Layout 1 Introduction Raphidiopsis raciborskii (Woloszynska) (Aguilera et al.) is a cyanobacterium belonging to the order Nostocales. Like other members of Nostocales, this species can potentially fix atmos- pheric nitrogen by forming heterocytes (Yema et al., 2016). Originally, this cyanobacterium was better known by the name Cylindrospermopsis raciborskii (Wołoszyńska) (Seenayya & Subba Raju) and distinguished from Raphidiopsis for the ab- sence of heterocytes in this latter genus. A number of works have sustained this separation, e.g., Li et al. (2016). Based on mor- phological and 16S rRNA gene comparisons, Moustaka-Gouni et al. (2009) suggested that Raphidiopsis mediterranea Skuja represented a non-heterocytous life-cycle stage of C. raciborskii. Successively, based on the analysis of 16S rRNA, 16S-23S ITS, and cpcBA-IGS sequences, and following the rule of priority, Aguilera et al. (2018) proposed the unification of the two genera in one unique genus, Raphidiopsis, which is the genus presently included in AlgaeBase (Guiry and Guiry, 2024), and also recog- nized in the Genome Taxonomy Database (GTDB) (Parks et al., 2022). Nevertheless, the taxonomic characterization of this genus and its species is still the subject of debate and research (Komárek, 2020; Sha, 2025). Raphidiopsis raciborskii is considered an invasive species originating from tropical countries (Sukenik et al., 2012). Differ- ent primary dispersion centers were proposed, including African lakes and Australia (Padisák, 1997; Haande et al., 2008) and America (Moreira et al., 2015). In Europe, the first records of R. raciborskii were documented in Lake Kastoria, Greece (Skuja, 1937). Successively, since the 1970s, the presence of this species has been increasingly reported throughout the European continent SPECIAL SECTION Potentially toxic cyanobacteria blooms in the Southern Alps and the Italian peninsula Recent spread of Raphidiopsis raciborskii in the lake district south of the Alps Martina Austoni,1 Adriano Boscaini,2 Fabio Buzzi,3 Leonardo Cerasino,2 Giorgio Franzini,4 Federica Giacomazzi,4 Manuela Marchesi,3 Chiara Zampieri,4 Nico Salmaso2,5 1CNR Istituto di Ricerca Sulle Acque (IRSA), Verbania; 2Research and Innovation Centre, Fondazione Edmund Mach, San Michele all’Adige; 3ARPA Lombardia, Settore Monitoraggi Ambientali Dipartimento di Lecco, Oggiono; 4ARPA Veneto (ARPAV), Servizio Laboratorio Provinciale di Verona; 5National Biodiversity Future Center (NBFC), Palermo, Italy ABSTRACT In recent years, there has been a rise in cyanobacterial blooms, and climate warming is believed to be a key driver sustaining these changes. Climate change may affect the geographic distribution of potentially toxigenic species and cyanobacteria, leading to the appearance of new threats in previously unexposed areas. Recently, the potentially toxic cyanobacterium Raphidiopsis (Cylindrospermopsis) raciborskii, known for forming blooms, has increased its presence, particularly in temperate regions. In this work, we expanded the knowledge about the distribution of R. raciborskii in Northern Italy. Specifi- cally, we reported new observations recorded during the last decade based on investigations carried out in the framework of scientific and government monitoring and large biogeographical surveys carried out on the whole Alpine Space area. The detection of R. raciborskii in Northern Italy highlights the importance of closely monitoring freshwater quality and implementing measures to prevent the spread of harmful organisms. Corresponding author: Martina Austoni, CNR Istituto di Ricerca Sulle Acque (IRSA), Verbania, Italy. E-mail: martina.austoni@cnr.it Key words: cyanobacterial blooms, biological invasions, climate change, biogeography. Authors’ contributions: all the authors made a substantive intellec- tual contribution. All the authors have read and approved the final version of the manuscript and agreed to be held accountable for all aspects of the work. Conflict of interest: the authors declare no potential