This method can help answer key questions in the Candida field about strain virulence and the contributions of specific genes to these processes. The main advantages of this technique are that it avoids concerns regarding animal use, is robust, fast, cheap, and provides high numbers of animals per experiment. Generally speaking, individuals that are new to this method will likely struggle a bit because they'll have some difficulty assessing worm viability prior to the beginning of the experiment or they might have some issues with inconsistent injection of the worms.
Order larvae from wholesalers and suppliers that do not introduce hormones, antibiotics, or other treatments to the larvae and which are able to ship and deliver live specimens. When the larvae arrive, check each container to confirm the health and viability of the larvae. Healthy larvae will be completely submerged beneath the bedding, moving, and possessing a yellow, light tan coloration with few larvae containing black marks or discolorations along the body.
The larvae can be stored in their containers in a ventilated space at ambient temperature for up to two weeks. To prepare the yeast cells for Galleria mellonella infection, grow the C.albicans strains to be injected overnight in three milliliters of fresh yeast-peptone-dextrose broth per strain in standard culture tubes on a rotating drum at medium speed and 30 degrees Celsius. The next morning, collect the cells by centrifugation, and wash the yeast three times in five milliliters of fresh PBS per centrifugation.
After the last wash, resuspend cells in one milliliter of fresh PBS and transfer the yeast into a microcentrifuge tube. Serially dilute the cells in additional PBS at one-to-100, one-to-1, 000, and one-to-100, 000 concentrations, and count the cells from the one-to-100 and one-to-1, 000 dilutions. Adjust the cells to a 2.5 times 10 to the seventh cells per milliliter of PBS concentration in a new microcentrifuge tube, and confirm the accuracy of the infectious dose by plating the appropriate volume of the one-to-100, 000 dilution to achieve 100 colony-forming units on one yeast-peptone-dextrose agar plate for each culture to be used in infection.
To infect the larvae with the Candida cells, first spread a small amount of waxworm bedding into one empty, 100-by-15 centimeter sterile Petri dish per biological replicate, and sterilize a 10-microliter syringe equipped with a 26-gauge needle three times with 10 microliters of 70%ethanol and three times with 10 microliters of PBS per wash. Next, vortex the inoculation dilution of C.albicans, and load 10 microliters of cells into the syringe. Open one container of G.mellonella larvae, and use a finger to carefully turn the bedding over to uncover larvae, selecting yellow, tan larvae of a similar size that are in good health with a lack of black pigmentation on the bodies, using the middle and index fingers and the thumb to pick up one larva in a manner similar to grasping a pencil.
Roll the larva onto its back so the legs are facing up, holding the full length of the body between the fingers and thumb so that the larva is unable to curl or pull away from the injection. With the other hand, rotate the syringe so the needle bevel is facing up, and slowly insert the entire length of the bevel into the body at the junction with the rearmost left leg, ensuring that the needle penetrates the body and does not simply push the body of the larva inwards. The most critical step in this procedure is injection of the waxworm in such a way that damage to the larval body is minimized and the full bolus is inoculated.
Then inject the full 10 microliters of Candida inoculum, and extract the needle. Co-house up to 10 larvae injected from the same Candida culture in each 100-by-15-millimeter Petri dish following inoculation, and incubate the larva at 37 degrees Celsius for eight days. Check the dishes every 24 hours to monitor death.
The mortality can initially be assessed by darkened pigmentation, the formation of black patches or bodies, and a lack of movement. To confirm mortality, use tweezers to gently roll moribund larvae onto their backs and lightly poke the larva's underside with the tweezers. Larvae that begin to pupate into moths can be included in the analysis but should be removed if they begin to molt.
No significant difference in the kinetics of G.mellonella death exists between larvae infected zero or 10 days after arrival. The characteristic signs of illness arise in larvae from both ages that succumb to the infection, beginning with discoloration that gives way to black patches before a total loss of motility and eventually death. Infection with C.albicans speeds up this process of discoloration and morbidity but does not significantly alter the cascade of phenotypic markers leading to death compared to uninfected or PBS-injected larvae.
Notably, the infection of G.mellonella with mating type-like homo-or heterozygotes from any of the tested clinically isolated C.albicans strains produces high rates of mortality compared to PBS-injected animals, while injections of prototrophic derivative strains attenuates the lethal effect in this model. It's important to remember to choose healthy larvae and to be gentle in handling the larvae. If multiple strains will be inoculated, take care not to let the Candida cells sit in the PBS for too long.
Following this procedure, other methods like immunological profiling and Candida localization within larvae can be performed to answer additional questions about the interactions between the host and fungal pathogen during an infection. After its development, this technique paved the way for researchers to explore Candida pathogenesis using high-throughput formats. Don't forget that working with Candida albicans requires care and that standard precautions, including the wear of personal protection equipment, should always be taken while performing this procedure.
