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Detection of a Planktothrix agardhii Bloom in Portuguese Marine Coastal Waters Catarina Churro 1, * 1 2

3 4

*

ID

, Joana Azevedo 2 , Vitor Vasconcelos 2,3 and Alexandra Silva 1,4

Laboratório de Fitoplâncton, Departamento do Mar e Recursos Marinhos, Instituto Português do Mar e da Atmosfera, Rua Alfredo Magalhães Ramalho, 6, 1449-006 Lisboa, Portugal; [email protected] Centro Interdisciplinar de Investigação Marinha e Ambiental, CIIMAR/CIMAR, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos, 4450-208 Matosinhos, Portugal; [email protected] (J.A.); [email protected] (V.V.) Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 4069-007 Porto, Portugal Centro de Ciências do MAR, CCMAR, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal Correspondence: [email protected] or [email protected]; Tel.: +351-21-302-7000

Academic Editor: Luis M. Botana Received: 3 October 2017; Accepted: 29 November 2017; Published: 3 December 2017

Abstract: Cyanobacteria blooms are frequent in freshwaters and are responsible for water quality deterioration and human intoxication. Although, not a new phenomenon, concern exists on the increasing persistence, scale, and toxicity of these blooms. There is evidence, in recent years, of the transfer of these toxins from inland to marine waters through freshwater outflow. However, the true impact of these blooms in marine habitats has been overlooked. In the present work, we describe the detection of Planktothrix agardhii, which is a common microcystin producer, in the Portuguese marine coastal waters nearby a river outfall in an area used for shellfish harvesting and recreational activities. P. agardhii was first observed in November of 2016 in seawater samples that are in the scope of the national shellfish monitoring system. This occurrence was followed closely between November and December of 2016 by a weekly sampling of mussels and water from the sea pier and adjacent river mouth with salinity ranging from 35 to 3. High cell densities were found in the water from both sea pier and river outfall, reaching concentrations of 4,960,608 cells·L−1 and 6810.3 × 106 cells·L−1 respectively. Cultures were also established with success from the environment and microplate salinity growth assays showed that the isolates grew at salinity 10. HPLC-PDA analysis of total microcystin content in mussel tissue, water biomass, and P. agardhii cultures did not retrieve a positive result. In addition, microcystin related genes were not detected in the water nor cultures. So, the P. agardhii present in the environment was probably a non-toxic strain. This is, to our knowledge, the first report on a P. agardhii bloom reaching the sea and points to the relevance to also monitoring freshwater harmful phytoplankton and related toxins in seafood harvesting and recreational coastal areas, particularly under the influence of river plumes. Keywords: Planktothrix agardhii; microcystins; mcyA; halotolerance; salinity; harmful algal blooms; cyanobacteria

1. Introduction Toxic cyanobacterial blooms have long been a recognized threat to freshwater ecosystems and human health, and their negative impacts have been reported and reviewed extensively [1–7]. These toxic proliferations are still a major concern in freshwater lakes and reservoirs and their complexity and unpredictable nature continue to challenge researchers and monitoring authorities [8]. Toxins 2017, 9, 391; doi:10.3390/toxins9120391

