Name: An Optimized Protocol for Candida albicans Infection in Schmidtea mediterranea to Study Fungal Pathogenesis and Host Defense (Video) | JoVE Journal
Uploaded: 2026-04-17T13:30:00.000+00:00
Duration: 6 min 53 s
Description: Learn an optimized protocol for Candida albicans infection in Schmidtea mediterranea to study fungal pathogenesis and host innate immune responses in vivo. This high-throughput method enables systematic analysis of infection dynamics.

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The authors thank all members of the Nobile, Oviedo, and Hernday labs for insightful discussions about the S. mediterranea-C. albicans infection model and are grateful to Edelweiss Pfister for lab management and planarian maintenance. This work was supported by the National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) awards R35GM156045 and R35GM124594 to C.J.N., and R35GM158501 and R01GM132753 to N.J.O. This work was also supported by the Kamangar family in the form of an endowed chair to C.J.N. N.M.S. was supported by the Center for Cellular and Biomolecular Machines (CCBM) National Science Foundation (NSF) Center of Research Excellence in Science and Technology (CREST) fellowships under awards NSF-HRD-1547848 and NSF-EES-2112675.
The funders had no role in the design of the study, data collection and analysis, interpretation of the data, writing of the manuscript, or the decision to publish the results. N.M.S. acknowledges the use of ChatGPT for assistance with editing the manuscript (checking spelling, grammar, formatting, and sentence structure).

C.J.N. is a co-founder of BioSynesis, Inc., a company developing diagnostics and therapeutics for biofilm infections. All other authors declare no competing interests.

Varying the infectious dose of wild-type C. albicans produces reproducible, dose-dependent differences in morbidity in S. mediterranea, underscoring the model&#8217;s sensitivity for quantitative assessment of fungal virulence. This protocol outlines strategies for establishing baseline infectious and lethal doses within laboratory-maintained planarian colonies and presents criteria for confirming successful systemic infection via soaking, integrating host health scoring with fungal burden measurements ...

