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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 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.

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 30−60 min at room temperature. Rinse thoroughly under running deionized water and dry them.
   NOTE: This process reduces the chance of fungi and bacteria growing on agarose.
3. Melt agarose at 2% in deionized water and immediately add 500 μL of the molten agarose to each well of the 24 well plates using a 1,000 μL pipette (Figure 3A,B). When the agarose begins to cool and clog the pipette tip, reheat the agarose and use a new pipette tip.
   NOTE: Avoid touching the walls of the well with the pipette tip because it may leave a piece of agarose on the wall where a female may lay eggs, complicating the imaging and counting process.
4. Before use, dry out any condensation on the well walls overnight on a lab bench (Figure 3C,D).
   NOTE: The timeline of the EAgaL plate assay from blood feeding to larval imaging is depicted in Figure 4, which is for colony mosquitoes (Ae. aegypti “Liverpool” reared under 14 h light:10 h dark light cycle, 27 °C, 80% relative humidity, 500 larvae in a 49.5 cm x 29.2 cm x 9.5 cm tray with 2 L of water), under insectary conditions. It is recommended that each laboratory test the EAgaL plate with the mosquitoes being used, especially when using different strains, different mosquito species, as well as different rearing protocols.

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 parameters^10,11. 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.

1. Remove any source of water and/or sugar from the female mosquitoes at least 16 h prior to blood feeding in order to enhance blood feeding.
2. Heat a water circulator for artificial feeding at 37 °C and feed female mosquitoes using vertebrate blood placed in artificial feeders for 15−30 min (Figure 5A).
3. Anesthetize mosquitoes with CO₂ or on ice, transfer them to a glass dish on ice, and select ones that are engorged with blood (Figure 5B) into a container provided with 30% sucrose water for more than 72 h, when females finish excretion and egg development.
   NOTE: If the females are provided with a lower concentration of sucrose water, remove the sucrose water and any wet surfaces after 48 h to prevent the females from ovipositing.

3. Oviposition

1. About 1 h prior to transferring mosquitoes to the plates, add 2−3 drops (~80−120 μL) of water into each plate well using a transfer pipette.
2. At least 72 h after blood feeding, knockdown mosquitoes with CO₂ or on ice, transfer them to a glass dish on ice, and individually place each mosquito on an inverted lid of the 24 well plate on ice (Figure 6A).
   NOTE: This procedure has been applied from Ioshino et al.⁹.
3. Once all 24 mosquitoes have been placed, cover the lid with an inverted plate bottom (Figure 6B), secure the lid and plate with a fresh, new rubber band and place in an environmental chamber (or rearing room) (Figure 6C) until mosquitoes recover (~10−15 min) and turn the plate right side up (Figure 6D).
4. Allow the female mosquitoes to oviposit for 24−48 h and remove females by releasing them from the plates into a large cage.
   NOTE: If it is important to keep track on individual females, anesthetize them by chilling the plates and remove them individually on ice. Delayed oviposition and dark excretions that interfere with egg counting were observed when females were transferred to the plates earlier than 72 h post blood meal (PBM) (Figure 7D).