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ccurrence, magnitude, and persistence of theseoccurrence, blooms are distribution, perceived to magnitude, be increasing and2 persistence of these blooms are pe Toxins distribution, 2017, 9, 12 of 13 Theyears occurrence, distribution, magnitude, persistence of these blooms are global perceived to been be increasing lobally over the last [8–13] and have been related toand increasing globally over human the last activities years and [8–13] and have related to increasing hum globally over the last years [8–13] and have been related to increasing human activities proliferations and global warming [9–14]. However, cyanobacterial proliferations warming are intricate [9–14]. phenomena However, cyanobacterial are intricate occurrence, distribution, magnitude, and persistence of these blooms are perceivedand to betheir increasing [9–14]. However, proliferations intricate phenomena and theirof occurrence ccurrence is over the warming result the [8–13] juncture of several factors, occurrence relatedisarebut the not resultexclusive of theand to, juncture several factors, related globally the lastof years and cyanobacterial have been related to increasing human activities global is the result of inputs, thecyanobacterial juncture of several related but not exclusive to,inputs, increasing/reduction ncreasing/reduction of nutrient increasing river factors, barriers, increasing/reduction increased water of nutrient stratification increasing river barriers, increa warming [9–14]. However, proliferations are intricate phenomena and their inputs, increasing river barriers, increased water stratification periods, changes inglobal the temperatures, a occurrence is the result ofcycle, the juncture of several factors, related notWhether exclusive to, eriods, changes inof thenutrient hydrological increasing global temperatures, periods, changes and inCO the 2but [9–16]. hydrological cycle, increasing hydrological cycle, increasing global temperatures, and Whether orchange not thismassive is connected of nutrient inputs, increasing river increased water stratification r not increasing/reduction this is connected to climate change massive cyanobacterial or notbarriers, blooms this is CO connected are no longer to climate seasonal, cyanobacterial blooms 2 [9–16]. to year, climate change massive cyanobacterial blooms are no all longer seasonal, allwater systems wh ersisting during all andhydrological also, not restrict toincreasing lentic water persisting systems during where rivers year, and and estuaries also, persisting not restrictduring to lentic periods, changes in the cycle, global temperatures, and CO 2 [9–16]. Whether and also, notreports restrict lentic water systems where rivers and estuaries have ofalso been cyanobacterial ave also beenthis affected [8,9,17,18]. The oftofreshwater cyanobacterial have also been blooms affected reaching [8,9,17,18]. the The sea reports freshwater or not isyear, connected to climate change massive cyanobacterial blooms are no longer seasonal, affected The of freshwater blooms reaching the sea are increasing and [19–22]. Furthe re increasing andduring have been mostand probably underestimated [19–22]. arecyanobacterial increasing Furthermore, and have thebeen effect most ofand these probably underestimated persisting all[8,9,17,18]. year, also,reports not restrict to lentic water systems where rivers estuaries have been most probably underestimated [19–22]. Furthermore, effect of these blooms marine and wildlife are l loomshave in the marine ecosystem and the impact in human and blooms wildlife inare thelargely marinethe unknown ecosystem [19–22]. and the impact in human also been affected [8,9,17,18]. The reports of freshwater cyanobacterial blooms reaching the seain the ecosystem the impact in human and wildlife are largely unknown [19–22]. most commonly he most commonly found freshwater toxins—microcystins—are The most produced commonly by found several freshwater bloomtoxins—microcystins—are produ are increasing and haveand been most probably underestimated [19–22]. Furthermore, the effectThe of these found freshwater toxins—microcystins—are produced by several bloom-forming cyanobacteria species, orming cyanobacteria species, such as the Microcystis, forming Anabaena, cyanobacteria Anabaenopsis, species, such and as Microcystis, Planktothrix, Anab blooms in the marine ecosystem and impact inPlanktothrix, human and wildlife are largely unknown [19–22]. such genera—Microcystis as Microcystis, Planktothrix, Anabaena, Anabaenopsis, andproduced Aphanizomenon. These genera—Microcystis phanizomenon. and toxins—microcystins—are Planktothrix—are Aphanizomenon. globally distributed These genera—Microcystis formbloomand Planktothrix—are globally The mostThese commonly found freshwater byand several and Planktothrix—are globally and form in lakes reservoirs alland over theInworld. loomsforming in lakes and reservoirs all over the world. In freshwater, blooms the main inblooms lakes concern and reservoirs fromand microcystin all over the world. freshwater, the main c cyanobacteria species, such as distributed Microcystis, Planktothrix, Anabaena, Anabaenopsis, In freshwater, thedialysis main concern intoxication is through ingestion dialysis ntoxication is through ingestion or of/withfrom contaminate intoxication drinking is water, through recreational ingestion contact or dialysis of/withof/with contaminate drinking w Aphanizomenon. These genera—Microcystis and microcystin Planktothrix—are globally distributed andorform contaminate drinking recreational contact and the animal poisoning [6,23]. These hepatotoxins are to be chemically nd animal poisoning [6,23]. These hepatotoxins are known to and be chemically animal poisoning stable in [6,23]. bothfrom These freshmicrocystin and hepatotoxins are known blooms in lakes and reservoirs all water, over the world. In freshwater, main concern marineintoxication water, to persist in sediments, to or accumulate in both freshwater marine water, and marine to persist in recreational sediments, andto accumulate in both freshwater and known to be chemically stable in both fresh and marine water, tofilter-feeders persist in sediments, to accumulate is through ingestion dialysis of/with contaminate drinking water, contact o transfer the food chainfreshwater [19–21,24–26]. In a recent review by to transfer Preece al.the [22]food the authors chain [19–21,24–26]. In a recent review in both andhepatotoxins marine filter-feeders and toetup transfer upstable the food chain [19–21,24–26]. In a by Preece et al. andup animal poisoning [6,23]. These are known to be chemically indescribe both fresh and eshwater blooms occurrences inby estuarine coastal indescribe North and blooms South occurrences America, Europe, in estuarine coastaland waters in North and marine water, to persist in sediments, to in freshwater both freshwater and marine and recent review Preece etand al.accumulate [22] the waters authors freshwater bloomsfilter-feeders occurrences inand estuarine frica, to Australia, Turkey, and Japan. In some of South the microcystins Africa, Australia, wereetTurkey, detected and in authors the Japan. water In some of the transfer up the food chain In reports, a recent review by Preece al. [22] the describe coastal waters in[19–21,24–26]. North and America, Europe, Africa, Australia, Turkey, and Japan. Inreports, some microcystins w nd were accumulating marine microcystins shellfish withwere animal deaths and were [22]. Some in countries, marine shellfish likeEurope, with animal deaths implicated [ freshwater blooms occurrences in estuarine and coastal waters inaccumulating North South America, of theinreports, detected inimplicated the water andand were accumulating in marine shellfish ustralia and Australia, USA,with haveanimal recently defined microcystins guidelines Australia for fish, and prawns have and recently molluscs to water microcystins guidelines for fish, Africa, Turkey, and Japan. In some of the reports, microcystins were detected indefined the deaths implicated [22]. Some countries, likeUSA, Australia and USA, have recently defined pply in some states [26,27]. In general, however, governments, apply food instandards some states regulations [26,27]. Inand general, legal [26,27]. however, food standa and were accumulating inguidelines marine shellfish with animal deaths implicated [22]. countries, like Ingovernments, microcystins for fish, prawns and molluscs to apply inSome some states general, uidelines for seafood safety do recently not yet include freshwater phytoplankton guidelines forfor related seafood toxins safety[23,27]. do In yet include freshwater Australia and USA, have defined microcystins guidelines fish, prawns and molluscs to do however, governments, food standards regulations and legal guidelines for not seafood safety not yet phytoplankton he same way, theinclude Portuguese only includes the obligation same way, monitoring Portuguese includes the obligation apply in some states freshwater [26,27].legislation In general, however, governments, food standards regulations and legalonly phytoplankton related toxins [23,27]. Inofthe the same way, marine thelegislation Portuguese legislation guidelines for seafood safety do not yetof include freshwater phytoplankton toxins [23,27]. In through hytoplankton related toxins in the seafood through the Law Decree phytoplankton n.