Plankton sits at the foundation of nearly every aquatic food web. These organisms, ranging from bacteria barely a fifth of a micron across to zooplankton visible to the naked eye, drive biogeochemical cycles and transfer energy upward to fish and other animals. Warming is expected to reduce plankton diversity and shift food web efficiency in favour of smaller organisms and pathogenic bacteria, potentially weakening the resilience of entire ecosystems. Yet most experimental studies on plankton responses to warming have been conducted in the Baltic Sea or in cold northern lakes, leaving the Mediterranean dangerously underexplored. Standardised approaches for studying heatwave effects on natural plankton communities in Mediterranean waters have simply been lacking.

The FUTURE framework was built by a multidisciplinary team spanning the University of Sassari and two institutes of the National Research Council of Italy: the Water Research Institute (CNR-IRSA) and the Research Institute on Terrestrial Ecosystems (CNR-IRET). The researchers selected two contrasting case studies in Sardinia, in the western Mediterranean. Lake Bidighinzu is an artificial reservoir approximately 40 metres deep whose primary function is storing drinking water. Cabras Lagoon, by contrast, is a shallow coastal lagoon with a maximum depth of about three metres, vital for extensive fishery activities and subject to strong salinity gradients. Both sites belong to the Long-Term Ecological Research network LTER-Italy and have decades of ecological data behind them, particularly on phytoplankton ecology.

Because long-term daily water temperature records were unavailable for the selected ecosystems, the team could not directly apply standard marine heatwave metrics based on the 90th percentile of a 30-year climatology. Instead, they evaluated the occurrence of atmospheric heatwaves using available air temperature data, noting that these metrics are also applicable to freshwater systems. Analyses of records from January 1990 to December 2023 for Cabras Lagoon and from January 1960 to December 2023 for Bidighinzu Lake, performed with the heatwaveR package in R, detected several atmospheric heatwaves in both ecosystems. These events occurred mainly in summer, with peaks of air temperature anomalies exceeding +8°C.

The framework integrates three complementary approaches. The first is a one-year monthly field monitoring programme designed to characterise the natural seasonal dynamics of plankton communities across temperature gradients. In the lake, sampling was conducted at the deepest point, with water collected using a Niskin bottle at depths from 0.30 metres down to one metre above the bottom. In the lagoon, sampling was performed at three points along the salinity gradient, from the sea inlet to the main freshwater inflow. Every month, the researchers measured water temperature, pH, dissolved oxygen, conductivity, salinity, turbidity and chlorophyll a with a multiparameter probe, checked and recalibrated against certified standards before each use. Water transparency was measured with a Secchi disc, and algal nutrients — nitrate, nitrite, ammonium, orthophosphate and silicate — were quantified spectrophotometrically following classical oceanographic methods.

The plankton itself was divided into size fractions and analysed with traditional techniques. Picoplankton, cells between 0.2 and 2 micrometres, was fixed with a formaldehyde and glutaraldehyde solution and quantified by flow cytometry, integrating light scattering signals with fluorescence from phycoerythrin, allophycocyanin and chlorophyll. Total bacterial cells were stained with SYBR Green I and counted on the same instrument. Nanoflagellates, between 2 and 20 micrometres, were examined by epifluorescence microscopy on DAPI-stained filters, with at least 30 random fields counted per slide at 1000× magnification. Phytoplankton and microzooplankton were analysed with the Utermöhl settling technique using an inverted microscope, and cell volumes were converted to carbon content using established geometric approximations. Mesozooplankton, larger than 200 micrometres, was collected with an 80-micrometre mesh net — fine enough to capture juvenile copepods and rotifers — and identified to species level where possible, with body measurements converted to biomass through published length-weight regressions.

The second approach is a laboratory heatwave simulation experiment. Two independent 14-day experiments were carried out in summer 2024: the lake experiment from 1 to 15 July and the lagoon experiment from 23 July to 6 August. Natural plankton communities were incubated in triplicate in 10-litre semi-transparent plastic buckets under four experimental conditions, combining two treatments. The first treatment was a +5°C increase in water temperature above the in situ environmental average, chosen because it represents the maximum sea surface temperature anomaly recorded in the Mediterranean and the maximum intensity detected during the severe marine heatwaves of the last decade. The second treatment was the presence or absence of mesozooplankton, designed to reveal whether heatwave effects on the plankton community act directly or are mediated indirectly through predator-prey dynamics.

