Name: protocol for plasmodium falciparum infections in mosquitoes and infection phenotype determination
Uploaded: 2007-07-04T00:00:00
Duration: 14 min 10 s
Description: Watch this Scientific Journal Video about Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination at JoVE.com

A. Preparation of gametocyte culture:
<ol>
<li>Bring fresh human blood and serum to 37oC in a water bath.</li>
<li>In a fume hood, under sterile conditions, pipet 1 mL of warm blood into an eppendorf tube. Spin the blood in a centrifuge for 5 minutes at 2200 rpm. Erythrocytes should be pelleted; remove and discard the clear serum. Wash erythocytes with an equal volume of new serum 3 times to obtain the whole blood for the gametocytes.</li>
<li>Retrieve the gametocyte culture from the CO2 incubator or candle jar. This culture should be maintained on RPMI media and fed to the mosquitoes on day 16 of the culture. To determine the gametocytemia % (on day 16 of the culture) make a thin smear on a glass slide with one drop blood and perform a Geimsa stain. Determine the number of gametes in a field of the parasite culture. It is usually in the range of 2 - 4%. Calculate the volume of whole blood that you will need to achieve the desired final % gametocytemia (0.3 -1 %).</li>
<li>Remove most of the media from your gametocyte culture using a pipet, taking care not to disturb or suck up the parasites (parasites will appear as black while the media will be light pink/red). Pipet the rest of the media and parasites into a 15 ml falcon tube. Spin in a centrifuge for 5 minutes at 2200 rpm. Parasites and erythrocytes will pellet, remove, and discard media supernatant without disturbing parasites.</li>
<li>Combine parasites with the volume of prepared whole blood (from step A2) to give you the desired final gametocytemia according to previous calculations. Pipet to mix thoroughly. (~1 minute)</li>
</ol>
This is now the sample you will feed to your mosquitoes. Keep at 37oC in a water-bath.
B. Feeding mosquitoes:
<ol>
<li>Put your Anopheles mosquitoes (~30-50) into wax-lined cardboard cups covered with mesh netting secured by a rubber band or cardboard lid supplied with cup. We use a battery-operated aspirator for this step.</li>
<li>Prepare the circulation water-bath by inserting nozzles on either side of glass feeders to rubber tubing. Connect as many feeders as you need in a series; one end of the series should be connected by tubing to the circulator of the water bath (the part that pushes water through the feeders), the other end of the series should consist of rubber tubing submerged in the bath to allow the circulated water to empty back into bath. Turn on the bath until the water is about 37oC and the water is circulating through the feeders and back to the bath. This is essential to keep the blood warm and keep the parasites alive.</li>
<li>Prepare the membrane (we use small squares of stretched parafilm) by stretching around the glass membrane feeder, using pressure to seal tightly to the feeder opening. This membrane will mimic the skin.</li>
<li>Place one feeder membrane-side down onto each mosquito cup and secure feeders using clamps or tape. The membrane should sit flush with the net so mosquitoes can access it from inside the cup.</li>
<li>Carefully pipette parasite-blood meal (from step A5) into the neck of the feeder, making sure the blood goes all the way through the neck into the reservoir, contacting the membrane so the mosquitoes can access the blood. We use about 200 &mu;l of blood per feeder, but this can vary.</li>
<li>Allow mosquitoes to feed. We allow them to feed for &asymp;30 minutes (variable, depending on theexperiment; for most experiments, occasionally monitoring how many have already taken a meal by looking at the blood content in the abdomen.)</li>
<li>After feeding, remove feeders from the mosquito cups. Turn off the water bath and disconnect feeders from tubing.</li>
<li>Remove the membrane and soak feeders in 10% bleach to decontaminate and rinse with water. Place contaminated material (membranes, paper towels, gloves, etc) into a small biohazard bag and deposit in a large biohazard receptacle.</li>
<li>Anesthetize mosquitoes by placing cups in refrigerator or cold room. Once mosquitoes are fully anesthetized, transfer them on a petri dish embedded in an ice bucket. Sort mosquitoes, looking carefully at the abdomens for any sign of a blood meal, and discard those that did not feed. Some mosquitoes will be fully engorged while others will show a narrow band of ingested blood; all are acceptable as "fed". Put the fed mosquitoes back into the cardboard cups.</li>
<li>Provide a sugar meal (a piece of cotton soaked in 10% sucrose) to mosquitoes, and incubate under normal insectary conditions for 7 days. Everyday, the mosquitoes should be monitored for the mortality rate. Also, the cotton should be made wet with the sucrose, so that they are not deprived of food. It is better, yet,to add a paper towel soaked in distilled water, to keep the mosquitoes moist. These mosquitoes are P. falcipraum infected, so all care must be taken so that no mosquitoes can escape from the cup. It is best to put the cups in a small cagefor double protection.</li>
</ol>
C. Dissecting mosquito midguts and estimating oocyst loads:
<ol style="margin-top: 0in">
<li style="tab-stops: list .5in">Aspirate mosquitoes (from step B) from cardboard cups using a battery-operated aspirator. Anesthetize mosquitoes, which are inside the removable aspirator reservoir, by moving to a cold room or burying the reservoir in ice for at least 5 minutes or until mosquitoes stop moving.</li>
<li style="tab-stops: list .5in">Transfer anesthetized mosquitoes from the reservoir to a glass petri dish embedded in ice. Spread the mosquitoes to a single layer and cover with a plastic petri dish lid. Mosquitoes that can not be accommodated on the dish remain in the reservoir, which is returned to cold room or ice.</li>
<li style="tab-stops: list .5in">Using fine-tipped forceps, transfer a single mosquito from the dish to 100 ul PBS contained on a glass slide mounted under a dissection microscope. Leave the others covered in the chilled petri dish.</li>
<li style="tab-stops: list .5in">Using fine-tipped forceps, hold mosquitoes at the thorax and the posterior abdomen. Pull the body apart until the midgut is exposed. Use forceps to remove excess tissue from gut.The gut looks like a white, sac-like body. Remove excess tissue from the gut.Mount the gut in one well of a 12-well glass slide and immerse in 5-10 &mu;l of 0.2% mercurochrome. Squash the carcass and discard on damp paper towel.Continue for other mosquitoes until the 12-well slide is full. Cover the mounted guts with a glass coverslip.</li>
<li style="tab-stops: list .5in">Examine guts under a light microscope, looking for round oocysts that stain bright pink against the clear/faint pink gut tissue. Count oocysts, record and continue to the next sample. Continue for all of the mosquitoes from all experimental treatments.</li>
<li style="tab-stops: list .5in">When finished, wipe slide clean with a paper towel. Place the paper towel with guts, and paper towel with carcasses, into small biohazard bag and discard in the trash. Gloves are worn throughout procedure. Care is taken not to let any infected mosquitoes escape.</li>
</ol>

