Biomonitoring survey of the hydrographical network in a MAB UNESCO Biosphere Reserve

The knowledge of microbial biodiversity (bacteria and protists) in aquatic ecosystems is far from complete. This is mainly due to the inadequacy of conventional taxonomic identifications, which are based on the discrimination of diacritical morphological traits. Further, investigations are mainly focused on specific lake and river typologies that are usually also of interest for economic exploitation, often disregarding the small and/or ephemeral water bodies. Nevertheless, due to their physiographical complexity and temporal dynamics, these neglected hydrographical elements can host a vast microbial diversity. In order to fill the gap in the biodiversity estimates in the Alpine region, we carried out a survey using high throughput sequencing (HTS) of 16S and 18S rDNA markers from water and sediments collected in the MAB UNESCO Alpi Ledrensi and Judicaria Biosphere Reserve (Project Acqua Viva). The study sites are located between Lake Garda and the Brenta Dolomites, and include natural environments in a semi- anthropized Alpine context with agricultural and tourist vocation. The survey was carried out during the summer 2019, covering 20 sites of different sizes and characteristics, including lakes, alpine pasture ponds and wetlands. The results allowed disclosing a high number of amplicon sequence variants (ASVs) belonging to a wide variety of bacterial and protists groups, and significant differences linked to lake and sample typologies. Despite a wide presence of potentially toxigenic cyanobacteria, microcystins and anatoxin-a were detected only in a few water bodies, including Lake Ledro, which showed the presence of both Planktothrix rubescens and Tychonema bourrellyi in the pelagic samples

Pregnancy and Maternal Behavior Induce Changes in Glia, Glutamate and Its Metabolism within the Cingulate Cortex

An upregulation of the astrocytic proteins GFAP and bFGF within area 2 of the cingulate cortex (Cg2) occurs within 3 hours of parturition in rats. These changes are the result of an interaction between hormonal state and maternal experience and are associated with increased dendritic spine density in this area. Here, we examined whether this upregulation of astrocytic proteins generalized to other glial markers and, in particular those associated with glutamate metabolism. We chose glial markers commonly used to reflect different aspects of glial function: vimentin, like GFAP, is a marker of intermediate filaments; glutamine synthetase (GS), and S-100beta, are used as markers for mature astrocytes and GS has also been used as a specific marker for glutamatergic enzymatic activity. In addition, we examined levels of proteins associated with glutamine synthetase, glutamate, glutamine and two excitatory amino acid transporters found in astrocytes, glt-1 and glast. S100beta immunoreactivity did not vary with reproductive state in either Cg2 or MPOA suggesting no change in the number of mature astrocytes across these conditions. Vimentin-ir did not differ across groups in Cg2, but expression of this protein decreased from Day 1 postpartum onwards in the MPOA. By contrast, GS-ir was increased within 24 h postpartum in Cg2 but not MPOA and similarly to GFAP and bFGF this upregulation of GS resulted from an interaction between hormonal state and maternal experience. Within Cg2, upregulation of GS was not accompanied by changes in the astrocytic glutamatergic transporters, glt-1 and glast, however, an increase in both glutamate and glutamine proteins were observed within the Cg2 of postpartum animals. Together, these changes suggest postpartum upregulation of glutamatergic activity and metabolism within Cg2 that is stimulated by pregnancy hormones and maternal experience

Potential impact of dreissenids species in relation to the first report of quagga mussel (Dreissena bugensis) at the end of winter 2022 in Lake Garda (Northern Italy)

Along with Dreissena polymorpha (zebra mussel), Dreissena bugensis (quagga mussel) is considered one of the most widespread and successful invasive species in Europe, Russia, and North America. The quagga mussel is a bivalve mollusc of the dreissenids family originating from the estuarine region of the rivers Dnieper and Southern Bug (Black Sea). The spread in Europe began after the 1940s and was associated with construction of canal-river systems. D. polymorpha was observed in Lake Garda since the end of the 1960s. In the last years, D. bugensis was found in the northern perialpine region and in late winter 2022 it was identified along the eastern shores of Lake Garda. Both species are defined as ecosystems engineers because they can strongly change the substrate structure, causing great economic damage in water pipes, potable water treatment plants, and port constructions. Moreover, they can affect resource availability for the other species. A few significant ecological differences between the two species suggest that the impact of quagga mussel could be even larger compared to that caused by zebra mussel. In this poster we will discuss the main expected ecological impacts following the introduction of quagga mussel into new aquatic habitats

Metabarcoding protocol: Analysis of Bacteria (including Cyanobacteria) using the 16S rRNA gene and a DADA2 pipeline (Version 1)

This protocol has been prepared as part of the Interreg Alpine Space project Eco-AlpsWater (ASP569) - Innovative Ecological Assessment and Water Management Strategy for the Protection of Ecosystem Services in Alpine Lakes and Rivers, Activity A.T1.3, Deliverable D.T1.3.2 – 1, https://www.alpine-space.eu/projects/eco-alpswater/en/hom

Metabarcoding protocol: Analysis of protists using the 18S rRNA gene and a DADA2 pipeline (Version 1)

