Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

1. Plant Cultures

1. Purchase seeds from any commercial vendor or collect from mature plants in the field.
   NOTE: This protocol is suitable for most commercially available milkweed species (e.g., Asclepias incarnata, A. syriaca, A. curassavica, Gomphocarpus physocarpus). Some seeds may need to be cold-stratified, and instructions from the seed supplier should be checked.
2. Plant seeds in a fine germinating soil (60–70% fine peat moss, perlite, vermiculite, limestone).
   1. Fill a standard seedling tray with germination mix soil; ensuring that the soil reaches the top of the wells. In each well, make an indentation to create a hole in the soil about a 3 cm deep.
   2. Place one seed in each hole and water very well with a watering can such that the soil covers the seeds and is saturated.
   3. Grow seeds in a greenhouse (see conditions below, 1.5). Water regularly, daily to every-other-day; enough to maintain the soil moisture to a moderate level.
3. When the seedlings have grown their first set of full leaves, re-pot seedlings in a general potting mix (50–60% peat moss, bark, and limestone) (Figure 1A).
   1. Use 4-inch round pots that fit with a tight seal with the cup cages. Fill with general potting soil up to about 5 cm below the rim.
   2. Create a hole in the soil deep enough to reach the bottom of the pot.
   3. With the hand, gently scoop the mature seedling from its well and place it deep inside the hole in the 4 inch pot. Cover the seedling with the soil. Water very well.
   4. Grow plants in the greenhouse and water regularly, daily to every other day; enough to maintain moderate soil moisture.
4. Greenhouse conditions.
   1. Set the greenhouse thermostats to maintain daytime temperatures between 18–28 °C and nighttime temperatures between 16–22 °C using the manufacturer's instructions.
   2. During winter months when the days are shorter, supplement the daylight with 600 W high-pressure sodium bulbs (12 h, 8 am – 8 pm).
5. Control unwanted pests (e.g., thrips, aphids) with a foliar organic soap solution, however, use these products with caution.
   1. Make the soap solution according to the manufacturer's recommendation and apply using a spray bottle.
   2. Leave the soap on the plants for 4–24 h. Gently rinse the plants with water to remove the soap 4–24 h post-application and rinse them with water a second time prior to use with laboratory aphid cultures.
6. Culture the average aphid population on plants that have grown at least 3–4 sets of full leaves and are at least 10 cm tall (Figure 1B).

2. Aphid Cultures

1. Start the laboratory aphid populations from an existing lab isoclonal population or start from the field-collected aphids following the directions below.
   1. When starting a laboratory population from an existing lab isoclonal population, transfer aphids as described in 2.3.1–2.3.3.
2. When starting the new isoclonal, field-collected aphid populations and place a single, reproducing, adult aphid on a suitable host plant as mentioned in Step 1.6.
   NOTE: Populations may be started from winged (alate) or unwinged (apterous) adults.
   1. Manually inspect plants from the greenhouse for unwanted pests prior to the use with laboratory aphids. Freeze any plants with unwanted aphids. If desired, use an ethanol vacuum flask to remove thrips or other pests.
      NOTE: Be sure to rinse plants that have been treated with soap prior to use as described in Step 1.5.2.
   2. Safely transfer a single adult aphid using a paintbrush or a mouth pipette created with 3/16" ID x 1/4" OD plastic tubing, a 1,000 µL pipette tip, and a 2,00 µL pipette tip (Figure 2A).
   3. Securely cover plants with aphids with a cup cage created with a plastic cup with the top cut off, covered with a fine mesh and secured with tape (Figure 2B).
   4. Place aphid-infested plants in a tray and keep in a controlled environmental chamber (16L:8D, 22 °C, 70% humidity).
3. To maintain the stock populations, transfer aphids to fresh, new plants weekly (2.2.1–2.2.3).
   1. Safely transfer 1–3 2^nd or 3^rd instar nymphs and 1 adult-aged aphids using a mouth pipette (Figures 2A, 3).
      NOTE: Stocks are best maintained by transferring unwinged individuals.
   2. Securely cover aphid-infested plants with a cup cage created with a plastic cup with the top cut off, covered with a fine mesh and secured with tape.
   3. Place plants in a tray and keep aphids in an environmental chamber (16L:8D, 22 °C, 70% humidity).
   4. Alternatively, if desired and if the host plant is of decent quality, use an ethanol vacuum flask to reduce populations leaving only one reproducing adult and two to three 2nd or 3rd instar nymphs.
4. To create same age populations for the use in experiments, place up to 5 adults (preferably unwinged) from the stock population onto a new host plant.
   1. Remove the adults 24 h later.
   2. About 5–7 days later, once the F₁ offspring have matured to adulthood, place up to 5 unwinged F₁ adults on a new host plant. Remove the adults 24 h later.
   3. Once the F₂ population has matured to adulthood, this population is ready to be used in experiments.
      NOTE: This process ensures that the experimental population is roughly the same age and are born of roughly same age mothers.
5. Confirm the genotypic differences between field-caught isoclonal lines using microsatellite genotyping (described below, Sections 3 & 4).

