Materials and Reagents

Test tubes (Kangsheng Medical Glass Factory, Kangsheng, catalog number: GT-16125 ), diameter x length: 16 mm x 125 mm; Volume: 18 ml
Conical flasks (Sichuan Glass Factory, Fuhai, catalog number: ZXP-250 ), volume: 250 ml
Soil matrix (Shandong Luhao Agricultural Science and Technology co., Itd, Luhao), volume: 50 L
Seedling pots, diameter: 13 cm (purchased from a local supermarket)
Capillaries (Drummond, catalog number: 3-000-203-G/X )
Centrifuge tubes 50 ml (KIRGEN, catalog number: KG2821 ); 1.5 ml (Axygen, catalog number: MCT-150-C )
Sterile Petri dishes (Biofound, catalog number: FM-90-G )
48-well cell culture plates (Corning, Costar, catalog number: 3548 )
Tissue culture-treated round coverslips (8 mm diameter) (Solarbio, catalog number: YA0353-100 )
Broad bean (Vicia faba) seeds (purchased from local farmers market)
A. pisum strain (originally collected from Yunnan, China and maintained in the lab of Prof. Zhiqiang Lu at Northwest A&F University)
Gram-negative bacteria Pseudomonas aeruginosa (PAO1, from Dr. Xihui Shen at Northwest A&F University); Gram-positive bacteria Micrococcus luteus (Kept in the lab of Prof. Zhiqiang Lu at Northwest A&F University)
NaCl (Guangdong Guanghua Sci-tech Co., Ltd, Huada, catalog number: 1.01307.040 )
Yeast extract (Oxoid, catalog number: LP0021 )
Tryptone (Oxoid, catalog number: LP0042 )
Phosphate-Buffered Saline (PBS) (Thermo Fisher, catalog number: 10010023 )
Agar power (Solarbio, catalog number: 9002-18-0 )
Grace’s medium (Sigma-Aldrich, catalog number: S0146 )
Phenylthiourea (Sigma-Aldrich, catalog number: P7629 )
Heat-inactivated fetal bovine serum (Biological Industries, catalog number: 04-011-1A )
Escherichia coli (K-12) and Staphylococcus aureus Alexa Fluor 594 BioParticles (Invitrogen, catalog numbers: E23370 and S23372 )
4% paraformaldehyde (Solarbio, catalog number: P1110 )
SF488 Phalloidin (Solarbio, catalog number: CA1646 )
Anti-fading reagent (Solarbio, catalog number: S2100 )
Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
Luria-Bertani liquid medium (see Recipes)
Luria-Bertani agar medium (see Recipes)
0.85% NaCl solution (see Recipes)
Hemolymph collection medium (see Recipes)

Equipment

Dissecting forceps (ideal-tek, catalog number: 5.SA)
Growth chamber (Ningbo Jiangnan Instrument Factory, model: GXZ-500B)
Ultra-cold storage freezer (Whirlpool Corporation, Sanyo, model: 09S338)
Ice machine (Scotsman ice systems Co., Ltd., Scotsman, model: MF36)
Laminar flow cabinet (Shiheng Instrument Equipment Co., LTD., Dinco, model: SW-CJ-2F)
Incubator (Taicang Experimental Equipment Factory, Peiying, model: HZQ-F100)
Eppendorf Biophotometer (Eppendorf, catalog number: 6133000044)
Laser scanning confocal microscope (Olympus, model: FV3000)

Software

ImageJ (NIH, Bethesda, MD, USA)
GraphPad Prism 5.0 (GraphPad, Inc., La Jolla, CA, USA)

Procedure

Pea aphid rearing (Figure 1)
1. Put the broad bean seeds into water for imbibing absorption of water adequately for 2 days.
2. Remove the seeds peels gently.
3. Plant one peeled seed about 1 cm depth in a seedling pot filled with soil matrix.
4. The planted seeds grow in the growth chamber at 21 ± 1 °C and 70 ± 5% relative humidity under a 16-h light (L): 8-h dark (D) photoperiod.
5. Seedlings with more than six leaves can be used.
6. Place ten adult aphids on each seedling through clamping the tentacles with tweezers and allow to produce offspring in the growth chamber.
7. Remove the adult aphids from the seedlings two days later.
8. Rear the nymphs on the seedlings for nine days until they reach wingless adults.
9. Use these newly emerged aphid adults to do the following experiments.
  
