as a toxicant of midge larvae. The mortality of 4th instar midge larvae after short exposure to high chloramine concentrations (LC50 values of 32 mg/liter for 75 min) suggested the efficacy of instituting a Shock Chloramination treatment program. Tanks were partially drained until they contained only 20 cm of water and were then temporarily disconnected. Chloramine was added to this water to produce a concentration of approximately 70 mg/liter for 1-2 h. Subsequently, all dead chironomids were flushed out, and the tank was refilled to attain the operational volume of water. A 2nd identical treatment of water in the tank was suggested 7 d later to kill midges from reproductive adults and egg-masses that survived the 1st treatment. This treatment program was tested in commercial covered tanks and gave complete control of these pests for 6-10 wk. These results suggest that this treatment program may effectively prevent midge outbreaks in Israel's drinking water supply system during the height of the summer.

Cloning and expression of the lepidopteran toxin produced by Bacillus thuringiensis var. thuringiensis in Escherichia coli.

The Bacillus thuringiensis var. thuringiensis strain 3A produces a proteinaceous parasporal crystal toxic to larvae of a variety of lepidopteran pests including Spodoptera littoralis (Egyptian cotton leaf worm), Heliothis zeae, H. virescens and Boarmia selenaria. By cloning of individual plasmids of B. thuringiensis in Escherichia coli, we localized a gene coding for the delta-endotoxin on the B. thuringiensis plasmid of about 17 kb designated pTN4. Following partial digestion of the B. thuringiensis plasmid pTN4 and cloning into the E. coli pACYC184 plasmid three clones were isolated in which toxin production was detected. One of these hybrid plasmids pTNG43 carried a 1.7-kb insert that hybridized to the 14-kb BamHI DNA fragments of B. thuringiensis var. thuringiensis strains 3A and berliner 1715. This BamHI DNA fragment of strain berliner 1715 has been shown to contain the gene that codes for the toxic protein of the crystal (Klier et al., 1982). No homologous sequences have been found between pTNG33 and the DNA of B. thuringiensis var. entomocidus strain 24, which exhibited insecticidal activity against S. littoralis similar to that of strain 3A.