Plate-Based Imaging Assay for Quantifying Photoperiodic Responses in Neurospora crassa Using Protoperithecia Development

Summary

The Protoperithecia Assay quantifies photoperiodic responses in Neurospora crassa by measuring protoperithecia formation under defined light–dark cycles. This plate-based method provides a simple and scalable approach for comparing photoperiod-dependent developmental responses across experimental conditions.

Abstract

Neurospora crassa is a well-established model organism for studying biological timing and light-regulated development. The Protoperithecia Assay (PPA) is a plate-based imaging method for quantifying photoperiodic responses by measuring protoperithecia formation as a developmental output. The goal of this protocol is to provide a reproducible and scalable workflow for assessing how defined light–dark cycles influence fungal sexual development. In this assay, fungal cultures are grown on synthetic crossing medium under controlled photoperiod conditions, followed by standardized removal of surface conidia to improve visualization. Protoperithecia are quantified by manual image-based counting, and average counts per plate are used for statistical comparisons across photoperiod conditions. Unlike prior approaches, the PPA integrates standardized culture conditions, controlled photoperiod exposure, and quadrant-based imaging to enable reproducible quantitative comparisons across strains. This workflow extends beyond a basic protocol by establishing a consistent and scalable framework for measuring photoperiod-dependent developmental output under defined experimental conditions. The method requires approximately one week from inoculation to analysis and can be applied across multiple strains under defined environmental conditions. The PPA provides a simple and accessible platform for comparative analysis of photoperiod-dependent development in fungi under controlled laboratory conditions.

Introduction

The protoperithecia assay (PPA) quantifies photoperiodic responses in the fungus Neurospora crassa. Many organisms measure day and night length to coordinate biological processes in a phenomenon known as photoperiodism^{1,2,3,4,5,6,7,8}. In this assay, the development of the female sexual structure, the protoperithecium, serves as a measurable, light-dependent proxy for photoperiodic response. Although photoperiodism is well characterized in plants through night-break experiments³, fewer standardized approaches exist for quantifying photoperiodic responses in non-photosynthetic eukaryotes such as fungi. N. crassa provides a tractable model system for this purpose due to its well-defined genetics and light-responsive developmental pathways. Photoperiod-dependent regulation of reproductive timing has been observed across organisms^{1,2,3,4,5,6,7,8}, supporting the relevance of light-driven physiological responses; however, mechanistic studies in fungi remain limited by the lack of reproducible assays.

Protoperithecia are premature female sexual structures in N. crassa. They arise from ascogonia, which develop coiled trichogynes that fuse with hyphae or conidia of the opposite mating type⁹. Following fusion, nuclei from the “male” partner enter the protoperithecium. The number of protoperithecia produced is seasonally regulated and strongly light-induced¹⁰. Photoperiodic regulation is central to ecology, chronobiology, and fungal physiology because many clock-controlled processes depend on light. Light influences numerous genetic pathways, including photoreceptors, kinases, and light-transduction mechanisms linked to sexual development¹¹. Protoperithecia are well-suited for measuring photoperiodic output because they are easily visualized under a microscope and respond sensitively to light.

Despite the importance of photoperiodism in fungi, few standardized and reproducible assays are available to quantify protoperithecia production under controlled light cycles. A medium-throughput, plate-based assay is valuable because fungi are inexpensive and easily maintained model organisms, enabling systematic analysis of light-induced sexual development and gene function. Most photoperiodism research has focused on plants, whereas equivalent quantitative frameworks for fungal systems remain limited. In other organisms, including insects, light-dependent phenomena such as diapause are well characterized⁷, but comparable approaches in fungi are lacking. This gap restricts reproducible comparison of photoperiodic responses across strains and experimental conditions.

The PPA builds on a 2004 study that measured protoperithecia production across increasing day lengths, from constant darkness to constant light, in the band (bd) mutant (ras-1^bd), which has been used in circadian studies^4,12. Photoperiod-dependent changes in protoperithecia production were observed in this background. The present workflow adapts and extends this approach using the reference strain FGSC 2489, which is associated with whole-genome sequencing and knockout resources^13,14. The assay quantifies the average number of protoperithecia produced under defined photoperiods over one week as a proxy for photoperiodic response. This approach standardizes culture conditions, imaging, and quantification, enabling reproducible comparison across experimental conditions and strains. Key methodological advances include standardized plate-based culture conditions, defined photoperiod exposure parameters, quadrant-based imaging, and a reproducible quantification workflow.

This protocol quantifies photoperiodic responses in N. crassa by measuring protoperithecia formation following exposure to defined light–dark cycles. For each photoperiodic condition, four biological replicates are used, each consisting of one independently inoculated synthetic crossing (SC) plate. Four images are collected per plate and averaged to generate a single value per biological replicate for statistical analysis. Plates are incubated for seven days under calibrated photoperiods (e.g., 12 h:12 h or 8 h:16 h light–dark cycles).

