The seeds of the copal tree copaíba (Copaifera langsdorffii), which grows to a height of 30 meters on the banks of the Paraná river in the Center-West of São Paulo, in a transition area between he Atlantic Rain Forest and the Cerrado (Wooded Savanna), contain a protein that inhibits the metabolism and growth of the larvae of the cowpea weevil (Callosobruchus maculatus), an insect that has a devastating action on farming. The study, by Sérgio Marangoni and his pupil for a doctorate, José Antônio da Silva, from the Institute of Biology of the State University of Campinas (Unicamp), also opens up prospects for the preparation of natural insecticides that are less toxic.
Also known as a borer, weevil and carpenter, the grain beetle has a life cycle of only 20 to 30 days. Even so, it manages to destroy about half Brazil’s annual crop of cow-pea (Vigna unguiculata), widely consumed in the north and northeast, and, in general terms, causes losses of 30% to 40% of the main grains harvested in the country – in particular corn, wheat, soybeans and rice. The insect attacks both the green pods and the stored grain. The attack begins in the field, with about 2% of the seeds infested, but the main loss takes place during storage. In the whole world, according to the Food and Agriculture Organization (FAO) of the United Nations, the losses it causes represent 10% of the total production of grain. In Brazil, the average loss comes to around 20%, but it some places it can reach 70%.
“The cowpea weevil”, comments Marangoni, “causes a direct reduction in weight and in the quality of the seeds, which are rendered useless both for consumption and for planting. This is a serious problem, as the main source of protein for the more needy populations is precisely the cow-pea.” The devastating action begins already in the field, with an attack on the grains of the cereals. In just one grain of beans, corn or soya, each female lays from 60 to 80 eggs. Then a geometric progression is unleashed, which insecticides are unable to stop. The attack goes on in the storage, goes through processing, and even reaches the industrialized product. That is why cowpea weevil are to be found even in closed packets of macaroni put away in cupboards: the eggs have survived the milling of the wheat and the manufacturing of the pasta and are only going to die in contact with the boiling water with which the food is prepared.
Marangoni and Silva set off from 20 years work in the Protein Chemistry Laboratory at Unicamp. In the course of three years, they tested many plants, until finding that the proteic extract from the seeds of the copal tree (copaíba), used as food for birds, proved to be resistant to the development of the insect’s larvae. The researchers have found several seed inhibitors, amongst them the purple orchid tree (Bauhinia variegata) and the flamboyant (Delonix regia).
Once the trail was found, they both felt encouraged to study the proteins of the seeds of the copal tree (copaíba), and, in 2000, they identified one that inhibits the action of serine protease trypsin, the grain beetle’s digestive enzyme. “In beans, the beetle’s natural food, there is a protein that inhibits the trypsin, but the beetle has now developed a resistance mechanism for it”, says Marangoni. But the protein that the two researchers discovered, called serine protease inhibitor, or TDI, blocks the grain beetle’s digestive process, and so it stops eating and wastes away until it dies, a few days later.
In the laboratory, once the interest for the copal tree (copaíba) had been defined, the seeds were ground. The trypsin inhibiting TDI was taken out of the resulting powder, and was used to produce artificial seeds, which were given to the grain beetle larvae to eat. Feeding on the false seed, the insect began to waste away and die, as the TDI inhibits the action of the proteolytic enzymes (or proteases) in the digestive tube.
The work strengthens the hypothesis that the trypsin inhibitors are connected with defense mechanisms in leguminous plants like beans. The next stage is to transport the gene of the inhibitor from the copal tree (copaíba) to transgenics. Another line is the use of the protein in the manufacturing of natural bait to catch the cowpea weevil. Under the guidance of Marangoni, Silva is studying the complete sequence of the protein with X-ray crystallography, in collaboration with the National Synchrotron Light Laboratory (LNLS), and is drawing up a three-dimensional model of the protein. He has already discovered that it is a small one. Little by little, the modeling is unveiling the mechanisms of the interaction of protein with the digestive enzymes, and is suggesting strategies.
For Silva, there is no concern over effects on human health, since TDI is from the same family of proteins that already exist in beans, with the advantage that this enzymatic inhibitor has proved to be more resistant in evolutionary distinct species. The problem is how to maintain the genetic modification.
“The originality of the work with the copal tree (copaíba) seed lies in the fact of finding in Brazilian flora an efficient agent against a scourge of our farming”, says Marangoni. The challenge of finding in Brazil’s biodiversity a natural product to fight a farm pest of such importance has been overcome and recognized, with this year’s report of the identification of the trypsin inhibitor in the Journal of Protein Chemistry . “At any rate”, he concludes, “a few years of research will still be needed until it is possible to reduce the grain beetle’s voracity.”
Isolation and Biochemical Characterization of Serine Proteinase Inhibitors from Seeds of the Copaifera langsdorffii. Study of Anti-fungicidal Action and Resistance to the Bruchidae Callosobruchus maculatus
Regular line of benefits for research
Sérgio Marangoni – Institute of Biology at Unicamp