conflict of in- terest. Funding: this work has been prepared with the partial support and co-financing of the European Regional Development Fund through the Interreg Alpine Space program, project Eco-AlpsWater (EAW, grant number ASP569). Availability of data and materials: all data generated or analyzed during this study are included in this published article. Acknowledgements: we are grateful to the Environmental Agen- cies of Northern Italy, APPA Trento, APPA Bolzano, ARPA Lom- bardia, ARPA Piedmont, and ARPA Veneto, which provided logistical support in the field and in the laboratory. We also thank the anonymous reviewers for their valuable comments and sugges- tions on an earlier version of the manuscript. Received: 9 October 2024. Accepted: 24 February 2025. Publisher’s note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affil- iated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. ©Copyright: the Author(s), 2025 Licensee PAGEPress, Italy Advances in Oceanography and Limnology, 2025; 16:13225 DOI: 10.4081/aiol.2025.13225 This work is licensed under a Creative Commons Attribution- NonCommercial 4.0 International License (CC BY-NC 4.0). Recent spread of Raphidiopsis raciborskii in the lake district south of the Alps 11 (Padisák, 1997; Sukenik et al., 2012; Antunes et al., 2015). In Italy, R. raciborskii was reported for the first time in blooms recorded in 1995 in Lake Trasimeno, in 2002 in Lake Albano, and in 2003 in Lake Cedrino (Manti et al., 2005) (Table 1A). Succes- sively, several other observations of R. raciborskii in Central Italy, Sardinia, Sicily and Emilia Romagna were reported (Manfredini and Ghion, 2005; Mattei and Stefanelli, 2008; Mugnai et al., 2008; Messineo et al., 2009, 2010; Manganelli et al., 2014; Elia et al., 2019; Del Pasqua et al., 2024). In Northern Italy, reports on the presence of R. raciborskii were limited to a few observa- tions in the eutrophic/hypereutrophic lakes Sartirana in 2007 and Castellaro in 2010 and 2012 (Manganelli et al., 2014) (Table 1A). It is interesting to observe that in 2009, a bloom of Raphidiopsis mediterranea Skuja was observed in Lake Annone ovest (Man- ganelli et al., 2014). Raphidiopsis raciborskii is part of the group of cyanobacteria responsible for the formation of cyanoHABs (Harmful Algal Blooms caused by Cyanobacteria). The toxigenic strains of this species are able to synthesize a wide range of secondary metabo- lites, including the cytotoxic Cylindrospermopsin (CYN), the neu- rotoxic Saxitoxin (STX), and analogs (responsible for the Paralytic Shellfish Poisoning, PSP, in humans through consump- tion of contaminated shellfish from marine environment) (Vico et al., 2020; Santos-Silva et al., 2024). The two classes of toxins are never produced simultaneously and are always produced individ- ually by strains developing on different continents. Strains pro- ducing CYN have been documented in Australia, New Zealand, and Asia (Vico et al., 2020). Exclusive STX producers have been identified in South America (González-Madina et al., 2022). No CYN or STX producers have been identified from strains isolated in Africa, North America, or most of Europe (Vico et al., 2020). In this work, we will expand the knowledge about the geo- graphical distribution of R. raciborskii in Northern Italy. Specifi- cally, we will include new detections recorded during the last decade based on investigations carried out in the framework of scientific and government monitoring, and biogeographical sur- veys carried out on the whole Alpine Space area (Project Eco- AlpsWater). Experimental approach The new records of Raphidiopsis raciborskii were docu- mented during the regular monitoring activities carried out by the Environmental Agencies of Northern Italy (Lombardy, Veneto, Trentino Alto Adige, and Friuli Venezia Giulia) and by investiga- tions by research institutions (CNR of Verbania, Piedmont). The observations of the phytoplankton samples were carried out ex- clusively with inverted microscopes and using the