  Figure 1. Pea aphid rearing. Put the broad bean seeds into water for imbibing absorption of water adequately for 2 days (A and B). Plant one peeled seed about 1 cm depth into a seedling pot filled with soil matrix and then the seed sprouts (C). Place adult aphids on each seedling through clamping the tentacles with tweezers (D and E) and allow them to produce offsprings (F). Remove the adult aphids from the seedlings two days later and the nymphs grow into wingless adults nine days later (G).
Bacterial infection
1. Prepare Luria-Bertani liquid medium, Luria-Bertani agar medium, 0.85% NaCl and sterilize these reagents in an autoclave.
2. Streak culture the P. aeruginosa and M. luteus (keep in a ultra-cold storage freezer at -80 °C) on Luria-Bertani agar plates respectively in an incubator at 37 °C overnight.
3. Pick a P. aeruginosa bacterial colony into a test tube containing 5 ml Luria-Bertani liquid medium and pick a M. luteus bacterial colony into a conical flask containing 100 ml Luria-Bertani liquid medium in a laminar flow cabinet.
4. Culture the bacteria in a bed temperature incubator at 37 °C with the rotate speed at 220 r/min.
5. Measure the absorbance of the culture at 600 nm on an Eppendorf biophotometer until the optical density reaches approximately 1 (Figure 2A).
6. Harvest the cells by centrifugation at 8,000 x g for 10 min and resuspend the pellets in sterilized 0.85% NaCl solution and wash the bacteria three times with 0.85% NaCl solution through centrifugation and resuspension.
7. Bring the P. aruginosa suspension to 2 x 10⁹ colony formation units (CFU)/ml (Figure 2B) and M. luteus cell suspension to 2 x 10¹⁰ CFU/ml.
8. Anesthetize the adult aphids on ice for ten minutes and prick through the abdominal wall into the hemocoel of the aphids about 0.5 mm deep with a capillary dipped in bacteria suspensions or sterilized 0.85% NaCl solution (Figures 2C and 2D).
  
  Figure 2. Bacterial infection. Culture P. aeruginosa in a test tube containing 5 ml Luria-Bertani liquid medium until the optical density at 600 nm reach approximately 1 (A). Harvest the P. aeruginosa cells by centrifugation and resuspend the cells to 2 x 10⁹ (CFU)/ml (B). Prick through the abdominal wall into the hemocoel of the aphids about 0.5 mm deep with a capillary dipped in the P. aeruginosa suspension (C and D).

Hemocyte phagocytosis assays
1. Anesthetize the adult aphids in a sterile Petri dish on ice for 10 min.
2. Remove one aphid legs gently with dissecting forceps and mixed the drops of hemolymph with a 5 μl Grace’s medium drop containing 1 μM phenylthiourea and 10% (vol/vol) heat-inactivated fetal bovine serum (FBS).
3. Collect the hemolymph from 20 aphids per test group as above and mix the collected hemolymph well with 2 μl of 1mg/ml E. coli or S. aureus Alexa Fluor 594 BioParticles.
4. Perform the operations of 5-14 steps in the dark.
5. Transfer the prepared samples to tissue culture-treated round coverslips (8 mm diameter) in a 48-well cell culture plate.
6. Allow the hemocytes to settle and adhere through incubating at room temperature for 1 h.
7. Wash the coverslips twice with 200 μl hemolymph collection medium.
8. Fix the washed hemocytes for 10 min with 200 μl 4% paraformaldehyde in PBS.
9. Wash the coverslips three times (10 min each) with 200 μl PBS.
10. Permeabilize the hemocytes with 200 μl 0.1% Triton X-100 in PBS for 10 min and wash twice with 200 μl PBS (10 min each).
11. Incubate the permeabilized hemocytes with 200 μl diluted SF488 Phalloidin by 1:200 in PBS for 1 h.
12. Wash the coverslips with 200 μl PBS three times (10 min each).
13. Mount the coverslips on slides using anti-fading reagent (3 μl per coverslip).
14. Observe and take photos under a laser scanning confocal microscope (Figure 3). The excitation and acquisition wavelengths of the bacteria were respectively 590 nm and 617 nm. The excitation and acquistion wavelengths of Phalloidin were respectively 495 nm and 519 nm.
  