The experimental set-up was carefully adapted to each ecosystem. In the lake, mesozooplankton for the zooplankton treatments was collected by two vertical net hauls from 0 to 10 metres, filtering roughly 600 litres of water, and added at natural densities of approximately 100 individuals per litre based on field observations. In the shallow lagoon, where the massive presence of the non-native ctenophore Mnemiopsis leidyi made net hauls impractical, the community was incubated as it is in the zooplankton treatments, while a 200-micrometre filtration excluded larger zooplankton only from the control units. Light conditions simulated summer with a photosynthetically active radiation of 2000 micromol photons per square metre per second in a 15:9-hour light-dark cycle. Temperature, pH, dissolved oxygen and conductivity were monitored daily in each unit, and water was sampled at the start, at regular intervals and at the end of each experiment for nutrients, plankton counts and molecular analyses. Evaporated water was replenished once per experiment with pre-filtered, autoclaved water from the original site.

The third approach deploys advanced molecular tools. Water samples were passed through a series of sieves and filters of 20, 3 and 0.22 micrometres porosity, and DNA was extracted with commercial kits. Eukaryotic and bacterial community composition were characterised by 18S and 16S rRNA gene amplicon sequencing respectively, processed through the DADA2 pipeline with taxonomic assignment against the PR2 and Silva databases. The antibiotic resistome — the total content of antibiotic resistance genes — was assessed by shotgun metagenomic sequencing, with reads analysed using the Resistance Gene Identifier against the Comprehensive Antibiotic Resistance Database. The abundance of resistance genes was expressed as gene copies per 16S rRNA gene copy. Metagenome-assembled genomes were reconstructed with tools including MaxBin2, Metabat2 and CONCOCT, then classified against the Genome Taxonomy Database to identify the bacterial hosts carrying resistance genes. Potentially pathogenic bacteria were flagged by selecting genera containing clinically relevant species, and resistance genes were ranked by risk class. Mesozooplankton specimens were additionally processed for Sanger DNA barcoding using 18S rRNA and cytochrome oxidase I markers to complement microscopic identification with molecular species profiles.

The researchers acknowledge limitations but argue these can be mitigated. Temperature control in indoor microcosms may be challenging, though stable power supplies and precise regulation equipment help. Microcosms cannot fully replicate natural spatial and trophic complexity, and the exclusion of higher trophic levels may influence cascading effects of warming through top-down control. Metagenomics provides limited resolution at species and strain level and cannot reliably assess changes in abundance and biomass alone — which is precisely why the framework couples molecular methods with traditional counting. The strength of the design lies in combining indoor heatwave experiments, which capture direct warming effects across trophic levels including bacteria and pathogens, with field monitoring that reveals plankton dynamics along natural temperature gradients. The protocol is flexible and scalable, adaptable to available resources and research objectives, and applicable regardless of ecosystem type. By filling a critical regional knowledge gap with a standardised, reproducible approach, the framework aims to provide harmonised information to authorities implementing adaptation strategies in climate-sensitive Mediterranean waters — from drinking water reservoirs to fishery lagoons — before the next severe heatwave arrives.

Subject of Research: Methodological framework for studying heatwave effects on Mediterranean plankton communities

Article Title: Methodological design to study the effects of heatwaves on natural plankton communities from Mediterranean vulnerable ecosystems

Article References: Pulina, S., Kamburska, L., Titocci, J., Padedda, B. M., Piscia, R., Rosati, I., Sabatino, R., & Di Cesare, A. (2026). Methodological design to study the effects of heatwaves on natural plankton communities from Mediterranean vulnerable ecosystems. One Ecosystem, 11, Article e183254. https://doi.org/10.3897/oneeco.11.e183254

Image Credits: AI Generated

DOI: 10.3897/oneeco.11.e183254

Keywords: heatwaves, plankton, Mediterranean, climate change, Sardinia, coastal lagoon, artificial lake, antibiotic resistance genes, metagenomics, mesozooplankton, ecosystem services, One Ecosystem