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. Author manuscript; available in PMC: 2023 Oct 25.
Published in final edited form as: Methods Mol Biol. 2023;2680:231–244. doi: 10.1007/978-1-0716-3275-8_14

A Planarian Model System to Study Host-Pathogen Interactions

Eli Isael Maciel 1,2, Ashley Valle Arevalo 1,2, Clarissa J Nobile 1,3, Néstor J Oviedo 1,3
PMCID: PMC10599129  NIHMSID: NIHMS1937556  PMID: 37428381

Abstract

This protocol is focused on using the recently established planarian infection model system to study host-pathogen interactions during fungal infection. Here, we describe in detail the infection of the planarian Schmidtea mediterranea with the human fungal pathogen Candida albicans. This simple and reproducible model system allows for rapid visualization of tissue damage throughout different infection timepoints. We note that this model system has been optimized for use with C. albicans, but should also be applicable for use with other pathogens of interest.

Keywords: Planarians, Candida albicans, Host-pathogen interactions, Fungal infections, Infection model systems

1. Introduction

Planarians are flatworms commonly used to study fundamental mechanisms regulating cellular decisions in an adult body [1]. They possess a complex body plan consisting of several tissue types, including a nervous system, excretory system, and digestive system [2–5]. In addition, they have the capacity to regenerate complete tissues and maintain high rates of cellular turnover and tissue homeostasis, resulting from their large population of adult pluripotent stem cells called neoblasts [5–9].

Recently, the planarian immune system has garnered scientific interest for its ability to quickly eliminate (within 12 days) a wide range of microbial pathogens, such as the bacterial pathogens Mycobacterium tuberculosis and Staphylococcus aureus, and the fungal pathogen Candida albicans [10–15]. Remarkably, planarians rely on an evolutionarily conserved innate immune system to clear these infections [11].

Three different methods (injection, feeding, and soaking) have been used in prior studies to infect planarians with microbial pathogens [10, 12, 14]. The infection by injection method introduces the pathogen inside the animal. In this case, a known concentration of the pathogen is injected directly into the animal at the body site of interest (see Fig. 1a). A notable advantage of this approach is that it provides an opportunity to initiate the infection at specific regions in the animal. However, a drawback is that injury is inflicted on the animal, which may trigger additional cellular responses, making it challenging to dissect the contributions of the immune and regenerative responses. The most popular method of infection is through feeding. Here, the planarians are offered food (e.g., liver paste) mixed with the pathogen for limited time periods (see Fig. 1b). Although this method does not involve injury, it is difficult to precisely determine the amount of pathogen ingested, which may lead to inconsistencies in infection levels across different animals. Another issue with the infection by feeding method is that planarians are natural scavengers and have evolved a large population of phagocytic cells within their digestive tracts that protect them against infection [11, 16]. Thus, immune responses to infection by feeding may differ compared to other methods since pathogens are exposed to phagocytic cells immediately after ingestion. These anatomical interactions with the pathogen suggest that the digestive route offers unique opportunities to assay pathogen clearance by the host, but it is more restrictive in evaluating pathogen virulence. Another method of infection involves soaking planarians in known concentrations of the pathogen for a limited time frame. Planarians live in water and adding pathogens to the media allows for the control of pathogen concentration and exposure time to the pathogen. In addition, with this method, pathogens can be easily and rapidly removed through the addition of fresh media. This “soaking method” is injury-free, simple, and consistent and allows for the visualization of the infection process accounting for the initial physical interactions between the host and the pathogen (i.e., pathogen adhesion) to the planarian epithelial surface and penetration to deeper host tissues [14]. The soaking method also facilitates the evaluation of pathogen clearance by the host, as well as virulence of the pathogen.

Fig. 1.

Fig. 1

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Infection methods to study host-pathogen interactions in the planarian model system. (a) Injection. An overnight C. albicans culture is pelleted and resuspended in planarian water. A few microliters of the pathogen mixture are picked up in a glass capillary tube connected to a nano-injection needle and injected directly into the planarian at a body site of interest. The infected planarians are then placed in a well and observed and/or collected at specific timepoints for CFU enumeration and downstream experimentation. (b) Feeding. An overnight C. albicans culture is diluted to the desired concentration and pelleted. The pellet is mixed with liver paste and dispensed inside the planarian well. The planarians are left with the liver solution for one hour and then washed. Animals successfully infected will have a slightly red body tint indicative that they have recently eaten. The planarians are then directly observed and/or collected at specific timepoints for CFU enumeration and downstream experimentation. (c) Soaking. An overnight C. albicans culture is diluted to the desired concentration and dispensed into the well containing planarians in a 4 mL total volume of planarian water. The planarians are left in the well with the microbial cell mixture of planarian water for 3 days, after which the planarians are washed, and their water changed daily. The planarians are then directly observed and/or collected at specific timepoints for CFU enumeration and downstream experimentation

