We thank Victor Marquez for providing DZNep.HCl and Scotty Bolling for manufacturing the bloodfeeders. The Mopti and SUA2La strains of An. gambiae were obtained from the Malaria Research and Reference Reagent Resource Center (MR4). This work was supported by the Fralin Life Science Institute and the grant from National Institutes of Health 1R21AI094289 to Igor V. Sharakhov.

NOTE: The three parts of the protocol describe the aqueous exposure of the DZNep drug to larval mosquitoes, a blood-based exposure of DZNep to adult females to study its effect of fecundity, and SAH hydrolase inhibition by DZNep measured using a simple colorimetric technique. A schematic representation of these assays is shown in Figure 1.
1. Immature Mosquito Development and Survivorship Assays
NOTE: This section explains the use of a water-soluble drug that affects mosquito larvae development and survival.
<ol>
	<li>Hatch A. gambiae eggs at 28 &#176;C in distilled water (dH2O) and rear them to 2nd instar larvae.</li>
	<li>Take a six-well DNAse, RNAse free cell culture plate and pour 10 ml dH2O to each well.</li>
	<li>Select mosquito larvae at 2nd instar and add 15 larvae per well. Before adding 2nd instar larvae to plate, put them on a paper towel for 2-3 sec to remove excess water.</li>
	<li>Label wells with color-coded tapes to randomize experiment. Label 2 wells per test concentration on each plate (Figure 2).</li>
	<li>Prepare a 1 mM DZNep hydrochloride (MW = 298.73) stock solution by dissolving 0.3 mg DZNep in 1 ml of dH2O. Add stock solution of DZNep.HCl to labeled wells to achieve a final concentration of 0.5 &#181;M and 5.0 &#181;M. 
	NOTE: The stock solution can be stored at 4 &#176;C for short term (1 day to a month) or -20 &#176;C for long term (longer than one month). 
	NOTE: Addition of the stock solution for aforementioned concentrations will not change the volume of water in the wells significantly. However in case of much higher concentrations, it is advisable to dissolve the compound in the larval medium to avoid any unnecessary dilution of the compound.</li>
	<li>Add equal amount of flake fish food to each well. Cover the plates and incubate them at 28 &#176;C in an incubator.</li>
	<li>Record the mortality for the DZNep-treated and untreated larval mosquitoes after a 24-hr exposure period. Remove and discard any dead larvae. Repeat this recording every day until all larvae die or pupate and emerge as adults. Record the size of larvae every second day, i.e., day 0, 2, 4, 6, 8, until they pupate.</li>
	<li>Analyze the larval development and mortality data using an adequate statistical analysis software. 
	NOTE: A bar graph can be used to represent the mortality data in Microsoft Excel. Multivariate Analysis of Variance (MANOVA) performed with a software such as JMP or SPSS will reveal if different doses of the drug result in significantly different survival of mosquito larvae.</li>
</ol>
2. Fecundity Assay by Administering Drug Through Artificial Feeder to Female Mosquitoes
NOTE: This section details the addition of the drug to blood and feeding it to adult female mosquitoes using an artificial feeder system.
<ol>
