This research was possible through funding from the University of Neuch&#226;tel. We would like to thank all the students that helped in improving this technique, namely our colleague Kevin Thievent. We would also like to thank the members of the Thomas Lab for making their laboratory available. We would like to thank Janet Teeple for her help with mosquito rearing. We would also like to thanks Loyal Hall in the laboratory of Pr. Tom Baker for his help in the preparation of the micro capillary glass tubes.

1. Saline Solution for Injection and Dissection
<ol>
	<li>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.</li>
	<li>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 &#8220;injection solution&#34;.</li>
	<li>Then, add 5 ml of 0.1% methyl green solution to 45 ml of the saline solution. This 0.01% methyl green &#8...

<ol>
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D., Moebius, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria+Immunity+in+Man+and+Mosquito:+Insights+into+Unsolved+Mysteries+of+a+Deadly+Infectious+Disease*.">Malaria Immunity in Man and Mosquito: Insights into Unsolved Mysteries of a Deadly Infectious Disease*.</a> Annu Rev Immuno. 32 (1), 157-187 (2014).</li><li>Schmid-Hempel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EVOLUTIONARY+ECOLOGY+OF+INSECT+IMMUNE+DEFENSES.">EVOLUTIONARY ECOLOGY OF INSECT IMMUNE DEFENSES.</a> Annu Rev Entomol. 50 (1), 529-551 (2005).</li><li>Hillyer, J. F., Schmidt, S. L., Christensen, B. 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The authors declare that they have no competing financial interests.

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 ...

Insects rely on immune responses to protect themselves against parasites and pathogens1-3 that breach through their cuticle or their midgut epithelium4. In mosquitoes, these responses are efficient against bacteria5, viruses6, filarial nematodes7, and malaria parasites1,8,9. In mosquitoes, a key immune response is the encapsulation of foreign particles with melanin10-12. This encapsulation may happen in the midgut or in the hemolymph circulating system10-12. This melanization response is the result of the pro-phenoloxidase cascade10-12, 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 systems13-18. It is used in Anopheles gambiae mosquitoes, the major vector of malaria in Africa, to study host-parasite interactions14-16,19. 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 detection20-23. 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 cascade10-12.
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 mosquito15,16,27, 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 mosquito15,16,27. A dye is added to this solution to ease bead selection. It is the same for the dissection solution used to retrieve the bead15,16,27.
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, immunosuppression29-31, or immune evasion31-34. 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 tools2,35,36. 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 success13,27,37. Such studies can be conducted in varying environments2,4,38. Stimulating immunity is also of interest to those focusing directly on immunopathology39,40.
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.

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	<tbody>
		<tr height="85">
			<td height="85" style="height:85px;width:301px;">Microcapillary glass tubes GB120TF-10</td>
			<td style="width:177px;">science-products.com</td>
			<td style="width:84px;">GB120TF-10</td>
			<td style="width:156px;">http://www.science-products.com/Products/CatalogG/Glass/Glass.html</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:301px;">Microcaps Capillary pipette bulb</td>
			<td style="width:177px;">Drumond</td>
			<td style="width:84px;">1-000-9000</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:301px;">negatively charged Sephadex CM C-25 beads</td>
			<td style="width:177px;">Sigma-Aldrich, Steinheim, Germany</td>
			<td style="width:84px;">C25120 SIGMA</td>
			<td style="width:156px;">need few to start</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:301px;">Methyl green</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:84px;">323829 ALDRICH</td>
			<td style="width:156px;">need few to start</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:301px;">Software ImageJ</td>
			<td style="width:177px;">opensource</td>
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inoculating anopheles gambiae mosquitoes with beads to induce and measure the melanization immune response

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.

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...

Watch this Scientific Journal Video about Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response at JoVE.com

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

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.

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

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is ...

