Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

1. Insect collection and mass rearing

1. Collect tortricid insects from fields following previously published References^8,12.
   NOTE: H. magnanima and A. honmai larvae are collected from damaged tea leaves (Figure 1A); adults are attracted using 4 W portable UV light (365 nm wavelength, see Table of Materials, Figure 1B).
2. Rear the collected larvae (Figure 1C,D) individually on a piece of artificial diet in a 1/2-oz cup for 2-3 weeks until adult eclosion (Figure 1E,F). Confirm the sex of pupae and adults by morphological traits (Figure 1G,I).
3. Mate the males and females in a plastic box (30 cm x 20 cm x 5 cm) to oviposit egg masses on a paraffin paper (Figure 1J-L). Place a female and two males in a 120 mL plastic cup with a piece of paraffin paper to establish a matriline.
   NOTE: It is important to make creases on the paraffin paper. For H. magnanima, paraffin paper needs to be on the bottom of the case. Meanwhile, for A. honmai, put a paper on the case ceiling to collect eggs (Figure 1L) since H. magnanima oviposit egg mass on the upper side of tea leaves, but A. honmai lays eggs on the underside of the leaves⁸. The procedures were also verified in other tortricid species, and it is better to place papers on both sides if the ecology and behavior of the target species are unclarified.
4. Cut out the egg masses (approximately 100-200 eggs per egg mass¹², Figure 1M) on the paraffin paper with scissors. Place the eggs in a 1/2 oz cup with dumped paper for 5-7 days.
   NOTE: The matured embryo exhibits a blackhead capsule (Figure 1N). The periods of embryogenesis are diversely attributed to tortricid species, but generally 5 days post oviposition (dpo) for H. magnanima and 4 dpo for A. honmai at 25 °C with 60% relative humidity, 16 h light/8 h dark cycles⁷.
5. Store the egg masses showing black head capsules at 4-8 °C for 7 days.
6. Slice ~60 g artificial diets using a grater for mass rearing. Place the egg mass filled with matured embryos on the sliced artificial diet in a plastic container (23 cm x 16 cm x 8 cm). Place paraffin papers on the egg mass with the sliced diets (Figure 10).
   NOTE: Below 30% relative humidity is considered over dry, while over 70% is too humid. Creating holes on the plastic container's lid is better to enable better ventilation. Fill the holes with cotton to prevent the escapes of small larvae.
7. To eliminate surface contaminants from the eggs, soak the egg mass into 3% formalin and 0.2% Benzalkonium chloride solution for 5 min, respectively¹². To eradicate gut or intracellular bacteria, utilize an artificial diet supplemented with 0.05% (w/w) Tetracycline hydrochloride or 0.06% (w/w) rifampicin instead of a normal artificial diet (see Table of Materials).
   NOTE: This step is optional. It is important to knead Silk Mate 2S and 0.05% (w/w) Tetracycline hydrochloride or 0.06% (w/w) rifampicin equably for consistency.
8. Collect the pupae from the plastic container and distinguish the sex based on morphological¹² characteristics (Figure 1G-I).
   NOTE: Generally, tortricids present 5-6 instars until pupation¹². The H. magnanima and A. honmai larvae take 3 weeks and 2 weeks, respectively, after hatching until pupation.
9. Place 15 males and 10 females in a plastic box (30 cm x 20 cm x 5 cm, Figure 1K) for mating with 25 °C, 16 L/8 D. Collect eggs per 5-7 days and repeat steps 1.4-1.9. for each generation.
   ​NOTE: If collecting newly oviposited egg masses for subsequent analysis is needed, set the dark period's start and end, e.g., from 9 AM to 5 PM. In this condition, H. magnanima and A. honami usually oviposit eggs after 5 h (2 PM).

