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Creating the 'Spitome'
NIAID Laboratory Studies Complex World of Insect Saliva

By Paul Williams

While the buzz around this summer's campfires may be that of lovelorn cicadas, Dr. José Ribeiro knows that it is the insects' bloodthirsty relatives that should receive the most attention from outdoor enthusiasts. As head of the vector biology section of the Laboratory of Malaria and Vector Research (LMVR) at the National Institute of Allergy and Infectious Diseases, Ribeiro studies the ongoing battle between humans and the more than 14,000 species of blood-sucking arthropods — mosquitoes, ticks and other bugs whose bites can spread potentially fatal diseases. His work focuses on the role of arthropod saliva in blocking the body's natural defenses and in aiding the transmission of disease-causing parasites.

A mosquito takes a blood meal.

"Over hundreds of millions of years, blood-feeding insects and ticks have evolved a magic potion that disarms our system for reducing blood flow to a wound," says Ribeiro. "The mission of my lab is to identify the many active substances found in their saliva and to determine how each one affects the body. With this knowledge we can uncover possible targets for new vaccines against bug-borne diseases."

Big Problems in Small Packages

Bloodsucking insects and ticks carry some of the world's most debilitating diseases. For example, malaria, which is spread by female mosquitoes, is responsible for more than 1 million deaths annually, according to the World Health Organization. WHO estimates that more than 40 percent of the global population — mainly those living in tropical areas of Africa, Asia and Latin America — are at risk of contracting the disease.

The vector biology section of the NIAID Laboratory of Malaria and Vector Research includes (from left) Dr. Nanda Gudderra, Dr. Ivo Francischetti, Dr. José Ribeiro and Dr. John Andersen.

In the United States, mosquitoes are responsible for the spread of West Nile virus, among other infections. In 2003, approximately 9,858 human cases of West Nile virus infection were reported to the Centers for Disease Control and Prevention — more than double the number of reported cases in 2002. The cases reported in 2003 spanned 45 states and the District of Columbia, resulting in 262 deaths.

Despite the rise of West Nile, mosquitoes are not the biggest bloodsucking offenders in the U.S. Lyme disease, a non-fatal, rash-inducing affliction spread by ticks, is by far the most common bug-borne illness with nearly 24,000 reported cases in 2002, according to the latest data from the CDC. This figure represents a nearly 30 percent increase from 2001. Ribeiro attributes the rising number of Lyme and other bug-borne diseases to human activity such as the expansion of residential developments into areas where animals used to roam freely.

"Insects and ticks are very adaptable, and we are very good at creating changes in the environment," he says. "For example, the deer population is exploding in and around the suburbs because the wolf, the deer's natural predator, is no longer there. This creates the perfect habitat for ticks and brings the potential for Lyme disease right to our backyards."

Part Syringe, Part Pharmacy

Ribeiro first became interested in studying arthropod saliva more than 20 years ago when he saw a sand fly feeding on his arm. (Sand flies are bloodsuckers that can spread leishmaniasis, a parasitic skin disease that occurs in tropical countries such as Brazil.) As he was looking at the bite's aftermath, he noticed that although the affected skin was red, it was not painful or itchy. From this observation, he realized that his immune system was not causing the redness. Rather, it was something introduced by the sand fly.

This revelation, he says, led to the discovery of maxadilan, the most potent known vasodilator. Vasodilators are chemical compounds that widen human blood vessels, increasing the flow of blood to a particular body part. Sand flies essentially spit maxadilan on human skin to bring blood closer to the surface, hence improving their chances of a hearty blood meal followed by a quick getaway.

In subsequent studies of arthropod saliva, Ribeiro found that while mosquitoes, ticks, sand flies and other bugs share the thirst for blood, they all evolved their feeding habits independently. As a result, each arthropod species has a unique salivary cocktail that enables it to not only maximize the size and speed of its blood feast, but also to drill through human skin without attracting attention.

For example, tick saliva contains at least one anti-clotting agent, one anti-platelet agent and one vasodilator. Together, these substances block the body's ability to stop a wound from bleeding. Ticks also secrete special chemicals that prevent the body from feeling pain at the feeding site.