In this chapter, we present detailed protocols for the three planarian infection methods introduced above, with a primary focus on the soaking method. We use C. albicans as the pathogen example in our protocols, but note that C. albicans could be exchanged for other pathogens of interest. C. albicans is a common human fungal pathogen that can cause a variety of infections, ranging from superficial skin infections to severe systemic infections [17, 18]. C. albicans is multimorphic and can develop several different cellular morphologies depending on its environment [19, 20]. One notable C. albicans morphological transition that is important for virulence is the yeast to hyphal transition, where round budding yeast-form cells transition into elongated filamentous cells (pseudohyphae and hyphae) and vice versa. This reversible morphological transition is important for C. albicans to adapt to multiple host environments as well as to cause host tissue damage [21].

2. Materials

2.1. Culturing of C. Albicans for Planarian Infection

  1. Schmidtea mediterranea planarian asexual strain (CIW4).

  2. C. albicans isogenic wild-type strain (SN250) [20].

  3. C. albicans nrg1/nrg1 hyper-filamentous mutant strain (TF125) [20].

  4. C. albicans efg1/efg1 nonfilamentous mutant strain (TF156) [20].

  5. YPD (liquid and agar medium): 2% Bacto peptone, 2% dextrose, 1% yeast extract, 2% agar (for agar media only).

  6. 20 mL test tubes.

  7. Short and long autoclaved toothpicks.

  8. Cuvettes and spectrophotometer.

2.2. Infecting Planarians Through Injection

  1. Planarian water (1X Montjuic saltwater solution): 1.6 mM NaCl, 1.0 mM CaCl2, 1.0 mM MgSO4, 0.1 mM MgCl2, 0.1 mM KCl, 1.2 mM NaHCO3; using ultrapure water bring up to 13.5 L.

  2. Overnight C. albicans culture.

  3. Glass capillary tubes.

  4. Glass needle puller (e.g., p-97 flaming/brown micropipette puller).

  5. Nanoject II Injector (Drummond).

  6. Kimwipes.

  7. Parafilm.

  8. Forceps.

  9. 6-well polystyrene non-tissue culture plate.

  10. 3 mL transfer pipette.

  11. Petri dishes.

2.3. Infecting Planarians Through Feeding

  1. Planarian water (1X Montjuic saltwater solution): 1.6 mM NaCl, 1.0 mM CaCl2, 1.0 mM MgSO4, 0.1 mM MgCl2, 0.1 mM KCl, 1.2 mM NaHCO3; using ultrapure water bring up to 13.5 L.

  2. Overnight C. albicans culture.

  3. Liver paste (derived fresh and organic from the butcher which is processed the same day with a blender, aliquoted, and stored at −80 °C).

  4. 6-well polystyrene non-tissue culture plate.

  5. 3 mL transfer pipette.

  6. Petri dishes.

2.4. Infecting Planarians Through Soaking

  1. Planarian water (1X Montjuic saltwater solution): 1.6 mM NaCl, 1.0 mM CaCl2, 1.0 mM MgSO4, 0.1 mM MgCl2, 0.1 mM KCl, 1.2 mM NaHCO3; using ultrapure water bring up to 13.5 L.

  2. Overnight C. albicans culture.

  3. 6-well polystyrene non-tissue culture plate.

  4. 3 mL transfer pipette.

  5. Petri dishes.

  6. Dissecting microscope.

2.5. Fixation After Infection Via Feeding

  1. 20 mL scintillation vials.

  2. NAC solution: 7.5 g N-acetylcysteine; diluted in 10 mL of ultrapure water.

  3. 1× PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4; using ultrapure water bring up to 1 L.

  4. 4% fixative solution: 1.1 mL 36.5% formaldehyde; diluted in 1× PBS.

  5. 1% SDS solution: 1 mL of 10% SDS and 9 mL of 1× PBS.

  6. 0.3% PBSTx: 100 mL of 10× PBS, 3 mL of Triton X-100; using ultrapure water bring up to 1 L.

2.6. Fixation After Infection Via Injection and Soaking

  1. 20 mL scintillation vials.

