An Optimized Protocol for Candida albicans Infection in Schmidtea mediterranea to Study Fungal Pathogenesis and Host Defense

My research focuses on Candida albicans infections and how host responses shape whether the outcome is clearance or disease progression. A key challenge is the lack of models for early fungal infection. This protocol uses planarian as a tractable, high-throughput solution.

The planarian system enables the study of disease initiation, progression, clearance, and lethality, with high-resolution measurement of innate immune responses. To begin, select Schmidtea mediterranea planarians approximately five millimeters in length under a stereomicroscope, and ensure animals are fully extended before measuring. Avoid using planarian smaller than five millimeters, as they may include individuals with blastemas or incomplete development that can produce inconsistent results.

Also, exclude planarian larger than five millimeters, as larger animals are less suitable for fluorescence immunostaining. Exclude animals with blastemas, tissue lacking pigmentation, or incomplete pigmentation development. Transfer healthy planarian meeting all inclusion criteria into two milliliters of fresh planarian water per well in a non-tissue-culture-treated six-well polystyrene plate.

Assign wells for negative controls consisting of uninfected or mock-treated animals and positive controls consisting of animals infected with wild-type Candida albicans. Remove all water from each well and replace with two milliliters of fresh planarian water to ensure uniformity across wells. Allow the animals to equilibrate in the new environment for one day before infection.

Tilt the six-well plate and gently remove all water from one well at a time, using aseptic technique. Immediately add the calculated volume of planarian water and Candida albicans culture to the well before proceeding to the next well. Record the date and time of inoculation.

Place the inoculated six-well plate in a dark, static location at room temperature. Incubate the planarian with Candida albicans for up to 72 hours, depending on the desired endpoint. At the first time point, 24 hours post-infection, tilt the plate slightly.

Then, using a sterile transfer pipette, aspirate the liquid containing Candida albicans culture and gently dispense it into an area of the well free of animals to mix the settled fungal layer. Repeat this step five to 10 times per well to break up the settled fungal layer, avoiding direct contact with the planarians. Observe fungal settlement against a dark background before and after the resuspending steps for best visibility.

Resuspend uninfected control wells under identical conditions. Repeat this procedure at 48 hours post-infection. At 72 hours post-infection, transfer planarians to fresh wells with two milliliters of clean water, minimizing fungal carryover by using minimal liquid.

Once animals are transferred, remove all water and replace it with fresh planarian water. Begin assessing host outcomes at one day post-infection and continue daily until the desired experimental endpoint. Quantify host damage using the standardized zero-to-three scoring system.

At three days post-infection, gently transfer planarian to a new six-well plate containing two milliliters of fresh planarian water per well, while avoiding transfer of residual fungal culture. Observe animals using light microscopy with a minimum 10-times objective. Record host outcomes using the standardized scoring sheet.

Additionally, anti-Candida immunostaining can be performed to observe fungal colonization of the host epithelium during infection and clearance. Fungal epithelial colonization was markedly reduced by four days post-infection, and by seven days post-infection nearly all surface-associated fungi were cleared under ID50 conditions. Uninfected controls remained entirely asymptomatic.

At approximately 7.0 times 10 to the power of seven Candida albicans cells per milliliter, about 30%were asymptomatic, 50%were symptomatic, and 20%were dead. At approximately 8.0 times 10 to the power of seven Candida albicans cells per milliliter, about 30%were asymptomatic, 20%were symptomatic, and 50%were dead. Median survival declined from about 90%at 7.0 times 10 to the power of seven Candida albicans cells per milliliter to about 50%at 8.0 times 10 to the power of seven cells per milliliter, and to less than 25%at 8.5 times 10 to the power of seven cells per milliliter.

Overall survival was about 50%in planarians infected with wild-type Candida albicans at approximately 8.0 times 10 to the power of seven cells per milliliter, compared to 100%survival in uninfected controls. This protocol enables in vivo study of host-pathogen dynamics by integrating imaging and molecular approaches in a genetically tractable host. Future applications include high-throughput mutant screening, comparative studies across microbial species, and adaptation for toxicology and drug efficacy testing.