Utermöhl method (i.e., “UNI EN 15204” and Wetzel and Likens, 2000). Genetic (by analysis of environmental DNA, eDNA) and cyanotoxins (by LC-MS) analyses, including the determination of STX and CYN, the two most common toxins produced by R. raciborskii, were carried out on 59 samples from lakes and rivers throughout the Alpine region as part of the Alpine Space project Eco-AlpsWater. Metabarcoding analyses were performed using the V3-V4 region of the 16S rRNA marker. The field and labo- ratory methods and the bioinformatic analyses of the data have been reported by Salmaso et al. (2022, 2024), Domaizon et al. (2022), Cerasino et al. (2017), and Jablonska et al. (2024). The 16S rRNA sequences are reported in the Zenodo repository (https://zenodo.org/records/5822484), and the raw sequences are reported in the public ENA archives (study accession number PRJEB49047). New evidence for the colonization of Raphidiopsis raciborskii in the southern perialpine region New records of Raphidiopsis raciborskii were documented in Lombardy (lakes Comabbio and Mantova Superiore) and Veneto (Lake Frassino) but not in Friuli Venezia Giulia, Piedmont, Trentino Alto Adige and Valle d’Aosta (Table 1B, Figure 1). Lake Comabbio is a small natural lake in Northern Italy that has only one emissary that connects it to Lake Varese after cross- ing the Palude Brabbia Nature Reserve (ZSC IT2010007). It is a eutrophic and polymictic low-depth lake with naturally high trophic levels mainly due to its morphology. In this lake, R. raci- borskii was identified for the first time during a research survey Table 1. A) First documentation of the presence of Raphidiopsis (Cylindrospermopsis) raciborskii in Italy. B) New records described in this work. Genetic analyses confirming the species determinations were performed only on strains collected in Lake Trasimeno (Mugnai et al., 2008). (A) Lake Locality Year Reference LM Metab. Trasimeno Central Italy, Umbria 1995 (Manti et al., 2005) × NA Albano Central Italy, Latium 1995 (Manti et al., 2005) × NA Cedrino Sardinia 2003 (Manti et al., 2005) × NA Valle Santa Northern Italy, Emilia Romagna 2003 (Manfredini & Ghion, 2005) × NA Biviere di Gela Sicily 2005 (Barone et al., 2010) × NA Lake Sartirana Northern Italy, Lombardy 2007 (Manganelli et al., 2014) × NA Lake Castellaro Northern Italy, Lombardy 2010 (Manganelli et al., 2014) × NA Lake Foschi Golf Club Northern Italy, Emilia Romagna 2022 (Del Pasqua et al., 2024) × NA Canale Gallego Northern Italy, Emilia Romagna 2022 (Del Pasqua et al., 2024) x NA Artificial lake (Paul Harris Park) Northern Italy, Emilia Romagna 2023 (Del Pasqua et al., 2024) x NA (B) Lake Locality Year Reference LM Metab. Frassino Northern Italy, Veneto 2018 This work × × Mantova Superiore Northern Italy, Veneto 2021 This work × × Comabbio Northern Italy, Lombardy 2015 (Austoni et al., 2024) × NA NA, not available; LM, light microscopy; Metab., identification of the genus, Raphidiopsis (Cylindrospermopsis), by metabarcoding (16S rRNA amplicon sequencing). M. Austoni et al.12 carried out by the CNR of Verbania in July 2015 (Austoni et al., 2024). The population identified in Lake Comabbio presented straight trichomes. Coiled trichomes were never observed. R. raci- borskii average cell density was 2.5×109 cell L–1 on July 24th (lit- toral zones) representing almost the total phytoplankton cell density. ELISA immunoassay analysis of the environmental sam- ples detected concentrations of STX ranging from 0.27 to 0.36 μg L–1, suggesting a possible association between Raphidiopsis and STX production, but further analysis via HPLC is necessary (Aus- toni et al., 2024) and this should be confirmed by LC-MS and ge- nomic analysis of isolated strains. In 2021, this species was detected for the first time in Lake Mantova Superiore during the regular monitoring activities carried out by ARPA Lombardia (Table 1B, Figure 1). Lake Mantova Su- periore is classified as polymictic. It is located inside the Natural Reserve ‘Valli del Mincio’, and it is