Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti

Castillo, Julio C.; Robertson, A.E.; Strand, Michael R.

doi:10.1016/j.ibmb.2006.08.010

Cited by 166 publications

(273 citation statements)

References 57 publications

(101 reference statements)

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“…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).…”

Section: Representative Resultssupporting

confidence: 70%

“…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).…”

Section: Representative Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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°C.…”

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confidence: 99%

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

Qayum

Telang

2011

JoVE

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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

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Section: Representative Resultssupporting

confidence: 70%

Section: Representative Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Qayum

Telang

2011

JoVE

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A NOVEL TYPE OF HEMOCYTES LOCALIZING MELANIZATION WITH HIGH‐SPREADING BEHAVIOR IN MYTHIMNA SEPARATA

Kato

Yoshida

Miura

et al. 2014

Arch Insect Biochem Physiol

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Lepidopteran larvae show a cellular response to invading foreign substances that are larger than hemocytes, for example, parasitoid eggs or larvae. This response is called hemocyte encapsulation and is often accompanied by phenoloxidase (PO)-catalyzed melanization. In the present study, we artificially transplanted endoparasitoid larvae and small glass fragments into the hemocoel of the common armyworm, Mythimna separata. We observed that the host larva showed a cellular response and that, 2-4 h after transplantation, melanin formation was spatially confined to the surface of the encapsulated substances. We further noted that specific morphological hemocytes surrounded by melanin formation became attached to the surface of the foreign substances. We designated these hemocytes hyperspread cells (HSCs) on the basis of their specific characteristics and circumferential spread. We confirmed the occurrence of prophenoloxidase (PPO)/phenoloxidase (PO) on the periphery of the HSCs and in the substance secreted around the HSCs by using anti-PPO antibody. We were unable to detect PPO-mRNA in HSCs by using in situ hybridization, although we showed that oenocytoids contained PPO-mRNA and PPO protein. We used light microscopy and scanning electron microscopy to discriminate five main types of circulating M. separata hemocytes. We observed that HSCs differed from plasmatocytes, but spread out well. Further, during the encapsulation process, HSCs appeared to provide a localized melanization spot on the surface of foreign invaders.

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Transstadial immune activation in a mosquito: Adults that emerge from infected larvae have stronger antibacterial activity in their hemocoel yet increased susceptibility to malaria infection

Brown

Shapiro

Thompson

et al. 2019

Ecology and Evolution

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Larval and adult mosquitoes mount immune responses against pathogens that invade their hemocoel. Although it has been suggested that a correlation exists between immune processes across insect life stages, the influence that an infection in the hemocoel of a larva has on the immune system of the eclosed adult remains unknown. Here, we used Anopheles gambiae to test whether a larval infection influences the adult response to a subsequent bacterial or malaria parasite infection. We found that for both female and male mosquitoes, a larval infection enhances the efficiency of bacterial clearance following a secondary infection in the hemocoel of adults. The adults that emerge from infected larvae have more hemocytes than adults that emerge from naive or injured larvae, and individual hemocytes have greater phagocytic activity. Furthermore, mRNA abundance of immune genes—such as cecropin A, Lysozyme C1, Stat‐A, and Tep1—is higher in adults that emerge from infected larvae. A larval infection, however, does not have a meaningful effect on the probability that female adults will survive a systemic bacterial infection, and increases the susceptibility of females to Plasmodium yoelii, as measured by oocyst prevalence and intensity in the midgut. Finally, immune proficiency varies by sex; females exhibit increased bacterial killing, have twice as many hemocytes, and more highly express immune genes. Together, these results show that a larval hemocoelic infection induces transstadial immune activation—possibly via transstadial immune priming—but that it confers both costs and benefits to the emerged adults.

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Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti

Cited by 166 publications

References 57 publications

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

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

A NOVEL TYPE OF HEMOCYTES LOCALIZING MELANIZATION WITH HIGH‐SPREADING BEHAVIOR IN MYTHIMNA SEPARATA

Transstadial immune activation in a mosquito: Adults that emerge from infected larvae have stronger antibacterial activity in their hemocoel yet increased susceptibility to malaria infection

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