Pest also die through the mouth

Through knowledge of the digestion of insects, researchers create genetically modified plants that resist attacks by undesired species and reduce food production losses, without harming the environment

It is not only fish that die through the mouth. Insects do too – and through the very food that they keep on eating. Out of every 10 kilograms of rice that could otherwise be harvested, the insects eat almost three. Every year they devour approximately 11 million tons (or 13%) of Brazilian agricultural production. To limit the losses, farmers use around 20,000 tons of insecticide, leaving residues on the crops, in the soil, in rivers and on the food itself. But it does not have to be like that. Another solution is to learn about the insects’ digestive mechanisms in order to interfere in their feeding habits. It is better ecologically and more economical.

The process that make this approach feasible was developed through studies conducted by the biochemist Walter Ribeiro Terra, of the Chemistry Institute of the University of São Paulo (USP). His discoveries now let plants be developed that are more resistant to attack by grasshoppers, caterpillars, flies, beetles, bugs, and fleas. The principle does away with chemicals that contaminate the environment: the plants themselves, given genes from another species, actively produce agents inhibiting the production of the pests’ digestive enzymes, substances that block the insects’ digestive systems, above all those of all agricultural pests, that thus die of starvation.

The most eloquent example of this approach is the genetically modified sugar cane, developed by the USP’s Luiz de Queiroz College of Agriculture (Esalq), in Piracicaba. To become resistant to one of the most terrible pests affecting this crop, the cane borer (Diatraea saccharalis), the plant is given two genes from two soybean species, which produce the so-called protease inhibitors (enzymes that digest proteins). With a broad field of action, these substances work in a similar manner to antibodies and block the metabolism of herbivorous creatures. This is one reservation: the genes inducing the production of these natural enzymes must come from  distant species because the inhibitors produced naturally by the plants lose efficiency through the adaptation mechanisms perfected by the insects.

In one of the corners of the Molecular Plant Biology laboratory, under the watchful eyes of molecular biologist Márcio de Castro Silva-Filho’s team, samples of the borer resitant cane are growing. The oldest plants get some room outside, in a greenhouse, and they will shortly be given the first larvae that will test their efficiency under more realistic conditions. Silva-Filho believes that the final results will be even better than those already achieved, since in the laboratory the caterpillars can find food all the time and have no predators. To explain what he has achieved so far, Silva-Filho shows two test tubes occupied by the cane borer larvae, part of one of his team’s experiments. One of the tubes is almost entirely filled by the larvae, fed for 26 days on a completely unmodified artificial diet. In the other, significantly fewer larvae have grown and they have reproduced more slowly because they have been fed on a diet containing the soybean inhibitors.

The research into sugar cane with soybean genes is the most advanced and promising of the studies on the plant improvement that Silva-Filho has been undertaking at Esalq. Since 1997, he has been working on the Characterization of the Biochemical, Entomological, and Molecular Interaction between Digestive Proteinase Inhibitors and Insects of the Order Lepidoptera project, with R$ 156,800 funding, plus US$ 121,200 from FAPESP. Similar research had been carried out on tobacco and corn, before tackling sugar cane, but the results were unsatisfactory: the pests proved resistant to the inhibitors tested.

At Esalq, Silva-Filho assembles the genes and tests the new cane jointly with the entomologist José Roberto Postali Parra, who prepares the special diets given to the caterpillars. To set up the experiments and interpret the results, both have been in contact with Terra, recently recognized as one of the Brazilian scientists most cited on the Web of Science, the database of scientific articles organized by the Institute for Scientific Information (ISI) (see Pesquisa Fapesp No. 52). “Through these partnerships, we have are part of a basic field, namely the characterization of insect enzymes and entomological research, using the inhibitors in artificial diets, and then we begin the molecular biology experiments, cloning genes and obtaining genetically modified plants”, explains Silva-Filho.

Together, the specialists are trying to untie one of the knots in this task – the complex interaction mechanisms between insects and plants. They have already discovered that an insect chooses a plant as a host. The process involves the synthesis of enzymes insensitive to the action of the inhibitors produced by the host plant and it only appears intentional, since it results from the simultaneous adaptation mechanisms of the animal sand plants. The researchers have also observed that some insects, such as the fall armyworm (Spodoptera frugiperda) and the tobacco budworm (Heliothis virescens), manage to have various plants as hosts because they are able to alter their enzyme production to suit the food they are eating. And what is more, the new proteases secreted in reaction to the inhibitor are still more efficient at degrading the plant’s proteins.