º 293/98related (1998)related [28] toxins transposed in seafood through the Law only includes obligation monitoring marine phytoplankton related toxins in seafood the Decree n.º 293/9 om the Directive n.º 293/98 91/492/CEE [29].includes The from Portuguese the legislation Directive concerning n.º 91/492/CEE 91/492/CEE [29]. The Portugues the European same way, the Portuguese legislation the obligation of monitoring marine (1991) [29]. Law Decree (1998)(1991) [28] only transposed from theEuropean European Directive yanobacteria and microcystins is only tothrough drinking water cyanobacteria safety (Law and Decree microcystins nº 306/2007 isrelated only [30] related to drinking phytoplankton toxins in related seafood the Law Decree n.º 293/98 (1998) [28] transposed Therelated Portuguese legislation concerning cyanobacteria and microcystins is only to drinking water water safety (Law ansposed the European Drinking Water Directive [31]) transposed thethe from legislation the European concerning the Directive Water [31]) Directive from from the European Directive n.º306/2007 91/492/CEE [29].and The Portuguese legislation concerning safety (Law Decree [30](1991) transposed from European DrinkingDrinking Water and [31]) and the le ualitycyanobacteria of bathing water [32,33], that was from the European quality of bathing Bathing water Water [32,33], Directive [34], wasfrom transposed from the European Bath microcystins is transposed onlythe related to of drinking water safety (Law nºthat 306/2007 [30]the European theand legislation concerning quality bathing water [32,33], thatDecree was transposed oorly transposed addresses this question. In the light of [34], whatpoorly appears to be poorly an [31]) additional addresses and this increasing question. concern, Inofthe lightappears of appears from theWater European Drinking Water Directive and the legislation concerning thewhat to Bathing Directive addresses this question. In the light what be an to be an additiona is important to bathing report freshwater cyanobacteria blooms coastal it isthe important waters and tofreshwater report to access freshwater recreational cyanobacteria quality of waterand [32,33], that was transposed from European Bathing Water Directive [34], inblooms additional increasing concern, it is in important to report cyanobacteria blooms coastalin coastal waters a nd seafood In the present work, we light describe the seafood occurrence andsafety. seafood of an aIn freshwater safety. In the cyanobacterium present wethe describe the occurrence of a fre poorlysafety. addresses this question. Inrecreational the of what appears to be additional and increasing concern, waters and to access and the present work, we work, describe occurrence loom it inismarine water and land-sea interface at a bloom shellfish harvesting bloom in marine and bathing water and located land-sea a shellfish harvesting and important report freshwater cyanobacteria blooms in coastal waters andarea tointerface access recreational of to a freshwater cyanobacterium in marine water and land-sea aton ainterface shellfishat harvesting he W coast of Portugal. We went further testing concentration the W coast andWe microcystin ofofwent Portugal. content Wetesting went in further the testing cell and concentration and m and seafood safety. In the present work, describe the occurrence a freshwater cyanobacterium and bathing area located on we thecell W coast of Portugal. further cell concentration water and shellfish, cyanobacteria strain in and a marine environment water and shellfish, withand contrasting cyanobacteria salinities viability in a marine environment w bloom in marine water and land-sea at shellfish, a shellfish harvesting bathing areainstrain located onenvironment microcystin content inviability theinterface water cyanobacteria strain viability a marine nd thethe presence ofwith microscystin related to evaluate potential and toxic presence impact in microscystin the genes ecosystem. to evaluate W coast of Portugal. We wentgenes further testing cell concentration andof microcystin content ingenes the contrasting salinities and the presence of the microscystin related torelated evaluate potential toxic potential toxic im water and shellfish, cyanobacteria strain viability in a marine environment with contrasting salinities impact in the ecosystem. Results 2. Results and the presence of microscystin related genes to evaluate potential toxic impact in the ecosystem. 2. Results In November of 2016, during the regular monitoring program In November for the screening of 2016, of during harmful the regular monitoring program for th 2. Results November of 2016,ofduring the regular monitoring program foragardhii the screening of of the harmful marine phytoplankton, In a high concentration the filamentous marine cyanobacteria phytoplankton, Planktotrix a high concentration filamentous cyanobac −1) was marine phytoplankton, asample high concentration ofprogram the filamentous cyanobacteria Planktotrix agardhii 4,960,608 cells· L−1) was observed in during a marinethe at the northwest (4,960,608 Portuguese cells· Lfor coast (geographical inofa marine sample at the northwest Portug In November of 2016, regular monitoring the observed screening harmful 1 ) was observed in a marine sample at the northwest Portuguese coast (geographical (4,960,608 cells ·L−W). oordinates: 39°21′00.4″ N 9°22′19.4″ The area is aof shellfish coordinates: harvestingcyanobacteria 39°21′00.4″ site and a recreational N 9°22′19.4″ sea W). The area is a shellfish harvesting s marine phytoplankton, a high concentration the filamentous Planktotrix agardhii −1) was coordinates: 39◦ 210 00.4” 9◦ 220 19.4” W).atThe area is afor shellfish harvesting site and a recreational sea each used for bathing surfing. beach used bathing andcoast surfing. (4,960,608 cells· Land observed in aN marine sample the northwest Portuguese (geographical beach used for bathing and surfing. The observed cyanobacteria from the group Oscillatoriales The with observed no heterocytes cyanobacteria nora akinetes, are from the coordinates: 39°21′00.4″ N are 9°22′19.4″ W). The area is a shellfish harvesting site and recreational sea group Oscillatoriales with no The observed cyanobacteria are from the group with no heterocytes norwithout akinetes,sheaths. The filam he filaments were sheaths. filaments The Oscillatoriales filaments were were blue-green, continually solitary and beach used forblue-green, bathing andsolitary surfing. and without the filaments blue-green, solitary without sheaths. filaments were attenuated The observed cyanobacteria are from the group Oscillatoriales no heterocytes nor akinetes, ttenuated at one extremity and were straight at the other, the and cells attenuated were 3.54 at ± with 0.365 oneThe extremity µm wide and and straight 2.65continually ± at the other, the cells were 3.54 ± 0 at one extremity and straight at the other, the cells were 3.54 ± 0.365 µm wide and 2.65 ± 0.467 µm apical cell was na 467 µ the m long with nowere constrictions at cross-cell apical 0.467 cellµ m was long narrowed with noatconstrictions onewere side ofcontinually the at cross-cell walls, the filaments blue-green, solitarywalls, and the without sheaths. The filaments constrictions at at cross-cell walls, the cell was narrowed at one the filament lament with calyptra andnorounded at the other side filament (Figure with 1a,f). calyptra These morphological rounded atof±the other side (Figure 1a,f) attenuated atlong onewith extremity and straight the other, the cellsapical were 3.54 ± 0.365 µand m wide andside 2.65 with calyptra and rounded at the other side (Figure 1a,f). These were haracteristics were inwith congruence with theatdescription of the characteristics cyanobacterium were Planktothrix in morphological congruence agardhii acharacteristics thethe description of the cyanobacteriu 0.467 µ m long no constrictions cross-cell walls, the apical cell was narrowed at onewith side of

