Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Blood-feeding procedures were not performed using live animals or human hosts and complied with the guidelines set by The Rockefeller University Institutional Animal Care and Use Committee (IACUC) and Institutional Review Board (IRB).

1. Meal preparation

1. Preparation of the phagostimulant, adenosine 5′-triphosphate
   1. Prepare a 25 mM solution of aqueous NaHCO₃ (molecular weight, MW = 84.006 g/mol). For 100 mL of 25 mM NaHCO₃, add 210 mg of NaHCO₃ to a volumetric flask and fill with double-distilled water (ddH₂O) to a total volume of 100 mL. Using a magnetic stir bar, thoroughly mix the solution until all the NaHCO₃ is dissolved.
   2. Reconstitute ATP disodium salt hydrate (MW = 551.14 g/mol) in the aqueous 25 mM NaHCO₃ to a final concentration of 200 mM ATP. For a total volume of 10 mL of 200 mM ATP in 25 mM NaHCO₃ buffer, add 1.1 g of ATP disodium salt hydrate to a volumetric flask and fill with 25 mM NaHCO₃ buffer to a total volume of 10 mL. Using a magnetic stir bar, thoroughly mix the solution until all the ATP is dissolved.
      NOTE: To minimize hydrolysis of ATP, it must be buffered by a salt solution such as NaHCO₃.
   3. Aliquot the ATP solution and store at -20 °C.
      NOTE: This stock solution of ATP is typically made fresh every six months and is used for all meals described below. To prevent degradation, ATP aliquots should not undergo multiple freeze-thaw cycles or be heated along with other meal components.
2. Preparation of the fluorescent tracer solution, fluorescein
   1. Prepare a 2% (w/v) stock solution of aqueous fluorescein. For a total stock solution volume of 10 mL, mix 0.2 g of fluorescein disodium salt with 10 mL of ddH₂O in a 15 mL conical tube wrapped in aluminum foil at room temperature. This stock solution of fluorescein can be used for dilution in all meals described below.
      NOTE: As fluorescein is light-sensitive, avoid exposure to light by wrapping containers in aluminum foil.
3. Preparation of animal-derived blood meals
   1. Calculate the number of meals needed to feed all mosquitoes; each Glytube holds a 2 mL meal and feeds approximately 25 mosquitoes. Prepare one additional meal to calibrate the standard curve for fluorescence readings. Unless stated otherwise, all steps in this section describe reagent amounts required to prepare one meal with a final volume of 2 mL.
   2. For animal-derived blood meals, transfer 1.98–2 mL defibrinated sheep blood into a 15 mL conical tube (see step 3.3 for desired volume of blood).
      NOTE: Commercially defibrinated sources of vertebrate blood, including from sheep, guinea pigs, and humans, may be used¹³. Prior to use, ensure that the purchased blood has not passed its expiry date and mix it well by inverting the bottle, especially if there is visible separation of blood components.
   3. For optimal feeding, add ATP to a final concentration of 1–2 mM after the sheep blood has been warmed to 45 °C in a water bath. For a final concentration of 1 mM ATP, add 10 μL of the 200 mM ATP stock solution to 1.99 mL of pre-warmed blood and mix. For a final concentration of 2 mM ATP, add 20 μL of the 200 mM ATP stock to 1.98 mL of pre-warmed blood and mix. If ATP is not to be added, warm 2 mL of defibrinated sheep blood.
   4. If fluorescence-based quantification of meal size is to be subsequently carried out, add fluorescein solution to a final concentration of 0.002% (2 μL of 2% fluorescein stock in 2 mL total meal volume). Reduce the volume of blood by the same amount as the fluorescein added. Retain 1 mL of the final meal formulation containing 0.002% fluorescein to generate the reference standard curve. Treat the retained volume identically to the meal that is being delivered to mosquitoes; expose to the same light and temperature conditions throughout the duration of the experiment, and subsequently freeze this along with the delivered meal.
4. Preparation of artificial blood meals
   1. Calculate the number of meals needed to feed all mosquitoes; each Glytube holds a 2 mL meal and feeds approximately 25 mosquitoes. Prepare one additional meal to calibrate the standard curve for fluorescence readings. Unless stated otherwise, all steps in this section describe reagent amounts required to prepare one 2 mL meal.
   2. To prepare artificial blood (adapted from Kogan (1990)²²), as in Table 1, first make a stock solution of 400 mM NaHCO_3. For a total volume of 10 mL of 400 mM NaHCO₃ (MW = 84.006 g/mol), add 336 mg of NaHCO₃ to a volumetric flask and fill with double-distilled water (ddH₂O) to a total volume of 10 mL. Using a magnetic stir bar, thoroughly mix the solution until all the NaHCO₃ is dissolved.