3. DNA Extraction

1. Preparation
   1. Use sterile techniques to prepare 1 L lysis buffer (0.1 M NaCl, 0.2 M sucrose, 0.1 M Tris (pH 9.1), 0.05 M EDTA, 0.05% SDS).
   2. Warm the heating block or water bath to 65 °C.
2. Tissue homogenization and lysis
   1. Place the aphid near the bottom of a sterile, 1.5 mL microcentrifuge tube.
   2. Place the sterile pestle in the tube with the aphid and immerse the bottom of the tube in liquid nitrogen.
      NOTE: Optimal tissue disintegration is achieved when the aphid is positioned between the pestle and side of the tube.
   3. Grind the aphid with the pestle to initially lyse cells.
   4. For a single adult aphid, use 200 µL (split into 2 x 100 µL aliquots) of the lysis buffer. Add the first aliquot to grind and resuspend the crushed aphid until the sample is visibly disintegrated, then use the second aliquot to wash off the pestle.
   5. Incubate the crushed aphids in lysis buffer at 65 °C in the water bath or heat block for 30 min.
3. DNA precipitation
   1. While the tube is warm, add 14 µL of 8 M KOAc. Invert the tube to mix.Store the sample on ice for 30 min.
      Note: The protocol can be paused here, and samples can be stored at -20 °C up to 24 h.
   2. Centrifuge at 13,000 x g for 15 min at room temperature. Transfer the supernatant to new 1.5 mL tube with a pipette. Be careful not to remove any of the pelleted debris.
   3. To improve DNA pellet visualization, add 2 µL glycogen (20 µg/mL) to the supernatant. If the sample size is large enough, omit this step.
   4. Add 200 µL of cold 100% molecular-grade ethanol to the supernatant. Invert tubes to mix and incubate at room temperature for at least 15 min.
      NOTE: The protocol can be paused here, and samples can be stored at -20 °C up to 24 h.
   5. Centrifuge at 13,000 x g for 15 min at room temperature. Remove ethanol by pipetting.
4. DNA wash and elution
   1. Add 200 µL of cold 70% molecular-grade ethanol. Then flick the tube to resuspend and wash the pellet.
   2. Centrifuge at 13,000 x g for 5 min. While visualizing the pellet, carefully remove the ethanol by pipetting and add 200 µL of cold 100% molecular-grade ethanol.
   3. Centrifuge at 13,000 x g for 5 min. While visualizing the pellet, carefully remove ethanol by pipetting.
      NOTE: Repeat the 100-70-100 ethanol wash if necessary.
   4. Air dry the pellets for 5–10 min with the tube laying horizontally open on a tissue paper.
   5. Resuspend the DNA pellet in 80 µL of low TE (10 mM Tris-HCl, 0.1 mM EDTA).
   6. Quantify the resuspended DNA using a spectrophotometer.
   7. Store at 4 °C.