ypically freshwater produceatthethe hepatotoxins freshwater organism to produce the hepatotoxins Microcys filament withorganism calyptraknown and to rounded othertypically sideMicrocystins. (Figure 1a,f). These known morphological characteristics were in congruence with the description of the cyanobacterium Planktothrix agardhii a typically freshwater organism known to produce the hepatotoxins Microcystins.

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in congruence with the description of the cyanobacterium Planktothrix agardhii a typically freshwater organism Toxins known 2017,to 9, 12produce the hepatotoxins Microcystins. 3 of 13

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Figure 1. Light microscopy photographs of Planktothrix agardhii strains observed in this study. (a,f)—

Figure 1. P.Light microscopy photographs of Planktothrix agardhii strains observed in this study. agardhii filaments observed in marine water from the sea pier; (b,c)—P. agardhii filaments observed (a,f)—P. agardhii filaments in marine wateragardhii fromstrain the sea pier; (b,c)—P. agardhii in freshwater from S.observed Domingos reservoir; (d,e)—P. IPMA2; (g,h)—P. agardhii strain filaments IPMA5; (i,j)—P. agardhii IPMA3. The arrow indicates the calyptra. bar IPMA2; 5 µ m. observed in freshwater from S.strain Domingos reservoir; (d,e)—P. agardhiiScale strain (g,h)—P. agardhii Figure (i,j)—P. 1. Light microscopy photographs of Planktothrix strains observed in this study. strain IPMA5; agardhii strain IPMA3. The arrowagardhii indicates the calyptra. Scale bar(a,f)— 5 µm.

this observation, site was monitored closely. Samples agardhii were collected three sites: P.Following agardhii filaments observed inthe marine water from the sea pier; (b,c)—P. filamentsatobserved at the sea pier (salinities 35 to reservoir; 37), beach(d,e)—P. (salinities from 7strain to 13)IPMA2; and river outfallagardhii (salinities from in freshwater from S. from Domingos agardhii (g,h)—P. strain 3 toIPMA5; 5).this One sample was taken in the freshwater reservoir located inland at the same water line Following observation, the site was monitored closely. Samples were collected at three sites: (i,j)—P. agardhii strain IPMA3. The arrow indicates the calyptra. Scale bar 5 µ m. (salinity 0). at the sea pier (salinities from 35 to 37), beach (salinities from 7 to 13) and river outfall (salinities Cell concentrations in waterthe were high atmonitored all the locations andSamples present were during all sampling period Following this observation, site closely. collected at three sites: from 3 to 5). One sample wasorders takenofin thewas freshwater reservoir located inland at the same water line but with very different magnitude, decreasing from the river outfall to the sea (Figure 2). at the sea pier (salinities from 35 to 37), beach (salinities from 7 to 13) and river outfall (salinities from (salinity 0). sea pier, cell concentrations showed high oscillations andsame were water between 3 At to the 5). One sample was taken in the freshwater reservoir weekly located (Figure inland 2a) at the line −1 and 174,244 cells·L−1 (Figure 2a). At the beach the cell concentrations were higher 4,960,608 Cell(salinity concentrations water were high at all the locations and present during all sampling period 0).cells·L in 6 cells·L−1 (Figure 2b). At the river than the sea pier, ranging from 296.229 × 106 cells· L−1 to 124.571 × 10the but with very different orders of magnitude, from river outfall to the period sea (Figure 2). Cell concentrations in water were high at decreasing all the locations and present during all sampling outfall, P. agardhii concentrations were the highest recorded, between 6810.3 × 106 cells·L−1 and 2328 × butpier, with very different orders of magnitude, decreasing from the river outfall to the sea (Figure 2).between At the sea cell concentrations showed high oscillations weekly (Figure 2a) and were 106 cells·L−1 (Figure 2c) with orders of magnitude similar to the freshwater reservoir (8935.7 × 106 At the sea pier, cell concentrations showed high oscillations weekly (Figure 2a) and were between − 1 − 1 4,960,608 cells ·L−1). and 174,244 cells·L (Figure 2a). At the beach the cell concentrations were higher cells·L 4,960,608 cells·L−1 and 174,244 cells·L−1 (Figure 2a). At the beach the cell concentrations were higher than the than sea pier, ranging from 296.229 × 106 cells·L−1 to 124.571 × 106 cells·L−1 (Figure 2b). At the the sea pier, ranging from 296.229 × 106 cells·L−1 to 124.571 × 106 cells·L−1 (Figure 2b). At the river river outfall, concentrations the highest between 6810.3 ×2328 106 ×cells·L−1 outfall,P.P.agardhii agardhii concentrations werewere the highest recorded,recorded, between 6810.3 × 106 cells· L−1 and 6 − 1 6 cells· −1 (Figure and 232810× 10 Lcells ·L 2c) (Figure 2c) with orders of magnitude similar to the freshwater with orders of magnitude similar to the freshwater reservoir (8935.7 × 106reservoir −1). (8935.7 ×cells· 106Lcells ·L−1 ).

Figure 2. P. agardhii cell concentrations in water samples. (a)—Sampling point 1: monitoring station of the Portuguese National Shellfish Monitoring System at the sea pier; (b)—Sampling point 2: River runoff at the beach between the sea and the river; (c)—Sampling point 3: river outfall near the beach. The arrow indicates the first observed occurrence; (*)—No sampling.

Figure 2. P. agardhii cell concentrations in water samples. (a)—Sampling point 1: monitoring station

Figure 2.ofP.the agardhii cell concentrations in water samples. (a)—Sampling point 1: monitoring station Portuguese National Shellfish Monitoring System at the sea pier; (b)—Sampling point 2: River of the Portuguese Shellfish Monitoring at the point sea pier; (b)—Sampling point 2: River runoff at theNational beach between the sea and the river;System (c)—Sampling 3: river outfall near the beach. runoff atThe thearrow beach between and the river; (c)—Sampling indicates the the first sea observed occurrence; (*)—No sampling.point 3: river outfall near the beach. The arrow indicates the first observed occurrence; (*)—No sampling.

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Filaments were isolated from the water to freshwater Z8 culture media. Cultures were established with success indicating P. agardhii freshwaterZ8species. were alsowere obtained Filaments were that isolated from is thepreferably water to afreshwater cultureCultures media. Cultures fromestablished all the locations including the seathat pier, that the cells were viable at the sea water with success indicating P. indicating agardhii is preferably a freshwater species. Cultures werewith a alsoof obtained all P. theagardhii locations including the sea pier, indicating cellsiswere viable atinthe salinity 35. Thefrom list of cultures established and used in that thisthe study displayed Table 1. sea water with a salinity of 35. The list of P. agardhii cultures established and used in this study is displayed in Table 1. Table 1. List of Planktothrix agardhii cultures established. River outfall corresponds to the sampling point 3, the sea pier corresponds to sampling point 1 and S. Domingos reservoir is the freshwater lake Table 1. point List of4.Planktothrix agardhii cultures established. River outfall corresponds to the sampling at sampling point 3, the sea pier corresponds to sampling point 1 and S. Domingos reservoir is the freshwater lake at sampling point 4.

Strain Number Strain Number IPMA1 IPMA IPMA2 1 IPMA 2 IPMA3 IPMA4 IPMA 3 IPMA5 IPMA 4 IPMA6 IPMA 5 IPMA 6

Origin Origin River outfall River Sea outfall Pier Sea outfall Pier River Sea outfall Pier River S. Domingos reservoir Sea Pier S. Domingos Domingos reservoir reservoir S.