   3. For the protein components of artificial blood, prepare stock solutions of 50 mg/mL of γ-globulins in 400 mM NaHCO₃, 35 mg/mL of hemoglobin in ddH₂O, and 300 mg/mL of albumin in ddH₂O. Protein stock solutions can be stored at 4 °C for up to 2 months. The final concentration of total human proteins in artificial blood is 125 mg/mL. This includes final concentrations of 15 mg/mL γ-globulins, 8 mg/mL hemoglobin, and 102 mg/mL albumin.
   4. For each 2 mL meal, combine 600 µL of γ-globulins, 460 µL of hemoglobin, 680 µL of albumin, and 250 µL of ddH₂O from stock solutions listed in Table 1. Wait to add 10 µL of 200 mM ATP stock solution until after the meal has been warmed to 45 °C, immediately before presenting the meal.
   5. If fluorescence-based quantification of meal size is to be subsequently carried out, add fluorescein solution to a final concentration of 0.002% (2 μL of 2% fluorescein stock in 2 mL total meal volume). Reduce the volume of ddH₂O in step 4.4 by the same amount as the fluorescein added. Retain at least 1 mL of the final meal formulation containing 0.002% fluorescein to generate the reference standard curve. Treat the retained volume identically to the meal that is being delivered to mosquitoes; expose to the same light and temperature conditions throughout the duration of the experiment, and subsequently freeze this along with the delivered meal.
5. Preparation of example protein-free saline meals (adapted from Duvall et al. (2019)⁷)
   NOTE: Protein-free saline meals can be prepared in multiple ways^7,27,32. The saline meal presented here is a protein-free version of the artificial blood recipe described above.
   1. Calculate the number of meals needed to feed all mosquitoes; each Glytube holds a 2 mL meal and feeds approximately 25 mosquitoes Prepare one additional meal to calibrate the standard curve for fluorescence measurements. Unless stated otherwise, all steps in this section describe reagent amounts required to prepare one 2 mL meal.
   2. To prepare the saline meal, make a stock solution of 400 mM NaHCO₃. For a total volume of 10 mL of 400 mM NaHCO₃ (MW = 84.006 g/mol), add 336 mg of NaHCO₃ to a volumetric flask and fill with ddH₂O to a total volume of 10 mL. Using a magnetic stir bar, thoroughly mix the solution until all the NaHCO₃ is dissolved.
   3. For each 2 mL meal, combine in a 15 mL conical tube 600 µL of 400 mM NaHCO₃ with 1.39 mL of ddH₂O. Wait to add 10 µL of the 200 mM ATP stock solution until after the meal has been warmed to 45 °C in a water bath.
   4. If fluorescence-based quantification of meal size is to be subsequently carried out, add fluorescein solution to a final concentration of 0.002% (2 μL of 2% fluorescein stock in 2 mL of total meal volume). Reduce the volume of ddH₂O in step 5.3 by the same amount as the fluorescein added. Retain at least 1 mL of the final meal formulation containing 0.002% fluorescein to generate the reference standard curve. Treat the retained volume identically to the meal that is being delivered to mosquitoes; expose to the same light and temperature conditions throughout the duration of the experiment, and subsequently freeze this along with the delivered meal.

2. Meal delivery to mosquitoes

1. Setting up mosquito containers for feeding
   NOTE: Mosquitoes can be fed in a variety of containers as long as the following criteria are met. Ensure that the container is large enough for mosquitoes to fly around in, but not so large that it will be difficult for the mosquitoes to locate the mesh surface and begin feeding. The mesh used to cover the container can vary in material and hole size. The holes must be large enough for the female mosquito’s stylet to pierce through, but not so large that the mosquito can escape. Secure the mesh firmly so that it is taut, and the Glytube can rest stably on its surface throughout the feeding period.
   1. An example container (Figure 1) is a modified 946 mL (32 oz) high density polyethylene (HDPE) plastic bucket. To replicate this setup, use a razor blade to cut a central hole of ~10 cm diameter in the bucket lid. To assemble the container for occupation by mosquitoes, secure a ~400 cm² square piece of white 0.8 mm polyester mosquito netting on top of the bucket, securely pushing the perforated lid down over it to snap tightly.
   2. Collect female mosquitoes that are at least 3 days post-eclosion to ensure that they are mature enough to blood feed. Optimal feeding rates are observed after 7 days³³.
