Department of Biology, University of Richmond
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Qayum, A. A., Telang, A. A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti. J. Vis. Exp. (51), e2772, doi:10.3791/2772 (2011).
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 pathogens1, 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.
1. Preparation in advance of hemocyte collection
2. Preparation of microinjector in advance of hemocyte collection
3. Preparation of female mosquitoes in advance of hemocyte collection
4. Hemocyte collection
5. Hemocyte fixation and staining
6. Representative Results
Examples of fixed and HEMA3 stained hemocytes collected from an Aedes aegypti adult female are shown in Figure 1A-C. Figure 1A shows a hemocyte that we identified as a prohemocyte based on its small size, spherical-spheroidal shape and high nuclear to cytoplasm ratio (550x magnification) 11. Figure 1B shows a hemocyte classified as an oenocytoid based on its spheroidal shape and high cytoplasm to nuclear ratio (550x magnification). Lastly, Figure 1C shows a granulocyte-type hemocyte (550x magnification). Granulocytes are more granular in nature and tend to be more amoeboid in shape and behavior as they adhere to glass surfaces. Using previously published criteria for hemocyte typing and recovery 11, our collection method yielded a similar number of total hemocytes and we likewise found that granulocytes make up the largest percentage of total hemocytes 11 (Fig 2).
Figure 1. Light microscope images of a representative HEMA3 fixed, stained prohemocyte (A), oenocytoid (B), and granulocyte (C) from an Ae. aegypti adult female. C = cytoplasm, N = nucleus G = granules.
Figure 2. Total counts ± SE of all hemocytes and granulocytes obtained per female mosquito by our hemolymph dilution and collection method.
The hemocyte collection method described here is modified from previously published methods and allows one to isolate clean preparations of hemocytes from Aedes mosquitoes with fewer steps. While our particular interest lies in characterizing hemocyte populations in Aedes mosquitoes, we believe this technique can be applied to other mosquito groups after initial trials are conducted to determine proper injection volumes. Our protocol uses anticoagulant buffers in place of saline solution and yields a population of hemocytes that accurately reflects the numbers in vivo. However, we have not yet determined suitability of our protocol for maintaining isolated hemocytes in culture.
Collection of hemolymph via a cut proboscis has been shown to yield the fewest number of circulating hemocytes and will not be further discussed here. Perfusion-based methods, while easy to conduct, have been criticized for allowing a greater level of contamination from internal tissues and scales 11. Recently, high injection/recovery methods were developed to improve recovery of hemocytes, while reducing levels of contaminates 11, but entails a number of injection and collecting steps. Our protocol obtained the same level of recovered hemocytes and reduced contaminants but with fewer injection and collecting steps, thereby offering a simple yet accurate method to obtain clean preparations of hemocytes. First, the simple step in our protocol of removing legs, wings and the tip of the abdomen in a container separate from where hemolymph is collected results in much cleaner preparations of hemocytes. Second, our method gains accuracy by utilizing glass needles held in a needle holder attached to a positive-displacement microinjector. While diluent can likewise be injected using hand-held needles or other hand-held delivery devices, use of a microinjector increases the accuracy of delivery volume and subsequently results in consistent volume of collected hemolymph. Our trials indicate that combined injection volumes of 9.5 to 10.5 μl (steps 4.1 and 4.4) allowed us to consistently collect 9-10 μl of hemolymph from injected female mosquitoes (unpublished data), thereby providing another measure of consistency in quantifying hemocyte populations. Our method simplifies overall collection by removing the need for other hand-held injection needles and/or glass capillary tubes to collect diluted hemolymph.
No conflicts of interest declared.
Authors would like to thank John Frey and Ben Peterson for mosquito rearing. We also thank Christine Davis and Gary Radice for help with hemocyte micrographs. This research was funded by University of Richmond Arts & Science summer fellowship to A. A. Qayum and faculty grant to A. Telang.
|Name of the reagent||Company||Catalogue number||Comments (optional)|
|Fetal bovine serum||Sigma-Aldrich||F0643|
|Hema3 stain kit||Fisher Scientific||123-869|
|Glass needles (borosilicate with filament)||Sutter Instrument Co.||BF100-78-10||1.0mm O.D. and 0.78mm I.D.|
|Needle puller||Sutter Instrument Co.||Model P-87|
|MicroInjector||Tritech Research, Inc.||MINJ-PD|
|Needle holder||Tritech Research, Inc.||MINJ-4|