Materials and reagents

Biological materials

Hosts for blood feeding

Uranotaenia lowii feed from anuran hosts in nature [9]. In contrast to mosquito species from genera like Aedes and Anopheles, which can often be raised using artificial blood diets [13], it is recommended to feed frog blood for the rearing of Ur. lowii. Field host surveys show that this species feeds on frogs from different families [9], so a variety of anuran species are likely to support a colony of Ur. lowii. We recommend, however, the following hosts to be used for blood feeding, as they successfully promote robust egg production: barking tree frog (Dryophytes gratiosus), Cuban tree frog (Osteopilus septentrionalis), cane toad (Rhinella marina), and southern toad (Anaxyrus terrestris) [12,11]. These anuran species can be procured from local shops or online stores (e.g., https://joshsfrogs.com/ or https://www.backwaterreptiles.com/).

Note: Protocols for using anurans for blood feeding must be approved by the appropriate Institutional Animal Care and Use Committee.

Uranotaenia lowii

This protocol describes the rearing and maintenance of Ur. lowii strain MFRU-FL (NCBI BioSample: SAMN33601576). It is to be expected, however, that these guidelines are transferable to other strains of this species. This protocol also provides a foundation for developing similar approaches to maintaining colonies of other Uranotaenia frog-biting species (i.e., de Silva et al. [14]). The colonies were originally established from egg rafts obtained from the USDA-ARS-CMAVE Mosquito and Fly Research Unit (Gainesville, FL, USA).

Reagents

1. Deionized water

2. Potbelly pig chow powder finely sieved (i.e., Mazuri^® Mini Pig Youth); grind using a porcelain mortar and pestle (Letoyi, catalog number: B08LB3TJL4) and sieve through a fine mesh strainer (Clscea, catalog number: B0CBDFW92V)

3. Bovine liver powder (i.e., MP Biomedicals, catalog number: 290039601)

4. Brewer’s yeast (i.e., MP Biomedicals, catalog number: 290331205)

5. White sugar (e.g., Domino Cane Granulated Sugar, catalog number: 83FF)

6. Bleach (Clorox, catalog number: 30966, concentrated regular bleach)

7. 70% ethanol (sterile 70% denatured ethanol) (Texwipe, catalog number: TX3265)

1. Liver powder suspension (larval food) (see Recipes)

2. Sucrose solution [10% (w/v) adult food] (see Recipes)

Recipes

1. Liver powder suspension (larval food)

   Reagents	Quantity or Volume
   Bovine liver powder	3 g
   Brewer’s yeast	2 g
   Deionized H2O	100 mL

   Note: Shake well before each use. Can be stored at 4 °C for a week.




2. Sucrose solution (adult food)

   Reagents	Quantity or Volume
   White sugar (e.g., Domino Cane Granulated Sugar)	10 g
   Deionized H2O	100 mL

   Note: Mix the reagents well before use.

Laboratory supplies

While we provide manufacturers’ names and models for some items, we are not endorsing or promoting those specific products. Instead, we offer them as examples and, when possible, suggest low-cost alternatives to facilitate accessibility for a wider range of laboratories.

1. Cotton balls, size: 5 cm, prepared using any cotton roll (e.g., Intrinsics, catalog number: 227200)

2. Transfer pipettes [Fisherbrand Transfer Pipette, 4.6 mL (Fisher Scientific, catalog number: 13-711-274MD)], or wood applicator sticks (i.e., Fisher Scientific, catalog number: 22-363-158), or plastic spoons obtained from stores for transferring egg rafts

3. Scissors (8-inch titanium scissors) (Westcott, catalog number: 13901)

4. Brown, non-white, seed germination paper (38-lb regular weight creped seed germination paper) (Anchor Paper Company, catalog number: NC1466201). Prepare strips of 17.5 cm length × 8 cm width using scissors

5. Labeling tape (Write-On label tape, white, 1/2" roll) (Research Products International Corp, catalog number: 560172)

6. Manual insect aspirator (Forestry Suppliers, catalog number: 53758)

7. Digital thermometer and hygrometer (i.e., ThermoPro TP49 Digital Hygrometer; temperature range: 14 °C–70 °C; humidity range: 10%–99%)

8. Oviposition and emergence cups: plastic cup (Rubbermaid 2-Cup, catalog number: 7J60)

9. Larval rearing trays; plastic trays, size: 38 cm length × 30 cm width × 7 cm depth (any polypropylene plastic tray can be used, e.g., United Scientific Supplies, catalog number: 81702)

10. Shipping supplies

    1. Cardboard box (small to medium to fit the Styrofoam box; Uline outer shipping carton with insulated Foam Shipping Kit, catalog number: S-12682)

    2. Styrofoam box with a lid (Uline Insulated Foam Shipping Kit, catalog number: S-12682)

    3. Bubble wrap (Uline Air Bubble Wrap Roll, catalog number: S-5120)

    4. Small Petri dishes (i.e., one 75 mm diameter petri dish can contain around 10 egg rafts) (non-sterile transparent Polypropylene Petri dish: United States Plastic Corp., catalog number: 89824)

    5. Germination paper cutout in circles (diameter determined by Petri dish diameter) (Anchor Paper Company, 38 lb regular weight creped seed germination paper, catalog number: NC1466201)

    6. Ice packs (two minimum) (Uline cold packs, catalog number: S-7890)

    7. Industrial tape 2 Mil (Uline, catalog number: S-423)

Equipment

1. Environmental chamber

   The use of environmentally controlled chambers (insectaries) dedicated to mosquito rearing is encouraged (e.g., Percival WE-35VL, Boone, IA). However, it is possible to maintain the colony in a small room in which temperature and humidity are maintained close to target values (temperature: 22–28 °C, humidity 50%–80%, 12:12 h light/dark photoperiod).

   Note: If there is no environmental chamber, the relative humidity within the cage can be increased by placing a humidifier (e.g., Vick humidifier, catalog number: V745A/V745-JUV; not restricted to any specific manufacturer) near the rearing cage and covering the entire setup with a plastic shelf cover (e.g., Formosa covers premium wire shelf cover, heavy-duty storage solution for wire shelving rack, #shelf 4919 pvc off white). The humidifier must be regularly filled with deionized water to regulate the relative humidity in the enclosure.

2. Frog restraining cage

   A cylindrical cage (height: 8 cm; diameter 12 cm) made entirely of mesh. The top mesh can be replaced by an acrylic circular sheet (i.e., PET Thick Plexiglass sheet; 3 mm, Uline Acrylic sheet, catalog number: S-22486) to observe blood-feeding behavior during experiments. The frog restraining cage should be made of a robust mesh that has holes big enough (1 cm) for the mosquitoes to enter but not too large for the frog to escape (e.g., Home Intuition Heavy Duty Plastic Gutter Guards Mesh, catalog number: B07D91Y8BC).

   Note: The use of a frog restraining cage is essential for limiting the movement of the frogs to increase the likelihood of blood feeding. Limited host movement promotes feeding opportunities and reduces frog defensive behaviors such as swatting, a common strategy used by anurans to repel attacking mosquitoes and midges (i.e., de Silva et al. [15]). Additionally, restraining the frogs prevents them from consuming mosquitoes. It is also advisable to provide a substantial meal for the frog before placing it in the restraining cage, so they are less motivated to eat the mosquitoes.

3. Mosquito rearing cage

   Mosquito cages that provide access through a sleeve (e.g., BioQuip lightweight aluminum collapsible cages; 46 cm × 46 cm × 46 cm; 2 mm × 2 mm mesh size; Mosquito Rearing Cage, catalog number: 4S4545).

Procedure