Candida albicans is a common opportunistic fungal pathogen that asymptomatically colonizes 50-70% of humans1,2,3. Typically, a harmless commensal, C. albicans can overgrow under conditions such as microbiota imbalance, antibiotic use, immunosuppression, epithelial barrier disruption, or the presence of medical devices, leading to diseases that range from superficial mucosal infections to life-threatening systemic candidiasis4,5. In immunocompromised individuals &#8211; including those with HIV/AIDS, patients undergoing chemotherapy or immunosuppressive therapies, individuals with chronic illnesses, or those in intensive care units &#8211; mortality rates can exceed 70%, underscoring the significant public health burden of invasive candidiasis6,7,8. Globally, invasive fungal infections affect more than 6.5 million people annually, with direct healthcare costs in the United States alone exceeding $8 billion per year for Candida-related infections9,10. Despite available antifungal treatments, morbidity and mortality rates associated with C. albicans infections have remained largely unchanged for decades, highlighting the urgent need to better understand host-pathogen interactions in fungal disease.
C. albicans employs a diverse repertoire of virulence factors that support colonization, persistence, and pathogenesis across a range of host environments. These include adhesion to host tissues, immune evasion, cell-wall remodeling, secretion of hydrolytic enzymes, morphological plasticity, and biofilm formation11,12,13,14,15,16. Collectively, these traits allow C. albicans to adapt to diverse host niches, modulate immune responses, and resist antifungal therapies16,17,18,19,20,21. Elucidating how these processes shape infection outcomes &#8211; including host morbidity, mortality, and pathogen clearance &#8211; is essential for developing more effective preventive and therapeutic strategies.
To investigate these host-pathogen dynamics, we established a model system using the planarian Schmidtea mediterranea as a host22,23,24. This model serves as a versatile platform for studying the multisystem host response to fungal infection, leveraging a soaking-based infection method that enables synchronous exposure of animals to C. albicans. The following section presents updated step-by-step procedures that refine this protocol, improve reproducibility, and facilitate consistent implementation across laboratories.
Planarians are free-living invertebrates with exceptional regenerative capacity, capable of rapidly replacing tissues lost to injury or infection25. They lack an adaptive immune system and rely entirely on conserved innate defenses &#8211; including pattern recognition receptors, antimicrobial peptides, mucus secretion, and phagocytic cells &#8211; to clear bacterial and fungal pathogens within days26,27,28,29. Because innate immunity represents the first line of defense across eukaryotes and remains understudied outside mammalian systems27, planarians offer an opportunity to examine these processes in vivo with cellular and molecular resolution. Their regenerative ability is driven by adult pluripotent stem cells, known as neoblasts, which constitute approximately 30% of adult cells and contribute to recovery after infection23,30. These features make S. mediterranea a powerful model for dissecting pathogen virulence and host defense at genetic, cellular, tissue, and organismal scales, enabling us to address questions that are often difficult to study using traditional mammalian systems.
In addition, planarians&#8217; small size (typically a few millimeters in length), low cost, and ease of maintenance31 support large-scale experiments while avoiding the financial, ethical, and regulatory constraints inherent to vertebrate models. Like C. albicans, S. mediterranea is genomically tractable, with a fully sequenced and annotated genome, and it supports advanced molecular and cellular techniques &#8211; including transcriptional profiling, high-resolution immunohistochemistry and histology, and robust RNA interference (RNAi)32,33,34,35,36,37,38. These tools enable parallel analysis of host and pathogen responses during infection.
Several alternative preclinical models have been used to study fungal infections. Invertebrate hosts such as Galleria mellonella (wax moth), Caenorhabditis elegans (roundworm), and Drosophila melanogaster (fruit fly), as well as the vertebrate Danio rerio (zebrafish), each provides distinct experimental advantages but also has notable limitations. These systems often rely on survival as the primary endpoint because infections rapidly induce host death, preventing detailed assessment of morbidity, recovery, and multisystem responses &#8211; features that the planarian model readily captures.
Mammalian models such as Mus musculus (mouse), Rattus norvegicus (rat), Oryctolagus cuniculus (rabbit), and Cavia porcellus (guinea pig) more closely recapitulate human physiology but are limited by high costs, small cohort sizes, ethical and regulatory requirements, and the challenge of distinguishing innate from adaptive immune responses. Moreover, decades of fungal pathogenesis research in mice have focused predominantly on late-stage systemic infection39,40,41,42,43, often overlooking early events such as epithelial barrier disruption and mucosal overgrowth &#8211; the most common routes of fungal disease initiation in humans44,45,46.
Studying host-pathogen interactions is therefore essential for understanding the biological processes that underlie fungal virulence, host defense, and disease progression. Invasive fungal infections represent an escalating global threat, with multiple species now exhibiting resistance to all major antifungal drug classes. More than 1 billion people are estimated to be affected by fungal infections, and factors such as climate change, widespread antimicrobial use, and the increasing number of immunocompromised individuals are accelerating the emergence of antifungal-resistant pathogens47,48,49,50. Understanding how pathogenic fungi colonize and interact with their hosts is critical for developing new preventive and therapeutic strategies.
The following describes a revised and standardized systemic infection protocol for S. mediterranea-C. albicans interactions, designed to promote reproducibility and to enable systematic investigation of fungal pathogenesis and host defense mechanisms. The original protocol was followed in prior publications22,23,24. Table 1 summarizes the key updates incorporated into this revised version, including improvements to fungal inoculation procedures, reduced well volumes, defined animal inclusion criteria, determination of infectious and lethal doses, and updated animal husbandry recommendations. This workflow provides detailed guidance for culturing C. albicans, infecting planarians through soaking exposure, and assessing fungal virulence and host responses using qualitative and quantitative readouts under symptomatic and lethal-dose conditions (Figure 1). The optimized procedures presented here incorporate variables empirically identified as key determinants of infection dynamics and host outcomes, thereby improving reproducibility and enabling systematic assessment of host&#8211;pathogen interactions.

