Wanted: Not Dead, But Alive - And Out Cold

Insects come in a variety of shapes and colors that dazzle our eyes. But there’s one insect that you almost always hear before you see it (if you see it at all), and chances are the sound of it coming is not music to your ears: mosquitoes! The only place most of us want to see a mosquito is crushed between our hands or flattened against a wall, and as a result we rarely get to examine these little buggers up close and in their preferred three dimensional form (as opposed to the 2-D configuration that is far less annoying). But mosquitoes transmit some of the most important human and animal diseases out there, and based on this one could argue that they deserve a closer look. So we took one … literally.

Diseases transmitted by biting insects represent some of the most significant threats to human health worldwide, and notable among these are the mosquito-borne arboviruses (arthropod-borne viruses) that cause diseases such as malaria, dengue fever, and yellow fever. Many species of mosquitoes feed on animals but won’t pass up a human snack when the opportunity arises. So arboviruses are often zoonotic pathogens, meaning that they can be transmitted from animal hosts to humans by our buzzing friends. West Nile virus is a classic and topical example here in North America. The virus can cause illness in humans but also infects a range of wild animals including birds, mammals, and reptiles. With regard to such infectious agents, a goal of NEON sampling is to gain insights into the environmental factors and mechanisms that influence the abundance of insect vectors and the proportion that are infected with pathogens. Testing for arboviruses in mosquito samples is complicated by the delicate nature of the viral genetic material, called RNA. Most arboviral tests work by detecting viral RNA within infected mosquitoes, but the reliability of these methods can be influenced by the condition of the RNA. In most cases, viral RNA in live, infected mosquitoes will be of the highest quality and will be readily detectable. Once a mosquito has died, however, any viral RNA within it will begin to degrade, and the rate and extent of decay is dependent on how long an infected mosquito has been dead and how it has been stored since buzzing its last buzz. Allowing dead mosquito samples to heat up, dry out, or become wet is believed to promote degradation of viral RNA and reduce reliability of test results: a negative result could mean mosquitoes were truly uninfected, or that they were infected but associated viral RNA was too degraded to be detected.

These realities present challenges for NEON as many of our sampling sites will be far from laboratory facilities where mosquitoes can be processed. In light of this we are exploring, as part of our field operations prototyping, the effects of various sample-handling methods on mosquito survival during the field-to-lab transit phase. The objective of these trials is to identify the storage and transportation options that maximize survival of collected mosquitoes and by extension, maximize reliability of our arboviral testing results. Unlike 99% of the general human population, our goal is to keep captured mosquitoes alive as long as possible. To this end we are trialing various sample-handling options and comparing the proportion of mosquitoes that are still alive once samples arrive back at the lab. But how does one count the number of live and dead mosquitoes without the live ones making a break for it? The answer: anesthetize them using a chemical called triethylamine (TEA). Mosquito samples (sometimes more than 500 insects buzzing around in a catch cup roughly the size of a soda can) are placed inside a zip lock bag with a cotton ball moistened with a few drops of TEA. After about 5 minutes the mosquitoes are out cold and can be sorted on a lab bench at a leisurely pace; no need to worry about samples flying away or biting you and transmitting an arboviral surprise. Under a microscope, live mosquitoes can be readily identified by their twitching limbs and the telltale abdominal expansion and contraction of a breathing insect. Using this technique we can safely and accurately count the number of live versus dead mosquitoes and thereby quantify the efficacy of different sample handling options in maximizing mosquito survival during transport. The better we are at getting mosquitoes from the field to the lab alive, the more reliable our arbovirus test results, and the more our data may reveal about environmental influences on key mosquito-borne diseases.