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ZOOLOGY

Vegetarian vampire

Mite transmits a virus that causes disease in orange trees when it feeds on cell juice

Mites of the genus Brevipalpus, which number over 200 known species, are nearly invisible to the naked eye. They measure only 0.3 millimeter in size and, if placed on a sheet of paper, they would be smaller than the period at the end of this sentence. They generally adapt well to various environmental and climatic conditions—they can withstand harsh winters, for example—but they prefer tropical areas with milder temperatures. They can infest more than a thousand plant species and become a pest when they spread in large numbers in crops of tea or grapes. One of the most harmful effects of these mites is that they spread disease-causing viruses in orchids, passion fruit, coffee and orange crops. Some viruses initially cause spots on leaves, fruit and branches, but they can kill the plant. Estimates indicate that in recent years, orange growers in the state of São Paulo, the world’s largest producer of orange juice, have spent $80 million per year to control Brevipalpus phoenicis, the species that spreads the citrus leprosis virus in orange trees.

In an effort to find more effective ways to minimize such losses, Brazilian agronomist Elliot Kitajima of the University of São Paulo (USP) and German zoologist Gerd Alberti of the University of Greifswald decided to research how the mites acquire this virus and then transmit it to plants. “We now have more accurate information on the steps involved in this mechanism of acquisition and transmission, although many details still need clarification,” says Kitajima, who conducts research in the Department of Phytopathology and Nematology at USP’s Luiz de Queiroz College of Agriculture (ESALQ).

In the case of citrus leprosis, like a vampire that becomes contaminated by sucking the blood of an infected person, the Brevipalpus mite becomes infected with the virus when it consumes the cell contents, or cell juice, of the leaves of a sick orange tree. The virus travels through the mite’s digestive system without reproducing, and it is then passed on when the Brevipalpus feeds on a healthy plant, as shown in the findings presented in a series of papers published in 2014 in the journal Zoologica. “The paper also presents a taxonomic review of Brevipalpus and assesses the economic losses caused by the mite,” Alberti says. “This is probably the most in-depth study of the internal structures of these mites ever conducted,” he says.

Kitajima has studied the viruses transmitted by Brevipalpus mites since the 1970s when he was at the University of Brasília, and he moved to ESALQ in 1995. In the mid-2000s, however, he came to an impasse. “We were able to visualize the virus inside the mite, but we didn’t know enough about its anatomy to pinpoint precisely where in the mite the virus could be found,” he says. In 2006 he decided to contact Alberti, one of the world’s leading authorities on the subject, and Alberti accepted the challenge of researching the internal details of Brevipalpus mites.

It took seven years of intensive, meticulous work. The researchers produced very thin sections of the mites and analyzed them using powerful electron microscopes. They also had to do a three-dimensional reorganization of the anatomical structures—“a veritable tomography,” says Kitajima—to find the exact location of the virus in the organs and tissues. “It’s as if we cut a sausage and observed the spatial distribution of the bits of fat, which would correspond to the viruses,” he notes by way of comparison.

Voyage through the body
Using this strategy, they confirmed that Brevipalpus ingests the virus when it uses its stylet—a needle-like extension of the mouth apparatus that has only now been described in detail in these mites—to pierce the leaves of orange trees. The virus enters the highly branched intestine (cecum) of the mite along with the food, and it lodges between membranes of adjacent epithelial or glandular cells—rather than inside of them, as had been suspected. This observation indicates that the citrus leprosis virus travels through the body of the mite but does not reproduce in its tissues. “We don’t know for sure how the virus exits the cecum to the space between the cells and moves on from there to the stylet canal, through which it is injected along with the mite’s saliva into the cells of healthy leaves,” explains the ESALQ researcher.

Kitajima and Alberti were assisted by the group headed by João Spotti Lopes, an entomologist at ESALQ, as they sought to achieve a better understanding of the mite’s feeding process. Laura Garita, a master’s student under Kitajima, was able to attach onto the back of the mite a fine gold wire connected to an electrical circuit attached to the plant. Whenever the mite fed, the circuit closed and the electrical current was recorded on a computer. This process enabled her to determine the duration of each feeding phase—insertion of the stylet, salivation and suction. The mite takes an average of four hours to acquire the virus when feeding on a contaminated leaf, and about two hours to inoculate the virus into healthy tissue during another meal. Although viruses do not reproduce in Brevipalpus, the mite can carry them for a long time (10 days), even if it does not have access to new virus sources. The researchers also observed that viruses transmitted by Brevipalpus appear to produce only localized lesions, probably because it doesn’t inoculate the virus into the plant’s vascular system.

“These data are critical for completing the epidemiology, in order to understand how the virus is spread in nature and guide more intelligent and effective pest control initiatives,” Kitajima points out. He explains that the viral activity is generally limited, and it spreads slowly. “In orchards, therefore, it would be sufficient to focus control efforts on contaminated and surrounding plants; there is no need to spray insecticide on the entire crop.”

In the collection of articles, Kitajima prepared one that is a review of the biology of Brevipalpus, and another on the mite-virus relationship. Alberti handled the ones that focus on anatomical details and the functioning of the digestive and reproductive systems.

Kitajima now plans to find and characterize other viruses transmitted by Brevipalpus mites, expand research on the taxonomy and evolution of these arachnids, and study potential biological controls (natural predators). “We are talking with growers and sharing our discoveries with them,” he says. “In the case of citrus leprosis, we want to find the best strategies for achieving maximum reduction of orange crop expenditures.”

Projects
1. Management of citrus leprosis (No. 08/52691-9); Grant mechanism Thematic project; Principal investigator Elliot Watanabe Kitajima (ESALQ/USP); Investment R$576,462.69 (FAPESP).
2. Characterization of plant viruses transmitted by the false spider mite Brevipalpus spp. (Tenuipalpidae) and studies on the virus/vector/plant relationship (No. 00/11805-0); Grant mechanism Thematic project; Principal investigator Elliot Watanabe Kitajima (ESALQ/USP); Investment R$362,063.71 (FAPESP).

Collection of scientific articles
ALBERTI, G. & KITAJIMA, E. W. (eds.). Anatomy and fine structure of Brevipalpus mites (Tenuipalpidae) – Economically Important Plant-Virus Vectors. Zoologica. v. 160. p. 1-192. May 2014.

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