TITLE: Use of the waxworm Galleria mellonella larvae as an infection model to study  Acinetobacter baumannii
AUTHORS: Kah Ern Ten, Nazmul Hasan Muzahid, Sadequr Rahman, tan.hocksiew

[DESCRIPTION]
Galleria mellonella larvae have been increasingly used in various scientific research, including microbial infection studies. They act as suitable preliminary infection models to study host-pathogen interactions due to their advantages, such as the ability to survive at 37 °C mimicking human body temperature, their immune system shares similarities with mammalians, etc. Here, we presented a protocol for simple rearing and maintenance of Galleria mellonella in science research laboratories without requiring special instruments and specialised training. This allows the continuous supply of healthy Galleria mellonella for research purposes. Besides, this protocol also provides detailed procedures on the (i) Galleria mellonella infection assays (killing assay and bacterial burden assay) for virulence studies and (ii) bacterial cell harvesting from infected larvae and RNA extraction for bacterial gene expression studies during infection. Our protocol could not only be used in the studies of A. baumannii virulence but can also be modified according to different bacterial strains. 

[GUIDELINES]
Guidelines:

Unhealthy Galleria mellonella larvae (with black spots/melanisation) should not be used for any experiments.
Extra precautions must be taken when uncapping needles. A puncture-resistant glove is recommended.
Always use RNase-free tubes when preparing reactions for RNA works. An RNase decontamination solution is highly recommended for surface decontamination of working areas to destroy RNases.
RNA should always be kept on ice to avoid degradation.

References:

Andrea A, Krogfelt KA, Jenssen H. Methods and challenges of using the greater wax moth (Galleria mellonella) as a model organism in antimicrobial compound discovery. Microorganisms. 2019;7(3):85.
Fredericks L, Lee M, Roslund C, Crabtree A, Allen P, Rowley P. The design and implementation of restraint devices for the      injection of pathogenic microorganisms into Galleria mellonella. PLOS ONE. 2020;15:e0230767.
Harding CR, Schroeder GN, Collins JW, Frankel G. Use of Galleria mellonella as a model organism to study Legionella      pneumophila infection. J Vis Exp. 2013(81):e50964-e.

[STEPS]
SECTION: Galleria mellonella rearing and maintenance
1. Research-grade Galleria mellonella larvae were ordered in bulk from Carolina Biological (US).
SECTION: Galleria mellonella rearing and maintenance
2. Set up Galleria mellonella housing according to Figure 2.
 
 
SECTION: Galleria mellonella rearing and maintenance
3. Fill 2/3 of the larvae jar with the freshly prepared medium.
SECTION: Galleria mellonella rearing and maintenance
4. Transfer healthy larvae from the container provided by the vendor individually into a new glass jar with fresh medium, and cover with a layer of cloth-type voile. 
 

SECTION: Galleria mellonella rearing and maintenance
5. Place the glass jars above a heating mat with temperature controlled at 32 °C ± 2 °C with humidity 44%-54% and keep them in a plastic storage box in a dark environment.

SECTION: Galleria mellonella rearing and maintenance
6. Add the fresh medium every 3 days and remove the sick/dead larvae from the jars to prevent the spread of diseases.
 

SECTION: Galleria mellonella infection assays: Sample preparation
13. Prepare bacterial overnight culture by inoculating 1 colony of Acinetobacter baumannii in 5 mL of Luria Bertani broth and incubate with shaking at   for 16-18 hours.

SECTION: Galleria mellonella infection assays: Sample preparation
14. Cut pipette tips can be prepared by following Fredericks, Lee (2), which will be used as larvae restraint devices. 
 

SECTION: Galleria mellonella infection assays: Killing assay
15. Pellet 1 mL of the overnight bacterial culture by centrifuging at 8300 rpm, 7 min.
SECTION: Galleria mellonella infection assays: Killing assay
16. Resuspend the bacterial pellet with 1 mL of sterile 1X phosphate buffer saline (PBS) (pH 7.4).

SECTION: Galleria mellonella infection assays: Killing assay
17. Repeat centrifugation (8300 rpm, 7 min) to pellet the bacterial culture.

SECTION: Galleria mellonella infection assays: Killing assay
18. Resuspend the bacterial pellet with 1 mL of sterile 1X PBS  (pH 7.4).

SECTION: Galleria mellonella infection assays: Killing assay
19. Measure the optical density of the bacterial culture and adjust it to the appropriate OD600 nm.
 

SECTION: Galleria mellonella infection assays: Killing assay
20. Wash the Hamilton syringe (model 725LT).  
SECTION: Galleria mellonella infection assays: Killing assay
21. Sterilise the larval prolegs with 70% ethanol using a cotton swab. 
 

