Ateneo - UPO

The course covers molecular methods applied to the study of ecosystems: molecular markers and population genetic diversity; species identification by DNA barcoding; metabarcoding and environmental DNA for biomonitoring; metagenomics and metatranscriptomics; differential transcriptomics and molecular stress biomarkers; elements of proteomics, metabolomics and ecological epigenetics; interpretation and use of molecular data in environmental and regulatory contexts. The course includes 32 hours of lectures and 16 hours of laboratory work based on provided protocols, covering the full analytical chain from field sampling to the interpreted result.

Lecture notes (Modules 1–12), available on the course platform before classes begin. These serve as the primary reference and are sufficient on their own for exam preparation.

Further Reading
Freeland J.R., Molecular Ecology, 3ª ed., Wiley-Blackwell, 2020.
van Straalen N.M., Roelofs D., An Introduction to Ecological Genomics, 2ª ed., Oxford University Press, 2012.
Taberlet P., Bonin A., Zinger L., Coissac E., Environmental DNA: For Biodiversity Research and Monitoring, Oxford University Press, 2018.

Supplementary materials: Laboratory protocols, data sheets, datasets from the bioinformatics workshop, self-assessment worksheets for each module, and primary articles for discussion—all provided by the instructor on the course platform.

The course provides the conceptual and experimental tools through which molecular methods address ecological questions: identifying the organisms present in an environment, measuring the genetic diversity of a population, describing the composition of a community based on environmental DNA, and detecting an organism’s response to stress before it becomes visible at the individual or population level. In addition to the theoretical component, the course includes a guided experimental lab session that takes students through the entire real-world analytical workflow, from the soil sample to the interpreted data.

Sustainable Development Goals (2030 Agenda)
SDG 15 — Life on Land. Molecular methods for measuring terrestrial biodiversity and soil biological quality.
SDG 6 — Clean Water and Sanitation. Environmental DNA and metabarcoding applied to water quality monitoring.
SDG 4 — Quality Education. Comprehensive and accessible course materials provided in advance, designed to ensure an equivalent learning experience for students who cannot attend classes regularly.

Basic knowledge of molecular biology (nucleic acid structure and replication, transcription, PCR), genetics (inheritance, polymorphism, allele frequencies) and ecology (population, community, ecosystem, diversity indices), as acquired during the Bachelor's degree. No prior computing skills are required: the bioinformatic tools used in the laboratory are introduced by the lecturer and used through guided procedures.

Lectures supported by complete handouts, made available on the course platform before the start of the course. Each module includes a set of self-assessment questions with commented answers. Laboratory sessions are run on provided protocols with pre-formatted data sheets. A bioinformatics workshop is run on a provided dataset. One primary research article per macro-topic is discussed in class. Course material is designed to be self-sufficient: students unable to attend regularly have a complete study path and sit the same examination.

Non-attending students. Attendance is not compulsory. The syllabus, the examination and the assessment criteria are identical for attending and non-attending students; no reduced syllabus and no alternative examination are provided. A laboratory data kit is published on the course platform together with the handouts, at the start of the course: photographic and short video documentation of the experimental work, the protocols distributed in class, the completed ecotoxicological endpoint sheets for the campaign including replicates and controls, extraction yields, gel images, raw Sanger electropherograms, the ASV table of the metabarcoding dataset with sample metadata, and blank data sheets. Non-attending students write the laboratory report on the same template and are assessed against the same rubric. In the oral examination, the laboratory component assesses the rationale of the protocols, the choice of controls and the interpretation of results; manual execution of the procedures is not assessed.

Other questions.
Office hours are held by appointment, which must be scheduled via email. All course materials are posted on the learning platform before classes begin.

Students with disabilities or specific learning disabilities are encouraged to contact the instructor and the University’s dedicated service to arrange for the necessary accommodations and exemptions, both for coursework and for exams: https://www.uniupo.it/it/servizi/servizi-studenti-disabili-e-dsa

Attendance at the lab is contingent upon completion of the safety training required by the University and the use of the personal protective equipment provided.

The first lab session includes a field trip to a local farm for sampling; transportation arrangements, insurance coverage, and the schedule will be communicated at the beginning of the course.