The overall goal of this inoculation technique is to stimulate the mosquito immune response and to measure the melanization reaction. This method can help answer questions in the field of ecological immunology, including the genetic basis for the cost of the human response and the influence of environmental factors. Though this method can provide insight into immunization response, it can also be applied to other systems such as anti-viral or anti-bacterial response in other invertebrate systems.Generally, individuals new to this method will struggle because of the insect damage that the injection can cause. Add one milliliter 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.Then, add five milliliters of 0.1%methyl green solution to 45 milliliters 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. Autoclave the solutions at 110 degrees Celsius for 25 minutes.To prepare the capillary, heat pull microcapillary glass tubes to obtain a very fine tip, slightly bigger than the smallest beads. Open and adjust each capillary by breaking the tip with tweezers or by sawing the tip off. To select beads with a capillary, pour 0.009 grams of negatively charged beads in a five centimeter Petri dish.Then add five milliliters of injection solution 30 minutes in advance. This colors the beads to facilitate bead selection for injection. Using a capillary mounted in a capillary pipette bulb, visually select the smallest bead in 0.1 microliters of the saline solution under a binocular microscope.Rear Anopheles gambiae at 26 plus or minus one degree Celsius and 70 plus or minus 5%relative humidity in a cycle of 12 hours light, 12 hours dark. Keep adult females and give them access to a 10%sugar solution. Using an insect aspirator, place each mosquito in a 50 milliliter tube.Chill each mosquito briefly by placing the tube in crushed ice. Place a mosquito on its right side under the binocular microscope. Properly creating a mosquito is critical and demands some training.It's very difficult to learn how to pierce through the insect cuticle and trust that the bead was injected. Inoculate the mosquito with a capillary by firmly piercing through the left side of the thoracic cavity cuticle and then injecting the liquid and the bead. Take care not to damage the flight muscles by holding the capillary as perpendicularly to the mosquito as possible.Remove the capillary following injection. Using featherweight entomology forceps, place each female individually in a 180 milliliter plastic cup with access to a 10%sugar solution. Check if the mosquitoes are alive 24 hours after the inoculation.If so, check their condition and keep only the mosquitoes that are able to fly. Freeze these mosquitoes at minus 20 degrees Celsius. They can be kept several days to months before dissection if needed.Retrieving the beads from the mosquito for the first time is not an easy task. It can be difficult to trust that there were actually a bead injected into the mosquito. On a glass microscope slide and in dissection solution, use forceps to separate the thorax from the head and abdomen on a mosquito that has been frozen.Open the thorax with forceps to retrieve the beads. Using a microscope equipped with a camera, take a digital image of each bead at a standard lighting setting and 400x magnification. Do not change the illumination between the different pictures to enable their comparison.To begin picture analysis, open the image analysis software. Click on file and then on open to find a bead image. Then click on analyze.Then click on set measurements. Tick the mean gray value box in the set measurements window. Next, click on the circle selection tool icon.Select the bead perimeter, remaining inside the bead and avoiding the luminous halo around it. Then click on the analyze menu and click on measure. Obtain the mean gray value and the size of the cross-sectional area of the bead.To obtain the melanization value of each bead, subtract the mean gray value from 256. The melanization measure is a value between zero and 256, with zero representing a totally white image. Pictured here are a non-melanized bead, a partially melanized bead, and a highly melanized bead.Mosquitoes do not all melanize the beads in the same way, as some beads are less covered with melanin than others. In this figure, each point shows the melanization of a single bead and each column of points represents an individual mosquito. The solid points represents the highest melanization value per mosquito.The cross points represent an intermediate melanization value. And the empty points represent the lowest melanization value. The solid lines represent the mean melanization per bead treatment.The average bead melanization value decreased from 71 to 50 when the number of beads inoculated increased. In this figure, the black squares represent the mean bead melanization as a function of the number of beads. The red points represent the mean of the highest melanization and the blue triangles represent the mean of the lowest melanization as a function of the number of beads.The lowest melanization decreased from 71 to 35, but the highest melanization stayed about constant. The variability of melanization increased with the number of beads. The melanization effort was not uniform amongst all beads in a given mosquito, as one bead was prioritized over others.Once much of this technique can be done in a few hours for hundreds of mosquitoes, if it is performed properly. The main advantage of this technique is that it measures the final phenotype and not just one step of the physiological pathway. Following this procedures, other methods like genograms can be performed in order to gain a better understanding of the human pathways.After watching this video, you should have a good understanding of how to inoculate mosquitoes and then dissect them to measure the melanization response.

inoculating-anopheles-gambiae-mosquitoes-with-beads-to-induce-measure

Research

JoVE Journal

Immunology and Infection

7.9K Görüntüleme.  Université de Neuchâtel. The overall goal of this inoculation technique is to stimulate the mosquito immune response and to measure the melanization reaction. This method can help answer questions in the field of ecological immunology, including the genetic basis for the cost of the human response and the influence of environmental factors. Though this method can provide insight into immunization response, it can also be applied to other systems such as anti-viral or anti-bacterial response in other invertebrate syst...