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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.
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.
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 strategy1,2,3. 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....
1. Plate preparation
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 .......
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.
We thank Texas A&M Agrilife Research Insect Vectored Diseases Grant Program for funding. We also thank the Adelman lab members for help on developing this method and suggestions when drafting the manuscript, as well as Kevin Myles lab members. We also thank the reviewers and editors for their help to make this manuscript better.
....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 |
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