Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Summary

Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

Abstract

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is based on the injection of non-pathogenic particles into insects, as the particles will be detected by the immune system and will induce the production of immune effectors. We focus here on the stimulation of the melanization response in the mosquito Anopheles gambiae. The melanization response results in the encapsulation of foreign particles and parasites with a dark layer of melanin. To stimulate this response, mosquitoes are inoculated with beads in the thoracic cavity using microcapillary glass tubes. Then, after 24 hr, the mosquitoes are dissected to retrieve the beads. The degree of melanization of the bead is measured using image analysis software. Beads do not have the pathogenic effects of parasites, or their capacity to evade or suppress the immune response. These injections are a way to measure immune efficacy and the impact of immune stimulations on other life history traits, such as fecundity or longevity. It is not exactly the same as directly studying host-parasite interactions, but it is an interesting tool to study immunity and its evolutionary ecology.

Introduction

Insects rely on immune responses to protect themselves against parasites and pathogens^1-3 that breach through their cuticle or their midgut epithelium⁴. In mosquitoes, these responses are efficient against bacteria⁵, viruses⁶, filarial nematodes⁷, and malaria parasites^1,8,9. In mosquitoes, a key immune response is the encapsulation of foreign particles with melanin^10-12. This encapsulation may happen in the midgut or in the hemolymph circulating system^10-12. This melanization response is the result of the pro-phenolo....

Protocol

1. Saline Solution for Injection and Dissection

1. Prepare the saline solution by adding NaCl, KCl, and CaCl₂ to distilled water to obtain 1.3 mM NaCl, 0.5 mM KCl, and 0.2 mM CaCl₂ at pH = 6.8.
2. Add 1 ml of 0.1% methyl green solution to 99 milliliters of the saline solution to color the transparent beads. This is the 0.001% methyl green “injection solution".
3. Then, add 5 ml of 0.1% methyl green solution to 45 ml of the saline solution. This 0.01% methyl green “dissection solution” is 10 times more concentrated in methyl green to facilitate the coloration of the beads and their observation during th....

Results

Mosquitoes did not all melanize the beads in the same way, as some beads were less covered with melanin than others (Figure 1). Indeed, some beads remained blue because of a lack of melanization, whereas others were completely dark (Figure 1). The melanization value was standardized by linear interpolation to a value between 0 (which corresponded to a blue and unmelanized bead) and 100 (corresponding to a dark and heavily melanized bead) (Figure 2.......

Discussion

This injection technique is useful to stimulate and study the melanization response in mosquitoes. For example, here we studied the effect of the immune stimuli load.

The critical step in this procedure is to properly inoculate the mosquito. Any excessive damage to the flight muscles or to the mosquito itself may prevent the mosquito from feeding or may kill it before the dissection. A second key step is to keep the mosquitoes on ice long enough to knock them out without killing them. A small .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Microcapillary glass tubes GB120TF-10	science-products.com	GB120TF-10	http://www.science-products.com/Products/CatalogG/Glass/Glass.html
Microcaps Capillary pipette bulb	Drumond	1-000-9000
negatively charged Sephadex CM C-25 beads	Sigma-Aldrich, Steinheim, Germany	C25120 SIGMA	need few to start
Methyl green	Sigma-Aldrich	323829 ALDRICH	need few to start
Software ImageJ	opensource		Version 1.47f7 or later