<ol>
	<li>Avila, S. L., Ferreira, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria+diagnosis:+a+review.">Malaria diagnosis: a review.</a> Braz J Med Biol Res. 29 (4), 431-443 (1996).</li><li>Bell, A. S., Ranford-Cartwright, 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=A+real-time+PCR+assay+for+quantifying+Plasmodium+falciparum+infections+in+the+mosquito+vector.">A real-time PCR assay for quantifying Plasmodium falciparum infections in the mosquito vector.</a> Int J Parasitol. 34 (7), 795-802 (2004).</li><li>Collins, K. M., Hochberg, L. P., Ryan, P. R., Collins, W. E., Wirtz, R. A., Ryan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Plasmodium+malariae+infection+in+mosquito+vectors.">Quantification of Plasmodium malariae infection in mosquito vectors.</a> Ann Trop Med Parasitol. 98 (5), 469-472 (2004).</li><li>Czeher, C., Labbo, R., Djibrilla, A., Here, L., Arzika, I., Duchemin, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ELISA+study+of+oocyst-sporozoite+transition+in+malaria+vectors.">ELISA study of oocyst-sporozoite transition in malaria vectors.</a> Parasite. 13 (3), 257-261 (2006).</li><li>Haghdoost, A. A., Mazhari, S., Bahadini, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+the+results+of+light+microscopy+with+the+results+of+PCR+method+in+the+diagnosis+of+Plasmodium+vivax.">Comparing the results of light microscopy with the results of PCR method in the diagnosis of Plasmodium vivax.</a> J Vector Borne Dis. 43 (2), 53-57 (2006).</li><li>Patsoula, E., Spanakos, G., Sofianatou, D., Parara, M., Vakalis, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-step,+PCR-based+method+for+the+detection+and+differentiation+of+Plasmodium+vivax.">A single-step, PCR-based method for the detection and differentiation of Plasmodium vivax.</a> Ann Trop Med Parasitol. 97 (1), 15-21 (2003).</li><li>Osta, M. A., Christophides, G. K., Kafatos, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+mosquito+genes+on+Plasmodium+development.">Effects of mosquito genes on Plasmodium development.</a> Science. 303 (5666), 2030-2032 (2004).</li><li>Srinivasan, P., Abraham, E. G., Ghosh, A. K., Valenzuela, J., Ribeiro, J. M., Dimopoulos, G., Kafatos, F. C., Adams, J. H., Fujioka, H., Jacobs-Lorena, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+Plasmodium+and+Anopheles+transcriptomes+during+oocyst+differentiation.">Analysis of the Plasmodium and Anopheles transcriptomes during oocyst differentiation.</a> J Biol Chem. 279 (7), 5581-5587 (2004).</li></ol>