<table><tbody><tr><td>1.7 mL microcentrifuge tubes&amp;nbsp;</td><td>VWR/Genemate</td><td>490004-436-BOXOF500</td><td>Clear, box of 500 pieces</td></tr><tr><td>20 mL scintillon vials</td><td>Fisher Scientific</td><td>12-100-006</td><td>Clear glass bottles for immunostaining</td></tr><tr><td>24-well non-treated polystyrene plates</td><td>Fisher Scientific</td><td>08-772-51</td><td>Immunostaining</td></tr><tr><td>5.0 mL microcentrifuge tubes&amp;nbsp;</td><td>Eppendorf/Sigma Aldrich</td><td>EP0030119487-200EA</td><td>Clear, box of 200 pieces</td></tr><tr><td>6-well non-treated polystyrene plates</td><td>Fisher Scientific</td><td>08-772-49</td><td>Infection assays</td></tr><tr><td>Agar</td><td>VWR</td><td>89405-066</td><td>Bacteriological grade for YPD medium</td></tr><tr><td>Applicator sticks</td><td>Thermo Fisher/Puritan</td><td>22-029-491</td><td>&lt;em&gt;C. albicans&lt;/em&gt; culturing</td></tr><tr><td>BioRender</td><td>BioRender Inc.</td><td>N/A</td><td>Figure design software used to generate schematic illustrations</td></tr><tr><td>Calcium chloride (CaCl2)</td><td>Sigma-Aldrich</td><td>C5080-500G</td><td>Planarian water/1x Montju&amp;iuml;c salts</td></tr><tr><td>&lt;em&gt;Candida albicans&lt;/em&gt; polyclonal antibody anti-Rabbit, IgG</td><td>Thermo Fisher</td><td>PA1-27158</td><td>Immunostaining</td></tr><tr><td>Centrifuge tubes</td><td>VWR</td><td>89039-664</td><td>&lt;em&gt;C. albicans&lt;/em&gt; harvesting</td></tr><tr><td>Culture tubes borosilicate glass 18x150mm</td><td>VWR</td><td>47729-583</td><td>&lt;em&gt;C. albicans&lt;/em&gt; culturing</td></tr><tr><td>Cuvettes</td><td>Fisher Scientific</td><td>14-955-127</td><td>To measure optical density of cultures</td></tr><tr><td>Dextrose (D-glucose)</td><td>Fisher Scientific</td><td>D16-3</td><td>YPD medium</td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>G33-4</td><td>Molecular biology grade to supplement for cryogenic storage of &lt;em&gt;C. albicans&amp;nbsp;&lt;/em&gt;</td></tr><tr><td>Graphing and data visualization tool</td><td>GraphPad/Prism</td><td></td><td>Statistical software and vizualization tool. Verison 10.6.1.</td></tr><tr><td>Graphing tool</td><td>R</td><td></td><td>Statistical software. Verison 4.4.3.</td></tr><tr><td>Incubator Shaker</td><td>Eppendorf</td><td>EPM1282-0004-1EA</td><td>New Brunswick Innova 44/44R for&lt;em&gt; C. albicans&lt;/em&gt; culturing</td></tr><tr><td>Magnesium chloride (MgCl2)</td><td>Sigma</td><td>M8266-100G</td><td>For planarian water&amp;nbsp; (1x Montju&amp;iuml;c salts)</td></tr><tr><td>Magnesium sulfate (MgSO4)</td><td>Sigma-Aldrich</td><td>M7506-500G</td><td>For planarian water&amp;nbsp; (1x Montju&amp;iuml;c salts)</td></tr><tr><td>Microscope Slides</td><td>VWR</td><td>16004-368</td><td></td></tr><tr><td>Multizoom microscope</td><td>Nikon</td><td></td><td>Nikon AZ100&amp;nbsp;</td></tr><tr><td>N-Acetyl-L-cysteine</td><td>Sigma Aldrich</td><td>A7250-50G</td><td>Mucolytic and sacrificial reagent&amp;nbsp; fixation</td></tr><tr><td>Paramaldehyde, 16% solution, molecular biology grade</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Fixative reagent</td></tr><tr><td>Pellet Pestle Cordless Motor</td><td>Fisher Scientific</td><td>12-141-361</td><td>Planarian homogenization</td></tr><tr><td>Peptone, bacteriological grade</td><td>HIMEDIA/VWR</td><td>89129-480</td><td>YPD medium</td></tr><tr><td>Pestles</td><td>Fisher Scientific</td><td>12-141-368</td><td>RNase-free disposable pellet for planarian homogenization</td></tr><tr><td>Petri Dishes, 100 x 15 mm</td><td>VWR</td><td>25384-342</td><td></td></tr><tr><td>Poly(vinyl alcohol)</td><td>Sigma-Aldrich</td><td>P-8136&amp;nbsp;</td><td>For Gelvatol</td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma Life Science</td><td>P9541-500G</td><td>Planarian medium/water&amp;nbsp; (1x Montju&amp;iuml;c salts)</td></tr><tr><td>Secondary Antibody Goat anti-Rabbit, IgG, Alexa Fluor 568</td><td>Fisher Scientific</td><td>A-11011</td><td>Immunostaining</td></tr><tr><td>SN250</td><td>Fungal Genetics Stock Center</td><td></td><td>Wildtype &lt;em&gt;C. albicans &lt;/em&gt;strain (https://www.fgsc.net/)</td></tr><tr><td>Sodium Azide</td><td>Fisher Scientific</td><td>S227-100</td><td>For Gelvatol</td></tr><tr><td>Sodium bicarbonate (NaHCO3)</td><td>Sigma-Aldrich</td><td>S5761-1KG</td><td>Planarian medium/water&amp;nbsp; (1x Montju&amp;iuml;c salts)</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma Life Science</td><td>S3014-500G</td><td>Planarian medium/water&amp;nbsp; (1x Montju&amp;iuml;c salts)</td></tr><tr><td>Spectrophotometer</td><td>Agilent</td><td>26354</td><td>BioTek Epoch 2 Microplate to measure optical density</td></tr><tr><td>Static Incubator</td><td>Fisher Scientific</td><td>15-015-2634</td><td>&lt;em&gt;C. albicans &lt;/em&gt;culturing</td></tr><tr><td>Transfer pipettes</td><td>Fisher Scientific</td><td>13-711-9CM</td><td>Sterile disposable graduated pippettes for transferring planarians</td></tr><tr><td>Tris Base</td><td>Sigma-Aldrich</td><td>252859</td><td>For Gelvatol</td></tr><tr><td>Yeast extract</td><td>Thermo Fisher</td><td>212750</td><td>YPD medium</td></tr></tbody></table>