SECTION: Galleria mellonella infection assays: Killing assay
22. Place the larval tail into the wider part of the cut tip, then insert the narrow part of the cut tip to trap the larvae.
  

SECTION: Galleria mellonella infection assays: Killing assay
23. Inject 10 µL of bacterial suspensions with desired cell density into the last left proleg of larvae. 
SECTION: Galleria mellonella infection assays: Killing assay
24. Place the larvae in a sterile Petri dish lined with filter paper.
SECTION: Galleria mellonella infection assays: Killing assay
25. Incubate the larvae at 37 °C in a standard bacterial incubator and score for survival every 1440 min. 

SECTION: Galleria mellonella infection assays: Killing assay
26. Repeat the experiments independently 3 times to get the data of 3 biological replicates (n=30). 
 

SECTION: Galleria mellonella infection assays: Killing assay
27. Perform the Kaplan-Meier survival curves and statistical analysis (log-rank test) using GraphPad Prism software.
 

SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
28. Adjust overnight bacterial culture (washed twice with sterile 1X PBS, pH 7.4) to appropriate OD600 nm. Inoculum is always confirmed via plating.

SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
29. Sterilise the larval prolegs with 70% ethanol using a cotton swab.
SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
30. Trap the larvae in the restraint devices.
SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
31. Clean the Hamilton syringe with diluted bleach and distilled water.
SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
32. Inject 10 µL of bacterial suspension into the last left proleg of the larvae. Place the larvae in a sterile Petri dish lined with filter paper and incubate at 37 °C in the standard bacterial incubator. 
SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
33. Measure and record the weight of a sterile microcentrifuge tube (1.5 mL) before the hemolymph collection.

SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
34. At each time point, randomly choose 3 larvae from the incubated larvae.
SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
35. Anaesthetise them on ice in a 15 mL centrifuge tube for 10 min.

SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
36. Sterilise the larval surface by immersing them in 70% ethanol for 30 s, followed by washing. 

SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
37. Make an incision by puncturing the cuticle between the second and third proleg using a sterile 27G Terumo needle. Squeeze the larvae with sterile plastic forceps (sterilised with diluted bleach and 70% ethanol) and collect the hemolymph immediately from the puncture site via pipetting. 
 

SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
38. Pool the hemolymph from 3 larvae into the weighted microcentrifuge tube. 
 

SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
39. Incubate with 1 µL of digitonin (5 mg/mL) at Room temperature to lyse haemocytes and release intracellular bacteria (3). 
 

SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
40. Perform 10-fold serial dilution on the collected hemolymph and plate 100 µL of the bacteria with appropriate dilution onto Leeds Acinetobacter agar.

SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
41. Incubate the agar plate at 37 °C for 1200 min.

SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
42. Count the number of bacterial colonies and calculate the CFU/larva by normalising to the weight of hemolymph extracted. 
 

SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
43. Perform the experiments in three independent replicates.
SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
44. Plot the bacterial growth curve in vivo using GraphPad Prism software.
SECTION: Bacterial harvesting and RNA extraction from infected larvae: Sample preparation
45. Inoculate a single colony of bacteria in 5 mL of Luria-Bertani broth and incubate for 16-18 hours  with continuous shaking at  . 

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Sample preparation
46. Incubate 40 healthy larvae (6th instar stage, 200-300 mg) with a creamy-white appearance at 37 °C 1200 min.
 



SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial cell harvesting from infected larvae
47. Spin 1 mL of the overnight bacterial culture at 8300 rpm, 7 min.

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial cell harvesting from infected larvae
48. Resuspend in 1 mL sterile 1X PBS.

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial cell harvesting from infected larvae
49. Spin the bacterial culture again at 8300 rpm, 7 min, and resuspend in 1 mL sterile 1X PBS.

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial cell harvesting from infected larvae
50. Adjust the bacterial culture to the appropriate optical density. Inoculum is always confirmed via plating.
SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial cell harvesting from infected larvae
51. Sterilise the larval prolegs with 70% ethanol using a cotton swab.
SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial cell harvesting from infected larvae
52. Inject 10 µL of bacterial suspension into the last left proleg of the larvae using a Hamilton syringe (model 725LT) with a 27G Terumo needle. 
SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial cell harvesting from infected larvae
53. During the incubation time, prepare a stop mix solution (95% absolute ethanol: 5% Tri-RNA) in a 2 mL RNase-free microcentrifuge tube and keep it at -20 °C. Pre-cool microcentrifuge to 4 °C. 
 

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial cell harvesting from infected larvae
54. At the desired time point, sterilise the larval surface by immersing the larvae in 70% ethanol for 30 s, followed by rinsing two times with sterile distilled water.