There can be some variations in the Plasmodium infection and infection phenotype determination and different labs may follow different techniques.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Anopheles</td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>mosquitos</td>
</tr>
<tr>
<td>Plasmodium falciparum</td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Parasite. Gametocytes with known gametocytemia.</td>
</tr>
<tr>
<td>15 mL conical tubes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>plastic
</td>
</tr>
<tr>
<td>Serum, blood</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>human, O+</td>
</tr>
<tr>
<td>0.2% mercurochrome</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>glass-slide</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>12 well</td>
</tr>
<tr>
<td>light microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>cell counter</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10% bleach</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>pipette tips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for tissue culture</td>
</tr>
<tr>
<td>parafilm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>mosquito feeder</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>glass membrane feeders, rubber tubing to fit feeders</td>
</tr>
<tr>
<td>Petri dishes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>slass</td>
</tr>
<tr>
<td>fine-tip forceps</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1X, sterile</td>
</tr>
<tr>
<td>circulating water bath</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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			pipetteman		</div>

protocol for plasmodium falciparum infections in mosquitoes and infection phenotype determination

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito.    This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed.  The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Watch this Scientific Journal Video about Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination at JoVE.com

Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito. This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed. The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the ...

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Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of the virus in the mosquito tissues. This protocol is routinely used for studying mosquito-virus interactions, especially for identification of novel host factors that are able to determine vector competence. The entire experiment must be conducted in a BSL2 laboratory. Similar to Plasmodium falciparum infections, proper attire including gloves and lab coat must be worn at all times. After the experiment, all the materials that came in contact with the virus need to be treated with 75% ethanol and bleached before proceeding with normal washing. All other materials need to be autoclaved before discarding them.

Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination

Once a gene is identified as potentially refractory for the dengue virus, it must be evaluated for it's role in preventing viral infections within the mosquito. This protocol illustrates how the extent of dengue infections of mosquitoes can be assayed. The techniques for growing up the virus in culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

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Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

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Clock Scan Protocol for Image Analysis: ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a closed or segmented convex-shaped region of interest, leading to the generation of an averaged integral radial pixel-intensity profile. This protocol was originally developed in 2006, as a visual basic 6 script, but as such, it had limited distribution. To address this problem and to join similar recent efforts by others, we converted the original clock scan protocol code into two Java-based plugins compatible with NIH-sponsored and freely available image analysis programs like ImageJ or Fiji ImageJ. Furthermore, these plugins have several new functions, further expanding the range of capabilities of the original protocol, such as analysis of multiple regions of interest and image stacks. The latter feature of the program is especially useful in applications in which it is important to determine changes related to time and location. Thus, the clock scan analysis of stacks of biological images may potentially be applied to spreading of Na+ or Ca++ within a single cell, as well as to the analysis of spreading activity (e.g., Ca++ waves) in populations of synaptically-connected or gap junction-coupled cells. Here, we describe these new clock scan plugins and show some examples of their applications in image analysis.

This paper describes two novel ImageJ plugins for &#39;Clock Scan&#39; image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software package.