  2. NAC solution: 9 g N-acetylcysteine; diluted in 10 mL ultrapure water.

  3. 1× PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4; using ultrapure water, bring up to 1 L.

  4. 4% fixative solution: 1.1 mL of 36.5% formaldehyde; diluted in 1× PBS.

  5. Formamide solution: 300 μL of formamide, 400 μL of H2O2, and 9.3 mL of 1× PBS.

  6. 0.3% PBSTx: 100 mL of 10× PBS, 3 mL of Triton X-100; using ultrapure water, bring up to 1 L.

2.7. Whole-Mount Immunohistochemistry

  1. Anti-Candida, 1:500.

  2. Goat anti-rabbit Alexa 568, 1:800.

  3. HRP-conjugated goat anti-rabbit antibody, 1:1000.

  4. PBSTB: 1.25 g of BSA; diluted in 50 mL of 0.3% PBSTx.

  5. FITC tyramide solution: 1:1000 FITC, 500 μL of 1 M imidazole, and 50 mL of 0.3% PBSTx.

  6. Quench solution: 600 μL of H2O2 in 6 mL of PBSTx.

2.8. Calculating C. albicans Concentration from Overnight Cultures

  1. Glass hemacytometer.

  2. Coverslips.

  3. Handheld cell counter.

  4. 70% ethanol.

3. Methods

3.1. Culturing C. albicans for Planarian Infections

  1. C. albicans strain SN250 is the isogenic wild-type reference strain used to compare mutant strains like the hyper-filamentous strain TF125 (nrg1/nrg1) and nonfilamentous strain TF156 (efg1/efg1) to assess the effects of filamentation on the infection assays [20].

  2. C. albicans strains are streaked and grown in yeast extract peptone dextrose (YPD) agar at 30 °C for 48 h (see Note 1).

  3. Single-colony overnights are set up shaking at 30 °C for 12–16 h prior to the infection assay. This culturing condition is commonly used to grow C. albicans wild-type strains, mutant strains, and clinical isolates for experimentation [22].

3.2. Infecting Planarians Through Injection

  1. Place planarians in a cold plate or ice block with a layer of parafilm and a damp Kimwipe for immobilization.

  2. Spin down overnight C. albicans cultures grown for 12–16 h at 4596 g for 5 min.

  3. Resuspend the pellet in planarian water.

  4. Construct glass injection needles from glass capillary tubes after breaking the tip using forceps (see Note 2).

  5. Place the glass needle in a Nanoject II Injector (Drummond) and backfill with mineral oil and a few microliters of the C. albicans cells.

  6. Inject planarians with two pulses (see Note 3).

  7. Place injected planarians into planarian water.

3.3. Infecting Planarians Through Feeding

  1. Grow overnight culture of C. albicans for 12–16 h.

  2. Allocate a concentration of ten million C. albicans cells to a tube and pellet using a centrifuge at 4596 g for 5 min.

  3. Decant the supernatant and keep the pellet. Mix the pellet with 50 μL of liver paste, and pipette the mixture to a petri dish containing the planarians.

  4. Allow the planarians to feed for 2–4 h. They will turn a slight pink/red color indicating that they have eaten.

  5. Once they have eaten, clean the petri dishes or move the planarians to a clean petri dish.

  6. The planarians will regurgitate some parts of the mixture eaten and will need to be cleaned again the next day.

3.4. Infecting Planarians Through Soaking

  1. Grow overnight culture of C. albicans for 12–16 h.

  2. Maintain ten planarians in 6 mL wells containing 3 mL of planarian water.

  3. Add the desired concentration of C. albicans cells and adjust the total volume to 4 mL with planarian water (see Note 4).

  4. Maintain planarians in the infected media for 3 days.

  5. After the 3-day exposure, wash the planarians daily with fresh water and observe them under the microscope to record any behavioral or macroscopic defects until they are collected for specific downstream experiments at various timepoints.

3.5. Fixation After Infection Via Feeding

  1. Collect planarians in 20 mL scintillation vials filled with 3 mL of planarian water.

  2. Sacrifice planarians by placing in 7.5% of NAC solution for 5 min at room temperature with rocking.

  3. Fix planarians by replacing liquid with 4% of formaldehyde in 1× PBS for 20 min at room temperature with rocking.

  4. Remove the fixative and replace with a 1× PBS wash.

  5. After the wash, permeabilize planarians using a formamide solution with 6% of H2O2 for 20 min (see Note 5).

  6. Wash the animals three times with 1× PBS and store at 4 °C for up to 7 days.

3.6. Fixation After Infection Via Soaking or Injection

  1. Place select planarians collected at specific timepoints of interest in a 20 mL scintillation vial filled with 3 mL of planarian water.