4. Microsatellite PCR and Sequencing for Aphid Genotyping

1. Order the appropriate F and R primers for microsatellite sequencing (Table 1²⁰).
   NOTE: Reverse primer sequences should be modified with 5'-6-FAM or 5'-5-HEX fluorescent labels to allow for multiplexed samples for microsatellite sequencing.
2. Perform PCR with single aphid DNA samples (described in Section 3) and fluorescently labeled microsatellite primers.
   1. Mix PCR reactions according to the manufacturer's protocol (0.2 µM each F/R primer, 2.5 mM MgCl₂, 50–200 ng DNA template).
   2. Use the following thermocycler settings: initial denaturation at 94 °C for 4 min, 35 cycles of 94 °C for 30 s, 58 °C for 35 s, 72 °C for 45 s, and a final elongation step at 72 °C for 10 min.
3. Combine PCR samples with different fluorescent tags to reduce the number of samples sequenced and sequence the microsatellite samples at a genotyping facility.
4. Analyze the .fsa raw sample files using microsatellite analysis software.

5. RNA Extraction

1. Collect aphids' samples for RNA extraction in 1.5 mL RNase/ DNase-free tubes and immediate freeze in liquid nitrogen.
   NOTE: If the following steps are not performed immediately, the samples can be stored at -80 °C.
2. Tissue homogenization
   1. With the sterile pestle in the tube with aphid, freeze in liquid nitrogen for 10–15 seconds, until the sample stop sizzling. Crush the aphid well with the pestle as described in step 3.2.
      NOTE: Optimal tissue disintegration is achieved when the aphid is positioned between the pestle and side of the tube.
   2. In the fume hood, add 800 µL of guanidinium thiocyanate-phenol-chloroform extraction reagent to the sample (1–5 adult aphids). Homogenize samples with the pestle and dispose of the pestle.
3. Phase separation
   NOTE: All steps should be performed in a fume hood.
   1. Incubate the homogenized samples for 5 min at room temperature. Add 160 µL of chloroform to sample. Shake vigorously by hand for 15 s.
   2. Incubate for 2-3 min at room temperature. Centrifuge for 15 min at 12,000 x g at 4 °C.
      NOTE: Following centrifugation, the mixture separates into 3 layers: a lower, red phenol-chloroform phase, an interphase and a colorless upper aqueous phase. The RNA remains exclusively in the aqueous phase. The volume of the aqueous will be ~480 µL.
4. RNA precipitation
   1. In a fume hood, transfer the aqueous phase to a fresh, RNase-free tube. Do not disturb the intermediate phase.
   2. Precipitate the RNA by adding 400 µL of isopropanol and incubate the sample at -20 °C for 10 min.
      NOTE: The protocol can be paused here, and samples can be stored at -20 °C up to 24 h.
   3. Centrifuge the sample for 10 min at 12,000 x g at 4 °C.
5. RNA wash and elution
   1. Remove the supernatant; watch for the RNA pellet.
   2. Wash the RNA pellet with 1 mL of 75% ethanol in DEPC-treated water. Mix by slow vortexing. Centrifuge for 5 min at 7,500 x g at 4 °C.
   3. Repeat steps 5.5.1–5.5.2 to help remove phenol contaminants.
   4. Remove the supernatant and air dry the pellet for 5-10 min with tube laying horizontally open on a sterile bench. Do not let the RNA pellet dry completely.
   5. Dissolve the RNA pellet in 30 µL of RNase-free or DEPC-treated water. Gently pipette up and down to mix. Incubate at 55–60 °C for 10–15 min.