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial cell harvesting from infected larvae
55. Extract the hemolymph from the infected larvae using a Terumo 27G needle by puncturing the larval cuticle between the second and the third prolegs. Collect the hemolymph immediately from the punctured site and pool it into the microcentrifuge tube with ice-cold 0.2 volume of stop mix solution. 
 

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial cell harvesting from infected larvae
56. Incubate the hemolymph-stop mix solution mixture at Room temperature for 5 min.

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial cell harvesting from infected larvae
57. Gently vortex for 5 s.

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial cell harvesting from infected larvae
58. Centrifuge at 2300 rpm, 5 min, 4 °C. Collect the supernatant and transfer it into a 1.5 mL RNase-free microcentrifuge tube. 
 

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial cell harvesting from infected larvae
59. Repeat step 58 until no or very little host pellet is obtained. 
 

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial cell harvesting from infected larvae
60. Pellet the bacterial cells from the supernatant by centrifugation at 10000 rpm, 15 min, 4 °C. Discard the supernatant.

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial cell harvesting from infected larvae
61. Immediately resuspend the bacterial cell pellet in 1 mL of tri-RNA. Homogenise the sample by gentle vortex for 5 s or via repetitive pipetting.

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial cell harvesting from infected larvae
62. Store at -80 °C or proceed to RNA extraction.

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
63. Thaw the sample on ice.
 



SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
64. Incubate at Room temperature for 5 min.

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
65. Add 200 µL of chloroform into the mixture. Mix by inversion until a milky pink mixture is obtained.
SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
66. Incubate at Room temperature for 5 min.

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
67. Centrifuge at 13000 rpm, 15 min, 4 °C.

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
68. Collect the aqueous transparent supernatant into a 2 mL RNase-free microcentrifuge tube.
 

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
69. Add an equal volume of 95% ethanol (molecular grade).
SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
70. Extract RNA according to the manufacturer’s manual (Monarch® Total RNA Miniprep Kit, NEB) as described below: 	
SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
71. Assess the RNA quality by gel electrophoresis (1% agarose gel) and Agilent TapeStation 2200 and measure the absorbance values (A260/230 and A260/280) and concentration using a BioDrop spectrophotometer.
SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
72. Store the RNA at -80 °C or place it on ice for immediate downstream applications.

SECTION: Galleria mellonella rearing and maintenance
3.1. Ingredients of artificial diet (per jar):  
 

SECTION: Galleria mellonella rearing and maintenance
3.2. Mix well in a clean plastic container using a spatula. 
 

SECTION: Galleria mellonella rearing and maintenance
6.1. Dead/sick worms should be placed in a Petri dish or plastic bag and frozen at -20 °C  . Discard as biological waste.
SECTION: Galleria mellonella infection assays: Sample preparation
14.1. Sterilise the cut pipette tips by immersing them in 70% ethanol  , then discarding the ethanol and autoclaving.

SECTION: Galleria mellonella infection assays: Sample preparation
12. Incubate 10 randomly chosen healthy 6th instar stage larvae (200-300 mg) with creamy-white appearance and no melanisation at 37 °C, without food, in a standard bacterial incubator for one day before the experiment.

SECTION: Galleria mellonella infection assays: Sample preparation
12.1. Pre-incubation at 37 °C allows the selection of more suitable larvae, where unhealthy larvae will show melanisation and/or death after the pre-incubation and will be excluded from the experiment.

SECTION: Galleria mellonella infection assays: Killing assay
20.1. Wash the Hamilton syringe (model 725LT) with diluted bleach.
SECTION: Galleria mellonella infection assays: Killing assay
20.2. Then, wash it with distilled water to remove the bleach. (1/2)
SECTION: Galleria mellonella infection assays: Killing assay
20.3. Wash it with distilled water to remove the bleach. (2/2)
SECTION: Galleria mellonella infection assays: Killing assay
20.4. Attach the needle (27G) to the syringe and attach the Hamilton syringe to the Hamilton repeating dispenser (PB600-1).
 

SECTION: Galleria mellonella infection assays: Killing assay
23.1. The needle should be visible through the larval cuticle after inserting it into the proleg.
SECTION: Galleria mellonella infection assays: Killing assay
23.2. The Hamilton syringe should be cleaned after each experimental group (step 20) to avoid being carried over to the next experimental group. 
 

