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-phenoloxidase cascade^10-12, and it can lead to the death of the parasites or to their phagocytosis. In adult mosquitoes, where the number of hemocytes cells is limited, melanization is a humoral response, like against plasmodium parasites or filarial nematodes ⁷. In some other insects, it is directly the hemocytes cells that gather around the parasite to melanize them⁷. Besides, melanin is also essential for several other physiological process like egg production and cuticle wounds healing⁷.

The stimulation of immune responses is used as a tool to study insect immunity in several agricultural and public health model systems^13-18. It is used in Anopheles gambiae mosquitoes, the major vector of malaria in Africa, to study host-parasite interactions^14-16,19. These techniques are based on the capacity of insects to detect parasites with their pattern recognition receptors (PRR)². Mosquitoes may also detect other molecules interfering with their biology such as pathogen-associated molecular patterns (PAMPs), or detect their own damaged cells due to the release of collagen and nucleic acids. The mosquito immune cells such as the hemocytes are used for detection^20-23. The main immune signaling pathways are Imd, Toll, JAK/STAT²⁴, and ribonucleic acid interference (RNAi)^25,26. Both Toll and Imd pathways influence the melanization response and interact with the pro-phenoloxidase cascade^10-12.

The standard tool used to stimulate the melanization response is the inoculation of a mosquito with a small bead into the hemolymph of the thoracic cavity. The degree of melanin encapsulation can then be measured¹⁹ after retrieving the bead through the dissection of the mosquito. In most studies, only one bead was injected per mosquito^15,16,27, but injecting more beads is possible in order to study the limits of the melanization response¹⁹. These beads are injected using an injection solution (physiological serum) to limit disturbance of the mosquito physiology and the desiccation of the mosquito^15,16,27. A dye is added to this solution to ease bead selection. It is the same for the dissection solution used to retrieve the bead^15,16,27.

The advantage of inoculating insects with non-pathogenic stimuli is the ability to focus on the direct effect on the immune response. There are no complicating effects due to parasite pathogenicity²⁸, immunosuppression^29-31, or immune evasion^31-34. Besides, the consequences of the stimulations on other life history traits, such as longevity or fecundity, can also be studied. Thus, researchers studying evolutionary ecology may require such tools^2,35,36. For example, immuno-challenged bumblebees have a shortened life span under starvation. Similar negative effects of immune stimulations and deployments have been observed in different invertebrate models, often resulting in a shorter lifespan or less reproductive success^13,27,37. Such studies can be conducted in varying environments^2,4,38. Stimulating immunity is also of interest to those focusing directly on immunopathology^39,40.

This protocol is based on the inoculation of beads with mosquitoes to stimulate the melanization response and directly measure the amount of melanin. This enables quantitative and qualitative study of the melanization response in different experimental settings. Such a tool can be extended to the stimulation of other immune responses, such as the antibacterial response to heat-killed bacteria⁴¹. It can also be conducted in many ecological settings.

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 the dissection.
   NOTE: Filter sterilize the buffer if necessary.

2. Capillary Preparation

1. Heat-pull microcapillary glass tubes to obtain a very fine tip slightly bigger than the smallest beads (φ = 40 µm)⁴².
2. Open and adjust each capillary by breaking the tip with tweezers or by sawing the tip off.

3. Mosquito Rearing

1. Rear A. gambiae at 26 ± 1 °C, 70 ± 5% relative humidity, and a 12:12 hr light:dark cycle. Keep adult females and give them access to a 10% sugar solution.

4. Bead Selections with the Capillary

1. Pour 0.009 g of negatively charged beads (40-120 µm in diameter) in a 5 cm Petri dish and add 5 ml of injection solution 30 min in advance.
   NOTE: The goal is to let the dye color the beads to make bead selection for injection easier. Autoclave the beads if they don't come in a sterile jar.
2. Using a capillary mounted in a capillary pipette bulb, visually select the smallest bead in 0.1 µl of the saline solution under a binocular microscope.
   NOTE: When inoculating several beads simultaneously, select three beads from a total volume of 0.1 µl.

5. Mosquito Handling and Inoculation

1. Using an insect aspirator, place each mosquito in a 50 ml tube.
2. Chill each mosquito briefly by placing the tube in crushed ice (2-5 min).
3. Place a mosquito on its right side under the binocular microscope.
4. Under a stereomicroscope, inoculate the mosquito with the capillary by firmly piercing through the left side of the thoracic cavity cuticle and then injecting the liquid and the bead.
   NOTE: Take care not to damage the flight muscles by holding the capillary as perpendicularly to the mosquito as possible.
5. Remove the capillary.
6. Using featherweight entomology forceps, place each female individually in a 180 ml plastic cup covered with mosquito netting; give them access to a 10% sugar solution.
   NOTE: Caution is required with the sugar solution, as the mosquito may get stuck on sugar solution drops.

6. Mosquito Dissection and Bead Photography

1. Check if the mosquitoes are alive 24 hr after the inoculation. If so, check their condition and keep only the mosquitoes that are able to fly.
2. Freeze these mosquitoes at -20 °C.
   NOTE: They can be kept several days to months before dissection, if needed.
3. Remove the mosquitoes' wings using tweezers under a binocular microscope at 32X magnification. Fix the wings onto glass slides with transparent tape. Write down the code used for each individual mosquito.
4. Put the glass slides in a scanner and scan them at 1,200 dpi.
   NOTE: Check that the code is readable for each individual mosquito.
5. On a glass microscope slide and in dissection solution, use forceps to separate the thorax from the head and abdomen.
6. Open the thorax with forceps to retrieve the beads.
   NOTE: Beads that do not move to the abdomen, are not in contact with each other and most of them float freely in the hemolymph. Be careful not to break the bead with the forceps, as a non-melanized bead may be tough to find. Wait for 1 to 5 min for beads to obtain color.
7. Transfer the beads in a droplet of dissection solution on a glass microscope slide, before taking the picture.
   NOTE: No need to wash the beads.
8. Using a microscope equipped with a camera, take a digital image of each bead at a standard lighting setting and 400X magnification.
   NOTE: Do not change the illumination between the different pictures to enable their comparison.

7. Picture Analysis: Bead Melanization

1. Open the image analysis software. Click on "file" and then on "open" to find a bead image.
2. Click on "analyze." Click on "set measurements." Tick the "mean grey value" box in the "set measurements" window.
3. Click on the circle selection tool icon. Select the bead perimeter; remain inside the bead and avoid the luminous halo around it.
4. Click on the "analyze" menu. Click on "measure." Obtain the mean grey value and size of the cross-sectional area of the bead.
   1. To obtain the melanization value of each bead, perform the following operation using a calculator or calculator software: "256 - mean grey value." The melanization measure is a value between 0 and 256, with 0 representing a totally white image.

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