4. Egg counting

1. Check each well of the 24 well plate, as sometimes mosquitoes lay eggs on the wall of wells and at the margin of the agarose/plastic surface, where they are difficult to resolve in photographs. Using a wet paint brush, move the eggs laid on the wall and edge of agarose to the flat surface of the agarose so that all eggs are in a uniform plane and do not overlap with each other.
   NOTE: The edge of the agarose is typically out of the camera’s focal plane due to the surface tension of the agarose solution.
2. Using forceps, remove any broken legs, wings, and other particles in the wells that may interfere with imaging eggs (Figure 7C).
3. Insert white paper underneath the plate to increase the contrast with dark mosquito eggs prior to imaging using a stereomicroscope illuminator (Figure 7A).
4. Take an image of each well using a compact digital camera in microscope mode, which allows the user to focus on objects as close as 1 cm. This capability allows the camera to be placed directly on the plate to image eggs without a tripod or a stand (Figure 7A). To distinguish individual eggs laid in clumps, use the fine or super-fine mode to capture high-resolution images so that close-up details can be seen.
   NOTE: Clear the memory card of the camera before use to prevent confusion between new and old images.
5. After photographing each well of a plate, take an image of the entire plate with an imaging order label to distinguish each plate later (Figure 7E).
6. Add a thin layer ~5 drops of water (~200 μL) with a transfer pipette to each well to prevent its agarose plug and the eggs from drying and to induce embryo development and hatching. Pay attention to the water levels for the first few hours and check every day, because there may be water loss due to absorption by the agarose or evaporation. Add water when its level is too low for larvae to hatch.
   NOTE: Evaporation of water occurs non-uniformly both within a plate and within a stack of plates. It has been observed that drying occurs in the following order: 1) corner wells (A1, A6, D1, D6) dry the fastest; followed by 2) wells at the outer edge of the plate (A2-A5, B1, B6, C1, C6, D2-D5); and last 3) wells inside. When plates are stacked, the top plate dries the fastest.
7. Transfer the images to a computer with ImageJ (Fiji) and rename the files for easier organization such as “[plate ID]_[well ID].jpg” (Figure 8A,B).
8. Create a spreadsheet file to record egg numbers (i.e., fecundity) and larval numbers and calculate hatch rate (i.e., fertility) (Figure 9E).
9. Open the images with ImageJ (Fiji)¹² and use the “multi-point” tool to mark each egg (Figure 9A−C); zoom-in or zoom-out using “+” or “–” key to count the eggs in clumps. After marking all the eggs, double-click the multi-point icon to bring up the number of marks (Figure 9D). Record the results in a spreadsheet.

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.

1. Prepare larval food by mixing 1/16 tablespoon (~168 mg) of ground fish food (i.e., normal mosquito larval diet) in 20 mL of water and waiting for large solid particles to settle (Figure 10A−D). Start adding food (the supernatant) to the wells that contain hatched larvae as soon as they appear, because they do not survive for long without food.
   NOTE: Excess addition of food particles may interfere with imaging and counting of hatched larvae.
2. Approximately 5–8 days after addition of water to the wells, cool the plate by covering with crushed ice for 15−20 min to anesthetize larvae.
3. Take images of each well while keeping the plates on ice as was done with the eggs. For larval images, provide a dark background by inserting a black material underneath the plate to help enhance the contrast. After photographing each well of a plate, take an image of the entire plate with an imaging order label to distinguish each plate later on.
   NOTE: Images are taken while the plates are on ice because larval movement may compromise the counting. The plates are reusable; freeze and remove the mosquitoes and agarose to clean for the next use. The EAgaL plate is not suitable for rearing larvae to advanced stages.
4. Open images with ImageJ (Fiji) and use the “multi-point” tool to count by clicking on each larva. Over the 3−5 days period some larvae may have molted, especially in wells with low numbers of larvae. Therefore, when counting, exclude the shed cuticles (i.e., exuviae), which look like head-only larvae with a little bit of body, or shrunken larvae (Figure 10E). Record the results in the spreadsheet.

6. Perform analysis

1. Having collected all necessary data for the fecundity and fertility analysis, perform appropriate statistical analysis and create graphs using preferred software.

Representative 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 the EAgaL plates after 72 h PBM. FeT-silenced mosquitoes also exhibited delayed excretion and small and light-colored eggs (Figure 12A,B). Example results can also be found in Tsujimoto et al.⁸.

Figure 1: Fly tubes commonly used for mosquito fecundity/fertility assays. (A) A single tube with damp cotton and circular filter paper with sponge cap. (B) 100 tubes in a rack (~30 cm x 30 cm). Please click here to view a larger version of this figure.

Figure 2: Drilling holes in a lid of 24 well tissue culture plate. (A) Drilling in process. (B) A lid with holes that prevent condensation. Please click here to view a larger version of this figure.

Figure 3: Applying agarose (2% in water) into the wells. (A) Applying agarose using a 1,000 µL pipette. Note that the tip is not touching the wall of the well. (B) A plate containing agarose on the bottom. (C) Wall of a well right after agarose solidified. Note the condensation on the wall. (D) Wall of a well when condensation evaporated. Please click here to view a larger version of this figure.