SECTION: Galleria mellonella infection assays: Killing assay
23.3. Two control groups should be used:
larvae injected with only sterile PBS (to assess physical trauma), 
larvae without receiving any injections (non-manipulated control to assess background mortality).
SECTION: Galleria mellonella infection assays: Killing assay
25.1. Larvae are considered dead when they are unresponsive to physical stimuli and melanised.
SECTION: Galleria mellonella infection assays: Killing assay
25.2. Remove larvae from the cocoon to check survival, and dead larvae should be removed from the plate at every time point to avoid the spread of diseases.
SECTION: Galleria mellonella infection assays: Killing assay
25.3. Stop the experiment when pupation occurs to avoid biases.
SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
32.1. Negative control group: larvae injected with sterile 1X PBS only.

SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
36.1. Washing with sterile distilled water to remove the residual ethanol. (1/2)

SECTION: Galleria mellonella infection assays: Bacterial burden assay (quantification of bacterial CFU in vivo)
36.2. Washing with sterile distilled water to remove the residual ethanol. (2/2)
SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial cell harvesting from infected larvae
52.1. Place the larvae in a sterile Petri dish lined with filter paper and incubate at 37 °C in the standard bacterial incubator for 180 min. 
 

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
63.1. Pre-cool the microcentrifuge to 4 °C.

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
70.1. Load the mixture onto the RNA purification column. 
SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
70.2. Centrifugation at 16000 x g, 30 s. Discard the flowthrough.
SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
70.3. On-column DNase I treatment: 
Add 500 µL RNA Wash Buffer and centrifuge for 30 s. Discard flow-through.
Add DNase I mixture (5 µL DNase I with 75 µL DNase I Reaction Buffer) onto the column.
Incubate for 15 min at Room temperature.

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
70.4. Add 500 µL RNA Priming Buffer and centrifugate at 16000 x g, 30 s. Discard flow-through.

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
70.5. Add 500 µL RNA Wash Buffer and centrifugate at  16000 x g, 30 s. Discard flow-through.
SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
70.6. Add 500 µL RNA Wash Buffer and centrifugate at 16000 x g, 2 min. 

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
70.7. Transfer the column to a 1.5 mL RNase-free microcentrifuge tube.
SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
70.8. Load 20 µL of RNase-free water onto the column and incubate for 5 min at Room temperature. 

SECTION: Bacterial harvesting and RNA extraction from infected larvae: Bacterial RNA extraction
70.9. Centrifugation at  16000 x g, 30 s to elute the RNA.
SECTION: Galleria mellonella rearing and maintenance
6.2.  Larvae should be transferred individually to a new fresh medium when the old medium is dirty.
SECTION: Galleria mellonella rearing and maintenance
7. Allow larvae to grow into the last instar stage (approximately 300 mg, 3 cm long). At this stage, no food is needed.
SECTION: Galleria mellonella rearing and maintenance
7.1. Transfer healthy, creamy-white larvae at the 6th instar stage to a new glass jar with fresh food and kept at Room temperature in a dark environment for experimental use. 
 


SECTION: Galleria mellonella rearing and maintenance
8. Transfer 50 pupae (brownish colour) or larvae in the pre-pupal stage (with thick cocoons) into a moth jar using blunt-end forceps and cover with filter paper and a perforated lid. 
 

SECTION: Galleria mellonella rearing and maintenance
9. Female moths will lay eggs around the filter paper. 
SECTION: Galleria mellonella rearing and maintenance
9.1. Replace the filter paper (that has eggs on it) with a new filter paper. This should be performed every 3 days to avoid the escape of newly hatched larvae. 
SECTION: Galleria mellonella rearing and maintenance
9.2. The moth jars should be cleaned after 2 weeks the first moth appeared to avoid the escape of newly hatched larvae. This can be done by placing the moth jars in a cold room (4 °C)  . Transfer the moths into a plastic bag and freeze them at -20 °C  . Discard as biological waste.

SECTION: Galleria mellonella rearing and maintenance
10. Cut the collected filter paper (with eggs) into smaller pieces and transfer it into the egg jar with food.
Discard areas with contamination. 
Cover the egg jar with a perforated lid. 
 

SECTION: Galleria mellonella rearing and maintenance
11. Egg jars should be monitored every 3 days to ensure a continuous food supply until they grow into the adult stage.
SECTION: Galleria mellonella rearing and maintenance
11.1. Separate the medium into half using blunt-end forceps and put it into two jars when it is too crowded. Top up the medium with freshly prepared food.
SECTION: Galleria mellonella rearing and maintenance
11.2. Transferring the larvae individually into a new jar with freshly prepared food might be necessary when the medium has fungal contamination or an unusual smell.
SECTION: Galleria mellonella rearing and maintenance
11.3. When the larvae grow bigger, transfer medium and large larvae (approx. 1.5 cm and 2 cm) to a new jar with food and cover with cloth-type voile and perforated lids.