Progetti: GRIMOBE (solo in inglese)
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ON THE COMPATIBILITY OF BACILLUS THURINGIENSIS ISRAELENSIS WITH THE ECOSYSTEMS OF THE PLAIN OF MAGADINO FOLLOWING LONG TERM APPLICATION FOR THE CONTROL OF AEDES VEXANS |
1. INTRODUCTION
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The water level of the Lago Maggiore is subject to extreme fluctuations caused by heavy and long lasting precipitation periods and by artificial regulation. Therefore sections of the Plain of Magadino adjacent to the northern lake shore are regularly flooded. A natural reserve, Bolle di Magadino, is located within this area. As a consequence of the frequent flooding, Aedes vexans, the so called flood water mosquito, has become an increasing threat for the local population as well as for the tourist industry. During the past ten years commercial preparations of Bacillus thuringiensis israelensis (Bti) have been used successfully to limit the number of mosquitoes. The Bti products contain along with the bacterial spores mosquitocidal proteins, designated as delta-endotoxins. Upon ingestion, the delta-endotoxins are activated by a multi-stage process, and they kill the mosquito larvae by destruction of their midgut epithelium. As any other insecticide B. thuringiensis products have to fulfil stringent biosafety requirements. Commercial formulations were tested on many organisms in both laboratory and field experiments. In general, no adverse effects could be found against non-target organisms including mammals, birds, fishes, aquatic vertebrates, terrestrial invertebrates or humans. The present knowledge indicates that B. thuringiensis products, applied at normal rates, fulfil all safety requirements. On the other hand, we have learnt from past experience with pesticides that a zero risk does not exist, and that negative interference with ecosystems can never be excluded, particularly after long term application. Therefore permanent safety monitoring for possible long term effects is justified. A study of the pertinent bibliography has been carried out, in order to review potential problems which could arise due to long term application of Bacillus thuringiensis israelensis in the environment. Based on the literature, a project has been designed with the goal to gain a complete set of information of the fate and dynamics of the spores and delta-endotoxin of Bti, which are deliberately released into the periodically flooded ecosystems of the Plain of Magadino. |
2. RESEARCH PLAN
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2.1. Determination of the persistence of the Bti spores in the environment It is well known that the bacterial spores can remain viable in the environment for many years. They are resistant against adverse environmental conditions such as high temperature and drought. If nutrients are present to support growth and multiplication then the spores will germinate. Following proliferation, the vegetative cells will return into the spore stage again as soon as the nutrients are depleted. Under such conditions even an increase in the number of spores could be possible. It will be investigated whether the number of spores and the delta-endotoxin of Bacillus thuringiensis israelensis, which have been released since 1988 decrease, accumulate, or proliferate in the environment. Bti spores, present in the Plain of Magadino, will be determined quantitatively. Methods described in the literature will be adapted and representative samples of soil, water and sediment which have been treated with Bti will be tested. After extraction from the soil or the sediment, the number of spores present in the samples can be determined by classical plate count methods adapted for the genus Bacillus. Spores of Bacillus thuringiensis will be distinguished from other sporulating colonies by optical means. An alternative to the classical enumeration of spores could be hybridisation whereby DNA probes, recognising a portion of a gene of Bti coding for a delta-endotoxin, are produced. Such a method has to be developed for our specific purpose. A further method to selectively recognise B. thuringiensis spores could be an immunological approach. The number of Bti spores determined in soil, water and sediments at the different sites within treated area will be mapped. The samples will be collected over a period of at least two years in order to study the fluctuation in their number.
2.2. Horizontal transfer of the genes coding for the delta-endotoxins It is well known that plasmids are transferred from one organism to another, especially if they are related. All the genes coding for the delta-endotoxins are present on a large, single plasmid of about 75 Md. It is likely that Bacillus thuringiensis israelensis cells are able to exchange plasmids with the other soil borne microflora, for example with cells of the closely related species Bacillus cereus. Transformation experiments with the common soil organism Bacillus cereus will be carried out in the laboratory with plasmids carrying cry genes of Bti. The rate of transformation will be established. Moreover, a technique of detection of B. cereus cells carrying the plasmidic cry gene has to be developed and tested with samples of soil, water or sediment, to demonstrate the possibility of transformation under natural conditions. The rates of plasmid exchange between Bti cells and the soil borne microflora will also be determined.
