We thank Drs. Anthony Cornel and Laura Norris at UC Davis and Dr. Katharina Kreppel at the University of Glasgow for providing mosquito specimens from Tanzania. We thank Ms.&#160;Smita Das and Dr. Douglas Norris from Johns Hopkins School of Public Health for sharing mosquito samples from Zambia. We also thank Mr. Lee V. Millon at the Veterinary Genetics Laboratory for training on assay design. This work was supported by the National Institute of Health grant R01AI 078183 and R21AI062929.

1. PCR Amplification
<ol>
	<li>Mix all the PCR primers (See supplemental Table S1) in a 1.5ml microtube. Each SNP has two primers (forward and reverse). Ensure that the stock concentration of each PCR primer is kept at 100 &#956;M. Make enough primer mix for 500-1,000 reactions to reduce pipetting error and time at the bench (See supplemental Table S2). If desired, prepare aliquots for 100-200 reactions per tube and store at -20&#176;C.</li>
	<li>Prepare PCR cocktail as described in the manufacturer&#8217;s protocol (Table 1). The volume for 96 reactions includes an overhang of 20%. Vortex the tube containing the PCR cocktail lightly and centrifuge for 30 sec&#160;at 2,000 x g.</li>
	<li>Dispense 3 &#956;l&#160;of the PCR cocktail into each well of a non-skirted 96 well plate. A repeater pipette can save time for this process.</li>
	<li>Add 2 &#956;l of the appropriate genomic DNA of mosquitoes in each well. 
	NOTE: Process of obtaining genomic DNA from mosquito tissues (head/thorax, abdomen or whole body) is described in other literatures29-32. Choose an appropriate DNA extraction protocol to differentiate those with Plasmodium in the blood (abdomen) from infective mosquitoes (Plasmodium in the head/thorax). We typically use DNA extracted from body parts (head/thorax or abdomen) of mosquitoes, so DNA concentration varies from 0.05-2ng/&#956;l. Although the total DNA input is lower than the manufacturer&#8217;s recommendation (5-10ng/rxn), we typically get robust SNP calls from 98% of DNA samples without whole genome amplification.</li>
	<li>Seal the plate, gently mix or vortex, and centrifuge for 1 min&#160;at 370 x g.</li>
	<li>Use the following thermocycler protocol to carry out PCR amplification: (1) 94&#176;C for 2 min, (2) 94&#176;C for 30 sec, (3) 56&#176;C for 30 sec, (4) 72&#176;C for 1 min, (5) repeat step (2)-(4) for 45 cycles, (6) 72&#176;C for 1 min, and 4&#176;C hold.</li>
</ol>
2. SAP Treatment
<ol>
	<li>Prepare the SAP enzyme solution in a 96 well plate (See Table 2). Process 96 samples in a plate. The volume for 96 reactions includes an overhang of 20%.</li>
	<li>Vortex the tube of SAP enzyme solution lightly and centrifuge for 30 sec&#160;at 2,000 x g. Dispense 2 &#956;l of the SAP enzyme solution into each well. Again, a repeater pipette can save time for this process.</li>
	<li>Seal the plate, gently mix or vortex, and centrifuge for 1 min&#160;at 370 x g. Incubate the plate as follows: (1) 37&#176;C for 40 min, (2) 85&#176;C for 5 min&#160;to inactivate the enzyme, (3) 4&#176;C hold. At completion of this step, store the plate at -20&#176;C before continuing. Process the stored plate within 2 weeks.</li>
</ol>
3. SNP Extension
<ol>
	<li>If the sample plate is frozen after SAP treatment, allow it to thaw to RT before proceeding.</li>
	<li>Prepare the extension primer mix (Supplemental Table S2). Each SNP has one extension primer. Keep the stock concentration of each extension primer at 500 &#956;M. Optionally, aliquot for 100-200 reactions per tube and store at -20&#176;C.</li>
