Materials and reagents

Biological materials

1. Insect vectors (Delphacodes kuscheli Fennah) obtained from a colony reared in the Vector's Laboratory of IPAVE, originally isolated from the MRCV-endemic area (Río Cuarto County, Córdoba Province, Argentina) and maintained since 2008

2. MRCV isolate (Spinareoviridae: Fijivirus) obtained from infected oat plants of the Río Cuarto endemic area and maintained in wheat (Triticum aestivum cv. ProINTA Federal) since 2008 by serial vector transmissions using D. kuscheli

3. Seeds of wheat (Triticum aestivum) and oat (Avena sativa) obtained by the Wheat Group of IPAVE and the Wheat Improvement, Biotechnology and Pathology Group of the INTA EEA Marcos Juárez (Córdoba, Argentina)

4. Maize seedlings and field trials provided by Syngenta Agro S.A

Trials and breeding room supplies

1. Transparent plastic bottles, 6 L (repurposed water containers) for the fabrication of mass rearing cages

Note: Each bottle features openings at both ends and has windows on the sides. Voile fabric covers all openings (except the bottom end) to ensure air circulation and prevent humidity condensation and insect escape. A small hole in the voile at the top end allows insects entry and remains sealed with a cotton plug (Figure 1a).

2. Sifted soil

3. Clear plastic trays (24.5 × 17.5 × 4.5 cm; L × W × H)

4. Fine-mesh white polyester fabric (voile), ~50 mesh (300 μm aperture)

5. Soft rubber hose (approximately 7.5 mm Ø)

6. 1,000 μL universal fit pipette tips (e.g., Corning, catalog number: CLS4868 or similar)

7. Fast-drying universal adhesive for fabric bonding (Uhu^® or similar product)

8. Scissors

9. Professional cutter with segmented blade, 25 mm

10. Cotton (as needed)

11. 500 mL reinforced PET bottles (approximately 0.5 mm wall thickness) for transmission cages

Note: These cages feature a cut-out bottom end (Figure 2a) and a voile-covered lateral window to facilitate ventilation and prevent humidity condensation (Figure 2b). The screw cap end serves as an entry point for insects (Figure 2d).

12. Test tubes, 13 × 100 mm (e.g., Pyrex, catalog number: PY-99445-12)

13. Cool box 54 L (69 × 35 × 45 cm; L × W × H) (e.g., Colombraro, catalog number: 4252)

14. Plasticized test tube rack for 24 test tubes, 4 rows × 6 tubes (e.g., Leone, catalog number: LE-7-104)

15. 40 cm long stakes, fabricated from steel wire (2.5 mm Ø)

16. Elastic rubber bands (approximately 50 mm Ø)

17. Cold packs

18. Commercial contact aerosol insecticide (pyrethroid-based, e.g., Raid^® Home and Garden) (store at room temperature and verify the expiration date before use to ensure maximum efficacy)

19. LED bulbs (9–13 W, e.g., Philips LED bulbs) in cool-daylight (6,500 K) and warm-white (3,000 K) spectra

20. T8 LED tubes (9 W, 60 cm, e.g., Philips LED tubes) in cool-daylight (6,500 K) and neutral-white (4,000 K) spectra

Equipment

1. Growth room at 23 ± 2 °C with 16/8 h light/dark photoperiod (cool and warm LED lamps and neutral white LED tubes can be used); alternatively, a plant growth chamber (e.g., Conviron or similar) may be used

2. Dehumidifier (Hitachi, model RD-1604LD)

3. Mouth aspirator

Note: This device comprises a soft rubber tube (50–60 cm long × 7.5 mm Ø) fitted with a 1,000 μL pipette tip at each end. A piece of voile fabric between one end of the tube and the pipette tip acts as a mesh barrier, preventing insects from entering the tube (Figures 1a and 3a).

4. Insect manipulation chamber (black acrylic or black-painted wood; approximately 50 × 30 × 30 cm or larger; H × W × D).

Note: The chamber consists of a black box with an open front for manipulation access and a translucent glass panel at the back to allow light transmission (Figure 3b).

5. Bedside lamp with LED light (Figure 3b)

Figure 1. Representative scheme of mass rearing of Delphacodes kuscheli. (a) Supplies for the fabrication of mass rearing cages and mouth aspirator: 6 L plastic containers, fine-mesh voile for ventilation windows, pipette tips, and soft rubber tubing. (b) Rearing cages with oviposition and nymph emergence of D. kuscheli conditioned under LED lighting (16/8 h light/dark photoperiod) to ensure optimal plant and insect growth. (Black stars: adult insects; grey stars: nymphs).

Figure 2. Field transmission of mal de Río Cuarto virus. (a, b) Schematic diagrams showing the fabrication of the transmission cages using 500 mL PET bottles. The design includes an opening covered with fine-mesh voile for ventilation. (c) Cages placed in the field over individual maize plants for controlled viral transmission. (d) Transfer of viruliferous insects from the glass tubes into the transmission cages. Note the use of two securing stakes (40 cm long × 2.5 mm Ø) and two elastic bands to ensure stability.

Figure 3. Materials for insect handling and light-assisted manipulation. (a) Mouth aspirator for D. kuscheli collection, showing the soft rubber tubing (40 cm long × 2.5 mm Ø), pipette tips, and a fine-mesh voile filter secured with an elastic band to prevent insect inhalation. (b) Insect manipulation chamber (black-painted wood) equipped with two LED lamps at the back. The light source facilitates the collection of insects by taking advantage of their positive phototaxis, directing them toward the back of the chamber for easier capture.

Software and datasets

1. Infostat (15) or similar platforms (e.g., Navure, https://www.navure.com/)

Procedure