Figure 4: Timeline from blood feeding (day 0) to larval imaging (day 10−13). Please click here to view a larger version of this figure.

Figure 5: Artificial blood feeding and selection of engorged females. (A) Blood feeding using artificial feeder. (B) Selection of engorged females on ice. Please click here to view a larger version of this figure.

Figure 6: Transfer of females to EAgaL plate. (A) Transfer anesthetized females to an inverted plate lid on ice. (B) Then carefully place the inverted agarose-containing plate onto the inverted lid and (C) remove plate with lid attached from the ice and keep it an inverted position until females have recovered from anesthesia. (D,E) Turn the female-containing plate around to the upward position. Note that lid and bottom part of the plate are held by a rubber band and that the bottom part is labeled. Please click here to view a larger version of this figure.

Figure 7: Imaging of each well after females were removed. (A) Digital camera on top of an egg-containing 24 well plate for imaging. (B) Typical image of a well. (C) A well containing a leg, which must be removed. (D) Sample image of a well in which a female had been placed at 48 h PBM. Note the dark excretion marks, which can complicate egg counting (arrows). (E) Entire plate showing an imaging order label prepared following the imaging of all wells of the plate. This helps recognize wells during analysis. Please click here to view a larger version of this figure.

Figure 8: Renaming the images for better organization. (A) Images of individual egg-containing wells including an image order label with the names automatically assigned by the camera. (B) The same images renamed with plateID_wellID.jpg format. Please click here to view a larger version of this figure.

Figure 9: Counting eggs using “Fiji” (ImageJ2) software. (A) Screenshot of Fiji software showing the “multi-point” tool highlighted (turquoise square). (B) A well image with Fiji count marks on the eggs. (C) Zooming in helps when counting larger egg groups. (D) Double-clicking the “multi-point” tool icon on the main window shows count (red circle). (E) An example of a spreadsheet with hatch rate calculation formula. Please click here to view a larger version of this figure.

Figure 10: Larval diet preparation and a well containing larvae and exuviae. (A) Ground fish food. (B) 20 mL of water in a cup. (C) Mixture of ground fish food and water. (D) Food/water settled for 15 min. Take supernatant of this mixture as larval food. (E) Well with molted larvae; representative exuviae are indicated by arrows. Please click here to view a larger version of this figure.

Figure 11: Count data from a gene silencing experiment. Mosquitoes injected with dsRNA against putative iron transporter (FeT) exhibited significant reduction of (A) egg number, (B) larval number, and (C) hatch rate in comparison to control (EGFP). Bars show mean ± SD.  Please click here to view a larger version of this figure.

Figure 12: Representative images of the wells showing additional phenotypes in gene silenced mosquitoes. dsFeT showed delayed excretion (dark brown marks) and small, lightly colored eggs. Two representative wells for each treatment are shown. Please click here to view a larger version of this figure.

	FT	EAgaL
(1) Prep	20 min 29.0 sec	3 min 49.3 sec
(2) F_in	3 min 55.0 sec	1 min 56.0 sec
(3) F_out/E_img	43 min 44.3 sec	15 min 28.3 sec
(4) L_img	38 min 03.5 sec	9 min 30.5 sec

Table 1: Time requirements for completion of the fly tube method (FT) in comparison to the EAgaL plate method. (1) Prep: Time required to place cotton, pour water in, and insert filter paper disc into Drosophila rearing tubes (FT) versus pouring of agarose into wells of 24 well plate (EAgaL). (2) F_in: Time required to place individual mosquitoes into rearing tubes (FT) or wells of the 24 well plate (EAgaL). (3) F_out/E_img: Time required to release mosquito into a larger cage, remove, unfold egg paper, and image the eggs on the paper (FT) or release mosquitoes in a larger cage and image every well of the 24 well plate (EAgaL). (4) L_img: Time required to image larvae hatched in a small container (FT) or each well of the 24 well plate (EAgaL) after cold anesthesia. A total of 24 tubes were used for FT.