	<li>Prepare extension mix as described in Table 3. The 96 reaction volume includes an overhang of 20%.</li>
	<li>Vortex the tube of extension cocktail lightly and centrifuge for 30 sec&#160;at 2,000 x g. Dispense 2 &#956;l of the extension cocktail into each well. Again, a repeater pipette can save time for this process.</li>
	<li>Seal the plate, gently mix or vortex, and centrifuge for 1 min&#160;at 370 x g. Thermocycle the plate as shown in Figure 1. At this point, proceed to mass spectrometry or store the plate at -20&#176;C up to 2 weeks.</li>
</ol>
4. Conditioning the SNP Extension Product to Optimize Mass Spectrometry Analysis
<ol>
	<li>If the sample plate is frozen after SNP extension, allow it to thaw to RT before proceeding.</li>
	<li>Transfer 15mg of resin from its container onto the dimple plate using an elongated spoon. Use the scraper to spread resin into the wells of the dimple plate. Sweep the scraper from side to side across the dimple plate to spread the resin. Make sure there is resin in each well.</li>
	<li>Scrape excess resin off the dimple plate using the scraper. Let the resin stand in the dimple plate for at least 20 min. While letting the resin stand in the dimple plate, add 41 &#956;l of HPLC grade water to each well of the sample plate.</li>
	<li>Gently place the sample plate upside-down, onto the dimple plate. Make sure the sample plate resets against the small rounded post of the dimple plate, which aligns the wells in the sample plate with the wells in the dimple plate.</li>
	<li>Holding the sample plate and the dimple plate together, gently flip them over so the resin falls out of the dimple plate into the wells of the sample plate. Tap the dimple plate so the resin falls out into the sample plate. Make sure all the resin in the dimple plate falls out into the sample plate wells. Centrifuge the plate at 3,200 x g for 30 sec.</li>
	<li>Rotate the sample plate on a rotator for 5 min&#160;at RT. Rotator must rotate the sample plate 360&#176; about the long axis.</li>
	<li>Centrifuge the sample plate at 3,200 x g for 5 min. After this step, store the sample plate at -20&#176;C up to 2 weeks before proceeding to the next step.</li>
</ol>
5. MALDI-TOF Mass Spectrometry
<ol>
	<li>Transfer the conditioned SNP extension product onto a bioarray such as SpectroCHIP as per manufacturer&#8217;s instructions. We use MassArray Nanodispenser for this step.</li>
	<li>Once the transfer is complete, define how assays and plates are to be set up in the assay database. Name the assay and plates such that each plate has unique identifier that links to specific assay design (Supplemental Table S3).</li>
	<li>After assay and plate have been defined, acquire spectra using a mass spectrometer.</li>
</ol>
6. Data Analysis
<ol>
	<li>Perform data analysis using a real-time PCR analysis data interpretation software such as Typer Analyzer or TyperViewer (Sequenom). Obtain data in .xml format which contains the sample layout, mass spectrogram for each well, the marker information including name, expected mass for each variant of the corresponding SNP, and any error or warning messages associated with each reaction. See Figure 2 for the resulting mass spectrogram.</li>
	<li>Carry out SNP genotype clustering analysis by setting the detection threshold (magnitude or signal to noise ratio) and tolerance/variance allowed for each genotype. Figure 3 shows an example of a 04707-KDR#2 SNP genotype clusters.</li>
	<li>Export the resulting SNP genotype calls into a spreadsheet program.</li>
</ol>