Date of Isolation Date of Isolation 29 November 2016 298November November2016 2016 0829 November November2016 2016 November2016 2016 298November November2016 2016 0815 November November2016 2016 1515 November

S. Domingos reservoir

15 November 2016

Figure 1 represents the specimens observed from both environmental samples and clonal cultures. Figure 1 represents the specimens observed from both environmental samples and clonal The morphology of the P. agardhii strains maintained in culture was as described previously for the cultures. The morphology of the P. agardhii strains maintained in culture was as described previously observed wild specimens. for the observed wild specimens. The The morphometry of of thethestrains 3.44±±0.464 0.464 and 3.64 ± 0.601 for width cell width morphometry strainswas wasbetween between 3.44 and 3.64 ± 0.601 for cell and and between 2.45 ± 0.492 and 2.57 ± 0.466 for the cell length (Figure 3). No significant differences between 2.45 ± 0.492 and 2.57 ± 0.466 for the cell length (Figure 3). No significant differences were were obtained between strains for (ANOVA;F5,F600 = 1.682; p > and 0.05)cell and cell (ANOVA; length (ANOVA; obtained between strains forcell cellwidth width (ANOVA; = 1.682; p ˃ 0.05) length F5, 5, 600 0.845; pp ˃>0.05). F5, 600600== 0.845; 0.05).

Figure 3. Morphometry Planktothrixagardhii agardhii strains inin this study. Narrow lineslines indicate the the Figure 3. Morphometry ofof Planktothrix strainsisolated isolated this study. Narrow indicate range of the measurements; (♦)—indicates the average with the value and the standard deviation. range of the measurements; ()—indicates the average with the value and the standard deviation.

Since morphology of Planktothrix is very similar between species the molecular analysis of the Since gene morphology of Planktothrix is verythe similar between the molecular analysis rpoC1 was performed and confirmed identification of species the cultures also as P. agardhii byof the rpoC1BLAST gene was performed and confirmed the identification the cultures also as agardhii by BLAST search. The phylogenetic analysis showed thatofour strains form a P.well-supported monophyletic clade in one of the showed unresolved groups of the P. agardhii/P. rubescens complex (Figure 4). clade search. The phylogenetic analysis that our strains form a well-supported monophyletic P. agardhii and P. rubescens are difficult to distinguish molecularly, differing morphologically by its and in one of the unresolved groups of the P. agardhii/P. rubescens complex (Figure 4). P. agardhii color, one is green and the other is red [9,35]. P. rubescens are difficult to distinguish molecularly, differing morphologically by its color, one is green P. agardhii growth, exposed to a range of salinities was evaluated using optical density and the other is red [9,35]. measurements over a 216 h period (Figure 5). The growth pattern for all the strains tested (IPMA 2; 3 P. agardhii growth, exposed to a range of salinities was evaluated using optical density and 5) was similar regardless the origin of the strain (sea pier, sampling point 1—Figure 5a; river measurements over point a 2163—Figure h period5b (Figure The growth pattern for allpoint the strains tested (IPMA2; outfall, sampling and the5). freshwater reservoir, sampling 4—Figure 5c). The 3 andstrains 5) was similar regardless the origin of 10. theGrowth strain (sea pier, was sampling 1—Figure grew well in salinities between 0 and inhibition visible point at salinities 20 and5a; 30 river outfall, sampling point for all the strains tested3—Figure (Figure 5). 5b and the freshwater reservoir, sampling point 4—Figure 5c).

The strains grew well in salinities between 0 and 10. Growth inhibition was visible at salinities 20 and 30 for all the strains tested (Figure 5).

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4. Phylogenetic of P. agardhii and closely related Planktothrixretrieved retrieved from from Figure 4. Figure Phylogenetic tree of tree P. agardhii strainsstrains and closely related Planktothrix GenBank, inferred by using the Maximum Likelihood method based on the Kimura 2-parameter Figureinferred 4. Phylogenetic tree P. agardhii strains and closely related retrieved from GenBank, by using theof Maximum Likelihood method basedPlanktothrix on the Kimura 2-parameter model of the rpoC1 genethe sequences. The percentagemethod bootstrap values of 1000 replicates are given at GenBank, inferred by using Maximum Likelihood onof the Kimura 2-parameter model of the rpoC1 gene sequences. The percentage bootstrapbased values 1000 replicates are given each node. GenBank accessionThe numbers are indicated after the species designation. The tree is model of the rpoC1 gene sequences. percentage bootstrap values of 1000 replicates are given at each node. GenBank accession numbers are indicated after the species designation. The at tree is unrooted and drawn to scale, with branch lengths measured the number of substitutions each node. GenBank accession numbers are indicated after the in species designation. The tree per is site. unrooted and drawn to scale, with branch lengths measured in the number of substitutions per site. The and analysis involved 23with nucleotide sequences. There were a total of 452 positions in unrooted drawn to scale, branch representative lengths measured in the number of substitutions per site. The analysis 23 The nucleotide sequences. There werethea cultures total of obtained 452 positions the involved finalinvolved dataset. grey box representative indicates the origin and strain number The analysis 23 nucleotide representative sequences. There were aoftotal of 452 positions inin this in the finalstudy. dataset. The grey box IPMA1—MG452723; indicates the origin and strain number of the cultures obtained Accession numbers: the final dataset. The grey box indicates the origin andIPMA2—MG452724; strain number of theIPMA3—MG452725; cultures obtained in IPMA4— this in this study. Accession numbers: IPMA1—MG452723; IPMA2—MG452724; IPMA3—MG452725; MG452726; IPMA5—MG452727; IPMA6—MG452728. study. Accession numbers: IPMA1—MG452723; IPMA2—MG452724; IPMA3—MG452725; IPMA4— IPMA4—MG452726; IPMA5—MG452727; IPMA6—MG452728. MG452726; IPMA5—MG452727; IPMA6—MG452728.

Figure 5. Growth curves of the Planktothrix agardhii strains exposed to several salinities. (a) P. agardhii collected pier; (b) agardhii P. agardhii IPMA3 collected at thesalinities. river outfall; P. agardhii FigureIPMA2 5. Growth curvesatofthe thesea Planktothrix strains exposed to several (a) P.(c) agardhii Figure 5. Growth curves of the Planktothrix agardhiiRange strains exposed to several salinities. (a) P. agardhii IPMA5 collected at the freshwater reservoir. of salinities tested: (∆) 0; (○) 2.5; (□) 5; (▲) IPMA2 collected at the sea pier; (b) P. agardhii IPMA3 collected at the river outfall; (c) P. agardhii10; (●) IPMA2 collected at the sea pier; (b) P. agardhii IPMA3 collected at the river outfall; (c) P. agardhii IPMA5 20;collected (■) 30. at the freshwater reservoir. Range of salinities tested: (∆) 0; (○) 2.5; (□) 5; (▲) 10; (●) IPMA5

collected at the freshwater reservoir. Range of salinities tested: (∆) 0; (#) 2.5; () 5; (N) 10; ( ) 20; 20; (■) 30. () 30.