A data logger is placed inside the growth chamber to continuously record light intensity, temperature, and humidity, thereby verifying stable environmental conditions. Light intensity is maintained above 1,000 lux, as lower levels reduce protoperithecia production. Excessive illumination is avoided to prevent increases in chamber temperature and confounding thermal effects. After incubation, macroconidia are gently removed with a moistened paper towel saturated with 70% ethanol. Four evenly spaced quadrants at the perimeter of each plate are imaged at 40× magnification using a stereo microscope under brightfield illumination. Protoperithecia are counted manually using an image-processing package, and average counts per plate are used for statistical comparisons across photoperiods.

Protocol

The reagents, software, and equipments used are listed in the Table of Materials.

1. Preparation of media and materials

(Estimated time: 1–1.5 h active time; overnight cooling optional)

1. Prepare synthetic crossing (SC) media
   1. Prepare SC medium: 0.2% sucrose, 5 µg/L biotin, 1.5% agar, and Westergaard’s salts (pH 6.5 with 1 N NaOH)¹⁵.
   2. Autoclave the medium. Allow it to cool to 50–55 °C until it reaches a pourable state.
      ​CAUTION: Use heat-resistant gloves when removing flasks from the autoclave.
2. Pour plates
   1. Pipette 10 mL of molten medium into 60 mm Petri dishes using a disposable pipette.
   2. Allow plates to solidify at room temperature for 30–45 min.
   3. Store plates upside down at 4 °C for up to 4 weeks.
      ​NOTE: Maintain plate hydration. Do not store plates for more than 4 weeks at 4 °C.
3. Prepare fungal strains
   1. Grow each strain in small glass test tubes containing complete or minimal medium prepared according to laboratory manual instructions¹⁵.
   2. Incubate tubes in constant darkness (DD) at 30 °C until visible mycelial coverage forms along the tube surface (typically 2–3 days).
      CAUTION: Handle fungal cultures using standard microbiological practices and work within a biosafety cabinet to minimize contamination.

2. Inoculation of plates

(Estimated time: 15–30 min per set of plates)

1. Transfer fungal conidia
   1. Inoculate each SC plate using sterile technique and an inoculation loop.
   2. Place conidia at the center of the plate to ensure uniform growth (Figure 1A). Conidia appear as powdery, light-colored spores covering the colony surface.
   3. Apply approximately 1–2 µL of concentrated conidial suspension (or an equivalent loopful) to standardize inoculation across plates.
2. Prepare plates for incubation
   1. Close plates securely to prevent contamination while allowing gas exchange.
   2. Label each plate with strain name, date, and photoperiod condition.

3. Photoperiod treatment setup

(Estimated time: 10 min setup + 7-day incubation)

1. Calibrate the growth chamber
   1. Set the chamber temperature to 25 °C.
   2. Set humidity to 50% (acceptable range: 40%–60%) to prevent plate drying.
   3. Program photoperiod cycles: EQ (equinox): 12 h light / 12 h dark and SDP (short day): 8 h light / 16 h dark
   4. Set light intensity to >1,000 lux at plate level using a calibrated light meter. Avoid intensities that increase the chamber temperature above 25 °C.
      NOTE: Ensure that chamber lights do not overheat the interior.
      ​CAUTION: Do not use Parafilm or plastic bags to maintain humidity, as they restrict gas exchange and impair fungal growth. If humidity control is unavailable, place a water reservoir inside the chamber.
2. Place plates in the chamber
   1. Arrange plates evenly on shelves to ensure uniform light exposure.
   2. Place a data logger inside the chamber to continuously monitor temperature and humidity and verify stable environmental conditions.
3. Begin the assay
   1. Incubate plates under the programmed photoperiod for 7 days without disturbance.

4. Monitoring and preparing plates for imaging

(Estimated time: 15–20 min per plate)

1. Remove plates from the chamber
   1. Remove plates after 7 days of incubation.
   2. Inspect plates for conidial growth and protoperithecia formation.
2. Clean plates for visibility
   1. Remove surface conidia from the plate lid and agar surface using torn paper towels (Figure 1B).
   2. Moisten a paper towel with 1–2 mL of 70% ethanol. Gently dab the plate surface 3–5 times to remove conidia. Allow plates to air-dry for 1–3 min before imaging (Figure 1C).
      CAUTION: Ethanol is flammable; keep away from heat sources.
      NOTE: Avoid applying excessive pressure. Do not drag the towel across the agar surface, as this may damage the medium or disturb protoperithecia.

5. Imaging protoperithecia

(Estimated time: 5 min per plate)

1. Prepare for imaging
   1. Set up a microscope with consistent illumination and magnification (typically 40×).
   2. Ensure the plate surface is dry and free of debris.
2. Capture images
   1. Image four evenly spaced quadrants at the plate perimeter using consistent magnification.
   2. Label each image with strain, photoperiod condition, plate number, and quadrant position.
   3. Save images in a consistent format (e.g., TIFF) for analysis.

6. Quantification and data analysis

(Estimated time: 10–20 min per plate for counting; 15–25 min for analysis)

1. Count protoperithecia
   1. Open each image in an image processing package.
   2. Use the Cell Counter plugin to manually count protoperithecia. Exclude bubbles, edge artifacts, and ambiguous structures. Count clustered protoperithecia as individual, distinguishable units.
   3. Record counts for each quadrant and calculate the average per plate.
2. Analyze data
   1. Import quantified data into Statistical analysis software.
   2. Perform statistical analysis using Welch’s t-test for comparisons shown in Figure 2.
   3. Generate graphs comparing photoperiod treatments, strains, or replicates.

