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>

<ol>
	<li>Gatton, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+mosquito+behavioural+adaptations+to+malaria+control+in+Africa.">The importance of mosquito behavioural adaptations to malaria control in Africa.</a> Evolution; International Journal of Organic Evolution. 67, 1218-1230 (2013).</li><li>Sternberg, E. D., Thomas, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+adaptation+to+temperature+and+the+implications+for+vector-borne+diseases.">Local adaptation to temperature and the implications for vector-borne diseases.</a> Trends in Parasitology. , (2014).</li><li>Rocca, K. A., Gray, E. M., Costantini, C., Besansky, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2La+chromosomal+inversion+enhances+thermal+tolerance+of+Anopheles+gambiae+larvae.">2La chromosomal inversion enhances thermal tolerance of Anopheles gambiae larvae.</a> Malaria Journal. 8, 147 (2009).</li><li>Coluzzi, M., Sabatini, A., Petrarca, V., Di Deco, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+differentiation+and+adaptation+to+human+environments+in+the+Anopheles+gambiae+complex.">Chromosomal differentiation and adaptation to human environments in the Anopheles gambiae complex.</a> Transactions of the Royal Society of Tropical Medicine and Hygiene. 73, 483-497 (1979).</li><li>Donepudi, S., Mattison, R. J., Kihslinger, J. E., Godley, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+Cancer+Therapeutics.">Update on Cancer Therapeutics.</a> Science Direct. 2 (4), 157-206 (2007).</li><li>Greer, E. L., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+methylation:+a+dynamic+mark+in+health,+disease+and+inheritance.">Histone methylation: a dynamic mark in health, disease and inheritance.</a> Nature Reviews. Genetics. 13, 343-357 (2012).</li><li>Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modifications+and+their+function.">Chromatin modifications and their function.</a> Cell. 128, 693-705 (2007).</li><li>Dawson, M. A., Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+epigenetics:+from+mechanism+to+therapy.">Cancer epigenetics: from mechanism to therapy.</a> Cell. 150, 12-27 (2012).</li><li>Clough, E., Tedeschi, T., Hazelrigg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+regulation+of+oogenesis+and+germ+stem+cell+maintenance+by+the+Drosophila+histone+methyltransferase+Eggless/dSetDB1.">Epigenetic regulation of oogenesis and germ stem cell maintenance by the Drosophila histone methyltransferase Eggless/dSetDB1.</a> Developmental Biology. 388, 181-191 (2014).</li><li>Conrad, T., Akhtar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dosage+compensation+in+Drosophila+melanogaster:+epigenetic+fine-tuning+of+chromosome-wide+transcription.">Dosage compensation in Drosophila melanogaster: epigenetic fine-tuning of chromosome-wide transcription.</a> Nature Reviews. Genetics. 13, 123-134 (2011).</li><li>Miranda, T. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DZNep+is+a+global+histone+methylation+inhibitor+that+reactivates+developmental+genes+not+silenced+by+DNA+methylation.">DZNep is a global histone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation.</a> Molecular Cancer Therapeutics. 8, 1579-1588 (2009).</li><li>Cui, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRIMA-1,+a+Mutant+p53+Reactivator,+Restores+the+Sensitivity+of+TP53+Mutant-type+Thyroid+Cancer+Cells+to+the+Histone+Methylation+Inhibitor+3-Deazaneplanocin+A+(DZNep).">PRIMA-1, a Mutant p53 Reactivator, Restores the Sensitivity of TP53 Mutant-type Thyroid Cancer Cells to the Histone Methylation Inhibitor 3-Deazaneplanocin A (DZNep).</a> J. Clin. Endocrinol. Metab. 99 (11), E962 (2014).</li><li>Fujiwara, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-Deazaneplanocin+A+(DZNep),+an+inhibitor+of+S-adenosyl-methionine-dependent+methyltransferase,+promotes+erythroid+differentiation.">3-Deazaneplanocin A (DZNep), an inhibitor of S-adenosyl-methionine-dependent methyltransferase, promotes erythroid differentiation.</a> The Journal of Biological Chemistry. , (2014).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+polycomb+group+protein+EZH2+is+a+novel+therapeutic+target+in+tongue+cancer.">The polycomb group protein EZH2 is a novel therapeutic target in tongue cancer.</a> Oncotarget. 4, 2532-2549 (2013).</li><li>Nakagawa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+therapy+with+the+histone+methyltransferase+EZH2+inhibitor+3-deazaneplanocin+A+inhibits+the+growth+of+cholangiocarcinoma+cells.">Epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A inhibits the growth of cholangiocarcinoma cells.</a> Oncology Reports. 31, 983-988 (2014).</li><li>Crea, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+disruption+of+Polycomb+Repressive+Complex+2+inhibits+tumorigenicity+and+tumor+progression+in+prostate+cancer.">Pharmacologic disruption of Polycomb Repressive Complex 2 inhibits tumorigenicity and tumor progression in prostate cancer.</a> Molecular Cancer. 10, 40 (2011).