A vacation visit in the early 1990s by then-agronomy student José Maurício Simões Bento to a sugarcane farm in Olímpia, in inland São Paulo State, resulted a few years later in the introduction of the first Brazilian commercial pheromone, a chemical substance identified in the female Migdolus fryanus beetle that it uses to attract males for mating. Synthesized in the laboratory, it is then used to combat the insect in sugarcane crops. “I went to visit a university acquaintance who was the farm’s agricultural manager, and the beetles were swarming at that time,” says Bento, who now heads the laboratory at the National Institute of Science and Technology (INCT) of Semiochemicals in Agriculture at the Luiz de Queiroz College of Agriculture, University of São Paulo (Esalq-USP) in Piracicaba. Until then there was no effective control for the pest, which can bore up to five meters down, attack sugarcane roots and cause serious damage to crops. The environmental conditions that Bento encountered on his visit were favorable, because the males only swarm to mate during a one-week period early in the rainy season. To minimize damage to the plants, the farmworkers would walk around the fields collecting the beetles. But one worker had a very special tactic. “Geraldo would sit in a shady spot, place the females in his pocket and wait for the males to come near. When they got close, they were collected and thrown into a bucket,” he recalls.
Back at the Federal University of Viçosa (UFV) in the state of Minas Gerais, where he was a student, Bento began to work on identifying the beetle’s sex pheromone, under the guidance of Professor Terezinha Della Lucia and Professor Evaldo Vilela, pioneers in the study of chemical signals in Brazil. “After extracting the pheromone from the Migdolus, we sent the samples to Brazilian chemist Walter Leal, who at the time was working at Japan’s National Institute of Sericultural and Entomological Science (NISES), and there he was able to identify the natural compound and synthesize it.” To this day, it is used in traps on Brazilian plantations. Since at the time no one disputed the intellectual property of the innovation, Fuji Flavor, the Japanese company that made the identification and synthesis, began to produce the synthetic pheromone. That same company subsequently made a cash donation towards construction of the initial phase of the building that would eventually house the INCT laboratories and facilities at Esalq, the host institution for the chemical ecology research network. The network also includes UFV, the Federal University of Paraná (UFPR) and the Federal University of Alagoas (Ufal). The general coordinator is Professor José Roberto Postali Parra of Esalq. Bento heads a team of 25, including students at the undergraduate, master’s, doctoral and post-doctoral levels, and he welcomes researchers from countries such as Colombia, Ecuador, Spain and the United States to his laboratories.
The two major lines of research at INCT of Semiochemicals involve methods of obtaining new pheromones from insects, and studies on plant volatiles, which include the chemical compounds produced by plants, and their interactions with insects harmful to agriculture and with natural enemies. One of the recent studies on plant volatiles involved genetic manipulation, which resulted in a plant that repels Diaphorina citri, an insect the size of a grain of rice that sucks on the branches of orange trees and is the vector for greening, currently the most devastating disease of citrus plants, including oranges, lemons, tangerines and limes. In Asia it is called Huanglongbing, or HLB, which means yellow dragon disease, because it causes yellowing of leaves. “A repellent compound found in guava trees was inserted into the genes of a lineage of orange trees that is being tested in a greenhouse at the Citriculture Defense Fund (Fundecitrus),” Bento explains. The beginning of the discovery goes back to a visit made by Brazilian researchers to Asian citrus-producing countries in 2004, when it was discovered that the pest had arrived in Brazil. Since it had already spread throughout Asia some time earlier, many producers were planting guava trees interspersed with orange plants in place of citrus.
“In visits to orchards, the researchers noted that in areas where there were guavas, the oranges either did not manifest the disease, or it took longer for them to manifest it,” he says. When he found out about this, Bento—who at the time was already working with plant volatiles—thought of the possibility that some kind of chemical compound was being released and was interfering in the insect’s behavior in that area. “We studied guava volatiles and discovered that there was a compound highly repellent to the citrus pest,” he says. In partnership with the research group at the Valencian Institute for Agricultural Research in Spain, the gene was superexpressed through genetic manipulation of the citrus plants—the compound was present in the plants, but only in small quantities. The genetically manipulated plants are being tested to assess whether they really do repel the insects. Since citrus plants are perennials, the research could take up to a decade to complete. But the potential of the repellent plant, which has already been patented by the group of Brazilian and Spanish researchers and by Fundecitrus, represents a major breakthrough on strategies to combat the pest. In addition to Spain, other institutions abroad are partners in the research, including the University of California at Davis and Pennsylvania State University, both in the United States, the Max Planck Institute in Germany, the University of Neuchatel in Switzerland, and more recently, Wageningen University in the Netherlands. These partnerships involve collaboration and student exchanges for training purposes.
