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

Summary

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.

Abstract

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.

Introduction

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'-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.

Protocol

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.

1. Hatch A. gambiae eggs at 28 °C in distilled water (dH₂O) and rear them to 2^nd instar larvae.
2. Take a six-well DNAse, RNAse free cell culture plate and pour 10 ml dH₂O to each well.
3. Select mosquito larvae at 2^nd instar and add 15 larvae per well. Before adding 2^nd instar larvae to plate, put them on a paper towel for 2-3 sec to remove excess water.
4. Label wells with color-coded tapes to randomize experiment. Label 2 wells per test concentration on each plate (Figure 2).
5. 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 µM and 5.0 µM.
   NOTE: The stock solution can be stored at 4 °C for short term (1 day to a month) or -20 °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.
6. Add equal amount of flake fish food to each well. Cover the plates and incubate them at 28 °C in an incubator.
7. 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.
8. 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.

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.

1. 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).
2. 2 hr prior to the blood feeding, remove the cotton balls to ensure females are hungry.
3. 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’ outlets (diameter= 10 mm) via connectors for inflow and outflow of water. Label the two feeders as “Control” and “5 μM DZNep.”
4. Using a commercially available heating element, set up the program for feeding. In “Menu” go to “Settings” and select “Setpoints”; preloaded programs will appear. Select “SP1” and set the temperature for 37 °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.
5. Fill a bucket with water and immerse the heating element into the water.
6. 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.
7. Assemble the system, connecting the feeders and heating element by the tubes. Once completed, switch on the system and select “SP1”. Press “Enter” to start the system.
   NOTE: The water will get heated to 37 °C and maintained at that temperature.
8. 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 °C.
9. Fill a flat bottom tray with 10% bleach solution that will be used to decontaminate any blood spills.
10. Add 2 ml of defibrillated sheep’s blood to a microcentrifuge tube. Label this as “Control”. Repeat the process for the experimental tube labeled as “5 μM DZNep”.Add 6 μl of DZNep stock solution (see step 1.5) to the experimental tube labeled “5 μM DZNep” Gently mix the blood and drug by inverting it several times. Incubate the tube 10 min at RT.
11. 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.
12. 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.
13. 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.
14. 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 ≤0.05).

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.

1. 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.
2. 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).
3. 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.
4. 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).
5. 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.
6. 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 µM to 0.002 µM in dH₂O.
7. 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.
8. Centrifuge the homogenate for 5 min at 10,000 x g at 4 °C. Transfer the supernatant to a clean 1.5 ml microcentrifuge tube. Use the supernatant as the enzyme source for the SAH biochemical assay.
9. For the blank treatment (i.e., no DZNep, no enzyme), add 50 µl 1.6 mM SAH, 50 µl 1.6 mM DTNB, and 100 µl 0.1 M Na₂HPO₄ (pH 8.5) to the individual wells of a 96-well, flat bottom microplate.
10. For the control (i.e., no DZNep), add 50 µl 1.6 mM SAH, 50 µl 1.6 mM DTNB, 50 µl 0.1 M Na₂HPO₄ (pH 8.5) and 50 µl enzyme (SAH hydrolase obtained from crude extract) to the individual wells.
11. For DZNep treatments, add 50 µl 1.6 mM SAH, 50 µl 1.6 mM DTNB, 50 µl enzyme and 50 µl selected DZNep concentration to individual wells. Prepare 4 replicates per treatment and control.
12. 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.
13. 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.

Results

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...

Discussion

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 2^nd 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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-well microplate	Fisher Scientific	12565561
Cell culture plate	CytoOne	CC7682-7506
Centrifuge	Sorvall Fresco	76003758	A different centrifuge can be used
Colored tape rolls	Fisher	S68134
Dissection microscope	Olympus	SZ
DTNB	Sigma Aldrich	D8130
DZNep.HCl	Sigma Aldrich	SMLO305
Egg dish cups
Filter papers	Fisher	09-795E
Glass feeders	Virginia Tech
Glass tissue homogenizer
Heating element	Fisher Scientific	NC0520091
Incubator	Percival scientific	I36VLC8	A different incubator can be used
Microcentrifuge tube, 2 ml	Axygen	22-283
Microcentrifuge tube, 1.5 ml	Axygen	MCT-150-C
Micropipette	Eppendorf	4910 000.069
Na2HPO4	Fisher Scientific	M-3154
NaH2PO4	Fisher Scientific	M-8643
pH meter	Mettler Toledo 7easy	S20
Plate reader	Spectramax	M2
SAH	Sigma Aldrich	A9384	store at -20 °C
Triton X-100	Sigma Aldrich	T8787