an optimized protocol for candida albicans infection in schmidtea mediterranea to study fungal pathogenesis and host defense

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

Candida albicans is a common opportunistic fungal pathogen that asymptomatically colonizes most humans. Although typically a benign commensal, dysbiosis caused by antibiotic use, immune dysfunction, or epithelial barrier disruption can trigger fungal overgrowth and infection, ranging from superficial mucosal disease to life-threatening systemic candidiasis. New preclinical infection models are needed to dissect C. albicans pathogenesis in vivo across distinct infection stages and with different measurable host outcomes. The planarian Schmidtea mediterranea was previously established as an invertebrate host for studying host-pathogen interactions during C. albicans infection. S. mediterranea relies entirely on conserved innate immune mechanisms capable of overcoming infection with pathogenic microorganisms, including bacteria and fungi. Planarians&#8217; remarkable regenerative capacity and accessible stem cell populations make this organism a tractable model to analyze early immune responses, tissue repair, and pathogen clearance in vivo. This model supports simultaneous analysis of fungal virulence and host transcriptional responses, providing valuable insights into infection dynamics. Here, an updated protocol with detailed modifications, standardized procedures, and optimized steps for infecting S. mediterranea with C. albicans has been presented, designed to enhance reproducibility and enable systematic studies of fungal pathogenesis and host defense.

An updated protocol for infecting planarians with C. albicans (Figure 1) and systematically quantifying host outcomes (Figure 2) is presented. This refined workflow supports robust assessment of multiple infection parameters, including morbidity (symptomatic disease severity), survival (lethality), fungal burden (CFUs), and spatial-temporal dynamics of pathogen colonization (fluorescence immunostaining). Unlike most invertebrate infection models, which ...

This protocol provides an optimized, detailed guide for using the planarian Schmidtea mediterranea as a model system to study host-pathogen interactions during fungal infection. The method builds on the previous procedure for infecting planarians with the human fungal pathogen Candida albicans, providing detailed guidance to enhance reproducibility and experimental consistency.

Candida albicans is a common opportunistic fungal pathogen that asymptomatically colonizes most humans. Although typically a benign commensal, dysbiosis ...

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.

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Learn an optimized protocol for Candida albicans infection in Schmidtea mediterranea to study fungal pathogenesis and host innate immune responses in vivo. This high-throughput method enables systematic analysis of infection dynamics.

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

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