Name: a protocol for collecting and staining hemocytes from the yellow fever mosquito aedes aegypti
Uploaded: 2011-05-16T12:00:00
Description: Watch this Scientific Journal Video about A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti at JoVE.com

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 &amp; Science summer fellowship to A. A. Qayum and faculty grant to A. Telang.

1. Preparation in advance of hemocyte collection
<ol>
 <li>Prepare a hemolymph diluent solution that consists of 60% Schneider's Medium, 10% fetal bovine serum (FBS), and 30% citrate buffer (anticoagulant; 98 mM NaOH, 186 mM NaCl, 1.7 mM EDTA, 41 mM citric acid, pH 4.5) 11. Make 1.0 mL aliquots that can be stored at -20&deg;C. Use each aliquot only once (see Materials).</li>
 <li>Prepare glass microscope slides (75 x 25 x 1 mm) by scoring a 1 cm diameter circle at one end using a glass-etching tool. ...

<ol>
	<li>Enayati, A., Hemingway, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria+management:+past,+present,+and+future.">Malaria management: past, present, and future.</a> Annu. Rev. Entomol. 55, 569-569 (2010).</li><li>Medlock, J., Luz, P. M., Struchiner, C. J., Galvani, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+transgenic+mosquitoes+on+dengue+virulence+to+humans+and+mosquitoes.">The impact of transgenic mosquitoes on dengue virulence to humans and mosquitoes.</a> The American Naturalist. 174, 565-565 (2009).</li><li>Baton, L. A., Garver, L., Xi, Z., Dimopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+studies+on+the+innate+immunity+of+disease+vectors.">Functional genomics studies on the innate immunity of disease vectors.</a> Insect Sci. 15, 15-15 (2008).</li><li>Thomas, R. E., Wu, W. K., Verleye, D., Rai, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midgut+basal+lamina+thickness+and+dengue-1+virus+dissemination+rates+in+laboratory+strains+of+Aedes+albopictus+(Diptera:+Culicidae).">Midgut basal lamina thickness and dengue-1 virus dissemination rates in laboratory strains of Aedes albopictus (Diptera: Culicidae).</a> J. Med. Entomol. 30, 326-326 (1993).</li><li>Bartholomay, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+infection+responses+in+the+haemocytes+of+the+mosquito,+Aedes+aegypti.">Profiling infection responses in the haemocytes of the mosquito, Aedes aegypti.</a> Insect Mol. Biol. 16, 761-761 (2007).</li><li>Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insect+cellular+immune+response.">The insect cellular immune response.</a> Insect Sci. 15, 1-1 (2008).</li><li>Hillyer, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+mortality+in+immune+challenged+mosquitoes+(Aedes+aegypti)+correlates+with+a+decrease+in+haemocyte+numbers.">Age-associated mortality in immune challenged mosquitoes (Aedes aegypti) correlates with a decrease in haemocyte numbers.</a> Cell. Microbiol. 7, 39-39 (2005).</li><li>Chen, C. C., Laurence, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+study+on+humoral+encapsulation+of+microfilariae:+establishment+of+technique+and+description+of+reaction.">In vitro study on humoral encapsulation of microfilariae: establishment of technique and description of reaction.</a> International Journal for Parasitology. 17, 781-781 (1987).</li><li>Beerntsen, B. T., Christensen, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dirofilaria+immitis:+effects+on+hemolymph+polypeptide+synthesis+in+Aedes+aegypti+during+melanotic+encapsulation+reactions+against+Microfilariae.">Dirofilaria immitis: effects on hemolymph polypeptide synthesis in Aedes aegypti during melanotic encapsulation reactions against Microfilariae.</a> Exp. Parasitol. 71, 406-406 (1990).</li><li>Hillyer, J. F., Schmidt, S. L., Christensen, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibacterial+innate+immune+response+by+the+mosquito+Aedes+aegypti+is+mediated+by+hemocytes+and+independent+of+Gram+type+and+pathogenicity.">The antibacterial innate immune response by the mosquito Aedes aegypti is mediated by hemocytes and independent of Gram type and pathogenicity.</a> Microb. Infect. 6, 448-448 (2004).</li><li>Castillo, J. C., Robertson, A. E., Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+hemocytes+from+the+mosquitoes+Anopheles+gambiae+and+Aedes+aegypti.">Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti.</a> Insect Biochem. Mol. Biol. 36, 891-891 (2006).</li></ol>

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 yield...

<table border="1"><tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 <td>Schneider's Medium</td>
 <td>Sigma-Aldrich</td>
 <td>S0146 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Sigma-Aldrich</td>
 <td>F0643 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hema3 stain kit</td>
 <td>Fisher</td>
 <td>123-869 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass needles 
 (borosilicate with filament)</td>
 <td>Sutter Instrument</td>
 <td>BF100-78-10</td>
 <td>1.0mm O.D. and 0.78mm I.D. </td>
 </tr>
 <tr>
 <td>Needle puller</td>
 <td>Sutter Instruments</td>
 <td>Model P-87</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MicroInjector</td>
 <td>Tritech Research</td>
 <td>MINJ-PD</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Needle holder</td>
 <td>Tritech Research</td>
 <td>MINJ-4</td>
 <td>&nbsp;</td>
 </tr>
</tbody> 
</table>

a protocol for collecting and staining hemocytes from the yellow fever mosquito aedes aegypti

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.

Watch this Scientific Journal Video about A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti at JoVE.com

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

Research

JoVE Journal

Immunology and Infection

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A Simple Chelex protokol til DNA-ekstraktion fra<em&gt; Anopheles</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Anvendelse af kapsler til negativ farvning af virale prøver inden for biokonduktion

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

Ekstraktion af Hemocytes fra Drosophila melanogaster larver for mikrobiel infektion og analyse

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Co farvning blodkar og Nerve fibre i fedtvæv

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

In Vitro ELISA test til vurdering af rabiesvaccine potens

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

Vektor kompetence analyser på Aedes aegypti Myg ved hjælp af Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Udbredelse af microsporidian parasit Edhazardia aedis i Aedes aegypti Myg

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Fodring og kvantificering af animalsk blod og kunstige måltider i Aedes aegypti Myg

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...