	<li>Set up adult mosquito cages with equal number of male and female mosquitoes for control and test cage. Provide 10% sugar solution soaked in cotton balls to both the cages till the females are ready for feeding (3-5 days is optimum for Anopheles gambiae).</li>
	<li>2 hr prior to the blood feeding, remove the cotton balls to ensure females are hungry.</li>
	<li>Assemble two artificial blood feeders that consist of an inverted wide bottom glass funnel (diameter = 50 mm, length = 70 mm) and a surrounding plastic sealed with industrial glue. Attach plastic tubes (diameter = 7 mm) to two feeders&#8217; outlets (diameter= 10 mm) via connectors for inflow and outflow of water. Label the two feeders as &#8220;Control&#8221; and &#8220;5 &#956;M DZNep.&#8221;</li>
	<li>Using a commercially available heating element, set up the program for feeding. In &#8220;Menu&#8221; go to &#8220;Settings&#8221; and select &#8220;Setpoints&#8221;; preloaded programs will appear. Select &#8220;SP1&#8221; and set the temperature for 37 &#176;C. 
	NOTE: The heating element ensures that the blood administered is maintained at a constant temperature. Additional program settings may be used for different mosquito or insect species.</li>
	<li>Fill a bucket with water and immerse the heating element into the water.</li>
	<li>Cut a square piece of Parafilm (50 mm x 50 mm) and stretch it to make a thin membranous film. Cover the bottom of the artificial feeders with the Parafilm. Cut another piece of Parafilm (50 mm x 10 mm), stretch it and seal the edges of the feeders.</li>
	<li>Assemble the system, connecting the feeders and heating element by the tubes. Once completed, switch on the system and select &#8220;SP1&#8221;. Press &#8220;Enter&#8221; to start the system. 
	NOTE: The water will get heated to 37 &#176;C and maintained at that temperature.</li>
	<li>Monitor the temperature of water using a thermometer in the water bucket. 
	NOTE: The connectors and tubes help maintain a constant circulation of water through the system so that the temperature of the blood remains at 37 &#176;C.</li>
	<li>Fill a flat bottom tray with 10% bleach solution that will be used to decontaminate any blood spills.</li>
	<li>Add 2 ml of defibrillated sheep&#8217;s blood to a microcentrifuge tube. Label this as &#8220;Control&#8221;. Repeat the process for the experimental tube labeled as &#8220;5 &#956;M DZNep&#8221;.Add 6 &#956;l of DZNep stock solution (see step 1.5) to the experimental tube labeled &#8220;5 &#956;M DZNep&#8221; Gently mix the blood and drug by inverting it several times. Incubate the tube 10 min at RT.</li>
	<li>Using a pipette, add 2 ml of control and test blood to the top of correspondingly labeled feeders. Discard the pipette in the bleach solution. Cover the cage with a dark bag and breathe air on cage periodically to encourage females for feeding.</li>
	<li>Let the mosquitoes feed for approximately 30 min. Once feeding is completed, switch off the heating element, take the feeders out and strip the Parafilm. Soak the feeder in bleach solution for about 5 min and rinse well in deionized water.</li>
	<li>Remove the feeders and put cotton balls with sugar water on the cages for 48 hr. Place an egg dish containing water and filter paper in the cages overnight to facilitate egg laying. Label each egg dish with the respective test or control concentration.</li>