2. Separation of eggs and pharate larvae from egg masses for fixation, permeabilization, and staining

1. Soak the egg mass into 1,000 µL of 1.2% sodium hypochlorite aqueous solution for 10 min or into 1,000 µL of 5 M potassium hydroxide aqueous solution for 30 min to separate the eggs.
2. Wash the separated eggs in 1,000 µL of PBSt (137 mM NaCl, 8.1 mM Na₂HPO₄, 2.68 mM KCl, 1.47 mM KH₂PO₄, 0.05% of Polyoxyethylene (20) Sorbitan Monolaurate [Tween-20] pH 7.4, see Table of Materials) following the previously published report⁷.
3. Soak eggs in a mixture of 500 µL of 100% heptane and 500 µL of 4% paraformaldehyde-PBSt solution (w/v). Mix for 10 min at 1,500 rpm using a vortex mixer.
4. Soak the eggs into a mixture of 500 µL of 100% heptane and 500 µL of 100% methanol. Mix for 10 min at 1,500 rpm.
5. Wash the eggs twice with 1,000 µL of 100% methanol and store at 4 °C in 100% methanol until further experiments (Figure 2A).
   NOTE: The eggs can be stored for at least 1 year at 4 °C.
6. Soak the eggs sequentially in 99%, 70%, 50% ethanol, and PBSt for 5 min each for hydrophilization following the previously published report⁷.
7. Wash the eggs with PBSt, immerse the eggs in 1 µg/mL of DAPI solution for 5 min, and then wash the eggs with 1x PBS twice. Soak the eggs in 20 µL of 1.25% (w/w) lactic-acetic orcein solution (see Table of Materials)¹¹ to visualize heterochromatin until the nuclei exhibit a brilliant red color (this varies from 5-60 min) (Figure 2B,C).
   NOTE: The lactic-acetic orcein staining periods depend on temperature and humidity. It is better to check for proper staining using a microscope with 4x-10x magnification.
8. Transfer the stained eggs using a pipette to a glass slide. Enclose the stained eggs with antifade reagent (see Table of Materials) and a cover glass⁷.
9. Extract pharate H. magnanima and A. honmai larvae (4-5 days post oviposition (dpo)) from the egg masses using forceps (Figure 2D). Bisect larvae on a glass slide (Figure 2E).
10. Fix any tissues (e.g., suboesophageal ganglion, thoracic ganglia, malpighian tubule, etc.) with a mixture of 1:3 (v/v) 99.7% acetic acid/100% methanol for 5 min. Stain those with 1.25% (w/w) lactic-acetic orcein solution¹¹ until the nuclei are stained (this can take 5-60 min, depending on the temperature and humidity).
11. Determine the sex of each specimen by observing the presence (female) or absence (male) of heterochromatin (the W chromosome, Figure 2F,G) under a microscope.
    ​NOTE: The sex is determined by visualizing the sex chromatin. Each cell represents the sex chromatin as a dot in females^11,12, while the remaining portion of pharate larval tissues (not fixed) should be immediately immersed in cell lysis buffer¹² (10 mM Tris-HCl, 100 mM EDTA, and 1% SDS, pH 8.0) or phenol-containing RNA extraction reagents for either DNA or RNA extraction. Before the dissections, dispense 20 μL of the reagents into 0.2 mL PCR tubes in advance (Figure 3A). Immerse and store samples at -80 °C until further extraction. The samples can be stored for at least 3 months, but it is better to proceed with the downstream experiments to prevent the degradation of nucleic acids.

3. DNA and RNA extractions from sex determined pharate larvae

1. Pool 12 sex-determined male or female pharate larvae (5 dpo embryo) and add either cell lysis buffer or RNA extraction reagents (see step 2.11 and Table of Materials) into one 1.5 mL tube.
   NOTE: Follow steps 3.2-3.7 for DNA extraction and steps 3.8-3.11 for RNA extraction.
2. Homogenize tissues in 600 μL of the cell lysis buffer (10 mM Tris-HCl, 100 mM EDTA, and 1% SDS, pH 8.0), and centrifuge the samples at 10,000 x g for 5 min at 4 °C.
3. Collect the supernatant (500 μL) using a pipette and incubate at 50 °C with 1.5 µL of Proteinase K (20 mg/mL, see Table of Materials) for 5 h on a heat block.
4. Treat the samples with 1.0 μL of 10 mg/mL of RNase solution (see Table of Materials) at 37 °C for 30 min.
5. Add 200 μL of protein precipitation solution (see Table of Materials) to the tubes⁷, followed by a centrifuge at 17,000 x g for 10 min at 4 °C.
6. Mix the supernatant (500 μL) with 500 μL of 100% isopropanol, and then centrifuge at 20,400 x g for 10 min at 4 °C.
7. Wash the pelleted DNA twice with 1,000 μL of 70% ethanol. Then, air-dry (5-10 min at room temperature), dissolve the DNA in 30 μL of 10 mM Tris-Cl buffer (pH 8.5).
8. Homogenize the tissues in 600 μL of RNA extraction reagents and add 240 μL of ultra-pure distilled water to the tube. Centrifuge the tubes at 12,000 x g for 15 min at 4 °C.
9. Mix the supernatant (600 μL) with 600 μL of 100% isopropanol and transfer the mixture to a silica spin column (see Table of Materials). Centrifuge the tubes at 17,900 x g for 1 min at 4 °C.
10. Wash the column with 750 μL of 70% ethanol, and centrifuge the column twice at 17,900 x g for 1 min each at 4 °C.
11. Load 15 μL of Ultra-pure distilled water to the column. Centrifuge the tubes at 17,900 x g for 1 min at 4 °C to elute the RNA.
12. Calculate and verify the quality and quantity of DNA and RNA using a UV-based spectrophotometer. Assess the purity of nucleic acids using the A260/A280 (Nucleic acids/protein) and A260/A230 (Nucleic acids/salts and other contaminants) ratios¹³.
    NOTE: For RNA extraction, the previously published procedure¹² was followed, which resulted in phenol contamination (Table 1). The extracted DNA and RNA from a single embryo or pharate larvae yields low-amount and low-quality samples. Typically, DNA extraction from 12 pharate larvae yields 100-600 ng, while RNA extraction using the current method generates 900-1,500 ng, as shown Table 1.
    NOTE: Steps 3.13-3.15 are optional for further assays of high throughput sequencings.
13. Calculate the amounts of DNA and RNA using a fluorescence-based spectrophotometer.
    NOTE: The ratio of RNA quantities (UV-based concentration (ng/μL)/fluorescence-based concentration (ng/μL)) was calculated to assess the quality for further experimental application. For example, when UV and fluorescence-based concentrations are 80 ng/uL and 60 ng/uL, respectively, the ratio will be 1.5 (80/60). Usually, ratios under 1.5 indicate sufficient purity for downstream applications using high throughput sequencing¹³.
14. Analyze the quality of RNA by using microchip electrophoresis¹⁴.
15. Utilize the qualified RNAs to prepare the RNA library using an RNA library preparation kit (see Table of Materials). Sequence the libraries using a platform suitable for the prepared libraries.