the largest of the three shallow fluvial eutrophic lakes in the province of Mantua, in Northern Italy. The three lakes are considered a wetland complex. In Lake Mantova Superiore, only straight trichomes were observed (Figure 2), and the average cell density of integrated samples was 1.1×106 cell L–1 during the sampling campaigns of August 2021 and 2023 (2.1×106 cell L–1 and 1.4×105 cell L–1, respectively). Lake Frassino is located in Veneto (Northern Italy), about 1 km south of Lake Garda. It is a small shallow lake fed directly by local rainfall and small inlet streams. It is a eutrophic water body with high nutrient concentrations and high levels of primary pro- duction in an area dominated by agricultural and urban use (Bar- bato, 1987). Further, Lake Frassino has been included in the list of biotopes of the Natura 2000 network and in the sites of com- munity importance (ZSC IT3210003). In this lake, R. raciborskii was found for the first time in 2018 during regular monitoring ac- tivities carried out by ARPA Veneto (Table 1B, Figures 1 and 3). Only straight trichomes were detected also in this lake. Maximum cell density in the integrated samples (0-6 m) was 3.7×105 cell L– 1 in late autumn 2018. In the following years, the maximum cell density values of R. raciborskii in the summer period were 1.4×107 cell L–1 (2020), 7.0×105 cell L–1 (2022) and 1.3×108 cell L–1 (2023) (maximum value for the period from 2018 to 2023). In the Alpine Region, two 16S rRNA sequences belonging to the genus Raphidiopsis (Cylindrospermopsis) were identified in the lakes Frassino and Mantova Superiore (Table 1B) as part of the metabarcoding survey carried out during the Eco-AlpsWater project (Salmaso et al., 2024). In the same framework, the PCR analyses by Jablonska et al. (2024) did not reveal the presence of CYN or STX genes in the samples collected in the same lakes. PCR analyses were not available for Lake Comabbio. Coherently, the LC-MS analysis of the samples from lakes Frassino and Mantova Superiore showed no measurable concentrations of CYN and STX (Salmaso et al., 2024). Discussion and Conclusions The investigations carried out in different lake typologies of the southern perialpine lake district allowed us to confirm and ex- tend the knowledge about the presence of R. raciborskii in the Italian peninsula and Europe. This species was primarily found in shallow, human-affected, eutrophic lakes during the warmer months. In view of their great importance for tourism, drinking water supply, and as sources of biodiversity, it is worth highlight- ing the non-detection of R. raciborskii in the surveys carried out in the largest lakes south of the Alps (Garda, Maggiore, Como, Iseo, and Lugano). With the exception of STX, which was detected in an envi- Figure 1. Map of the Italian regions in which cyanobacterium Raphidiopsis raciborskii was found. Red dots represent sites where phy- toplankton samples containing R. raciborskii were collected. Left: Lake Comabbio (Lombardy); upper right Lake Frassino (Veneto); right below: Lake Mantova Superiore (Lombardy). Recent spread of Raphidiopsis raciborskii in the lake district south of the Alps 13 ronmental sample from Lake Comabbio (Austoni et al., 2024), here the analyzed samples tested negative for the presence of the two common cyanotoxins produced by selected strains of R. raci- borskii (STX and CYN), as well as for selected genes that are part of the operons encoding these toxins. However, besides Lake Comabbio, confirmation of cyanotoxin production in R. raci- borskii is needed in cases where measurable concentrations of CYN have been reported in environmental samples, e.g., in North America (Florida) (Burns, 2008), Africa (Odokuma and Isirima, 2007; Mowe et al., 2014) and Europe (Manti et al, 2005; Koko- ciński et al., 2009; Messineo et al., 2010), especially when several potential CYN producers were reported simultaneously (e.g. Kokociński et al., 2009; Messineo et al., 2010). 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