  Figure 3. The confocal images of hemocytes with F-actin stained by SF-488 Phalloidin (A), E. coli AlexaFluo 594 BioParticles (B) and the overlay of hemocytes and E. coli. Scale bars: 5 μm.
15. Calculate the bacterial fluorescence intensities in phagocytosing hemocytes with ImageJ software (The calculation processes were shown as Figure 4).
16. Take the phagocytic index (PI) as the phagocytic capacity of hemocytes according to a previous description ( Melcarne et al., 2019b).
  
  Figure 4. The calculation processes of the bacterial fluorescence intensities in phagocytosing hemocytes with ImageJ software. The image (the image is from Figure 3B) was loaded into ImageJ software (A). The phagocytizing bacteria were circled (B) and the bacteria outside of the hemocyte were cleared out (C). The image type was set as 8 bit and inverted (D). Adjusted the threshold and chose the “Default” options as “B&W” (black and white) (E). Got the analyzed result after set the measurements and the IntDen (integrated density) value was represented the fluorescence intensity (F).

Data analysis

The data analysis of hemocyte phagocytosis assay is referred to our recent article ( Ma et al., 2020). The intensity of excitation light and the magnification times were the same during all observation processes. The photos taken from about twenty different views per coverslip were used to calculate the bacterial fluorescence intensities in phagocytosing hemocytes (the calculation processes were shown as Figure 4).

PI calculation: Fraction of hemocytes phagocytosing (f) = number of hemocytes in fluorescence positive gate/total number of hemocytes. Phagocytic index (PI) = [mean fluorescence intensity of hemocytes in fluorescence positive gate] x f. Every experiment was performed three independent biological repetitions. All PI data was plotted using GraphPad Prism 5.0. Student’s t-test was used to determine other statistical values, which were presented as the mean ± SEM.

Notes

Remove the legs of pea aphids gently to prevent linking of other tissues.
Put the coverslips in the center of the cell culture plates. Make sure all the collected hemolymph and bacteria mixed samples bestrew the coverslips, but don’t flow to the lacuna neighbor to the wall of cell culture plate.

Recipes

Luria-Bertani liquid medium
Dissolve 5 g NaCl, 5 g Yeast extract and 10 g Tryptone in 1 L ddH₂O and mix well
Sterilize the medium in an autoclave
Luria-Bertani agar medium
Dissolve 5 g NaCl, 5 g Yeast extract, 10 g Tryptone, 15 g Agar power in 1 L ddH₂O and mix well
Sterilize the medium in an autoclave
0.85% NaCl solution
Dissolve 0.85 g NaCl in 100 ml ddH₂O and mix well and sterilize the solution in an autoclave
Hemolymph collection medium
Grace’s medium containing 1 μM phenylthiourea and 10% (vol/vol) heat-inactivated fetal bovine serum (FBS)

Acknowledgments

This protocol is adapted from the publication of Ma et al. (2020). Funding for the study was provided by the National Natural Science Foundation of China grants (31970467 and 31772530) and the Fundamental Research Funds for the Central Universities (Z1090219001).

Competing interests

The authors declare no conflicts of interest.

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