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Biology

Reversing the game: plants against the sauba ant

Natural defenses, made from substances extracted from vegetal, kill off the leaf-cutting ants and the fungi that they grow to eat

MIGUEL BOYAYANThe leaf cutter ant, Atta sexdens, one of the most damaging to Brazilian agricultureMIGUEL BOYAYAN

In order to control ants, use plants. Researchers at the Federal University of São Carlos (UFSCar) and at the State Paulista University (Unesp) in Rio Claro have discovered substances, extracted from leaves and seeds, which kill off leaf-cutting ants such as the leaf cutter ant (Atta sexdens), an old and persistent pest of Brazilian agriculture. The same compounds were lethal against the fungi that these insects grow and that serve as their food. Tested with success in the laboratory, the products have now begun to be submitted to field tests, and if everything goes well, within a few years they will be available on the agricultural insecticides and pesticides market, whose size amounts around R$ 900 million per year.

Coordinated by João Batista Fernandes of the Chemistry Department of UFSCar, who has dedicated ten years towards the search for a natural insecticide to control the leaf cutter ant, the project began with the study of plants popularly known to be toxic for ants. An example is sesame (Sesamun indicum), which farmers use it in jointly with what they want to protect. Consequently, the researchers verified that some plants are truly avoided by the leak-cutting ants: for example, they generally do not cut the leaves of the castor bean plant.

Having centered on fifteen plants, they concluded that four substances contained in some of them had greater potential: ricinine, extracted from the leaves of the castor bean plant (Ricinus communis) sesamine, from the seed of the sesame; coumarin present in the majority of citrus fruit trees; and fatty acids of various plants. The group verified that ricinine kills the ants, whilst the sesamine, coumarin and the fatty acids act against the fungi. “We found other substances that appeared to have the same degree of efficiency, but which don’t exist in such high concentrations”, says Fernandes.

Prohibited poisons
A herbivorous insect that is dominant in various South American ecosystems, the leaf-cutting ant causing considerable damage. A three-year old adult leaf cutter ants’ nest can house up to five million ants, cover an area of a hundred square meters and consume a ton of fresh vegetal material per year, damaging pasture land, crops (such as sugarcane, and citrus trees) and reforested areas, in particular those with eucalyptus. The damage can reach as high as 20% of the infested area. Today, the combat of this pest is carried out with highly pollutant and toxic insecticides – the base for the commercialization of some of them being compounds such as DDT, Aldrin and BHC, this having been banned since 1992.

In search of more selective and less aggressive insecticides to the environment and to man, the researchers began by studying the behavior of the ant’s nest. Afterwards, in the laboratory, they dealt with the extraction of substances to verify which of them were attractive and which worked as repellents to the insects, in order to then develop appropriate insecticides and fungicides.

Digestion by fungus
Besides, it was very important for the team to understand the dynamics of the life of the leaf-cutter ants. They are social insects and contrary to the common ant, they are not predators of other ants: around fifty million years ago, explains Fernandes, the ants of the Attini lineage evolved towards the growing of fungi, with which they live in symbiosis. They cut vegetal material, which they use as a substrate for the development, within the nest, of the fungus Leucoagaricus gongylophorus. Or that is to say, they don’t eat the pieces of leaf that they bring to the nest, but leave them there to be degraded by the fungus, this being the main food of the queen and the only one for the larvae. The adult ants feed themselves on substances from the sap of plants, and apparently, also from the results after the degradation of the vegetal material by the fungus.

In order to decompose this vegetal material, the fungus secretes a mixture of enzymes (xylanases, amylase, cellulase and pectinase) which transform the polysaccharides present in the leaves into soluble sugars (glucose and xylose, principally), essential nutrients to the ants. “The inhibition of these enzymes would reduce the formation of nutrients and would inhibit or even eliminate the ants’ nest”, explains the pharmacologist Maurício Bacci Júnior, a researcher with the Study of Social Insects Center (CEIS) and a professor at the Biochemistry and Microbiology Department of Unesp in Rio Claro.

Requested patent
Close to four years were spent by the group in the extraction of the active ingredients of the leaves of the castor bean tree and of the sesame seeds, and as well in bio-experiments to prove its insecticide and fungicide action. From each part of the plant – leaf, stalk, bark, flower, seed, fruit and root -, the researchers made four different extracts. Afterwards, they processed the extracts that demonstrated activity, until they obtained pure ricinine and sesamine. The fatty acids, for their part, were removed from the leaves of various vegetables or obtained from the triglycerides present in certain seeds.