Results

Following inoculation with the wild-type strain, conidia covered the SC surface within 36–48 h. After 7 days of photoperiod exposure, a dense conidial layer formed and was removed with a moistened paper towel saturated with 70% ethanol (Figure 1). This step exposed underlying protoperithecia without disrupting their structure. Under microscopy, protoperithecia appeared as dark, circular structures distributed across the plate surface. These structures exhibited a spherical morphology and wer...

Discussion

The goal of the PPA is to use sexual development in fungi as a proxy for determining whether an organism perceives a specific photoperiod. By quantifying the average number of protoperithecia produced under different light cycles, photoperiodic responses can be assessed and compared across conditions. Consistent with previous observations^4,16, N. crassa exhibits higher protoperithecia production under equinox-like photoperiods, whereas short-day conditio...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar	BD	90000-786	For Synthetic Crossing Media/Minimal Media
Alcohol burner flame	Wards	470346-104	For sterilizing inoculation loop
Ammonium iron sulfate hexahydrate	Supelco	FX0245-1	For Synthetic Crossing Media/Minimal Media
Ammonium nitrate (NH4NO3)	BeanTown Chemical	BT134170-250G	For Minimal Media
Autoclave	VWR International	76683-456	For sterilizing glassware and media
Biological Safety Cabinet	ThermoFischer Scientific	1323TS	For reduccing contamination of the plates, and the lab.
Biotin	VWR	97061-444	For Synthetic Crossing Media
Boric acid (H3BO3)	Electron Microscopy Sciences	100503-696	For Synthetic Crossing Media/Minimal Media
Calcium chloride (CaCl2)	AVANTOR PERFORMANCE MATERIAL LLC	JT1311-1	For Synthetic Crossing Media
Calcium chloride dihydrate (CaCl2·2H2O)	Sigma-Aldrich	223506-25G	For Minimal Media
Citric acid monohydrate (C6H8O7·H2O)	Sigma-Aldrich	1002440500	For Synthetic Crossing Media/Minimal Media
Copper sulfate pentahydrate (CuSO4·5H2O H2O)	BeanTown Chemical	BT121245-1KG	For Synthetic Crossing Media/Minimal Media
Ethanol	Decon Labs	71001-754	Used to remove conidia from plate surface and sterilizing inoculation loop
FIJI/ImageJ	National Institutes of Health (NIH)	N/A	For analyzing images/counting
Gloves	SW Safety Solutions	76382-944	For safety
GraphPad Prism	GraphPad Software, LLC	N/A	For performing statistical tests and creating figures
Growth Chamber	Caron Products & Services	6320 & 6340 Series	Programmable for photoperiod cycles
HOBO Data Logger	Onset Computer Corporation	MX2202	For light intensity and temperature logging
Inoculation loop	VWR International	10806-354	For transferring fungal material to petri dishes
Magnesium sulfate heptahydrate (MgSO4·7H2O)	BeanTown Chemical	BT145380-1KG	For Synthetic Crossing Media/Minimal Media
Manganese sulfate monohydrate (MnSO4·H2O H2O)	Sigma-Aldrich	1059410250	For Synthetic Crossing Media/Minimal Media
Microscope (Leica S6 D Greenough Stereo Microscope)	Leica Microsystems	10446297	For imaging
Neurospora crassa FGSC 2489	Fungal Genetics Stock Center	74-OR23-1VA, JCM19069	Fungal strain
Paper Towels	Kimberly-Clark Professional, Scott and Kleenex	6666114	For pouring media
Permanent Marker	Sharpie	30072	For labeling plates
Petri Dishes	VWR International	25384-164	For plating Synthetic Crossing Media
Pipettes	VWR International	75816-090	For pouring media
Potassium dihydrogen phosphate (KH2PO4)	BeanTown Chemical	BT225070-500G	For Synthetic Crossing Media/Minimal Media
Potassium nitrate (KNO3)	Sigma-Aldrich	221295-100G	For Synthetic Crossing Media
Small Disposable Glass Test Tubes	VWR International	470211-698	For initial fungal strain growth
Sodium chloride (NaCl)	VWR International	BDH9286-500G	For Synthetic Crossing Media
Sodium citrate dihydrate (Na3C6H5O7·2H2O)	VWR	97061-008	For Minimal Media
Sodium hydroxide (NaOH)	AVANTOR PERFORMANCE MATERIAL LLC	JT5635-2	For Synthetic Crossing Media
Sodium molybdate dihydrate (Na2MoO4·2H2O)	Supelco	1.06524.1000	For Synthetic Crossing Media/Minimal Media
Sucrose	VWR International	BDH9308-500G	For Synthetic Crossing Media/Minimal Media
Zinc sulfate heptahydrate (ZnSO4·7H2O)	BeanTown Chemical	BT122545-500G	For Synthetic Crossing Media/Minimal Media