The authors have nothing to disclose.

The MDA is composed of five major steps: PCR amplification, shrimp alkaline phosphatase (SAP) reaction, SNP extension, extension product conditioning, and matrix-assisted laser desorption/ionization &#8211; time of flight (MALDI-TOF) mass spectrometry33-37. The first PCR amplification step amplifies DNA flanking each SNP so that enough template DNA will be available at the SNP extension step. The SAP reaction neutralizes unused dNTPs which can interfere with the following SNP extension step. SNP extension involves a single extension primer per SNP locus and mass-modified terminator nucleotides. The 3&#8217;-end modified nucleotides prevent subsequent nucleotides from being incorporated into the extension primer. Thus the mass of the single base indicates the genotype of the corresponding SNP.
The most critical step in ensuring the success of this approach is having a good dataset of existing polymorphisms in target organisms. We used well characterized SNPs for species identification (N&#62;900) 10,12,38-40 and kdr (N&#62;1,000)15,18. The species specific SNPs for Plasmodium were identified from sequence alignment of the cytochrome b sequences of 123 P. falciparum, 97 P. vivax, 11 P. malariae and 18 P. ovale isolate sequences from Africa which were available on Genbank. Cytochrome b sequences from 7 host species (human, chicken, cow, dog, goat, pig and sheep) were collected from Genbank for host-specific SNP identification. Based on this large body of sequence data, we were able to design a robust diagnostic assay using an assay designer software.
The number of markers each reaction can accommodate is determined by the biochemical compatibility of the primer mix given a tolerance to primer dimer potential (0.9 of 0-1 scale). Authors used the default value (1; most strict) for false priming potential. Relaxation of this parameter can potentially increase the number of SNPs that can be assayed in a single reaction in addition to the SNPs included in this study.
The PCR amplification steps are robust and typically do not require adjustment from the initial assay design. The SNP extension mix (Table 3), however, may need modification in relative quantity of each primer using mass spectrometry to ensure a sufficient signal to noise ratio is achieved for each primer. The provided SNP extension primer recipe is the result of these adjustments. Compatibility among extension primers may limit the total number of SNPs that can be multiplex in a single reaction. Since the reaction volume is small (5-8 &#181;l), the sample plate needs to be properly sealed to prevent evaporation during amplification steps.
This platform can simultaneously score up to 35 SNPs per reaction, while other SNP genotyping platforms can accommodate over one thousand SNPs per reaction41. With the advances in genome sequencing technology, one can acquire millions of SNPs using next-generation sequencing 29,42,43. However, the technique used here provides an ideal application for studies that require genotyping a relatively small number of markers for many (100s-1,000s) individuals. Additional discussion of the design constraints of different SNP genotyping platform is covered in previous studies 41.
The MDA is aimed at characterizing epidemiologically important traits of malaria vectors and provides an ideal protocol for medium-throughput SNP genotyping assays analyzing thousands of mosquitoes collected from natural populations. The MDA presented here provides (1) over a 90% reduction in labor time, (2) a 60% reduction in reagent costs, and (3) reduction in false positives/negatives by utilizing multiple markers. The proposed assay can enhance epidemiological studies by greatly facilitating data collection. Also, it can improve genome-wide association studies by characterizing the mosquito samples with respect to population origin, insecticide resistance, parasite susceptibility and host choice. Finally, this MDA may provide inspiration and a template for designing other multiplex assays using a MALDI-TOF based SNP genotyping platform.

Anopheles arabiensis, Anopheles coluzzii and Anopheles gambiae are the major vectors responsible for malaria transmission in Africa1. These three species are morphologically indistinguishable2 and can only be distinguished by molecular assays3-9. In addition, there are many downstream assays routinely conducted to aid epidemiological and population genetics studies. These include (1) a genotyping assay for speciation islands10-12, (2) a genotyping assay recognizing non-synonymous SNPs in the 1014th amino acid codon position of para gene (the knock-down resistance, or kdr, SNP)13-18, (3) parasite detection PCR19-23, and (4) screening for host-specific DNA in mosquito midguts24,25.
We developed a multi-detection assay (MDA) that combines all these assays into a single multiplex reaction with the goal of analyzing epidemiologically important characteristics of malaria vectors in Africa. The MDA assay includes multiple markers for detecting (1) species (A. arabiensis, A. gambiae, A. coluzzii, or other (none of the three)), (2) insecticide resistance represented in kdr SNPs (both L1014Fand L1014S), (3) presence of two major malaria parasites, Plasmodium falciparum and P. vivax, and (4) blood source from one avian and six mammalian hosts.
Traditionally, these assays were conducted in separate polymerase chain reactions. When all these assays are done using a conventional PCR platform, it requires conducting 8-10 PCR reaction assays and accompanying gel electrophoresis steps. Each individual PCR reaction from preparation to documenting results takes 4-5 hr, while the MDA method presented here takes about 5 hr in totality. This is equivalent to a 90% savings in labor costs alone. The MDA presented here costs $5 per sample to genotype all 33 SNPs. This is considerably less expensive than the single agarose gel-based assays, which costs about $1.50 per sample. Assays detecting all the characteristics covered by the MDA would require a minimum of 8-10 separate agarose gel-based assays at a cost of $12-15 per sample. Moreover, the MDA greatly reduces the chance of generating a false positive or negative by utilizing at least three markers for each parasite or host source detection.
The platform we utilized is not limited to malaria vectors but can be used in a wide variety of applications such as medicine, veterinary medicine, and basic biology26-28. In-depth association studies or population genetics studies involving a large (in order of 100s) number of samples require cost-effective assays screening for multiple markers simultaneously. Most studies that utilize two or more separate PCR assays could implement the MDA for quicker results at a lower cost.