	<li>Remove the egg dish next day and examine the number and structure of eggs under a stereo microscope. Obtain images using the Q-colors5 software. Count the number of eggs. Analyze difference in the number of eggs obtained from test and control cages. 
	NOTE: An unpaired t-test can be used to determine if the number of eggs are significantly different (p &#8804;0.05).</li>
</ol>
3. Biochemical Assay of Mosquito Enzyme Inhibition by Drug
NOTE: This section describes an enzyme activity assay using DTNB as an indicator to determine the effect of DZNep on SAH hydrolase inhibition in adult male and female mosquitoes.
<ol>
	<li>Prepare a 0.1 M Na2HPO4 (dibasic sodium phosphate) solution by adding 14.19 g of Na2HPO4 to 1 L of dH2O. Next, prepare a 0.1 M NaH2PO4 (monobasic sodium phosphate) solution by adding 13.79 g of NaH2PO4 to 1 L of dH2O.</li>
	<li>Titrate the 0.1 M Na2HPO4 solution with the 0.1 M NaH2PO4 solution to pH 8.5. 
	NOTE: This will be the working stock solution of 0.1 M Na2HPO4 (pH 8.5).</li>
	<li>Prepare a homogenization solution of 0.1 M Na2HPO4 (pH 8.5) by adding 0.3% Triton X-100. 
	NOTE: The homogenization solution can be stored at 4 oC.</li>
	<li>Prepare a 1.6 mM SAH (MW = 384.4) solution by dissolving 6.15 mg of SAH in 10 ml of 0.1 M Na2HPO4 (pH 8.5).</li>
	<li>Prepare a 1.6 mM DTNB (MW = 396.4) solution by dissolving 6.3 mg of DTNB in 10 ml of 0.1 M Na2HPO4 (pH 8.5). Keep the fresh DTNB solution on ice until use.</li>
	<li>Prepare a 1 mM DZNep.HCl (MW = 298.73) solution by dissolving 0.3 mg of DZNep in 1ml dH2O. Next, dilute the 1 mM DZNep solution into a series of concentrations from 1,000 &#181;M to 0.002 &#181;M in dH2O.</li>
	<li>To measure the SAH hydrolase inhibition by DZNep, prepare a crude enzyme extract of mosquitoes: Homogenize 10 non-blood-fed adult mosquitoes in 1 ml of ice-cold 0.1 M NaH2PO4 (pH 8.5), containing 0.3% Triton X-100, using a glass tissue homogenizer. Transfer the homogenate to a 1.5ml microcentrifuge tube.</li>
	<li>Centrifuge the homogenate for 5 min at 10,000 x g at 4 &#176;C. Transfer the supernatant to a clean 1.5 ml microcentrifuge tube. Use the supernatant as the enzyme source for the SAH biochemical assay.</li>
	<li>For the blank treatment (i.e., no DZNep, no enzyme), add 50 &#181;l 1.6 mM SAH, 50 &#181;l 1.6 mM DTNB, and 100 &#181;l 0.1 M Na2HPO4 (pH 8.5) to the individual wells of a 96-well, flat bottom microplate.</li>
	<li>For the control (i.e., no DZNep), add 50 &#181;l 1.6 mM SAH, 50 &#181;l 1.6 mM DTNB, 50 &#181;l 0.1 M Na2HPO4 (pH 8.5) and 50 &#181;l enzyme (SAH hydrolase obtained from crude extract) to the individual wells.</li>
	<li>For DZNep treatments, add 50 &#181;l 1.6 mM SAH, 50 &#181;l 1.6 mM DTNB, 50 &#181;l enzyme and 50 &#181;l selected DZNep concentration to individual wells. Prepare 4 replicates per treatment and control.</li>
	<li>Read the optical density (OD) of the SAH enzyme samples at 405 nm for 5 min at 20 sec intervals using a 96-well microplate reader.</li>
	<li>Subtract the blank OD from control and treatment OD obtained from each well. Calculate the percent remaining SAH hydrolase activity using the following equation: % Residual Activity = (Treatment OD/Control OD) x 100.</li>
</ol>