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A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

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Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus particles are transmitted during a blood meal via the saliva of an infected mosquito. Forced salivation assays allow determining the vector potential on the basis of single mosquitoes, avoiding the use of animal experiments. The method is suitable to analyze a large number of mosquitoes in one experiment within a short period of time. Forced salivation assays were used to analyze 856 individual mosquitoes trapped in Germany, including two different Culex pipiens pipiens biotypes, Culex torrentium as well as Aedes albopictus, which were experimentally infected with Zika virus (ZIKV) and incubated at 18 &#176;C or 27 &#176;C for two and three weeks. The results indicated the lack of vector competence of the different Culex taxa for ZIKV. In contrast, Aedes albopictus was susceptible to ZIKV, but only at 27 &#176;C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.

For efficient control of mosquito borne virus transmission, the knowledge of the vector potential of respective mosquitoes is of particular interest. We describe forced salivation as a method to analyze vector competence of Aedes albopictus and three different Culex taxa for the transmission of Zika virus.

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Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease transmitted through an infectious bite from the female Anopheles mosquito. Malaria control will eventually rely on a multitude of approaches, which includes ways to block transmission to, through and from mosquitoes. To study mosquito stages of malaria parasites in the laboratory, we have optimized a protocol to culture highly infectious Plasmodium falciparum gametocytes, a parasite stage required for transmission from the human host to the mosquito vector. P. falciparum gametocytes mature through five morphologically distinct steps, which takes approximately 1-2 weeks. Gametocyte culture described in this protocol is completed in 15 days and are infectious to mosquitoes from days 15-18. These protocols were developed to maintain a continuous cycle of infection competent gametocytes and to maintain uninterrupted supply of mosquito stages of the parasite. Here, we describe the methodology of gametocyte culture and how to infect mosquitoes with these parasites using glass membrane feeders.

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

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Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female ...

Determining Temperature Preference of Mosquitoes and Other Ectotherms

Most insects and other ectotherms have a relatively narrow optimal temperature window, and deviation from their optima can have significant effects on their fitness, as well as other characteristics. Consequently, many such ectotherms seek out their optimal temperature range. Although temperature preferences of mosquitoes and other insects have been well studied, the traditional experimental setup is performed using a temperature gradient on an aluminum surface in a highly enclosed space. In some cases, this equipment restricts many natural behaviors, such as flying, which may be important in preference selection.
The objective of this study is to observe insect preference for air temperature by using a two-chamber apparatus with sufficient room for flight. The two chambers consist of independent temperature-controlled incubators, each with a large aperture. The incubators are connected by these apertures using a short acrylic bridge. Inside the incubators are two netted cages, linked via the apertures and bridge, allowing the insects to freely fly between the different conditions. The acrylic bridge also acts as a temperature gradient between the two incubators.
Due to the spacious area in the cage and easy construction, this method can be used to study any small ectotherm and/or any manipulation which may alter temperature preference including sensory organ manipulation, diet, gut flora, and endosymbiont presence at biosafety levels 1 or 2 (BSL 1 or 2). Additionally, the apparatus can be used for the study of pathogen infection using further containment (e.g., inside of a biosafety cabinet) at BSL 3.

Insects have an optimal environmental temperature range which they seek to remain within, and many external and internal factors can alter this preference. Here, we describe a cost-effective and simple method to study temperature choice, which allows insects to freely exhibit their natural behaviors.

Most insects and other ectotherms have a relatively narrow optimal temperature window, and deviation from their optima can have significant effects on ...

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes

Transgenic mosquitoes often display fitness costs compared to their wild-type counterparts. In this regard, fitness cost studies involve collecting life parameter data from genetically modified mosquitoes and comparing them to mosquitoes lacking transgenes from the same genetic background. This manuscript illustrates how to measure common life history traits in the mosquito Aedes aegypti, including fecundity, wing size and shape, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These parameters were chosen because they reflect reproductive success, are simple to measure, and are commonly reported in the literature. The representative results quantify fitness costs associated with either a gene knock-out or a single insertion of a gene drive element. Standardizing how life parameter data are collected is important because such data may be used to compare the health of transgenic mosquitoes generated across studies or to model the transgene fixation rate in a simulated wild-type mosquito population. Although this protocol is specific for transgenic Aedes aegypti, the protocol may also be used for other mosquito species or other experimental treatment conditions, with the caveat that certain biological contexts may require special adaptations.

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes

The present protocol describes how to measure common life parameter data in Aedes aegypti mosquitoes, including fecundity, wing size, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These measurements can be used to assess the fitness of transgenic mosquitoes.

Transgenic mosquitoes often display fitness costs compared to their wild-type counterparts. In this regard, fitness cost studies involve collecting life ...