</li><li>Vella, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EZH2+down-regulation+exacerbates+lipid+accumulation+and+inflammation+in+in+vitro+and+in+vivo+NAFLD.">EZH2 down-regulation exacerbates lipid accumulation and inflammation in in vitro and in vivo NAFLD.</a> International Journal of Molecular Sciences. 14, 24154-24168 (2013).</li><li>Tan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+disruption+of+Polycomb-repressive+complex+2-mediated+gene+repression+selectively+induces+apoptosis+in+cancer+cells.">Pharmacologic disruption of Polycomb-repressive complex 2-mediated gene repression selectively induces apoptosis in cancer cells.</a> Genes &amp; Development. 21, 1050-1063 (2007).</li><li>Chiang, P. K., Cantoni, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perturbation+of+biochemical+transmethylations+by+3-deazaadenosine+in+vivo.">Perturbation of biochemical transmethylations by 3-deazaadenosine in vivo.</a> Biochemical Pharmacology. 28, 1897-1902 (1979).</li><li>Lu, Y. X., Denlinger, D. L., Xu, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycomb+repressive+complex+2+(PRC2)+protein+ESC+regulates+insect+developmental+timing+by+mediating+H3K27me3+and+activating+prothoracicotropic+hormone+gene+expression.">Polycomb repressive complex 2 (PRC2) protein ESC regulates insect developmental timing by mediating H3K27me3 and activating prothoracicotropic hormone gene expression.</a> The Journal of Biological Chemistry. 288, 23554-23564 (2013).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="668">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Name of the Reagent/Equipment&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:192px;">Company</td>
			<td style="width:133px;">Catalgue Number</td>
			<td style="width:125px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well microplate</td>
			<td>Fisher Scientific</td>
			<td>12565561</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture plate</td>
			<td>CytoOne</td>
			<td>CC7682-7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Sorvall Fresco</td>
			<td>76003758</td>
			<td>A different centrifuge can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Colored tape rolls</td>
			<td>Fisher</td>
			<td>S68134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection microscope</td>
			<td>Olympus</td>
			<td>SZ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTNB</td>
			<td>Sigma Aldrich</td>
			<td>D8130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DZNep.HCl</td>
			<td>Sigma Aldrich</td>
			<td>SMLO305</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Egg dish cups</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter papers</td>
			<td>Fisher</td>
			<td>09-795E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass feeders</td>
			<td>Virginia Tech</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass tissue homogenizer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating element</td>
			<td>Fisher Scientific</td>
			<td style="width:133px;">NC0520091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Percival scientific</td>
			<td>I36VLC8</td>
			<td>A different incubator can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tube, &#160;2 ml</td>
			<td>Axygen</td>
			<td>22-283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tube, 1.5 ml</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette</td>
			<td>Eppendorf</td>
			<td>4910 000.069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>M-3154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaH2PO4</td>
			<td>Fisher Scientific</td>
			<td>M-8643</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH meter&#160;</td>
			<td>Mettler Toledo 7easy</td>
			<td>S20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate reader</td>
			<td>Spectramax</td>
			<td>M2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SAH</td>
			<td>Sigma Aldrich</td>
			<td>A9384</td>
			<td>store at -20C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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...

Watch this Scientific Journal Video about Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes at JoVE.com

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 ...

toxicological-assays-for-testing-effects-an-epigenetic-drug-on

Research

JoVE Journal

Biology

8.4K vistas.  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

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

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

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

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

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

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of ...

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 ...

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced, as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population ...

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 ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

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.

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, ...

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 ...