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The authors have nothing to disclose.

There are several steps vital to the successful application of this protocol. For the larval assay, care should be taken to correctly label and replicate each test concentration. Randomizing test samples and adding the designated amount of drug to respective test wells is an important part of this experimental set up. Before adding 2nd instar mosquito larvae to a 96-well microplate, each larva can be put on a paper towel for 2-3 sec to remove excess water. To prevent desiccation, larvae must be immediately tra...

Malaria is responsible for the highest number of insect-related deaths in the world. An estimated 219 million cases occur annually worldwide, resulting in approximately 660,000 deaths, primarily in Africa 1. Despite concerted efforts, malaria programs face several challenges. While insecticidal treated bednets and indoor residual spraying form key components of the program, resistance to insecticides in local populations impede these efforts 2. The rapid increase in insecticide resistant mosquito populations is largely attributed to the ability of the malaria mosquitoes to adapt quickly to changes in their environment and exploit different niches 3,4,5. To overcome the existing mechanisms of insecticide resistance, the exploration of novel insecticide targets and next generation compounds is warranted. A simple, step-by-step protocol for determining the efficacy of experimental insecticides on the various life stages of malaria mosquitoes would significantly enhance these efforts.
Pharmacological studies of drug effects on cell lines and animal models have established the use of epigenetic drugs as a useful tool for modulating the genetics and physiology of cells and organisms. DNA methylation and histone modification are two epigenetic mechanisms that affect gene expression in multicellular organisms without changing the underlying DNA sequence 6. Post translational modifications such as methylation play a crucial role in maintaining cellular integrity and gene expression, and may affect several fundamental processes 7,8,9. Research in some insect species have highlighted the importance of epigenetics in processes involving oogenesis and stem cell maintenance 10, as well as dosage compensation 11 . However, such aspects in disease vectors are yet to be explored. Using a compound to modulate this system in mosquitoes may provide us with insights into the novel insecticide target pathways. 3-Deazaneplanocin A (DZNep) is a known histone methylation inhibitor, which impact on various types of cancers have been studied 12,13,14,15,16. DZNep is a stable water-soluble epigenetic drug that indirectly inhibits histone lysine N-methyltransferase (EZH2), a component of the polycomb repressive complex 2 (PRC2) in mammalian cells. PRC2 plays an important role in regulating the growth of stem cells in multicellular organisms, and histone methylation is a key aspect of the PRC2 mediated gene silencing. In immunocompromised mice, cells pre-treated with DZNep have been shown to be less tumorigenic 17. This drug is becoming used for studying other diseases, such as non-alcoholic fatty liver disease, in which EZH2 is implicated 18. DZNep is an established S-adenosylhomocysteine (SAH) hydrolase inhibitor 19,20. The inhibition of SAH hydrolase results in an accumulation of SAH and, in turn, leads to the inhibition of methyltransferase activity by limiting available methyl donor groups. SAH is an amino acid derivative utilized by many organisms, including insects, in their metabolic pathways. A recent study has shown that DZNep in low doses may affect diapause and delay development in insects 21.
Here, a robust protocol to investigate the effects of a water-soluble compound on various life stages of mosquitoes is developed. The three parts of this protocol include instructions for examining the effects of a water-soluble compound on immature mosquitoes, adult blood-feeding females, and enzyme activity of adult male and female mosquitoes. First, DZNep is dissolved in water to study immature mosquito development and survival. This is performed at two concentrations to compare any differences arising from 10-fold increase in drug exposure. To explore the effect of drug on adult female mosquitoes, DZNep is added to defibrillated sheep blood and fed the blood artificially to females. Subsequently, the outcome of the drug on fecundity is examined. Finally, an enzyme activity assay is performed using 5,5&#39;-dithiobis-(2-nitrobenzoic acid) (DTNB) as an indicator to determine the effect of DZNep on SAH hydrolase inhibition in adult male and female mosquitoes. While this protocol is developed with a malaria mosquito, Anopheles gambiae, it can be easily adapted to studying the effects of compounds of interest on any species of mosquito or other insects. The techniques detailed in this protocol may not be efficiently applied to a drug with limited or no solubility in water or aqueous media.

<table><tbody><tr><td>96-well microplate</td><td>Fisher Scientific</td><td>12565561</td><td></td></tr><tr><td>Cell culture plate</td><td>CytoOne</td><td>CC7682-7506</td><td></td></tr><tr><td>Centrifuge</td><td>Sorvall Fresco</td><td>76003758</td><td>A different centrifuge can be used</td></tr><tr><td>Colored tape rolls</td><td>Fisher</td><td>S68134</td><td></td></tr><tr><td>Dissection microscope</td><td>Olympus</td><td>SZ</td><td></td></tr><tr><td>DTNB</td><td>Sigma Aldrich</td><td>D8130</td><td></td></tr><tr><td>DZNep.HCl</td><td>Sigma Aldrich</td><td>SMLO305</td><td></td></tr><tr><td>Egg dish cups</td><td></td><td></td><td></td></tr><tr><td>Filter papers</td><td>Fisher</td><td>09-795E</td><td></td></tr><tr><td>Glass feeders</td><td>Virginia Tech</td><td></td><td></td></tr><tr><td>Glass tissue homogenizer</td><td></td><td></td><td></td></tr><tr><td>Heating element</td><td>Fisher Scientific</td><td>NC0520091</td><td></td></tr><tr><td>Incubator</td><td>Percival scientific</td><td>I36VLC8</td><td>A different incubator can be used</td></tr><tr><td>Microcentrifuge tube, 2&amp;nbsp;ml</td><td>Axygen</td><td>22-283</td><td></td></tr><tr><td>Microcentrifuge tube, 1.5&amp;nbsp;ml</td><td>Axygen</td><td>MCT-150-C</td><td></td></tr><tr><td>Micropipette</td><td>Eppendorf</td><td>4910 000.069</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>M-3154</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>M-8643</td><td></td></tr><tr><td>pH meter&amp;nbsp;</td><td>Mettler Toledo 7easy</td><td>S20</td><td></td></tr><tr><td>Plate reader</td><td>Spectramax</td><td>M2</td><td></td></tr><tr><td>SAH</td><td>Sigma Aldrich</td><td>A9384</td><td>store at -20 &amp;#176;C</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>T8787</td><td></td></tr></tbody></table>