The obtaining of the active ingredients was based on a methodology of solvent fractionation: “It is a technique based on the existence of at least two immiscible solvents and on the solubility of the solutes in these solvents”, explains Fernandes. Applied for the first time for obtaining ricinine and sesamine, this technique allowed for the extraction of the active compound in a shorter time, with less cost and a degree of purity much superior in relation to common methodologies such as column chromatography with a solid support column containing silica or albumin.

In the Fernandes’s, both this methodology and the traps that are being developed and the forms of individual or associated application of the compounds, are being described in detail so that they may request patents. The traps are in the form of use of the insecticide with solid support – in general, citrus fruit pulp with a bait for the insects – mixed with the active ingredient and offered at the mouth of the ants’ nest or on the ant trails.

Synthetically manufacturing the active substances with insecticide and fungicide potential, have also shown good results. The tests with compounds based on sesamine, starch, flavanoids, coumarin and derivatives of ricinine, show that they have efficient action against ants and fungi.

“The synthesis of some of these substances is being carried out starting from low cost commercial products and by using simple processes”, sums up Fernandes, avoiding to give more details about the secret to be patented. “In some tests we proved that the action is equal to or better than the natural substances, mainly in combating fungi”, he emphasizes. In a scientific paper published in 2001 in the magazine Pest Management Science, the group detailed out the activity of the synthetically sesamine based compounds on the leaf cutter ant and on the fungus.

Feasible production
With the advances already obtained, the researchers are clearly unveiling the possibility of production on an industrial scale, but as yet they have not closed an agreement with an industrial company for the co-development of the products.

Having proven the efficiency of the new insecticides in the laboratory, the group is beginning environmental tests in March in order to evaluate factors such as temperature variation, humidity or light intensity that may interfere on the biological effect of the compounds. Only after this stage will the products manufactured by the scientists from UFSCar and Unesp be ready to control the leaf-cutting ants.

Within the tribe
In the group, Maurício Bacci Júnior took on the responsibility of the genic study. He analyzed the evolution of the Attini tribe, which has close to two hundred species, of which the most evolved are the leaf cutter ants (fourteen species of the Atta gender) and the quenquens (Acromyrmex gender). From the study, which he concluded this month, he is proposing the existence of a new species of leaf-cutting ant, derived from the Atta sexdens , and he describes genetic variations in other species.

He also identified, on the evolutionary scale of the Atta , the closest parent of thesexdens that have not been designated as a pest though they also eat fresh vegetable material: it is the Atta robusta : “Its occurrence is limited to the states of Rio de Janeiro and Espírito Santo, contrary to the Atta sexdens , which occurs throughout all of the country. In this manner, comparative studies between the two species could identify the characteristics which have turned the Atta sexdens into a pest”.

Bacci analyzed nuclear genes and mitochondria in order to distinguish the ants. For this he selected molecular markers from the sequencing of the DNA. “Among these markers a region of the mitochondrial genome showed itself to be extremely useful.” While two genes permit the establishing of evolutionary relationships between different genders and species, with a chosen region one can differentiate populations of the same species that live in different geographical regions.

Another of his discoveries refers to the fungi. After bringing together eighteen fungi used by different leaf-cutting ants , Bacci studied their characteristics. The comparison between DNA ribosome sequences suggests that they are genetically similar. “These results demonstrate that ants which present differences of habitat, favorite foods and occurrence as agricultural pests, survive by using of identical fungi. Thus, these fungi become the preferred targets for the control of the leaf-cutting ants, since, probably, the control mechanism that reaches one fungus will be efficient to combat the vast majority of them.”

The projects
1. Vegetal Species and Synthetic Natural Products for the Control of Leaf-cutting Ants (nº 00/12538-5); Modality Thematic Project; Coordinator João Batista Fernandes – Natural Products Laboratory of the Federal University of  São Carlos; Investment R$ 919,304.64
2. Study of the Evolution of the Derived Attini and the Isolation of their Symbiotic Fungi (nº 97/13383-0); Modality Young Researcher Program; Coordinator Maurício Bacci Júnior – Social Insects Study Center of the Paulista State University in Rio Claro; Investment R$ 212,334.43

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