No microcystins were detected in the tissues from mussels and the biomass from water and cultured strains. Furthermore, the microcystin related gene (mcyA) was also not detected in water nor cultures, indicating that no toxic strains were present in the environment.

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3. Discussion The possible human health risk of freshwater blooms in coastal environments and consequent contamination of marine shellfish has recently received attention with the death of a large number of sea otters with liver failure in Monterey Bay, California after ingestion of microcystin contaminated shellfish [36]. Furthermore, recreational exposure incidents of acute illness in humans have been reported for the La Plata estuary [7]. In Portugal, the reports of freshwater cyanobacterial blooms in estuaries were related with the cyanobacterium Microcystis. Until now, were reported in Minho estuary where a Microcystis bloom was transported through the river [37] and in Guadiana estuary where Microcystis blooms often develop and accumulate in the upper part of the estuary [38,39]. Microcystins have never been detected in the lower part of the estuaries, marine water nor marine shellfish [37,38]. In this work, we found that the bloom-forming cyanobacterium Planktothrix agardhii is reaching sea waters at high cell concentrations and that the surrounding area at the land-sea interface is heavily loaded with P. agardhii cells. The cell densities found in the water, from the river outfall, beach and sea were above the cell limit of 2 × 106 cells·L−1 recommended by WHO, from which microcystin concentration in water can exceed the WHO guideline of 1 µg·L−1 of safe daily ingestion by an oral root [3]. Gible and colleagues [21] found that mussels fed with 2 × 109 cyanobacterial cells·L−1 , with an average of 5.6 µg·L−1 total microcystins for 24 h could accumulate 4 to 6 ng of microcystins per g of tissue. Mulvenna and colleagues [40] calculated derived guideline values for microcystins in seafood of 51 µg of microcystins per Kg. Given the cell concentrations obtained, our results indicate that this area is vulnerable to shellfish contamination by freshwater toxins if present, despite no microcystins nor microcystin related genes were observed. Concerning WHO recommendations for bathing waters, the cell concentrations in our study at the sea pier, were below 2 × 107 cells·L−1 guideline that represents a low risk of adverse effects from cyanobacteria exposure [3]. However, at the beach and in the river outfall cell concentrations were above the 1 × 108 cells·L−1 guideline which constitutes a high risk according to WHO [3]. The studied site is not only a shellfish harvesting area but also a recreational sea beach used for bathing and known as a good site for surfing after what is called “Praia dos Supertubos”. The recreational use of this beach goes beyond the seasonality of the summer and is used by surfers, surf schools, and surf competitions during all year around. P. agardhii is considered a freshwater cyanobacterium, but, is a resilient and persistent species known to tolerate a wide range of temperatures and light intensities, meaning that has been found in a wide range of environments prevailing all year. It has been found blooming under ice-covered lakes in Poland, mixed in the water column in eutrophic lakes or to form metalimnetic blooms in more oligotrophic and/or water stratification conditions [41–45]. In this study, the possible source of the cells is the freshwater reservoir located 5 km upstream as it contains elevated cell densities of P. agardhii. Most probably the cells found in the sea belong to a population continually renewed by the input from the river. The salinity tolerance tests showed that all strains grown in salinities of 10, including the freshwaters reservoir strain. These results point out that P. agardhii may have the capability of growing in the river outfall, where salinities vary from 3 to 5. Furthermore, the ability of the freshwater strain also to develop in moderate salinities of 10 implicates a continuous flow of P. agardhii to the sea water, as the species proliferate through transitional waters. This resilience to salinity has been reported by several authors who verified a salinity growth range between 0 and 7.8 [35,46,47]. Blooms of P. agardhii have been reported for low salinity waters, such as, the Bolmon lagoon that has salinities from 5 to 10, Olivier pond with salinities between 2.8 and 3.9 and Albufera de Valencia with salinities between 1 and 3 [47–49]. In addition, a massive bloom of the closely related P. rubescens in Lake Occhito was reported to reach the sea contaminating mussel farms in Italy [50]. In the light of our and the aforementioned results, it is important to understand the halotolerance of this species and differences between toxic and non-toxic strains to understand their ability to colonize more saline environments. The Portuguese legislation has the obligation to monitoring cyanobacteria and Microcystins only related with drinking water safety for freshwater reservoirs and the legislation concerning seafood safety does not include freshwater toxins [28,30]. Given the results obtained in this study, the P. agardhii

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present in the environment was probably a non-toxic strain, but the concentrations were alarming for coastal areas especially from a species that is very often reported as toxic. Toxic and non-strains of cyanobacteria are morphologically indistinguishable and toxin analysis must be mandatory. We believe that cyanotoxin occurrence and concentration in marine waters and shellfish are pertinent to be monitor routinely as these blooms are unpredictable. Also, according to Portuguese legislation for recreational waters quality [32,33], sampling should only be carried out when a proliferation is observed, with no guideline for concentration levels. In the case of our study, per example, visual inspection of the water is not adequate since, usually, P. agardhii blooms do not form aggregates, scums or foams at the surface being generally dispersed through to the water column in high cell densities [3]. This work reinforces the need for a regular surveillance plan to monitor the presence of cells and toxins in marine water for recreational and environmental purposes as well as for seafood safety. It also demonstrates the relevance of monitoring transitional waters with a sequence of stations upstream enough to act as early-warning stations of blooms reaching coastal areas. 4. Materials and Methods 4.1. Location and Sampling Cyanobacteria were first observed in Marine waters from the northwest Portuguese coast in the beginning of November of 2016. The observation site is part of the Portuguese National Shellfish Monitoring System that is sampled every week for the screening of harmful marine phytoplankton and related toxins [51]. The region surrounding the sampling site is represented in Figure 6. It is near the outfall of the river S. Domingos that is characterized by a water dam 5 km upstream and the tributary of two other rivers (Figure 6). Between November and December of 2016, the samples were taken every week at three different locations at the site (Figure 6). At sampling point 1 (sea pier) both water and mussels were sampled. At sampling points 2 at the beach in the river run-off and 3 river out-fall, only water was sampled (Figure 6). The freshwater reservoir locates 5 km upstream in the river was sampled once to investigate the presence of cyanobacteria (Figure 6). All the water samples were taken at the surface in low tide, transported to the laboratory in refrigerated conditions and the salinity measured with a hand-held visual refractometer, Index Instruments LTD. A part was filtered for toxin and molecular analysis, part was preserved for cell counting and another part was kept fresh for filament isolation and culture establishment.