   3. Place female mosquitoes into the container and cover with mesh. If the container is densely populated with mosquitoes, increase the number of Glytubes used. Optimal feeding is achieved with ~25 mosquitoes/Glytube. This reduces competition for access to the feeding membrane.
   4. Set aside a control group of unfed mosquitoes that will not be offered a meal. In the weight measurement protocol, weigh the unfed group separately and use this weight to estimate weight gain in the experimental group that fed on a meal. In the fluorescence-based quantification protocol, add the unfed group of mosquitoes to the wells for the standard curve calculations and for negative controls. To match baseline mosquito tissue autofluorescence in the experimental group, ensure that the standard curve and negative control wells contain an unfed mosquito.
2. Constructing and setting up the Glytube (adapted from Costa-da-Silva et al. (2013)²⁰)
   1. As depicted in Figure 1, to generate a heat source, fill a 50 mL conical tube with 40 mL of 100% glycerol. Seal the open conical tube with a 5 cm × 5 cm piece of parafilm and repeat with an additional piece of 5 cm × 5 cm parafilm to minimize the chance of leakage. Optionally, the parafilm can be held in place using rubber bands. Invert the tube to ensure that there are no holes or gaps.
   2. To create the meal delivery device, cut a centered hole of 2.5 cm diameter in the screw cap of the conical tube using a sharp razor blade or, for better consistency, a lathe. Stretch a 5 cm × 5 cm piece of parafilm evenly so that it roughly doubles in size. The parafilm should be thin enough that mosquitoes can easily pierce through it, but there should be no leaks. Seal over the outer surface of the screw cap to fully cover the hole and set the cap aside.
      NOTE: To increase attraction to the Glytube, prior to stretching the parafilm, perfume it with human odor by gently rubbing it on a patch of human skin with no cosmetics applied, taking care that no holes are created. This is recommended if the experiment is not aimed at investigating the sensory cues required for mosquitoes to approach the meal.
   3. Heat both the sealed tube of glycerol and the meal (with all components except ATP) in a 42–45 °C water bath for at least 15 min. Do not pre-heat ATP; add it immediately before starting the experiment.
   4. Add ATP to the warmed meal and vortex thoroughly. Pipette 2 mL of the warmed meal into the inner chamber of the screw cap and gently place the inverted, warmed, glycerol-filled 50 mL conical tube in it. Partially screw the cap with the meal onto the glycerol-filled tube—just enough to prevent leakage of the meal or the glycerol.
      NOTE: The meal volume used can range between 1 mL and 2.5 mL. Lower volumes may be especially useful when meals are used to deliver compounds that are scarce or expensive. It is important to work quickly at this step so that the meal does not cool down to ambient temperature and reduce the likelihood of maximal feeding. The rate of cooling will depend on the ambient temperature of the room where these steps are conducted, but they should typically be completed within 5 min at 25 °C.
   5. Place the assembled Glytube on top of the mosquito container and allow the mosquitoes access to feed for at least 15 min to achieve maximal feeding rates.
   6. For optimal feeding, place mosquito containers inside a chamber equipped with a CO₂ pad, and allow at least 15 min of acclimation at 25–28 °C and 70–80% humidity prior to delivering the meal. The assay chamber used here is a simple and low cost modification of a previously published setup¹⁶. It uses a translucent polypropylene storage box of size 36 cm L × 31 cm W × 32 cm H with a removable lid. A 1.5 cm diameter hole made in the chamber wall allows CO₂ delivery through silicone tubing. The CO₂ diffusion pad is affixed to the inner center of the lid for delivery of purified air and CO₂ to condition the chamber atmosphere during the trial.
      NOTE: Make sure host cues (heat and CO₂, with optional host odor¹⁶) are present so that the mosquitoes are attracted to the membrane feeder. If mosquitoes are not crowding underneath the Glytube, check that CO₂ is properly delivered and that the meal and Glytube are sufficiently warm. If an external CO₂ source is not available, CO₂ can be delivered via puffs of exhaled human breath.
   7. After feeding, the Glytube cap can be discarded as biohazard waste or reused after soaking in a low percentage bleach solution and thoroughly rinsing in water.

3. Quantification of consumed meals

1. Weighing mosquitoes to be used for further experiments
   NOTE: Weighing mosquitoes to quantify meal size allows them to be used for further live experimentation, but this method requires taking weight measurements from a group of 5 mosquitoes. Since weights of individual mosquitoes are difficult to precisely measure using most laboratory balances, variability in individual meal size cannot be easily quantified by measuring weights. Weighing is only recommended for situations in which females visibly engorge on the meal.
