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disease control

The transformation of insects

A transgenic mosquito and an irradiated mosquito are the new anti-dengue fever weapons

LEO RAMOSThe reduction of the dengue fever-transmitting mosquito population, currently the only way to control the disease, is the objective of two projects developed by Brazilian researchers. One of them works in the city of Piracicaba, São Paulo State, and the other, in the town of Juazeiro, State of Bahia. The two researchers have the same goal: the large-scale production of males of the Aedes aegypti mosquitoes – the transmitters of dengue fever – in a laboratory. The idea is to breed males incapable of reproducing healthy offspring and then to release them into the environment to compete with wild mosquitoes for the females of the species. However, the researchers are resorting to two different strategies to achieve their aim. In São Paulo, the insects are bombarded with gamma rays to make them sterile, while in Bahia, the decision was to breed transgenic mosquitoes (see Pesquisa FAPESP no. 180). These mosquitoes receive a modified gene that produces a fatal protein for the offspring. This protein is the result of the mating with normal females found in the environment. The idea behind these strategies is the mass release of transgenic and irradiated mosquitoes, both incapable of procreating in regions infested with the Aedes aegypti.

Dengue fever is one of the major public health problems in the world. According to the World Health Organization (WHO), 50 million people are infected every year. Of these patients, 550 thousand have to be hospitalized and 20 thousand people die. The continuous release in sufficient numbers of these infertile insects is expected to mitigate the problem, by reducing the native population of the Aedes to a level below what is necessary to transmit the disease. In higher numbers, these mosquitoes will have a competitive advantage over the fertile wild mosquitoes, which will have fewer opportunities to mate and generate offspring. There is no risk in releasing them into the environment because only the females transmit the dengue fever virus.

The more recent of the two projects is the result of a partnership between the Center of Nuclear Energy in Agriculture (Cena), of the University of São Paulo (USP), in Piracicaba, and Bioagri, a private group that owns analyses laboratories. Professor Valter Arthur, from Cena’s Department of Radiobiology and Environment, says that the idea for the project came up some four months ago, during a conversation with Márcio Adriani Gava, technical director of Bioagri. “He contacted me because he wanted to enroll in a doctoral program under my supervision,” he says. “I knew that the Bioagri laboratory was raising mosquitoes to test the efficiency of insecticides and we have worked with insect irradiation for more than 30 years at Cena. So I proposed setting up a partnership.”

The intention was to benefit from the experience and the premises of Bioagri’s facility- located in the municipal region of Charqueada, 20 kilometers from Piracicaba – and Cena’s mosquito breeding expertise and equipment. “We have been raising insects for fifteen years for manufacturers of insecticides and larvicides to test the efficacy of their products,” says a Gava. “In addition to raising the Aedes aegypti, we also raise the Culex quinquefaciatus and the Anopheles aquasalis.” The facility in Charqueada breeds an average of three thousand individual insects of each species every month.

Cena, in turn, has had gamma ray irradiators since 1968. This lead-coated, cylinder-shaped machine is a little over two meters high with a diameter of approximately 80 centimeters. The inside contains cobalt-60 pellets that emit gamma rays inside an internal chamber measuring 17 by 13 centimeters, where samples of mosquitoes, fruit or seeds are placed. At Cena, the equipment is used for sterilizing insects and for conducting research into food preservation and disinfection, quarantine treatment of farm product pests, and seeds to increase the yield.

068-071_Mosquitos_199The process that leads to the sterilization of the Aedes begins at Bioagri. Mosquito populations are maintained in cages in the nurseries for mating and egg-laying purposes. To this end, the cages are equipped with small water containers and a strip of paper is placed on the rim. This is where the mosquitoes lay their eggs. Three times a week the egg-covered papers are removed and placed on a tray with water; the tray goes into another cage. After three or four days, the eggs hatch, and develop into larvae. After eight days on average, the larvae develop into pupae; the pupae are collected and separated according to their gender. This is done by putting them inside a device that consists of two parallel acrylic plates. As the female pupae are bigger, they are unable to fall through the space between the two plates. The male pupae are placed inside a container with water, which is then placed into Cena’s irradiator. Then the gamma rays are irradiated on these insects.

Ideal dose
According to Arthur, the ideal situation would be to irradiate the adults, whose reproductive and other organs are totally formed, to thus reduce the radiation effects. But it would be too difficult and complicated to place five or ten thousand live insects into a small container. This is why the scientists chose to use the pupae – the pupa is the phase immediately prior to the adult phase. “At the beginning of the project, the objective was to establish the ideal radiation dose,” says Arthur. “The amount of energy would have to be weak enough to keep the insects alive and strong enough to provoke changes in their biological system, to make them infertile. In addition, the sterile male would have to maintain the same characteristics of the wild mosquitoes, to be able to compete for the females on equal terms. The male would have to copulate with the female and the female would have to lay eggs that wouldn’t hatch.”