Assessment consists of three elements, announced in the first lecture and published with the course material. (1) A laboratory report on the provided template (maximum 6 pages), due within 15 days of the end of the laboratory, graded pass/fail against a published rubric: a pass is a prerequisite for admission to the examination and does not contribute to the final mark; one revision is allowed within 10 days. Students who did not attend the laboratory write the report using the laboratory data kit published on the course platform, on the same template and against the same rubric. (2) A final multiple-choice test (30 questions, four options, 45 minutes, no penalty for wrong answers): a minimum score of 18/30 grants access to the oral examination and does not contribute to the final mark. (3) An oral examination consisting of three questions, one for each macro-topic of the course, which determines the final mark in full, according to the published rubric.

Oral Exam Rubric. 18–20: Demonstrates knowledge of the fundamental elements of the three major topics and the logic behind the methods, using appropriate terminology. 21–23: Correct and well-organized presentation; can explain the purpose of each technique and the data it produces. 24–26: connects content across modules, recognizes the limitations of a technique, and interprets a simple result. 27–29: argues for alternative methodological choices and critically discusses a case study. 30 and 30 with honors: complete mastery, interpretive autonomy, and connection to primary literature.

Report Evaluation Grid. A report is considered acceptable if it includes all required sections, presents data with units of measurement and the number of replicates, contains at least one correct and legible figure, addresses the three guiding questions in the Discussion section, and includes a properly formatted bibliography.

Lectures (32 hours, 16 sessions of 2 hours each)
1. What is molecular ecology (2 h)? Levels of biological organization from the gene to the community. Which ecological questions can be answered at the molecular level and which cannot. Types of markers and their temporal and taxonomic resolution.

2. The Sample Before the Data (2 h). Sampling design, replication, and statistical power. Preservation of environmental matrices and degradation of nucleic acids. Chain of custody and traceability. What determines the quality of molecular data.

3. Genetic Diversity and Population Structure — I (2 h). Polymorphism and heterozygosity. Hardy-Weinberg equilibrium and its violations. Genetic drift, effective population size, and bottlenecks.

4. Genetic Diversity and Population Structure — II (2 h). Gene flow and F-statistics: calculation and interpretation. Population connectivity and conservation genetics. Sexual determination through the tree of life: chromosomal and environmental determination (ESD/TSD), sex-specific markers, sex ratio as an ecological endpoint, and its alteration by contaminants.

5. Molecular species identification (2 h). The logic of DNA barcoding. Marker selection: COI in animals, rbcL and matK in plants, ITS in fungi, 16S and 18S rRNA. Reference databases: BOLD, GenBank, SILVA, UNITE. Sanger sequencing and interpretation of the electropherogram.

6. Limitations of molecular identification (2 h). Barcode gaps and their absence. Nuclear pseudogenes of mitochondrial origin (NUMTs). Introgression, hybridization, mitochondrial inheritance, and discrepancies with nuclear phylogeny. Cryptic species. Completeness and quality of reference databases as limiting factors.

7. Metabarcoding and environmental DNA (2 h). Environmental DNA: origin, status, persistence, and transport in various matrices. Primer design and amplification bias. Library preparation and sequencing platforms. Applications: water biomonitoring, early detection of invasive species, diet analysis, and soil communities.

8. From Amplicon to Community Table (2 h). Quality filtering, denoising, OTUs, and ASVs. Taxonomic assignment and its uncertainties. Negative and positive controls, contamination, index hopping. Relative abundance: what can and cannot be inferred about actual abundance.

9. Metagenomics (2 h). Shotgun sequencing and comparison with the amplicon-based approach. Read quality control and removal of host sequences. Assembly, binning, and metagenome-assembled genomes (MAGs): what conclusions can be drawn and with what degree of uncertainty.

10. Metatranscriptomics and function (2 h). Functional annotation and reference databases. Distinction between taxonomic profile, functional potential, and actually expressed function. The soil microbiome as a quality indicator: genes and functions used as descriptors.

11. Gene Expression and Differential Transcriptomics (2 h). Regulation of gene expression in response to stressors. Designing a differential transcriptomics experiment: replication, confounders, normalization. Significance and limitations of a log2 fold change; multiple testing and FDR control. Functional enrichment analysis and its interpretive pitfalls.