Another research study with promising results, conducted jointly with UFPR, focuses on identifying chemical compounds present in the pheromones secreted by the moth known as the sugarcane borer (Diatraea saccharalis), whose larvae damage the interior of the plant stem. “We’re very close to doing precommercial studies in the field, the stage prior to release to producers,” Bento says. For the coffee berry borer, the principal pest of the coffee plant, the researchers are working on two approaches: pheromones and plant volatile substances. “We’re at a fairly advanced stage in identifying an attractant for this pest, which has no commercial chemical product to combat it.” The only one that was available on the market was withdrawn because of its high toxicity. Volatiles are also being studied for insects that attack corn and tomatoes.
Léo Ramos“The first option for combating pests has always been a chemical one, and now we’re offering an alternative that is viable from an ecological standpoint, because it takes the very compounds that insects and plants produce and uses those to control them through behavior,” Bento says. In addition to the environmental benefits, these compounds also mean cost savings for the producer, as shown in the example of using traps to monitor the citrus fruit borer since the early 2000s. The borer is a moth that lays its eggs in the fruit. When the caterpillar emerges, it penetrates the orange, which rots and drops. Chemical sprays are insufficient for combating the pest. During Bento’s doctoral studies under the guidance of Professor Parra at Esalq, he identified the compound present in the moth pheromone. “I brought the material to Professor Leal in Japan, who was a participant in another study, and he made the identification and synthesized the compound,” he says.
Today the tablet with the synthetic hormone is produced in Japan and sent to Brazil, where the firm Bug Agentes Biológicos, in Piracicaba, handles the task of placing it inside traps called Ferocitrus, which are sold to citrus producers by Coopercitrus, the Rural Growers Cooperative. The traps are made of a sheet of cardboard folded into a triangle, in which the interior of the walls has an adhesive membrane and pheromone-releasing tablets. The attracted male insects get stuck in the device. One trap covers an area of 10 hectares containing some 2,000 to 3,000 plants. If between 0 and 5 insects are caught, it means no control of any kind is needed. Between 6 and 8 means wait another week and check again to see if the number stays the same. If it is more than 9, it is time to resort to chemical control. “The trap is used today by most Brazilian citrus growers, and through this simple measure, there has been a 50% reduction in the use of insecticides, he reports. The average annual crop loss when using this strategy is one fruit per plant. Before, the loss could be as high as 350 fruits per plant—one-third of what an adult plant produces.
Ongoing observation of insect behavior and insect-plant interactions is required in order to obtain pheromones and volatile substances. The cycle begins when the plant releases odors that enable herbivorous insects to locate it in order to feed. As soon as the herbivores attack, the plant automatically emits a response, also in the form of specialized odors and compounds, primarily of the terpene class. These odors attract other insects that are predators or parasitoids of the herbivores. To collect the volatiles, one needs to know the specific hours of the day when they are produced. One study conducted at the Esalq laboratories, for example, assessed whether feeding by the fall armyworm (Spodoptera frugiperda), one of the principal corn pests, during nighttime and daytime periods induces different mixtures of HIPVs (herbivore-induced plant volatiles), and whether the parasitoid wasp Campoletis flavicincta is attracted by these mixtures. “We confirmed that such feeding does indeed induce different compositions of HIPV mixture at different times of the day, mainly by altering the proportions of compounds in the mixture,” says post-doc Maria Fernanda Gomes Peñaflor, a co-author of a paper published in the Journal of Pest Science in March 2012. “This modification in the mixture of HIPVs affects the wasp’s response, so that it is attracted only by the mixture released when the worm feeds at night.”