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Figure Figure6.6. Schematic Schematic representation representation at at scale scale of of the thesampling sampling site siteat atthe thenorthwest northwestof ofthe thePortuguese Portuguese coast. 1)—Monitoring station coast. ((●1)—Monitoring station of of the the Portuguese Portuguese National National Shellfish Shellfish Monitoring Monitoring System System at at the thesea sea ◦ 0 ◦ 0 pier 21 00.4” N 22 19.4” W), ((●2)—Sampling 2)—Sampling point 2, river pier(39 (39°21′00.4″ N 99°22′19.4″ river run-off run-off at at the the beach beachbetween betweenthe thesea sea ◦ 210 03.0” N 9◦ 220 04.7” W), ( 3)—Sampling point 3 at the river outfall near the beach and andthe theriver river(39 (39°21′03.0″ N 9°22′04.7″ (●3)—Sampling point 3 at the river outfall near the beach ◦ 210 04.6” N 9◦ 210 58.6” (39 ( 4)—Sampling point (39°21′04.6″ N 9°21′58.6″ W), (●4)—Sampling point 44 near near the the water water dam dam discharged dischargedat atthe the freshwater freshwater ◦ 190 58.1” N 9◦ 190 07.5” W), (−)—Sea Piers reservoir S.S. Domingos (39(39°19′58.1″ reservoirlocated located55km kmupstream upstreamofofthe theriver river Domingos N 9°19′07.5″ W), (−)—Sea Piers and andwater waterdam, dam,grey greylines linesrepresent representwater waterlines. lines.

4.2. 4.2.Filament FilamentIsolation Isolationand andCulture CultureEstablishment Establishment Cyanobacteria Cyanobacteriafilaments filamentswere wereisolated isolatedwith withaamicropipette micropipetteunder underthe theinverted invertedmicroscope microscopeLeica Leica®® DMi8 DMi8 from from water water fresh fresh samples samples concentrated concentrated with with aa10 10µm µ mnet. net. Cultures Cultures were were established established by by transferring the isolated filaments into Z8 culture medium [52] at salinities 35 and 0. Successful transferring the isolated filaments into Z8 culture medium [52] at salinities 35 and 0. Successful cultures ofof 5 µmol ·photons ·m−m2−2 ·s·− cultureswere weremaintained maintainedatat1919±±11◦ C °Cwith witha alight lightintensity intensity 5 μmol· photons· s−11 and and aa 12:12 12:12hh light:dark light: darkcycle. cycle. 4.3. 4.3.Cyanobacteria CyanobacteriaIdentification Identificationand andQuantification Quantification For Forthe thequantification quantificationof ofcyanobacteria, cyanobacteria, the thesamples sampleswere werepreserved preservedwith withneutralized neutralizedLugol’s Lugol’s iodine solution, settled down in sedimentation chambers (25 mL) and counted iodine solution, settled down in sedimentation chambers (25 mL) and counted using using an aninverted inverted ® microscope × magnification microscopeLeica Leica® DMi8 DMi8 at at400 400× magnification following following the the procedure procedure of of the theUtermöhl Utermöhltechnique technique described in the European Standard EN15204 [53]. The number of cells in oscillatoriales cyanobacteria described in the European Standard EN15204 [53]. The number of cells in oscillatoriales cyanobacteria was wascalculated calculatedby bydividing dividingthe themeasured measuredfilament filamentlength lengthby bythe themean meancell celllength. length. ® For the morphological characterization, the cyanobacteria were analyzed For the morphological characterization, the cyanobacteria were analyzedusing usingaaLeica Leica® DMi8 DMi8 inverted × magnification. inverted microscope microscope atat1000 1000× magnification. The The morphological morphological characters characters evaluated evaluated were: were: cell cell dimensions dimensions (length (length and and width), width), filament filament color color and andshape, shape, constrictions constrictions at at the the cell cellwall, wall,presence presenceof of sheath, shape of apical cell, presence/absence of calyptra and necredia [35,46,54,55]. Photographs were sheath, shape of apical cell, presence/absence of calyptra and necredia [35,46,54,55]. Photographs ® taken digital camera. cell measurements were performed Leica®using Lasx were with takena Leica with a DFC550 Leica® DFC550 digital The camera. The cell measurements were using performed software. least 50 At measurements were done were for calculating the size of the the size cellsof and Leica® LasxAtsoftware. least 50 measurements done for calculating thefilaments. cells and Differences in cell dimensions (length and width) were tested with a one-way Analysis of Variance filaments. Differences in cell dimensions (length and width) were tested with a one-way Analysis of (ANOVA) for p < 0.05.for p < 0.05. Variance (ANOVA) For For the themolecular molecular identification, identification, the the DNA DNA was was extracted extracted from from the the cultures cultures using using the the DNeasy DNeasy ®. Plant Mini Kit, Quiagen The total DNA concentration was quantified using the ® Plant Mini Kit, Quiagen . The total DNA concentration was quantified using the Qubit™ Qubit™ ® . A DNA fragment of 608 bp within the Fluorometric Thermo Fisher FisherScientific Scientific ®. A Fluorometric Quantitation, Quantitation, Thermo DNA fragment of 608 bp within the rpoC1 0 ) and RPOR rpoC1 was amplified the primers RPOF (50 -TGGTCAAGTGGTTGGAGA-3 gene gene was amplified with with the primers RPOF (5′-TGGTCAAGTGGTTGGAGA-3′) and RPOR (5′-