In the light of these tricks, the results achieved with genetically modified sugar cane can be considered encouraging, even though at first sight they may seem modest. The inhibitors produced by the soybean genes caused a protein shortage that substantially hindered the development of the Diatraea. The time taken in the larval stage increased by 30% and pupal stage by 10%. There was also an increase of 25% in the mortality rate, which will affect the reproduction rates. According to Parra, the total development cycle of the borer increased by 20%, rising from around 60 to 72 days, before reaching adulthood.

“We don’t want to wipe the insects out”, says Silva-Filho. Total extermination would be a very drastic measure, like chemical insecticides, which would swiftly lead to breeding generations that are ever more resistant to human intervention. “Our objective is to control the proliferation of these pests and delay for as long as possible the establishment of resistance, which is already happening with some genetically modified plants”, he says. British researchers at the University of Durham, in the UK, showed in 1987 that this is feasible when they genetically modified a variety of tobacco. They inserted genes from cowpea (Vigna ungiculata) into the plant, which disrupted the digestion and growth of Heliothis virescens tobacco budworm.

Terra even thinks it possible to develop drugs able to interfere in the insects’ digestion mechanisms, which have no effect on other living creatures. Isolating and comparing enzymes – sometimes quite similar to those of other organisms, such as amylase and trypsin, produced by the human pancreas – he and the biochemist Clélia Ferreira observed that some insects – such as those of the order Lepidoptera, which includes butterflies and moths and Coleoptera, such as beetles – have very particular mechanism for secreting the enzymes. The differences between the digestive process of a butterfly and a bedbug, for example, can be greater than those between a fish and man. In quantitative terms, insects produce a number of enzymes similar to the number released by humans – around 14, of which the USP team has characterized 12, in different species.

Knowledge of how many, how, and when the enzymes are formed and act on every corner of the insects’ digestive apparatus has given clear indications of what may or may not work in attacking them. A relevant item of information is in respect of the peculiarities of a tube with very thin and porous walls, the peritrophic membrane, which is similar to the cling film used to keep food in the refrigerator. The food begins to be broken down in this compartment, under the action of enzymes, before reaching the cells where the nutrients are absorbed (see illustration).

Terra’s team observed that the pores of the peritrophic membrane are seven to eight nanometers wide (a nanometer is one billionth of a meter). “Knowing the size of the peritrophic membrane’s pores, we see that some substances cannot have the desired toxic effect if they are too big for the insects to absorb”, explains Terra. This observation explains, for example, why the crystals of the toxin of the bacillus thuringiensis, one of the most important bacteria in controlling the various species of caterpillar, are only effective if their size is reduced, after partial digestion inside the peritrophic membrane. When this partial digestion does not take place – and the enzymes of some insects are unable to break them down – the toxin has no effect at all.

In the 60s, insecticides ruled, and nobody challenged them, when Terra began working in this field. If, at the beginning, few people took the approach of concentrating solely on the biology of insects seriously, now it is quite different. Terra’s work continued silently over this long period, and became more significant as pressure to conserve the environment grew and the search for alternatives to chemicals began. The United States, for example, has just banned the use of a commonly used pesticide, based on chloropicrin, a raw materials also used in Brazil, because it represents a risk to the nervous systems of children.

Nowadays, both the reasons and the difficulties are reasonably clear. In experiments, such as those carried out with sugar cane, the most complicate thing is not identifying the useful genes to make up new plant varieties, but picking the most appropriate proteins to become the target of the compounds produced by the plants. And this certainty can only be achieved through deep knowledge of the insect physiology, which also unveils the durability of the digestive tube of these creatures.

The work on the caterpillars of the sugar-cane borer is going well, but Terra has already said that it is not enough to discover the enzyme blockers for everything to be decided. The insects, as he has showed, have a sophisticate digestive mechanism and they can secrete different enzymes to digest a single nutrient. If one of the enzymes is blocked by some inhibitor, produced by the plant itself or applied by man, the substitute enzymes swing into action, with the same result but with different properties.

This ability to dodge the attackers has not yet been observed with sugar-cane borers. It was clear, however, in the studies of the Heliothis virescens tobacco budworm. The soybean trypsin inhibitor, added by the USP team to its diet, did not work simply because the caterpillar produced new enzymes outside its power. Terra is now trying to cause the caterpillar to express the entire enzyme repertoire, until he can find an effective inhibitor for all of them. “When we adopt a pest control strategy, we have to take account of this sophistication of the insects’ digestive system”, suggests the researcher.