6. RNAseq De Novo Transcriptome Assembly, Annotation, and Differential Expression Analysis

1. Analyze RNA sample concentration and quality using a chip-based capillary electrophoretic system.
   NOTE: A chip-based capillary electrophoresis system is the preferable method of choice than analyzing with a spectrophotometer because it provides a more accurate and sensitive measure of RNA concentration and quality.
   1. If samples are of suitable quality (≥ 250 ng total, RIN (RNA Integrity Number) ≥ 5), perform RNA sequencing.
      NOTE: Importantly, because this sequencing data will be used for both expression profiling and de novo transcriptome assembly, more read depth will result in a higher quality transcriptome. For a reasonably comprehensive assembly using Illumina sequencing technology, 100–200 million 100 bp, paired end reads would be a recommended starting point. Total mRNA library preparation and RNA sequencing were performed by a sequencing facility.
2. Check the quality of reads using Fast QC²².
3. Combine all sample reads and assemble the transcriptome de novo using Trinity^23,24 (Trimmomatic quality filtering enabled).
4. Refine the assembly
   1. Use Transdecoder²⁵ to identify open reading frames (ORFs) that are a minimum of 100 amino acids in length.
   2. Perform homology searches for the translated ORFs against Pfam²⁶ and UniProt²⁷ databases using BLASTP²⁸ and HMMER²⁹, respectively.
   3. Remove bacterial transcripts (any translated sequence whose best BLAST hit was to a bacterial gene with a bit score of over 300 and a minimum amino acid sequence identity of 50%).
   4. Collapse any complete, translated ORFs that are at least 99% identical at the amino acid level using CD-HIT³⁰.
   5. Collapse the remaining, incomplete ORFs that are at least 95% identical at the nucleotide level using CD-HIT³⁰.
   6. Assign the remaining nucleotide sequences with unique, species-specific identifiers (e.g., APHNE 0001).
5. Assess the completeness of the refined assembly, using BUSCO (http://busco.ezlab.org/) and the Arthropoda gene dataset³¹.
6. Transcriptome annotation
   1. First, annotate the refined transcriptome using HMMER against the Pfam database^26,29.
   2. Second, annotate the transcriptome using BLASTP against the UniProt database^27,28.
   3. Third, annotate the transcriptome using BLASTP against the coding sequences of selected insects with published, annotated genomes.
   4. Last, annotate the transcriptome using BLASTP against the pea aphid protein database only.
   5. Use Trinotate to generate GO annotations from UniProt accessions.
   6. Use Trinotate to organize all the annotation results into a SQLite database and generate an annotation report.
7. Differential expression analysis
   NOTE: Using the refined transcriptome as a reference, align and quantify each library separately.
   1. Use Trimmomatic to quality-filter and trim the original read files³².
      Note: If performing this step after a Trinity assembly, one may instead use the Trimmomatic output from that step.
   2. Perform local alignments for each sample using Bowtie2³³.
   3. Extract the read counts from each sample individually using SAMtools³⁴.
   4. Calculate the differential expression between samples of interest using DESeq2 with the default parameters and a parametric fit³⁵.

7. qPCR Verification of Differentially Expressed Genes

NOTE: If users are interested in differentially expressed genes from their RNAseq experiments, the following protocol can be used to verify patterns of differential expression.

1. Generate RNA samples as described above (step 5).
2. Quantitate RNA extractions using a spectrophotometer to check for the quality and obtain the concentration.
3. Synthesize cDNA samples using a commercially available kit as per the manufacturer's recommendation.
4. Determine the primer efficiencies for genes of interest to ensure accurate two-fold PCR amplification.
   1. Based on original RNA concentrations, perform serial dilutions (10¹) to obtain 3 cDNA concentrations.
   2. Using a quantitative PCR master mix, mix triplicate qPCR reactions according to the manufacturer's protocol using three primer concentrations (e.g., 100 nM, 200 nM, 300 nM) with three serially diluted cDNA concentrations (e.g., 0.1 ng/µL, 10 ng/µL, 100 ng/µL).
   3. For each target gene, calculate the slope (m) of the line created using the mean C_t values for each sample as the dependent variables and the log (cDNA concentration) as the independent variables (three points total).
   4. Use the following equation to calculate the primer efficiency (E) where m is the slope calculated in 7.4.3:
      E = 10^(-1/m)
      NOTE: Primer efficiencies between 90-110% are suitable for analyses. This process ensures equal amplification of all genes included in the calculations.
5. Use the ΔΔC_t method with a housekeeping gene to quantify the differential expression for genes of interest³⁶.