<table><tbody><tr><td>MassARRAY Analyzer Compact</td><td>Sequenom</td><td>MT9</td><td>MALDI-TOF mass spectrometry for genomic applications to analyze nucleic acids.</td></tr><tr><td>MassARRAY Nanodispenser</td><td>Sequenom</td><td>RS1000</td><td>Transfers completed iPLEX reaction products to the SpectroCHIP</td></tr><tr><td>iPLEX Gold Genotyping Reagent Set</td><td>Sequenom</td><td>10158</td><td>Reagents used for iPLEX assay including SAP kit.</td></tr></tbody></table>

a multi-detection assay for malaria transmitting mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Species identification: 
The following 5 SNPs together identify three species (A. arabiensis, A. coluzzii and A. gambiae) (Table 4). If a sample is not one of the three species, the three SNPs (01073-213, 04679-157 and 10313-052) fail to amplify.
kdr genotype for inferring insecticide resistance: 
The 1014th codon of the para voltage-gated sodium channel corresponds to kdr. Two SNPs on the 2nd and 3rd base of the 1014th codon determine the three possible amino acids. The possible combinations of the genotypes are listed in Table 5. This SNP works for three species of African malaria vectors: A. arabiensis, A. coluzzii and A. gambiae. These SNPs fail to amplify in A. darlingi, a Brazilian malaria vector. This is not surprising given that mitochondrial genome sequence similarity between A. darlingi and A. gambiae is only 88.9% while mitochondrial genome sequence similarity among three African malaria vectors are over 98%. Other species have not been tested. We were successful in amplification from samples collected in Mali, Cameroon, Tanzania and Zambia.
Plasmodium detection: 
If intact Plasmodium DNA is present in the mosquito tissue, we expect to detect P. falciparum or P. vivax specific DNA among the DNA sample extracted from the mosquito. The species specific SNPs were identified from sequence alignment of the cytochrome b sequence of 123 P. falciparum, 97 P. vivax, 11 P. malariae and 18 P. ovale isolate sequences from Africa available on Genbank. We designed 6 SNPs which produce unique SNP genotypes to distinguish P. falciparum or P. vivax from other Plasmodium species (Table 6). We have tested this assay on mosquito samples collected in Mali, Cameroon, Tanzania, Zambia and Brazil and confirmed that this particular set of markers works regardless of mosquito species.
Bloodmeal detection PCR:
Cytochrome b sequences from 7 host species (human, chicken, cow, dog, goat, pig and sheep) were collected from Genbank and consensus sequences were generated. SNPs informative for each host were then selected for genotyping. We tested this with blood sources from 7 different animals (Table 7).
Because PCR fragments are short (80-120 bp), this works on somewhat degraded DNA or on partially digested bloodmeals from mosquito tissue. The probability of false positive calls is greatly reduced by having multiple markers per host type.
<img alt="Figure 1" src="/files/ftp_upload/52385/52385fig1.jpg" /> 
Figure 1. Thermocycler program for SNP extension step. The amplification cycle is total of 200 (5x40).
<img alt="Figure 2" src="/files/ftp_upload/52385/52385fig2.jpg" /> 