toxicological assays for testing effects of an epigenetic drug on development, fecundity and survivorship of malaria mosquitoes

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making malaria control an omnipresent problem in tropical countries. The emergence of insecticide resistant populations warrants the exploration of novel drug target pathways and compounds for vector mosquito control. Epigenetic drugs are well established in cancer research, however not much is known about their effects on insects. This study provides a simple protocol for examining the toxicological effects of 3-Deazaneplanocin A (DZNep), an experimental epigenetic drug for cancer therapy, on the malaria vector, Anopheles gambiae. A concentration-dependent increase in mortality and decrease in size was observed in immature mosquitoes exposed to DZNep, whereas the compound reduced the fecundity of adult mosquitoes relative to control treatments. In addition, there was a drug-dependent decrease in S-adenosylhomocysteine (SAH) hydrolase activity in mosquitoes following exposure to DZNep relative to control treatments. These protocols provide the researcher with a simple, step-by-step procedure to assess multiple toxicological endpoints for an experimental drug and, in turn, demonstrate a unique multi-prong approach for exploring the toxicological effects of water-soluble epigenetic drugs or compounds of interest against vector mosquitoes and other insects.

Figure 1 is a schematic representation of the assays; it describes the various steps of the procedure listed in this article. As the protocol is based on different life stages of mosquitoes, there is no particular sequence to be followed for the experiments detailed here. The user may choose to conduct one or more assays at the same time, depending upon sample availability.

Figure 2 demonstrates the plate set up for the immature mosquito development an...

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Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes

A protocol is developed to examine the effects of an epigenetic drug DZNep on the development, fecundity and survivorship of mosquitoes. Here we describe procedures for the aqueous exposure of DZNep to immature mosquitoes and a blood-based exposure of DZNep to adult mosquitoes in addition to measuring SAH hydrolase inhibition.

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making ...

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Biology

8.4K Views.  Virginia Tech. A protocol is developed to examine the effects of an epigenetic drug DZNep on the development, fecundity and survivorship of mosquitoes. Here we describe procedures for the aqueous exposure of DZNep to immature mosquitoes and a blood-based exposure of DZNep to adult mosquitoes in addition to measuring SAH hydrolase inhibition. 

Video: Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new infections each year. Despite decades of research, there is still no vaccine against malaria, highlighting the need for novel control strategies. One innovative approach is the use of genetically modified mosquitoes to effectively control malaria parasite transmission. Deliberate alterations of cell signaling pathways in the mosquito, via targeted mutagenesis, have been found to regulate parasite development 1. From these studies, we can begin to identify potential gene targets for transformation. Targeted mutagenesis has traditionally relied upon the homologous recombination between a target gene and a large DNA molecule. However, the construction and use of such complex DNA molecules for generation of stably transformed cell lines is costly, time consuming and often inefficient. Therefore, a strategy using locked nucleic acid-modified oligonucleotides (LNA-ONs) provides a useful alternative for introducing artificial single nucleotide substitutions into episomal and chromosomal DNA gene targets (reviewed in 2). LNA-ON-mediated targeted mutagenesis has been used to introduce point mutations into genes of interest in cultured cells of both yeast and mice 3,4. We show here that LNA-ONs can be used to introduce a single nucleotide change in a transfected episomal target that results in a switch from blue fluorescent protein (BFP) expression to green fluorescent protein (GFP) expression in both Anopheles gambiae and Anopheles stephensi cells. This conversion demonstrates for the first time that effective mutagenesis of target genes in mosquito cells can be mediated by LNA-ONs and suggests that this technique may be applicable to mutagenesis of chromosomal targets in vitro and in vivo.