   1. Cold anesthetize mosquitoes by moving their container to a 4 °C cold room or placing it on ice.
   2. Weigh groups of 5 females from the unfed cohort (i.e., mosquitoes that were never offered a meal) and calculate their average weight as the estimate of the “pre-feeding” weight. The average weight of an unfed mosquito depends on genotype, sex, and rearing conditions. Unfed female Ae. aegypti mosquitoes reared with ad libitum access to sucrose typically weigh approximately 2 mg each.
   3. From the experimental cohort (i.e., mosquitoes that were offered a meal), sort females into “fed” and “not fed” piles based on the abdominal distension observable by eye⁷. Divide each of the “fed” and “not fed” piles, respectively, into groups of 5 mosquitoes for weighing. Mosquitoes within each group of 5 should be derived from the same experimental cohort for taking group weight measurements. Calculate the average weight per female from each of the “fed” and “not fed” piles of the experimental group.
2. Fluorescence measurement for end-point analysis^7,27,34
   NOTE: To obtain precise meal size measurements from individual mosquitoes that are no longer required for further live experimentation, store the mosquitoes and the remaining 1 mL of meal containing 0.002% fluorescein at -20 °C immediately after feeding. The experiment can be paused here. This method is outlined in Figure 2.
   1. To generate a reference standard curve, prepare a serial dilution of the same meal containing 0.002% fluorescein that was offered to the experimental group of mosquitoes. There will be a total of 8 standard curve solutions. In each of these solutions, the final volume of meal containing 0.002% fluorescein will be 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078125, or 0 µL, and each will be in 1x phosphate-buffered saline (PBS) for a total volume of 100 µL (e.g., 5 µL of meal containing 0.002% fluorescein in 95 µL of 1x PBS).
   2. To make the first solution of the standard curve, add 50 µL of meal containing 0.002% fluorescein to 950 µL of 1x PBS and vortex thoroughly (final volume: 5 µL of meal containing 0.002% fluorescein in 95 µL of 1x PBS). To make the rest of the standard curve solutions, perform a 2-fold dilution for each step by taking 500 µL from the previous tube and adding it to a new tube containing 500 µL of 1x PBS. Vortex well before preparing the next 2-fold dilution.
   3. To prepare wells to be used for generating a reference standard curve, pipette 100 µL of each of the standard curve solutions into each of the 8 wells in the first column of a 96-well PCR plate. Add 1 unfed control mosquito to each of the same 8 wells in the first column of the plate. Repeat in the second column of the plate for a replicate measurement.
      NOTE: If experimental groups are offered different meal types, a separate reference standard curve must be prepared for each meal type.
   4. Add 100 µL of 1x PBS in each remaining well for the unfed control and experimental groups. If tissue is to be disrupted in subsequent steps using a bead mill homogenizer or vortex, add one 3 mm borosilicate solid-glass bead to each well.
   5. As a negative control, add 1 unfed mosquito to each well in the next 2 columns of the plate. The fluorescence measured in this group sets a baseline cutoff to account for tissue autofluorescence and will be used to determine whether a mosquito in the experimental group fed on the meal.
   6. Add 1 mosquito per well to the remaining wells from the experimental groups that were offered a meal.
   7. Seal the plate carefully and disrupt the tissue by manual grinding. The abdomen should be thoroughly homogenized to release the meal. Methods to disrupt tissue include using a bead mill homogenizer with 3 mm borosilicate solid-glass beads (30 Hz for 30 seconds), vortex mixer with 3 mm borosilicate solid-glass beads, or a pestle grinder without beads.
   8. Centrifuge the plate at 2000 rpm for 1–2 min to collect the lysate.
   9. Prepare a black 96-well plate with 180 µL of 1x PBS in each well.
   10. Transfer 20 µL of lysate to each well with 180 µL of 1x PBS and mix. If available, use a multi-channel pipette at this step for increased speed and better consistency.
   11. Measure fluorescence intensity of each well using a plate reader on the 485/520 excitation/emission channel. Generate the reference standard curve by plotting the known volume of meal against the corresponding fluorescence intensity measurement.
   12. Using the reference standard curve generated, extrapolate the meal volume ingested by each of the experimental group mosquitoes. Subtract the average fluorescence intensity reading of the negative control group of unfed mosquitoes from the fluorescence intensity reading of each experimental group individual to correct for baseline tissue autofluorescence.