Figure 2. Mass-spectrogram for the MDA. The X axis is mass (Da) of SNP extension products and the Y axis is signal intensity. The Red lines indicate the expected mass of Unincorporated Extension primer, SNP allele 1 and allele 2 for the selected marker. Gray lines indicate expected mass of other markers in the MDA. This plot is generated from data analysis software (Typer Analyzer or Typer Viewer) from the output data file after Step 5. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52385/52385fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52385/52385fig3.jpg" /> 
Figure 3. Individual SNP genotype cluster view The X axis is the arctangent value (labeled as &#8220;Angle&#8221;) of the signal intensities of SNP allele 1 and SNP allele 2. The Y axis is the magnitude of genotype call signal. This plot is provided in the data analysis software (Typer Analyzer or Typer Viewer). Any particular data can be selected with a click of a mouse button and selected sample data is indicated by circle. Clustering analysis provided in the software clusters genotypes with a user-defined variance threshold and automatically colors three genotypes of a biallelic SNP. Example shown here indicates homozygous T (TT) individuals as blue triangles, heterozygote (TA) as green squares and homozygous A (AA) individuals as orange inverted triangles. Red circles represent No call (no amplification). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52385/52385fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td rowspan="2">Reagent</td>
			<td rowspan="2">Concentration in 5 &#956;l</td>
			<td>Volume </td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>(1 rxn)</td>
			<td>(96 rxns)</td>
		</tr>
		<tr>
			<td>Water (HPLC grade)</td>
			<td>NA</td>
			<td>0.8 &#956;l</td>
			<td>92.2 &#956;l</td>
		</tr>
		<tr>
			<td>10x PCR Buffer with 20 mM MgCl2</td>
			<td>1x (2 mM MgCl2)</td>
			<td>0.5 &#956;l</td>
			<td>57.6 &#956;l</td>
		</tr>
		<tr>
			<td>MgCl2 (25 mM) **</td>
			<td>2 mM</td>
			<td>0.4 &#956;l</td>
			<td>46.1 &#956;l</td>
		</tr>
		<tr>
			<td>dNTP mix (25 mM each) ***</td>
			<td>500 &#956;M</td>
			<td>0.1 &#956;l</td>
			<td>11.5 &#956;l</td>
		</tr>
		<tr>
			<td>Primer mix (500 nM each)</td>
			<td>100 nM</td>
			<td>1.0 &#956;l</td>
			<td>115.2 &#956;l</td>
		</tr>
		<tr>
			<td>PCR Enzyme (5 U/&#956;l)</td>
			<td>1.0 U/rxn</td>
			<td>0.2 &#956;l</td>
			<td>23.0 &#956;l</td>
		</tr>
		<tr>
			<td>Total Volume:</td>
			<td></td>
			<td>3.0 &#956;l</td>
			<td>345.6 &#956;l</td>
		</tr>
	</tbody>
</table>
Table 1. Multiplex PCR cocktail recipe. The volume of each reagent for the PCR extension step for one reaction and 96 reactions with a 20% overhang.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Volume (1 rxn)</td>
			<td>Volume (96 rxn)</td>
		</tr>
		<tr>
			<td>Water (HPLC grade)</td>
			<td>1.53 &#956;l</td>
			<td>176.3 &#956;l</td>
		</tr>
		<tr>
			<td>SAP Buffer (10x)</td>
			<td>0.17 &#956;l</td>
			<td>19.6 &#956;l</td>
		</tr>
		<tr>
			<td>SAP enzyme (1.7 U/&#956;l)</td>
			<td>0.30 &#956;l</td>
			<td>34.6 &#956;l</td>
		</tr>
		<tr>
			<td>Total Volume</td>
			<td>2.00 &#956;l</td>
			<td>230.4 &#956;l</td>
		</tr>
	</tbody>
</table>
Table 2. SAP enzyme solution. The volume of each reagent for the Shrimp Alkaline Phosphatase treatment for one reaction and 96 reactions with a 20% overhang.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Conc. In 9 &#956;l</td>
			<td>Volume (1rxn)</td>
			<td>Volume (96 rxns)</td>
		</tr>
		<tr>
			<td>Water (HPLC grade)</td>
			<td>NA</td>