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(50 -GCCGTAAATCGGGAGGAA-30 ) [56]. The amplifications were performed in a reaction mixture containing DreamTaq PCR Master Mix (ThermoFisher Scientific® ) with 1 U of Taq DNA polymerase using a T100™ Thermal Cycler (BioRad) programmed with a PCR cycle consisting of an initial denaturation step at 94 ◦ C for 3 min, followed by 40 cycles of 20 s at 94 ◦ C, 30 s at 55 ◦ C and 20 s at 72 ◦ C and a final extension step of 5 min at 72 ◦ C. The amplified fragments were visualized under UV light after electrophoretic analysis performed in 1% w/v agarose gel with GreenSafe Premium™ DNA staining (NZYTech® ), at 80 V in 0.5× Tris-borate EDTA (TBE) buffer for 45 min. The amplified PCR products were purified and sequenced by Sanger sequencing in the commercial platform GATC Biotech® . Sequences of the 608 bp fragment of the rpoC1 gene were analyzed with the Basic Local Alignment Search Tool (BLAST™) to access similarity and identity of the sequences with the sequences in the database. A total of 189 sequences of the partial rpoC1 gene from Planktothrix species were retrieved from the database. The nucleotide alignment was performed with MUSCLE [57] using MEGA7 [58]. A total of 23 sequences were chosen as representatives of each biological nucleotide sequence. A phylogenetic tree was constructed using the Maximum Likelihood method based on the Kimura 2-parameter model [59] using MEGA7 [58]. There were a total of 452 positions matrix in the final dataset and all positions containing gaps and missing data were eliminated. Node support was estimated using 1000 bootstrap replicates. The sequences obtained in this study were deposit in the GenBank® genetic sequence database [60]. Accession numbers: IPMA1—MG452723; IPMA2—MG452724; IPMA3—MG452725; IPMA4—MG452726; IPMA5—MG452727; IPMA6—MG452728. 4.4. Toxin Analysis Microcystins were extracted from (1) 15 g of mashed mussel tissue; (2) the filters (GF/C, microfibre filters, Whatman® ) with the biomass of 2 L of water and (3) lyophilized P. agardhii cultures biomass (100 mg dry weight) with 50% methanol for 2 h under magnetic stirring. The extracts were sonicated (ice bath, 60 Hz, 1 min pulses) with an ultrasonic probe, centrifuged (4495 g, 4 ◦ C, 5 min) and the pellets were re-extracted overnight by the same procedure. Supernatants of the two extractions were combined and subjected to rotary evaporation at 35 ◦ C to eliminate methanol. The resulting aqueous extracts were cleaned-up by solid phase extraction on SPE cartridges (Sep-Pak C18, Phenonenex) previously activated with 20 mL of ethanol and equilibrated with 20 mL of distilled water. The microcystin containing fraction was eluted with methanol at 80% (v/v) and the methanolic fraction evaporated. The resulting solution was filtered through 0.45 µm syringe filters and analyzed by HPLC-PAD. Microcystins were identified by their characteristic absorption maximum at 238 nm and quantified using commercially available MC-LR standard (Alexis® Biochemicals). The samples were injected in an HPLC system from Waters® Alliance e2695 coupled with a PDA 2998 equipped with on a Merck® Lichrospher RP-18 endcapped column (250 mm × 4.6 mm i.d., 5 µm) equipped with a guard column (4 × 4 mm, 5 µm) both kept at 45 ◦ C. The PDA range was 210–400 nm with a fixed wavelength of 238 nm. The linear gradient elution consisted of (A) methanol + 0.1% trifluoroacetic acid and (B) H2 O + 0.1% TFA (55% A and 45% at 0 min, 65% A and 35% B at 5 min, 80% A and 20% B at 10 min, 100% A at 15 min, 55% A and 45% B at 15.1 and 20 min) with a flow rate of 0.9 mL·min−1 . The injected volume was 20 µL. The system was calibrated by using a set of 7 dilutions of MC-LR standard (0.5 to 20 µg·mL−1 ) in methanol 50%. Each vial was injected in duplicate and every HPLC run series of ten samples was constituted with a blank and two different standard concentrations. Empower 2™ Chromatography Data Software was used for calculation and reporting peak information. The detection and quantification limits of MC-LR that can be detected in water and cyanobacteria biomass are 0.3 µg·mL−1 and 0.5 µg·mL−1 , based on a signal-to-noise ratio of 3 and 10. All HPLC solvents were filtered (Pall® GH Polypro, 47 mm, 0.2 µm) and degassed by ultrasound bath. DNA from the cultures and water filtered biomass (with the filtrate of 1 L of water samples) was extracted as mentioned above. A PCR reaction was performed to check for the presence of a fragment of the mcyA gene related with microcystin production. For the reaction, the primers MAPF

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(50 -CTAATGGCCGATTGGAAGAA-30 ) and MAPR (50 -CAGACTATCCCGTTCCGTTG-30 ) [61] were used in a mixture containing DreamTaq PCR Master Mix (ThermoFisher Scientific® ) with 1 U of Taq DNA polymerase using a T100™ Thermal Cycler (BioRad) with a thermocycling profile consisting of an initial denaturation step at 94 ◦ C for 3 min, followed by 35 cycles of 20 s at 94 ◦ C, 20 s at 60 ◦ C, and 20 s at 72 ◦ C and a final extension step of 5 min at 72 ◦ C. DNA from the P. agardhii strain CCALA159, that is a microcystin producer [62], was used has a positive control for the amplification. The PCR reactions were checked for fragment amplification under UV light after electrophoretic analysis performed in 1% w/v agarose gel with GreenSafe Premium™ DNA staining (NZYTech® ), at 80 V in 0.5× Tris-borate EDTA (TBE) buffer for 45 min. 4.5. Salinity Tolerance Experiments A 96-well microplate bioassay was used to evaluate P. agardhii growth in a salinity gradient (30 to 0). Culture Z8 media was prepared using natural seawater. Aliquots (100 µL) of exponential growing stock cultures were added to microplate wells previously filled with Z8. The total assay volume in each well was 200 µL. Three replicates were used for each experimental condition and 7 for the control condition (salinity 0). The plates were sealed with Parafilm™ to reduce evaporation. Cuts in the Parafilm™ were made to allow gas exchange and avoid condensation. Sealed plates were placed in the culture chamber under the same light and temperature conditions as described for culture maintenance. Optical densities of each well were measured daily for 9 days at 655 nm using a microplate absorbance reader BioRad 680XR. Mean values and the coefficient of variation (standard deviation/mean) of optical density measurements from replicates were calculated and used to estimate culture growth over a 216-h period. Some of the strains maintained in culture (IPMA1; 4 and 6) formed clumps, so, it were not tested for salinity tolerance. Acknowledgments: This work was supported by the project SNMB—INOV: Innovation for a more competitive shellfish sector co-financed by the Portuguese Government, Operational Program (OP) Mar 2020, Portugal 2020 and European Union through the European Structural Funds and Investment Funds (FEEI) and European Maritime and Fisheries Fund (EMFF). The authors also acknowledge the Centre of Marine Sciences (CCMAR), University of Algarve and Interdisciplinary Centre of Marine and Environmental Research (CIIMAR) of the University of Porto and the Funding of RD Units Strategic Plan from the Portuguese Foundation for Science and Technology (FCT) through the projects UID/Multi/04326/2013 and UID/Multi/04423/2013 respectively. Author Contributions: C.C. conceived and designed the experiments and performed the sampling, C.C. and J.A. performed the experiments; C.C., J.A., A.S. and V.V. analyzed the data; A.S. and V.V. contributed with reagents, materials, analysis tools and facilities; C.C., J.A., A.S. and V.V. wrote the paper. Conflicts of Interest: The authors declare no conflict of interest and the founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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