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 invo...

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 cor...

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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, ...

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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 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 ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Immunology and Infection

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

Summary

Explore More Videos

Chapters in this video

Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

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

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

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

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

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

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

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

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