Oligonucleotides can be used to site specifically substitute a single nucleotide of transfected target genes in both Anopheles gambiae and Anopheles stephensi cells.

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new ...

Immunology and Infection

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 ...

Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections.   Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

Genetics

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. ...

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 ...

Fluorescence In Situ Hybridization to Study Mosquito Spermatogenesis

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural changes to form haploid spermatozoa. Besides the biological aspect, studying spermatogenesis is of paramount importance for understanding and developing genetic technologies such as gene drive and synthetic sex ratio distorters, which, by altering Mendelian inheritance and the sperm sex ratio, respectively, could be used to control pest insect populations. These technologies have proven to be very promising in lab settings and could potentially be used to control wild populations of Anopheles mosquitoes, which are vectors of malaria. Due to the simplicity of the testis anatomy and their medical importance, Anopheles gambiae, a major malaria vector in sub-Saharan Africa, represents a good cytological model for studying spermatogenesis. This protocol describes how whole-mount fluorescence in situ hybridization (WFISH) can be used to study the dramatic changes in cell nuclear structure through spermatogenesis using fluorescent probes that specifically stain the X and Y chromosomes. FISH usually requires the disruption of the reproductive organs to expose mitotic or meiotic chromosomes and allow the staining of specific genomic regions with fluorescent probes. WFISH enables the preservation of the native cytological structure of the testis, coupled with a good level of signal detection from fluorescent probes targeting repetitive DNA sequences. This allows researchers to follow changes in the chromosomal behavior of cells undergoing meiosis along the structure of the organ, where each phase of the process can clearly be distinguished. This technique could be particularly useful for studying chromosome meiotic pairing and investigating the cytological phenotypes associated with, for example, synthetic sex ratio distorters, hybrid male sterility, and the knock-out of genes involved in spermatogenesis.

Author Spotlight: Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito  

Given their simple anatomy, Anopheles testes offer a good cytological model for studying spermatogenesis. This protocol describes whole-mount fluorescence in situ hybridization, a technique used to investigate this biological process, as well as the phenotype of transgenic strains harboring mutations in the genes involved in sperm production.

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact with&#160;the insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects along&#160;a range of insecticide concentrations.
This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. ...

Identifying Kinases Rewiring in Malaria Parasite: A Chemical Genetics Approach

Understanding the Development of Compensatory Pathways in a Mutant Malaria Parasite Harbouring Hypomorphic Allele of Plant-Like Kinases

One of the mechanisms for subverting the effect of drugs by&#160;the malaria parasite is through rewiring of its transcriptome. The effect is more pronounced for target genes belonging to the multigene family. Plasmodium falciparum protein kinases belonging to the CDPK family are essential for blood stage development. As such, CDPKs are considered good targets for the development of anti-malarial compounds. The chemical genetics approach has been historically used to elucidate the function of protein kinases in higher eukaryotes. It requires the substitution of gatekeeper residue for another amino acid with a different side chain through genetic manipulation. Amino acid substitution at the gatekeeper position modulates the activity of a protein kinase and changes its susceptibility to a specific class of compounds known as bumped kinase inhibitors (BKIs) that help in the functional identification of the target gene. Here, we have exploited the chemical genetics approach to understand compensatory mechanisms evolved by a mutant parasite harboring a hypomorphic allele of cdpk1. Overall, our approach helps in identifying compensatory pathways that may be simultaneously targeted to prevent the development of drug resistance against individual kinases.

Author Spotlight: Identifying Compensatory Pathways in Malaria Parasites Containing Hypomorphic Allele of Essential Protein Kinases

Chemical genetics involves the substitution of a gatekeeper residue with an amino acid containing a different side chain at the target locus. Here, we have generated a mutant parasite containing a hypomorphic allele of cdpk1 and identified compensatory pathways adopted by the parasite in the mutant background.

Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes

Summary

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Chapters in this video

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

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

Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

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

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

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

Understanding the Development of Compensatory Pathways in a Mutant Malaria Parasite Harbouring Hypomorphic Allele of Plant-Like Kinases