2.3. Persistence of the delta-endotoxins in the environment Bti produces four delta-endotoxins encoded by the genes cry IVA,B,C,D, as well as the haemolytic factor encoded by the gene cyt A. All 5 proteins constitute the parasporal crystal, which is formed during the sporulation stage of Bti. Several studies have shown, that the parasporal crystals are rapidly degraded in the environment by proteolysis and by indigenous microorganisms. It has also been demonstrated that the parasporal bodies are removed from the feeding zones of the mosquito larvae within hours by adsorption to soil particles. The degree of persistence of the insecticidal components will be determined in the soil, water and sediments of the Plain of Magadino. This will require the development of immunoassays based on the Enzyme-Linked Immunosorbent Assay (ELISA). The feasibility of different methods will be first tested under laboratory conditions using both, the delta-endotoxin complex from sporulated Bti cultures as antigens as well as monoclonal or polyclonal antibodies produced against this toxin. As a next step the delta-endotoxin content of samples from the Plain of Magadino will be determined. The quantitative detection of delta-endotoxin will be mapped. The samples will be taken over a period of at least two years in order to study the fluctuation in the amount of the toxin.
2.4. Development of resistance of the mosquito larvae against Bti Resistance of lepidopteran insects against delta-endotoxins of Bacillus thuringiensis has been demonstrated. Although no resistance against the delta-endotoxin complex of Bacillus thuringiensis israelensis has been detected so far, its long term use against mosquitoes in the Plain of Magadino could reveal first signs of resistance. In fact, there are indications that in 1997 and 1998 i.e. after nine and ten years of consecutive treatment, the product seems to have lost some of its initial activity. Should this be the case we would be confronted with a serious situation because of lack of an alternative. The tests for resistance of the mosquito larvae against Bacillus thuringiensis israelensis will be initiated with a diploma thesis. Based on the results obtained, a series of follow-up investigations could become necessary.
2.5. Non-target organisms In parallel with the above mentioned research, the object is to prepare a monitoring program, specific to the examination of treatment effects (safety), on non-target organisms. Diptera seem to be ideal for this purpose. The applied methodology should provide results that can be compared with studies performed using the same group on agroecosystems, with the scope of identifying suitable long-term bioindicators. This part of the project will be developed at a later stage (early 1999). |
3. TIME SCHEDULE
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The project outlined above will be carried out within a PhD thesis. The investigation of the fate of Bti and its mosquitocidal metabolites in the environment will require a time frame of three and a half to four years. Laboratory work, to become familiar with B. thuringiensis israelensis and with the basic methodology will already be initiated during the last part of the current year. This first part of the project (phase 1) will last approximately 1 year. The pertinent literature has been screened and summarised. The literature study will be presented in a separate report. The potential of resistance development after a ten-year application of Bti will be investigated within a diploma thesis wich has already started and will last approx. 1.5 year (E. Flacio, university of Milano). Regarding the subject of "non target organisms" ideally one should foresee a PhD thesis in collaboration with prof. Bigler (FAL Reckenholz). This argument will be taken into consideration preliminary by the Bolle di Magadino Foundation. This would be put into practice beginning on 1.1.2000. |
4. CONCLUSIONS
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The large scale
production of B. thuringiensis has slowly, but steadily increased over the past
thirty years. This is in contrast to chemical insecticides which have peaked
within a few years then collapsed due problems of safety or resistance. At
present, the annual world-wide production of B. thuringiensis may be in the
range of 3'000 tons whereby the majority of the products are used in the control
of Lepidoptera pests in agriculture and forestry, as well as in the control of
mosquitoes and blackflies which are vector of human infectious diseases.
November 1998 |