This protocol has been prepared as part of the Interreg Alpine Space project Eco-AlpsWater (ASP569) - Innovative Ecological Assessment and Water Management Strategy for the Protection of Ecosystem Services in Alpine Lakes and Rivers, Activity A.T1.3, Deliverable D.T1.3.2 – 2, https://www.alpine-space.eu/projects/eco-alpswater/en/hom

Plankton DNA extraction from Sterivex filter units

The objective of this protocol is to provide a reliable and replicable method for the DNA extraction of lake micro-plankton to be used for downstream DNA analysis. This protocol is one of those proposed by the Eco-AlpsWater consortium to promote the implementation of High Throughput Sequencing (HT S) of environmental DNA (eDNA) in the biomonitoring and ecological assessment of water bodies. The extraction is performed from samples filtered through Sterivex cartridges (Sterivex™ GP 0.22μm) and stored at -20°C, as described in the protocol dx.doi.org/10.17504/protocols.io.xn6fmhe, and with the use of the DNeasy® PowerWater Sterivex Kit (QIAGEN) with specific modifications adapted to plankton DNA extraction. The application proposed here, in the context of EcoAlpsWater, aims at comparing DNA inventories to traditional phytoplanktonic inventories and at characterizing more broadly the micro-planktonic diversity through eDNA analysis (including bacteria). This protocol is part of the deliverables provided by the WP1 of the Eco-AlpsWater project. All members of the EcoAlpsWater consortium (http://www.alpine-space.eu/projects/eco-alpswater/en/home) contributed to the optimization of this protocol

Mitigating harmful cyanobacterial blooms: strategies for control of nitrogen and phosphorus loads

Harmful blooms of cyanobacteria (CyanoHABs) have increased globally and cyanotoxins associated with some CyanoHAB species pose serious health risks for animals and humans. CyanoHABs are sensitive to supply rates of both nitrogen and phosphorus, but sensitivity may vary among species (e.g., between diazotrophic and non-diazotrophic species) and a range of physiographic and environmental factors. A sustainable approach to manage CyanoHABs is therefore to limit the supply of nitrogen and phosphorus from catchments to receiving waters. Alternative approaches of within-lake treatment have increased risks and large capital and operational expenditure. The need to manage catchment nutrient loads will intensify with climate change, due to expected increases in nutrient remineralization rates, alteration in hydrological regimes, and increases in lake water temperature and density stratification. Many CyanoHAB species have physiological features that enable them to benefit from the effects of climate change, including positive buoyancy or buoyancy control, high replication rates at elevated water temperature, and nutrient uptake strategies adapted for the intermittency of nutrient supply with greater hydrological variability expected in the future. Greater attention needs to be focused on nonpoint sources of nutrients, including source control, particularly maintaining nitrogen and phosphorus in agricultural soils at or below agronomic optimum levels, and enhancing natural attenuation processes in water and solute transport pathways. Efforts to achieve effective catchment management and avert the dire ecological, human health and economic consequences of CyanoHABs must be intensified in an era of anthropogenically driven environmental change arising from increasing human population, climate change and agricultural intensification

Draft genome sequence of the anatoxin-a producing cyanobacterium Tychonema bourrellyi B0820 isolated from the epilimnion of the deep Alpine Lake Garda

We report the draft genome sequence of strain B0820 of the cyanobacterium Tychonema bourrellyi isolated from the epilimnion of Lake Garda and assembled from a metagenome of a non-axenic culture. The strain analyzed was shown to produce anatoxin-a, a potent neurotoxin that can cause fatal intoxication in exposed organism

A comparative study of the metabolic profiles of common nuisance cyanobacteria in southern perialpine lakes

This work allowed the comparison of the metabolic profiles of the most important cyanobacteria species in southern perialpine lakes, namely Aphanizomenon flos-aquae, Dolichospermum lemmermannii, Microcystis aeruginosa, Planktothrix rubescens, and Tychonema bourrellyi. Monospecific cultures were obtained from samples of 3 different natural lakes (Garda, Idro, and Caldonazzo). LC-MS/MS analyses were conducted on strains. A first set of experiments was aimed at assessing the presence of the best known toxins (microcystins, nodularins, (homo)anatoxin-a, cylindrospermopsins, paralytic shellfish poisons) in the cultures. Results of this screening study revealed that M. aeruginosa and P. rubescens produced toxic peptides (microcystins), T. bourrellyi produced toxic alkaloids (anatoxin-a and possibly some paralytic shellfish toxins), Aph. flos-aquae and D. lemmermannii did not produce any of the analyzed toxins. M. aeruginosa and P. rubescens showed typical microcystin production with LR form dominant in the former, and RRdm form dominant in the latter. A second set of experiments was aimed at comparing the capability of the 5 cyanobacterial species to produce peptidic secondary metabolites. For this purpose, an untargeted peptidomic analysis was conducted on the strains. The analysis allowed revealing globally 328 metabolites, spanning in a mass range between 400 and 2000 Da. The majority of compounds with masses in the 500-1200 Da range (corresponding to the majority of peptidic secondary metabolites) resulted to be produced by M. aeruginosa and P. rubescens strains, thus indicating a higher ability of these species to produce non-ribosomal peptides compared to the others. 27 metabolites out of 328 could be putatively assigned to specific classes of compounds: microcystins, aeruginosins and anabaenopeptins were the most represented classes of compounds, and were mostly found in M. aeruginosa and P. rubescens strains