12. Biomarkers and Molecular Ecotoxicology (2 h). Molecular biomarkers: validity criteria, specificity, and relationships with dose and effect. Adverse Outcome Pathways: structure and use. Elements of molecular ecotoxicology; the link between molecular response and population-level endpoints.

13. Environmental proteomics and metabolomics (2 h). Analytical approaches and their resolution. Additional information compared to the transcriptome and relative cost. Specific challenges related to environmental matrices and non-model organisms.

14. Ecological epigenetics and multi-omic integration (2 h). DNA methylation and other epigenetic marks. Phenotypic plasticity and transgenerational effects: evidence and interpretive caveats. Multi-omic integration: strategies, design requirements, and practical limitations.

15. Interpretation of community data (2 h). Alpha and beta diversity. Rarefaction and normalization of sequencing depth. Cluster analyses (PCoA, NMDS) and their interpretation. Hypothesis testing on community data and choice of distance metric.

16. From Data to Decision (2 h). Weight-of-evidence approach. Use of molecular data in the context of environmental regulation. Discussion of case studies and summary of the analytical workflow covered in the laboratory.


Laboratory (16 hours, 4 sessions of 4 hours each)
The laboratory follows a single overarching theme: the characterization of soil from a production environment (winery), incorporating a standardized ecotoxicological assay and the molecular identification of the organisms present. All activities are conducted according to the provided protocol, with pre-prepared data sheets to be filled out.

L1 — Field Sampling (4 h). Field trip to the farm. Definition of the sampling plan on-site: sampling points, replicates, depths, and control samples. Collection of soil samples, georeferencing, labeling, and completion of the chain of custody. Basic characterization of samples (moisture, pH). Deliverables: completed sampling plan and sample data sheet.

L2 — Assay Setup and Extraction (4 h). Preparation of eluates. Setup of the oligochete annelid assay (avoidance test per ISO 17512-1 or OECD 207). Extraction of nucleic acids from soil and tissue, quantification, and quality control. Deliverables: assay setup sheet, extraction sheet (yield, A260/280 ratio).

L3 — Endpoint reading and amplification (4 h). Session scheduled at least 14 days after L2, as required by the duration of the assay. Reading of ecotoxicological endpoints (avoidance, mortality, biomass) and calculation. Barcoding PCR and gel electrophoresis. Sample preparation for sequencing. Deliverables: endpoint worksheet with calculations, annotated gel image.

L4 — Bioinformatics workshop (4 h). Editing of Sanger electropherograms; BLAST and BOLD queries; alignment and phylogenetic tree. Analysis of a provided metabarcoding dataset: ASV table, alpha and beta diversity, sorting, and figure generation. Integrated interpretation of molecular and ecotoxicological data. Deliverables: taxonomic assignment table with explicit criteria, two figures, and an interpretation paragraph.

The course provides the conceptual and experimental tools through which molecular methods address ecological questions: identifying organisms present in an environment, measuring the genetic diversity of a population, describing the composition of a community based on environmental DNA, and detecting an organism’s response to stress before it becomes visible at the individual or population level. In addition to theoretical instruction, the course includes a guided experimental lab session that takes students through the entire real-world analytical workflow, from soil sample to interpreted data.

Dublin Descriptors
Knowledge and understanding. By the end of the course, students will be familiar with the molecular markers used in ecology and their scope of application; the theoretical foundations and limitations of DNA barcoding, metabarcoding, and environmental DNA analysis; the principles of metagenomics and metatranscriptomics; the molecular mechanisms of stress responses; and the fundamentals of molecular ecotoxicology.

Ability to apply knowledge and understanding. Following provided protocols, students perform the main laboratory procedures in molecular ecology—extraction of nucleic acids from environmental matrices and tissue, amplification of diagnostic markers, setup and interpretation of a standardized ecotoxicological assay—and interpret the output of a standard bioinformatics pipeline applied to a provided dataset, producing accurate tables and figures.

Independence of judgment. The student recognizes the limitations of the validity of molecular data—adequacy of controls, contamination, amplification bias, completeness of the reference database—and distinguishes conclusions supported by the data from those that go beyond it.

Communication skills. The student writes a scientific report following a given structure, including original tables and figures and a properly formatted bibliography, and delivers an organized oral presentation using appropriate terminology.

Learning ability. The student is able to locate and read a primary article on molecular ecology in English and place its methods within the context of those covered in the course.