  2. Sacrifice planarians by removing the planarian water and replacing it with 9% of NAC solution for 5 min at room temperature with rocking.

  3. Remove the NAC solution and add 4% of fixative for 15–20 min at room temperature with rocking.

  4. Remove the 4% of fixative and rinse two times with 1× PBS.

  5. Remove the 1× PBS, and permeabilize the planarians with 1% of SDS for 20 min at room temperature with rocking.

  6. Rinse the planarians three times with 1× PBS.

  7. Bleach planarians in 6% of H2O2 in 1× PBS for 2–4 h under a light source (see Note 5).

  8. Remove the bleaching solution, and rinse planarians with 1× PBS three times, and store at 4 °C for up 7 days.

3.7. Whole-Mount Immunohistochemistry

  1. Transfer planarians from 1× PBS solution to 0.3% of PBSTx solution (see Note 6).

  2. After 20 min, replace the PBSTx with PSTB for 4 h at room temperature with rocking.

  3. After the 4 h, transfer planarians to a 24-well plate.

  4. Incubate planarians with the anti-Candida (1:500) antibody for 4 h at room temperature or 8 h at 4 °C overnight.

  5. Wash planarians every 20 min for 2.5 h with PSTB.

  6. After all the washes, add the Alexa 568 (1:800) secondary antibody, and incubate for 4 h at room temperature or 8 h at 4 °C.

  7. Remove the secondary antibody, and wash planarians every 20 min for 2.5 h. Mount and observe in the microscope the stained planarians (see Fig. 2).

Fig. 2.

Fig. 2

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Immunohistochemistry of C. albicans using different infection methods in the planarian model. (a) Outline of the planarian anatomy in its position in the subsequent images. (b) Injection. The planarian was injected in the parapharyngeal area with 6000 wild-type C. albicans cells, fixed, and stained 6 hours post infection. Using this infection method, a small number of C. albicans cells are shown to adhere to the indicated area. (c) Feeding. The planarian was fed ten million wild-type C. albicans cells/mL and stained 1 day post infection. Using this infection method, C. albicans cells can be observed throughout the digestive tract of the planarian. (d) Soaking. The planarian was soaked in 15 million wild-type C. albicans cells/mL and stained at 3 days post infection. Using this infection method, C. albicans cells can be observed to adhere throughout the planarian epithelial layer and different morphologies of C. albicans cells can be observed (see Note 7)

3.8. Calculating C. albicans Concentration from Overnight Cultures

  1. Clean glass hemocytometer with alcohol and let dry. Moisten a coverslip with a small amount of water prior to adhering to the hemocytometer.

  2. Using aseptic technique, make a 1:20 dilution of the overnight C. albicans culture and add 10 μL of the dilution to the hemocytometer. Allow cells to flow through and fill chamber(s).

  3. Place the hemocytometer on a microscope, and using the 20× objective, focus on the grid lines of the hemocytometer.

  4. Count the number of C. albicans cells in all four outer squares and divide by four. This is the mean number of cells per square.

  5. Calculate the number of cells per square × 104 = the number of cells/mL of overnight culture. Multiply by 20 to account for the original 1:20 dilution. This will be the working concentration.

  6. Clean the hemocytometer with 70% ethanol, and dispose of coverslip.

4. Notes

  1. Recommendations for Growing C. albicans Strains. Do not use C. albicans colonies that are greater than 7 days old or store plates at 4 °C as C. albicans acquires aneuploidies under these conditions. Likewise, new plates should be streaked from glycerol stocks rather than re-streaked from existing plates. Alternative growth media can be used for these experiments, although the concentrations used in the protocol will need to be optimized for the specific media chosen. We recommend YPD media since it is a rich medium that supports the growth of all C. albicans strains and has been optimized for these infection assays.

  2. Recommendation for Glass Capillaries. The dimensions of the pulled capillaries are as follows: outer diameter is ~1.15 mm, and inner diameter is ~0.5 mm.

  3. Recommendations for Using the Different Planarian Infection Methods. When using the injection method, a few microliters can be used to infect all planarians using a nanoinjector dispensing two pulses ~32 nanoliters/pulse.

