Aspergillus Germination Dynamics

Custom R scripts for analyzing phenotypic heterogeneity and germination dynamics in Aspergillus niger spores using oCelloScope time-lapse imaging.

📚 Overview

This repository contains R scripts and data-processing pipelines developed during my PhD research to study fungal spore germination dynamics. The goal was to analyze phenotypic heterogeneity in swelling and germ tube formation across various A. niger spore populations, using method based on relative changes in spore morphology.

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📚 Background & Scientific Rationale

Phenotypic heterogeneity plays a central role in the germination behavior of Aspergillus spores. Earlier methods (Chapters 2–4) relied on fixed area and circularity thresholds, which introduced bias across species and subpopulations. The improved pipeline (Chapter 5) defines germination relative to initial spore size and shape, accommodating asynchronous germination and subpopulation differences.

This method:

Removes the need for species-specific thresholds

Is robust against size-related artifacts

Is applicable to mixed populations (e.g., environmental samples or co-cultures)

The revised pipeline enhances reproducibility and reflects biological variation, not just population averages.

🧪 Key Concepts:

• Spore phenotypic heterogeneity affects germination timing and dynamics.
• Germination is analyzed in two phases: swelling and germ tube formation.
• Maturity of spores (e.g., 8-day-old vs. 38-hour-old) significantly impacts nutrient response.
• Compatible solute-deficient mutant (SJS128) Hypothesised to have the same germination dynamics as immature A. niger reference strain.

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🧬 Data & Conditions

The datasets were generated using oCelloScope time-lapse imaging to track the germination of individual spores across 24 hours.

Strains:

• N402 – reference strain mature spores (8 days)
• N402 38h – Immature spores (harvested at 38 hours)
• SJS128 – Mutant lacking biosynthesis of compatible solutes genes

Nutrient Treatments:

• Amino acids: Proline, Alanine, Arginine
• Sugar: Glucose
• Each tested at 10 mM and 1 mM

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🔍 Analysis Pipeline

1. Preprocessing

• Clean and merge .csv files exported from oCelloScope.
• Track spores by ObjectId and calculate:
  • Initial area
  • Initial circularity
  • Relative increases over time

2. Germination Phase Assignment

• Resting (R): No significant area or circularity change
• Swelling (S): Area ↑ ≥ 10%, circularity ~ stable
• Germ Tube (G): Area ↑ ≥ 50%, circularity ↓ ≥ 10%

3. Metrics Extraction

Using the germinationmetrics R package:

• Germination % (Pmax)
• Time to 50% germination (t50_coolbear)
• Germination heterogeneity (GermUncertainty, d)
• Coefficient of Velocity of Germination (CVG)
• Weighted Germination Percentage (WGP)

4. Curve Fitting & Visualization

• Modeled germination using 4-parameter Hill functions (FourPHFfit.bulk)
• Swelling and germ tube formation plotted with overlaid replicate dots

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🧠 Acknowledgements

Some part of this pipeline builds on the germinationmetrics R package (Aravind et al., 2024), originally designed for plant seed germination.

✉️ Contact

For questions or collaborations:

Maryam Ijadpanahsaravi

✉️ m.ijadpanahsaravi@uu.nl

🔬 PhD Candidate, Microbiology, Utrecht University