In the case of sex pheromones, you need to pay attention to mating times, which generally occur at dawn or dusk. “These are the times when the males and females synchronize from a biological standpoint,” Bento says. The odors, which are chemical signals produced by the female, attract only the males of the same species and are produced in small quantities, though they can be detected by the insects at long distances. “The compounds travel through the air on the wind and, when they come into contact with the male insect’s antennae, the sensilla (sensory structures on the antennae) are able to enzymatically recognize the chemical molecules. At that point, an electrical impulse travels from the antenna to the brain of the insect, which is stimulated and responds to the compound. The odor is collected in the laboratory at certain times. Pure, humidified air passes over the females, appropriately housed in structures made of glass, which absorbs the odor they exhale. This odor is retained in a tiny adsorbent polymer (in this process, the molecules and ions are retained on the surface of the material), which is then washed with a solvent—a process known as elution—that removes the chemical compound produced.
The compound is identified using a gas chromatography technique, which separates the different compounds in the sample, based on the physiochemical characteristics of each compound—such as molecular mass or polarity. After separation, the substances can be visualized in the form of peaks on a chromatogram. By combining gas chromatography with other analytical techniques, such as mass spectrometry, the chemical structure of the compounds can be identified. To determine whether the male responds to any of the compounds, antennae removed from the insect are connected to electrodes and analyzed in an instrument called an electroantennogram detector. The signals emitted by the antenna are electrical stimuli to each chemical compound appearing on the chromatogram. When the sensilla of the antenna recognize the chemical molecules of interest—for example, the odor of a sexual partner—an electrical impulse is produced. “The signal is amplified and compared with the molecules shown on the chromatogram, and this enables us to select the compound of interest.”
Researchers are able to identify—in that mixture of chemical compounds—exactly what substance the insect responds to. Once it is identified, they synthesize the substance and proceed through the other stages to produce the trap and manage the insects in the field. Few centers in the world have mastered this technique for identifying chemical compounds. “In some cases, one mixture produces 50 to 100 compounds, and the insect normally responds to just one, two or three substances at most,” he points out.
In the case of volatile substances, the principle for obtaining and identifying the compounds is the same one that applies to pheromones. The plants are placed in a glass chamber to prevent contamination, then clean, humidified air is passed through it, and the gases are collected in tiny polymers. Then the compounds are eluted by solvents and identified. At this stage there are differences between the plants attacked by insects and the ones that were not visited. The compounds produced after the attack can be synthesized and, depending on their purpose, will be used in agriculture.
One behavioral study conducted in the laboratories of the INCT of Semiochemicals at Esalq led to a surprising discovery: that insects are able to detect a drop in atmospheric pressure—which generally precedes rain or a storm. Publication of the news in the journal PLoS One had an international impact and was highlighted in Nature and Science. “We noticed in our experiments that the insects sometimes produced compounds, and other times they didn’t, even when replicating all the conditions,” Bento says. The researchers noted that, on the days when they didn’t respond to stimuli, it got windy or rained heavily a few hours after the experiment. Prior to that time, no research group had studied the effect of atmospheric pressure on the general behavior of insects. “We arrived at the conclusion that when pressure falls, insects ceased their sexual activity or emitted fewer sex pheromones and mated less frequently, because in nature they need to anticipate weather changes and the dangers of storms as a way of reducing mortality and ensuring the perpetuation of the species.” The studies under natural conditions were conducted in the laboratories at Piracicaba and, to prove the hypothesis, a study was done in partnership with Canadian researchers. “There, we manipulated atmospheric pressure in a barometric chamber that they use for bird studies.” The studies were done with three different orders of insects: Lepidoptera (moths), Coleoptera (beetles) and Hemiptera (fleas).
Technological bases for identification, synthesis and use of semiochemicals in agriculture (nº 2008/57701-2); Grant mechanism Thematic project – INCT; Principal investigator José Roberto Postali Parra/USP; Investment R$1,261,009.47 and US$338,475.42 (FAPESP).
PELLEGRINO, A.C. et al. Weather forecasting by insects: modified sexual behaviour in response to atmospheric pressure changes. PLoS One. V. 8, e75004. Oct. 2, 2013.
PEÑAFLOR, M.F.G.V. and BENTO, J.M.S. Herbivore-induced plant volatiles to enhance biological control in agriculture. Neotropical Entomology. V. 42, p. 331-43. Aug. 2013.