Applied work such as that of the genetically modified sugar cane represents only part of the Insect Biochemical Laboratory’s research, which FAPESP has financed to the tune of approximately R$ 600,000 since 1993. A project underway since 1998, The Digestion of Insects: A Molecular, Cellular, Physiological and Evolutionary Approach, has benefited from R$ 121,600, plus US$ 382,000, and should be completed in October 2001. This time, information is being looked for on the enzyme secretion mechanisms and the molecules’ nature processed by  key digestive enzymes, such as trypsins and glycosides, which act on carbohydrates, family of compounds that includes starch, sugar and cellulose.

Terra takes into account both the need to better understand the more common pests in Brazil and establish comparison standards between species (see box). Terra has shown that the organization of the digestive system depends more on the phylogenetic position than on feeding habits. Or, in other words, the digestive system becomes more sophisticated as insects have a higher place on the evolutionary scale. In practice, the more evolved, the more voracious they get.

It is impressive. While a beetle eats an average of 0.3 time its weight, an Erinyis ello moth larva can eat around 2.4 times its own weight in a day. It is as if a 75-kg person ate  180 kilograms of food a day. “The ability to process a large amount of food allows the insect to grow and reproduce rapidly”, says the researcher. Thus, the more evolved insects can breed several generations a year and leave descendants, ensuring the survival of the species, even with a high mortality rate. “These results can only be achieved with a quite efficient digestive system”, emphasizes Terra. The disadvantage, he reminds us, the gluttons live for a shorter time. A cockroach, which is less evolved, can live for up to five years, while a common fly lives for only six weeks.

The research conducted by USP attests that there is correlation between the evolutionary stage and the complexity of the insects’ digestive systems. With the more primitive, such as grasshoppers (of the order Orthoptera), the young closely resemble the adult insects, and they compete for food. They have a simpler digestive system, concentrating all the digestive enzymes in a single compartment.

In more evolved insects, like moths and butterflies (Lepidoptera) and flies (Diptera), the young, that is to say, the larvae, are very different from the winged adult. At each stage, they live in different environments and they do not compete for food. Their digestion is well compartmentalized, and only the enzymes responsible for the initial breakdown of the food, such as amylase and trypsin, penetrate inside the peritrophic membrane. Other enzymes, that continue processing the food acting on smaller molecules, work in the fluid between the peritrophic membrane and the intestinal cells. There is also a third group, that of the enzymes found on the surface of the cells, that completes the digestion producing compounds that can be readily absorbed.

A comparison of the digestive dynamics of the two groups, the more and the less evolved, has led to important discoveries. This was how Terra noted that the regions responsible for secreting and absorbing water change in such a way, that in the more evolved group, there always be a flow of fluid running against the flow of food. “This circulation allows enzymes to be saved  and they are recycled instead of being excreted”, he comments. In his view, the action of the enzymes limited to specific compartments and the counter flow of fluids increases the efficiency of the digestive system and the good use of the food.

Similar studies, looking for alternatives to the use of insecticides, attest to the soundness of the work of the USP team. This is true of biological control that introduces natural predators into crops being attacked by pests. In the state of São Paulo, the use of the fly Cotesia flavipes in combating the sugar cane borer brought production losses down from 11% in 1980 to 2.5% in 1990. This saved US$ 37.5 million in this period in sugar and alcohol production, according to the State of São Paulo Cane, Sugar and Alcohol Producers Cooperative (Copersucar). But this method, like that of insecticides, has its limitations; the fly only works when the caterpillar is exposed.

Genetically modified plants, on the other hand, promise to attack the insect inside the cane and further reduce losses, which have been unchanged for a decade. For Terra, genetically modified plants are not only more efficient for the economic standpoint. They  also offer less risk of ecological imbalance than insecticides. Recent experiments with soybeans, however, have not left a very positive image of genetically modified plants. There is still resistance, but the USP professor guarantees that, strictly speaking, these plants do not differ to any great degree from grafts, commonly used in farming, such as hybrid corn, nowadays widely used and quite different from the species from which they originated

Genetically modified sugar cane growing at the Esalq, even if all goes as expected, will have to submit to other tests before it reaches commercial planting. One of them is the large-scale test, which requires approval from the National Technical Commission on Biological Safety (CTNBio), the government body that controls planting genetically modified plants in this country. Another challenge will be the substitution of the promoters, regions of the DNA that activate the genes responsible for expressing the inhibitors. The initial test made use of promoters that activated the production of inhibitors in all parts of the plant and at all stages of development. The researchers want to be more specific. “The plant, in order to behave naturally, must produce inhibitors only when it suffers a lesion”, comments Silva-Filho.

