Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates (EAgaL Plates)

Summary

Described is a time and space-saving method to count eggs and determine hatch rates of individual mosquitoes using 24 well tissue culture plates, which can substantially increase the scale and speed of fecundity and fertility assays.

Abstract

Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness parameters, in particular egg production and hatch rates at the individual level, conventional methods have put a substantial burden on investigators due to high labor intensity and laboratory space requirements. Described is a simple method using 24 well tissue culture plate with agarose in each well and digital imaging of each well to determine egg numbers and hatch rates at an individual level with substantially reduced time and space requirements.

Introduction

The control of mosquitoes to protect humans from vector-borne pathogens is an important public health goal, mainly due to the lack of effective vaccines for most of the pathogens carried by mosquitoes. Many studies aim to reduce mosquito fitness in conjunction with a field-applicable population reduction strategy^1,2,3. This includes extensive studies to create transgenic mosquitoes and/or CRISPR/Cas9 knockout lines. Such population modification approaches require a detailed assessment of individual fitness parameters⁴. Conventional laboratory technique....

Protocol

1. Plate preparation

1. Drill holes in 24 well tissue culture plate lids (4−6 holes per well) using a household drill with a ~1.6 mm (1/16 in) bit (Figure 2).
   NOTE: These holes prevent water condensation from agarose to accumulate on the lid, where mosquitoes may lay eggs. The standard size of female Ae. aegypti ("Liverpool" strain) is ~3.11 mm wingspan. Reducing the size of the holes is recommended when using smaller mosquitoes to avoid escape from the plate.
2. On the day prior to the oviposition experiment, wash and rinse the plates thoroughly and soak them in 1−5% bleach for 30−....

Results

Mosquitoes were injected with dsRNA targeting a candidate iron transporter (FeT) or control gene (EGFP), blood-fed, and measured for fecundity and fertility output using the EAgaL plate method, following the procedure described above.

Mosquitoes in which FeT expression was silenced following dsRNA injection exhibited a significant reduction in both egg number and hatch rate (Figure 11A−C). All control and treatment mosquitoes were placed in .......

Discussion

The EAgaL plate drastically reduces labor, time, and space to conduct individual fecundity and fertility assays in Aedes aegypti when compared to the FT method. Preliminary comparison between the FT method and the EAgaL plate resulted in shorter times for all steps (imaging technique was applied to the FT method) (Table 1). As a reference, an estimate of startup and per assay (one 24 well EAgaL plate versus 24 FTs) costs are provided in Table 2.

Materials

Name	Company	Catalog Number	Comments
1.6 mm Φ drill bit			alternatively heated nails can be used
1000 μL pipette tips (long)	Olympus plastics	24-165RL
24-well tissue culture plate	Thermo Scientific	930186	clear, flat-bottom with ringed lid plates
Agarose	VWR	0710-500G
Compact digital camera	Olympus	TG-5/TG-6
Computer (Windows, Mac or Linux)
Deionized water
Fiji (imageJ) software			download from: https://fiji.sc/
Forceps	Dumont		sharp forceps may break mosquito's body
Glass Petri dishes	VWR
Household bleach
Household electric drill
illuminator for stereomicroscope (gooseneck)
P-1000 pipette	Gilson
paint brushes
Rubber bands
SD card			to record digital camera images (DSHC, SDXC should be better)
Spreadsheet software (Microsoft Excel)	Microsoft		Any spreadsheet software works
TetraMin fish food	Tetra		ground with coffee grinder, blender or morter & pestle
Transfer pipetts	VWR	16011-188