			<td>0.6190 &#956;l</td>
			<td>71.3 &#956;l</td>
		</tr>
		<tr>
			<td>iPLEX Buffer Plus (10x)</td>
			<td>0.222x</td>	
			<td>0.2000 &#956;l</td>
			<td>23.0 &#956;l</td>
		</tr>
		<tr>
			<td>iPLEX Termination mix</td>
			<td>1x</td>
			<td>0.2000 &#956;l</td>
			<td>23.0 &#956;l</td>
		</tr>
		<tr>
			<td>Extension Primer mix</td>
			<td></td>
			<td>0.9400 &#956;l</td>
			<td>108.3 &#956;l</td>
		</tr>
		<tr>
			<td>iPLEX enzyme</td>
			<td>1x</td>
			<td>0.0410 &#956;l</td>
			<td>4.7 &#956;l</td>
		</tr>
		<tr>
			<td>Total Volume</td>
			<td></td>
			<td>2.0000 &#956;l</td>
			<td>230.4 &#956;l</td>
		</tr>
	</tbody>
</table>
Table 3. SNP Extension cocktail The volume of each reagent for the SNP extension step for one reaction and 96 reactions with a 20% overhang.
<img alt="Table 4" fo:content-width="6.5in" width="650" src="/files/ftp_upload/52385/52385table4.jpg" /> Table 4. SNP genotypes for each species. 28S IGS-540, 28S IGS-649 and 01073-213 are located on the X chromosome, 04679-157 is on chromosome 2 and 10313-052 is on chromosome 3. Hybrid genotype (heterozygote) is highlighted in yellow. Fixed variant in A. coluzzii is marked in light blue and fixed variant in A. gambiae is marked in dark blue.
<img alt="Table 5" fo:content-width="6.5in" width="650" src="/files/ftp_upload/52385/52385table5.jpg" /> Table 5. SNP genotypes for kdr. Two SNPs KDR#1 and KDR#2 constitute a genotype for the 1014th codon of para gene. The first base is invariable so it is not included in the assay. Susceptible homozygous genotypes are marked in blue. The most resistant L1014F homozygote genotype is marked in dark blue. Yellow color indicates heterozygote. The L1014S, intermediately resistant genotype, is shown in orange.
<img alt="Table 6" fo:content-width="6.5in" width="650" src="/files/ftp_upload/52385/52385table6.jpg" /> Table 6. SNP genotypes for P. falciparum and P. vivax. Total 6 SNPs are used to determine the presence of malaria parasite. Pl-0041, Pl-1269 and Pl-1549 contain P. falciparum specific alleles (G, T and T respectively). Simultaneous amplification of the three alleles indicates presence of relatively intact P. falciparum DNA in sample DNA. Amplification of alternative alleles (A for Pl-0041 and Pl-1269) indicates presence of other Plasmodium species (e.g., P. vivax, P. ovale or P. malariae) in the sample. Pf-1549 is located in regions with many more P. falciparum specific flanking regions thus this SNP is only amplified when P. falciparum is present. Pl-0071, Pl-1245 and Pl-0157 contain P. vivax specific alleles (G, G and T respectively). Amplification of alternative alleles (A, A and C respectively) indicates presence of other Plasmodium DNA in the sample. Uninfected mosquitoes will have no amplification on all 6 markers.
<img alt="Table 7" fo:content-width="6.5in" width="650" src="/files/ftp_upload/52385/52385table7.jpg" /> 
Table 7. SNP genotypes for 7 hosts: Human, Chicken, Cow, Dog, Goat, Pig and Sheep. &#8220;NC&#8221; stands for &#8220;No Call&#8221; (no amplification) in the following table. Note that some non-specific amplification may occur at some loci, but not for all the SNPs of a particular host. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52385/52385table7.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Multi-detection Assay for Malaria Transmitting Mosquitoes at JoVE.com