  4. Recommendations on C. albicans Cell Concentrations to Use in the Soaking Infection Assay. We have tested a range of different infection concentrations from 5 to 20 million cells/mL for the three strains described in this chapter. For the hyper-filamentous strain, we determined that five million cells/mL cause 50% death of the planarians (see Fig. 3a). We determined that 15 million cells/mL are required of the wild-type and the nonfilamentous C. albicans strains to maintain survival of the planarians after 3 days of infection (see Fig. 3c). To comparatively visualize the different morphologies of these C. albicans strains during the infection process and to assess virulence, the use of the same number of C. albicans cells between strains is recommended. We typically use an infection concentration of 7.5 × 106 cells/mL for each strain to allow for enough planarians to survive throughout infection with any of the three C. albicans strains used in this chapter to conduct different downstream experiments, such as immunohistochemistry assays, transcriptional profiling experiments, and protein assays (see Fig. 3b). If the desired assay is simply to determine virulence via an endpoint survival assay, a concentration of 20 million C. albicans cells/mL and above is ideal (see Fig. 3d).

    When using the soaking infection method, once the C. albicans cells have been added, make sure to swirl the plate for 10 seconds. Lastly, when removing the infected media, make sure the animals are not left too long without water. Additionally, just transferring the animals to clean wells is a lot easier than cleaning the wells used for infection. We recommend using 3 mL transfer pipettes to move the planarians between wells. If a planarian gets stuck inside the transfer pipette, flick the region of the transfer pipette to detach the planarian. All procedures before, during, and after infection with C. albicans should be performed in water at room temperature, which is the ideal condition for planarians.

  5. Recommendations on Soaking and Injection Fixation. During fixation, some planarians may start to float with many air bubbles surrounding them. Swirl the plate to help them to sink. If the planarians continue to float after the bleaching step, perform additional 1× PBS washes.

  6. Recommendations on Immunohistochemistry. Use a black background underneath the plate to help in visualizing the bleached planarians. When removing liquid from the plate, tilt the plate at a 45° angle and retrieve the solution slowly from the top of the well, being careful not to touch the planarians.

  7. Recommendations on Visualization of C. albicans. Depending on the infection method chosen, visualization of the infection will vary (see Fig. 2). Using the injection method, very few wild-type C. albicans cells are observed to adhere to the planarians (see Fig. 2b). In the feeding method, wild-type C. albicans cells can be observed throughout the digestive tract in the yeast-form cell morphology that was introduced (see Fig. 2c). In the soaking method, wild-type C. albicans can be observed adhered to the epithelial layer of planarians and can be observed to transition from the yeast form to the hyphal form (see Fig. 2d).

  8. Recommendation on Observing C. albicans Yeast and Hyphal Morphologies Using the Planarian Soaking Infection Method. An isogenic C. albicans wild-type strain should be used as a reference strain to compare to the mutant strains of interest. In this example, we use the isogenic wild-type strain SN250, the hyper-filamentous strain TF125, and the nonfilamentous strain TF156 to infect the planarians using the soaking method. This method is optimal for visualizing the infection process in the planarians as well as changes in C. albicans cellular morphologies during the infection (see Fig. 4).

Fig. 3.

Fig. 3

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Planarian survival upon infection with different C. albicans strains (wild-type, hyper-filamentous, and nonfilamentous) 3 days post infection. (a) Planarian survival after infection with 5 × 106 cells/mL of C. albicans cells of the three different strains. (b) Planarian survival after infection with 7.5 × 106 cells/mL of C. albicans cells of the three different strains. (c) Planarian survival after infection with 15 × 106 cells/mL of C. albicans cells of the three different strains. (d) Planarian survival after infection with 20 × 106 cells/mL of C. albicans cells of the three different strains

Fig. 4.

Fig. 4

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Different C. albicans cellular morphologies are observed using the soaking infection method 3 days post infection. (a) Hyphal cells are observed in the hyper-filamentous mutant strain. (b) Both yeast-form and hyphal cells (yellow arrows) are observed in the wild-type strain. (c) The yeast-form growth morphology is observed in the nonfilamentous mutant strain (see Note 8)

Acknowledgments

We thank Edelweiss Pfister for lab management and planarian maintenance in the Oviedo lab and all members of the Oviedo and Nobile labs for insightful discussions on protocols and optimization procedures. This work was supported by the National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID) award R21AI125801, and the National Institute of General Medical Sciences (NIGMS) award R35GM124594 and by the Kamangar family in the form of an endowed chair to CJN. This work was also supported by the NIGMS award R01GM132753 to NJO. AVA was supported by diversity supplement fellowship R21AI125801–02S1 to parent grant R21AI125801. EIM was supported by the National Science Foundation (NSF) graduate fellowship award 1744620. The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.

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