To this end, the researchers tested radiation doses of 10, 20, 30, 40, 100 grays (Gy) and so on, up to 150 grays. A gray is a unit of the International Measuring System; it represents a quantity of absorbed radiation (or dose) per unit of mass. In the Cena experiment, an average of 300 to 500 pupae were used for each dosing. These insects were then placed back into the cages to develop into infertile adult males. “We found that the ideal amount of energy, which would be more efficient for our objectives, was 30 Gy,” says Arthur. “This is a relatively small dose. For comparative purposes, approximately 5 thousand Gy will kill a butterfly.”

Arthur makes a point of clarifying that the irradiated insects, fruits, or other products do not pose any risk of contaminating people’s health or the environment. “Many people believe that irradiated material is the same as radioactive or contaminated material,” he says. “But there is no such relationship. The irradiated material receives the energy, which interacts with the matter and then dissipates. The material is not contaminated by the radioactive material of the cobalt-60 pellets. The same effect is produced on a person who goes through an X-ray. The person gets the radiation, but is not contaminated.”

At Bioagri, cages with cloth screens are used to breed the mosquitoes

LEO RAMOSAt Bioagri, cages with cloth screens are used to breed the mosquitoesLEO RAMOS

The transgenic mosquitoes used in the project that is being carried out in Juazeiro do not pose any risk to the environment nor to the town’s population. This is guaranteed by the researchers involved in the project and by the National Biosafety Technical Commission (CTNBio), which approved the experiment in December 2010. The research project is being conducted via a partnership between the Institute of Biomedical Sciences (ICB) of the University of São Paulo (USP), the Biofábrica Moscamed organization and the Oxford Insect Technologies (Oxitec) company, an English concern. This research project is at a more advanced stage than the project that is being conducted in Piracicaba, as the genetically modified Aedes aegypti have already been released into the environment.

Growing with antibiotics
According to retired USP professor Aldo Malavasi, founder and director of Moscamed, where the insects are raised, approximately 15 million transgenic male mosquitoes of the OX513A strain, developed by Oxitec, were released into the environment between February 2011 and July 2012. “The mosquitoes were released in the districts of Itaberaba and Mandacaru of Juazeiro,” he says. “Three other regions have recently joined the project, to confirm the possibility of eliminating the Aedes in these places.” So far, the results have been encouraging. “The mosquito population has decreased by 80% to 90%,” says biologist Margareth Capurro, a professor at ICB, who is coordinating the project. “This is less than the level necessary to transmit the dengue fever virus.”

The male mosquitoes developed by Oxitec receive a gene that produces the lethal protein. To prevent the transgenic males from dying when they are still in the pupae phase, the mosquitoes grow and develop in contact with the antibiotic tetracycline. Without this antidote, which represses the synthesis of the mortal substance, no mosquitoes would survive to be released into the environment. Once they have been released, they copulate with wild females and the offspring of this mating inherit the lethal protein. “As tetracycline is not found in nature, the offspring die when they are still in the larvae or pupae phase,” Margareth explains. “This is why the mosquito population will drop.”

The initial encouraging results of the transgenic mosquito will lead the project into the next steps. According to Malavasi, the next step will be to test the transgenic mosquitoes in a medium-sized city. “Jacobina was the city of choice, in common agreement with the Health Bureau of the State of Bahia, which will provide the funding. Jacobina, in the northwest of the state, has 80 thousand inhabitants and a very high incidence of dengue fever,” he says. “The new production facility, which was built with funds provided by the State Bureau of Science, Technology and Innovation, was inaugurated on July 7. We will breed 4 million males per week, which is enough to reduce the city’s Aedes population. This major experiment is still waiting for the approval of CTNBio.

The Piracicaba project is lagging behind. According to Gava, field tests will have to be conducted before the irradiated mosquitoes are released into the environment. “We need to verify how the sterile Aedes will disperse in the environment and how competitive it will be with the wild strain,” he adds. The project is currently being developed by Cena and Bioagri only, with no funds from other institutions. “We estimate that we will need R$ 500 thousand to make the project feasible, which entails building a laboratory for the large-scale production of the Aedes,” says Arthur.

Although the Piracicaba and Juazeiro projects are using different technologies, they can complement each other to result in major advances in the control of the Aedes and, consequently, of dengue fever. Another advantage of this kind of control is that less chemical products, insecticides and larvicides need to be used, which results in less damage to human health and to the environment. Similar studies are being conducted all over the world. According to Arthur, the sterilization technique used on the Aedes is a pioneering technique in Brazil, but several projects of this kind conducted in other countries have already been published in scientific journals, showing the efficiency of this control method. The transgenic mosquitoes produced by Oxitec have already been tested in other countries. For example, in 2010, three million genetically modified males were released in the Caribbean’s Cayman Islands. The results were similar to the ones in Juazeiro: 80% of the local wild mosquito population was destroyed in the region where the mosquitoes had been released. Similar results were obtained in Malaysia, which led other countries, including India, Thailand, the United States, and Vietnam, to become interested in the experiments.