A Multi-detection Assay for Malaria Transmitting Mosquitoes | Biology | Video

A Multi-detection Assay for Malaria Transmitting Mosquitoes

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

a-multi-detection-assay-for-malaria-transmitting-mosquitoes

Nanyang Technological University

Research

Education

JoVE Journal

JoVE Core

Biology

Evolution

Speciation and Diversity

SCIENTISTS IN ACTION

13.1K Views.  School of Veterinary Medicine, University of California - Davis. Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

Video: A Multi-detection Assay for Malaria Transmitting Mosquitoes

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

Immunology and Infection

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 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 ...

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

Biochemistry

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectrometer in conjunction with Multivariate Data Analysis (MVDA). 3D7 P. falciparum were cultured to 10% parasitemia ring stage parasites and used to spike fresh donor isolated RBCs to create a dilution series between 0&#8211;1%. 10 &#181;L of each sample were placed onto the center of the ATR diamond window to acquire the spectrum. The sample data was treated to improve the signal to noise ratio and to remove the contribution of water, and then the second derivative was applied to resolve spectral features. The data were then analyzed using two types of MVDA: first Principal Component Analysis (PCA) to determine any outliers and then Partial Least Squares Regression (PLS-R) to build the quantification model.

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

Here, we present a protocol for the detection and quantification of Plasmodium falciparum in infected aqueous red blood cells using an attenuated total reflection infrared spectrometer and multivariate data analysis.

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total ...

Chemistry

Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations

Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA sequence variation on a large scale. Specifically, detecting insertions and deletions (indels) at gene-edited sites generated by CRISPR guide RNA (gRNA)/Cas9-mediated non-homologous end-joining (NHEJ) is important for assessing the fidelity of the mutagenesis and the frequency of unintended changes. We describe here a protocol for digital-droplet PCR (ddPCR) that is well-suited for high-throughput NHEJ analysis. While this method does not produce data that identifies individual sequence variation, it provides a quantitative estimate of the sequence variation within a population. Additionally, with appropriate resources, this protocol can be implemented in a field-site laboratory setting more easily than next-generation or Sanger sequencing. ddPCR also has a faster turn-around time for results than either of those methods, which allows a more quick and complete analysis of genetic variation in wild populations during field trials of genetically-engineered organisms.

This protocol provides the steps from DNA extraction to experimental set-up for digital droplet PCR (ddPCR), including analysis for the identification and quantification of non-homologous end-joining (NHEJ) events at target sites following gRNA-induced Cas9 cleavage and DNA repair. Other uses of this method include applications such as polymorphism detection and gene-editing variant verification.

Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA ...

Genetics

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This parasitic disease requires a protozoan parasite, an Anopheles mosquito vector, and a vertebrate host. The Anopheles genus comprises more than 500 species, of which 60 are known as vectors of the parasite. The Plasmodium parasite genus consists of 250 species, and 48 of these are involved in disease transmission. Furthermore, the Plasmodium falciparum parasite has contributed toward an estimated 99.7% of malaria cases in sub-Saharan Africa in recent years. 
Gametocytes form part of the sexual stage of the parasite and are ingested by the female mosquito upon feeding on an infected human host. Further development of the parasite within the mosquito is enhanced by favorable environmental conditions in the midgut of the mosquito. Here, the fusion of the female and male gametes takes place, and the motile ookinetes originate. The ookinetes enter the midgut epithelium of the mosquito, and mature ookinetes form oocysts, which, in turn, produce motile sporozoites. These sporozoites migrate to the mosquito's salivary glands and are injected as a mosquito takes a blood meal. 
For drug discovery purposes, mosquitoes were artificially infected with gametocyte-infected blood in the standard membrane feeding assay (SMFA). To detect infection within the mosquito and/or to assess the efficacy of antimalarial compounds, the midguts of the female mosquitoes were removed post infection and were stained with mercurochrome. This method was used to enhance the visual detection of oocysts under the microscope for the accurate determination of oocyst prevalence and intensity.

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

The standard membrane feeding assay (SMFA) is regarded as the gold standard for the assessment and identification of potential antimalarial compounds. This artificial feeding system is used to infect mosquitoes to further evaluate the effects of such compounds on the intensity and prevalence of the Plasmodium falciparum parasite.

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This ...

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

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

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.

A Multi-detection Assay for Malaria Transmitting Mosquitoes

Samenvatting

Meer video's verkennen