Bulk costs
EAgaL plate			Fly tubes
Item	Price	Quantity	Item	Price	Quantity
24-well plates	$45.61	Case of 50	Chromatography paper sheets	$103.63	46 × 57 cm 100 Sheets/PK
Agarose	$250.97	500 grams	Fly tube racks + tubes	$68.45	5 trays of 100
Olympus TG-6	$375.00		Fly tube plugs	$66.10	Case of 200
			Cotton balls	$104.27	Case of 2000
Startup total	$671.58		Startup total	$342.45
Unit cost (a 24-well plate or 24 tubes)
EAgaL plate			Fly tubes
Item	Price	Note	Item	Price	Note
a 24-well plate	$0.91	bulk price/50	Chromatography paper sheets	$0.16	641.7 sets of 24 tubes worth(2)
Agarose per plate	$0.15	500g= 1667 plates(1)	Fly tube racks + tubes	$3.29	(bulk price/500) × 24
			Fly tube plugs	$7.93	(bulk price/200) × 24
			Cotton balls	$1.25	(bulk price/2000) × 24
Unit total	$1.06		Unit total	$12.63

Table 2: Cost comparison between the EAgaL plate and FT. Top: Bulk costs for startup (assuming a drill and a drill bit can be provided by a researcher). Bottom: Estimated costs for one 24 well plate (EAgaL plate) and 24 FT. (1) Assuming a plate requires slightly more than just enough for a 24 well plate (12mL), 15 mL of 2% = 0.3 g of agarose per plate, a total of 500 g of agarose can make 1,667 plates. (2) One sheet of paper can make 154 of ~38 mm diameter discs. With 100 sheets, 641.7 (15,400/24) sets of 24 tubes can be made.

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.

There are a few points to consider when using the EAgaL plates. An initial concern was that mosquitoes placed in such a small space may not lay all eggs due to limited movement. To determine if this was the case, the mosquitoes were transferred collectively to a larger cage with an oviposition cup lined with a paper towel with water for an additional 48 h after they spent 48 h in the EAgaL plates. The mosquitoes did indeed lay additional eggs, but the average number of eggs per female was only 2.1, which does not result in any difference in the outcome of any statistical analysis in most, if not all, cases. These numbers are from more than 500 mosquitoes tested (data not shown). However, this may be solely for the Ae. aegypti “Liverpool” mosquito strain with the conditions described. Each laboratory may need to test whether this is the case for its mosquitoes and conditions.

For imaging, the single image of a camera attached to a stereomicroscope did not cover an entire well even at the lowest magnification. This required obtaining multiple images per well and in turn patching the images, or keeping track of eggs overlapped in multiple images of the same well. Both approaches severely complicated the analysis and significantly increased the labor involved. Moreover, due to the nature of a stereomicroscope, the camera angle is always slightly left or right from the perpendicular angle, which makes the left or right side wall block a part of the agarose surface.

Contamination by microorganisms, especially fungi, can be a problem during the assay. Although bleaching can minimize the contamination before the oviposition, fungi may be present in the insectary environment and carried by the mosquitoes themselves. In such cases, keeping insectary spaces clean may reduce the incidence. It is best for every lab to test an EAgaL plate to detect any potential issues.

Note that the EAgaL plate method was not designed to maintain mosquito cultures beyond the early larval stages. The average number of larvae per well was typically over 60, and it is not unusual to have more than 100 larvae per well. This creates crowded conditions, which result in a delay in development, lower pupation rate, and very small adults, which may compromise downstream studies.

Currently this method has only been tested with Ae. aegypti. However, it is currently being tested to expand its application to other species of Aedes and even other genera of mosquitoes such as Anopheles and Culex.

Because of the reduced time and space requirements for the EAgaL plate method, the fecundity and fertility assays can be scaled up to semi-high throughput (i.e., 5–10 plates or more per experiment). This feature of the EAgaL plate method may be extremely useful to assess the important fitness parameters of mosquitoes for insecticide testing, sterility evaluation, transgenesis, and gene editing studies.

Materials

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