A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito <em>Aedes aegypti</em>

Abstract

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens 1, 2 . The innate immune system of mosquitoes consists of several lines of defense 3 . Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5,6 . Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7 . The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells.Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8 , and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10 . These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11 . More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11 . While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.Protocol

Larval nutritional stress affects vector immune traits in adult yellow fever mosquito Aedes aegypti (Stegomyia aegypti)

“…Classification features used to identify prohaemocytes, oenocytoids and granulocytes were previously described (Qayum & Telang, 2011). Here, we measured the number of haemocytes collected from females derived from well‐ or malnourished larvae.…”

“…Haemolymph was collected from 1‐day‐old female Ae. aegypti derived from well‐ or malnourished larvae using previously described methodology (Qayum & Telang, 2011). Our method of collecting haemolymph is modified from established perfusion methods (Beerntsen & Christensen, 1990; Hillyer et al , 2004), but we use anticoagulant buffers in place of saline solution and rely on fewer steps as well as smaller injection volumes compared with other established injection‐based methods (Castillo et al , 2006) to obtain slide preparations of haemocytes.…”

“…Each slide containing haemocytes was prepared using Hema 3 (Fisher Scientific, Pittsburgh, PA, U.S.A. ) (Hillyer et al , 2005). Full details of our method to prepare haemocytes have been recently described (Qayum & Telang, 2011). For total haemocyte and granulocyte counts, haemolymph from 10 females per feeding regimen per replicate was collected for haemocyte preparations ( n = 60, 10 females × 3 replicates × 2 food treatments).…”

Use of <em>Galleria mellonella</em> as a Model Organism to Study <em>Legionella pneumophila</em> Infection

“…Previous videos have shown various methods for extracting hemocytes from insects 54,55 however, the method presented here is simple and suitable for immediate processing. Once extracted, hemocytes can be easily quantified, used for immunofluorescence, transmission electron microscopy 36 or flow cytometry 56 or cultured and infected ex vivo 3 allowing the response of the cells to infection to be investigated in detail.…”

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

“…This method proves efficient for isolating sufficient hemolymph volumes containing large numbers of hemocyte cells that can be stored or processed for further experimentation. In addition, hemocyte collection from adult mosquitoes includes a time-consuming step that involves the careful removal of legs, wings and the tip of the abdomen 16 . A similar step is not required in our protocol, thus isolation of hemocytes from slug caterpillars is a simpler and quicker process.…”