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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 techniques to assess the fitness of female mosquitoes includes the individual containment of mated, blood-fed female mosquitoes in 100 mL containers5, modified 50 mL conical tubes, or tubes for Drosophila rearing modified by providing moist surfaces using damp cotton and filter paper discs for oviposition (i.e., egg papers)1,2,6,7. Such methods require a relatively large space (e.g., 30 cm x 30 cm x 10 cm: W x L x H for up to 100 Drosophila tubes) (Figure 1), and the manipulation of individual egg papers for counting eggs and hatching larvae, which can be labor intensive. This manuscript presents a method to count mosquito eggs and determine hatch rates using 24 well plates and agarose as an oviposition surface to circumvent these issues8.
Concurrently, Ioshino et al.9 described a detailed method using 12 and 24 well plates to perform egg counting obtained from individual females. Their protocol represented a significant improvement from conventional methods in saving time and space9. However, the protocol they described continues to use wet filter paper as a surface for oviposition, which requires unfolding each individual paper to get counts, as eggs are often found underneath or in folds. Their protocol also did not include the use of imaging technologies or a method for larval counting.
Presented is an improved method to perform fitness assays for egg number (i.e., fecundity) and hatch rate (i.e., fertility) using agarose as an oviposition surface in a 24 well tissue culture plate format for Ae. aegypti that oviposit on moist surfaces. These plates were named “EAgaL” plates, from Egg, Agarose, and Larva. These 24 well plates provide individual mosquitoes with a minimal surface to lay eggs, thus simplifying and drastically reducing the time and effort needed to count and maintain eggs and hatched larvae for a few days. The EAgaL plate uses translucent agarose for the oviposition surface, which eliminates the need for handling egg papers and finding the eggs and larvae when hatched; photographing each well establishes a long-term archived record of the results and separates the counting process in both time and space from the rearing/handling process, where time is often limited.
1. Plate preparation
2. Mosquito feeding
NOTE: It is critical to use mosquitoes reared under uniform conditions for all treatment groups and control groups, because larval nutrition has an impact on mosquito fitness parameters10,11. Rear larvae under uncrowded conditions with sufficient food. Let female mosquitoes eclose in the presence of males so that mating is ensured, and mature for at least 3 days.
3. Oviposition
4. Egg counting
5. Fertility assessment
NOTE: In 2 days, first instar larvae may begin to eclose in the wells. Wait for 3−5 more days before imaging/counting to ensure that all viable eggs hatch.
6. Perform analysis
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.
The authors declare no conflicts of interest for this study.
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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