"If you see a tick feeding on you, it can be very surprising because you do not feel a thing," says Ribeiro. "Ticks can feed on us for days without being noticed because their saliva contains enzymes that destroy our body's pain producers, including bradykinin, ATP, ADP, serotonin and histamine."

Cracking the Genetic Code

So how do you extract spit from a tiny arthropod? According to Ribeiro, it depends on the arthropod. For mosquito saliva, scientists put the insect under a stereoscope (a microscope that combines two different views of the same object to produce a three-dimensional effect), remove its legs and wings, and place its mouthparts in a tube along with mineral oil and a dash of insecticide or serotonin to prompt salivation. A similar process is used for ticks. Sand flies are too small to have their mouthparts manipulated under a stereoscope, so researchers collect their salivary glands by dissection.

With each sample, Ribeiro and his five-person team create a "spitome," a full genetic transcript of the substances found in the saliva. After isolating these substances, the lab then works to identify the role of each one. Because most spitomes contain between 20 and 40 distinct chemical compounds, determining the function of each substance is painstaking work.

"While we can identify the compounds in arthropod saliva rather quickly, the process of finding out how these compounds affect our bodies is quite complex," says Ribeiro. "Because the potions in arthropod saliva are so diverse, we do not know even 20 percent of what these chemicals do to us."

Mining Drugs from Bugs

To determine the function of substances found in a particular spitome, Ribeiro turns to Dr. Jesus Valenzuela, head of the vector molecular biology unit of LMVR. Valenzuela develops experimental DNA vaccines to search for salivary genes that could one day be used to block the transmission of disease-causing parasites from arthropods to humans. While most existing vaccines use genetically engineered proteins to protect against disease, DNA vaccines work by directing the body's cells to produce proteins similar to those introduced by a virus or bacteria. Armed with these proteins, the immune system can recognize and neutralize any subsequent encounters with disease-causing invaders.

The lab's vector molecular biology unit includes (from l) Dr. Jesus Valenzuela, Dr. Luiz Oliveira, Dr. Sreenath Subrahmanyam and Amy Seitz.

Valenzuela and his team test their vaccines by injecting the DNA into laboratory animals and studying their immune responses after an arthropod's bite. So far, this approach has led them to identify and isolate a salivary gene from the sand fly that protects rodents against leishmania major, a common form of the skin disease. This is a particularly important and timely discovery as leishmaniasis is a growing nuisance for U.S. soldiers in sand-fly rich Afghanistan and Iraq — where troops disdainfully refer to the disease as the "Baghdad boil."

Valenzuela says isolating this salivary gene could lead to a leishmaniasis prevention strategy for humans that combines a vaccine against the disease with a helper vaccine that alerts the immune system to a sand fly's presence and kills the disease-carrying parasites in its saliva. In the meantime, he and his team will continue to study the salivary genes of different species of sand fly in hopes of finding vaccine targets against other forms of leishmaniasis. They will also examine tick saliva to identify genes that may help block the transmission of Borrelia burgdorferi, the bacterium in tick spit that causes Lyme disease.

Love Thy Enemy

While sucking blood and carrying disease hasn't won them many human friends, mosquitoes, ticks and other arthropods have proven that they are resilient creatures. They have fed on blood since the time of dinosaurs whose thick, leathery skins were the primary obstacle to taking a meal. When dinosaurs became extinct and mammals rose to prominence, the dinner menu changed significantly and arthropods were forced to develop chemicals against the clot-inducing platelets found in mammal blood. Anything less would have meant extinction.

There is a lot to learn from these bloodsuckers. As Ribeiro pointed out in a 1995 study, Nobel prizes have been awarded to scientists who discovered a few chemical compounds, while ticks and insects have been using Nobel prize-quality compounds to survive for more than 50 million years. "We know that arthropods are winning the war against us because they are still alive and feeding," he says. "Our goal is to unlock the secrets of their evolution and use that information to improve public health."

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