One of the reasons why he is participating in the Sugar Cane Genome Project, financed by FAPESP, is precisely this: he wants to find a promoter more appropriate to the his necessities, to avoid excessive gene secretion from interfering in other processes in the plant and in the environment. It is not just a question of improving the process of cane production. It is a priority. He intends to spare Brazil the problems ran into in the United States with large-scale planting of genetically modified corn, which led to the deaths of monarch butterflies that fed on the corn pollen. ‘We are taking into account that the environment is a complex system and the less interference the better”, says Silva-Filho.

From the standpoint of the researchers, the new plants will not cause any harmful effects to other living creatures if they are modified to affect very specific mechanisms in the insects, which are getting to be understood in greater detail every day. “The result depends on the choices made”, emphasizes Terra. In other words, the future depends both on common sense and on knowledge of what and how to modify in living creatures. The studies in progress on the digestive enzyme structures and their expression mechanisms, are finally suggesting a more encouraging prospect in which only the insects will affected in the struggle to produce more food.

The largest group in the animal kingdom

Described since antiquity, when the term entomology was used for the first time by Aristotle, insects represent a class, Insecta, that includes around 70% of all animal species; approximately 1.2 million of them. One way of dealing with this diversity has been to choose species according to their position on the evolutionary tree, since differences are related precisely to their stage in evolution. Terra works with 18 species located at strategic points on the evolutionary tree, which represent the main orders of the class Insecta. The ecological role of each species also played a part in the choice, since of this total, two thirds are agricultural pests – man’s biggest competitors for food. They account for around 10% of the insect species and can devour as much as a half of a crop.

Also joining this assemblage is a species of interest to medical research, since it transmits the protozoa that cause Chagas disease, while the others were included to complete the evolutionary picture. From a broader standpoint, the chosen species fit essentially into two categories, the least and the most evolved. Among the more primitive insects studied, with partial metamorphosis, is the locust Abracris flavolineata of the order Orthoptera, which also includes grasshoppers and 2% of all insect species. Also in this category are the Rhodnius prolixus bugs, a type of insect that transmits Chagas’ disease, and the Dysdercus peruvianus that feeds on cottonseed, of the order Hemiptera, which covers 7% of insect species, to which the cicada also belongs. Less primitive is the Tenebrio molitor beetle, a pest for flours, often used as bait by anglers, and the click beetle  Pyrearinus termitilluminans, that feeds on other insects, both of the vast Coleoptera order, which includes 30% of insects.

Walter Ribeiro Terra, 55 years old, took his degree in Biology at the University of São Paulo in 1968. he completed his doctorate in Biochemistry in 1972 and became a professor in 1977, at the same university. He has been the professor in charge of the Biochemistry Department of USP’s Chemistry Institute since 1990.
Clélia Ferreira, 45 years old, graduated in 1976 in Biology at USP, where she also did a doctorate in a Biochemistry, which she completed in 1982 and became a professor in 1985. She joined the Biochemistry Department of USP’s Chemistry Institute in 1984, where she has been an associate professor since 1994.
Márcio de Castro Silva Filho, 39 years old, is an agronomic engineer, a graduate of the Federal University, where he also took a master’s degree in Genetics and Plant Improvement in 1989. He studied in Belgium, from where he returned in 1994 with a doctorate in the Molecular Biology of Plants from the University of  Louvain. He has been a professor of genetics at USP’s College of Agriculture  (Esalq) since 1994.

The projects
The Digestion of Insects:  A Molecular, Cellular, Physiological and Evolutionary Approach (02/11938-5); Modality: Thematic Project; Coordinators: Walter Ribeiro Terra and Clélia Ferreira; Investments: R$ 121,600 plus US$ 382,000.
2. Biochemical, Entomological and Molecular Characterization of the Interaction between Inhibitors of Digestive Proteinases and Insects of the Order Lepidoptera  (97/04934-3); Modality: Thematic Project; Coordinator: Márcio de Castro Silva Filho; Investments: R$ 156,838.45 plus US$ 121,201.90