Detection of a Planktothrix Agardhii Bloom in Portuguese Marine Coastal Waters

toxins

Article Detection of a Planktothrix agardhii Bloom in Portuguese Marine Coastal Waters

Catarina Churro 1,* ID , Joana Azevedo 2, Vitor Vasconcelos 2,3 and Alexandra Silva 1,4

1 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] 2 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.) 3 Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 4069-007 Porto, Portugal 4 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 www.mdpi.com/journal/toxins Toxins 2017, 9, 12 Toxins 2017, 9, 391 2 of 13 2 of 13 occurrence, distribution, magnitude, and persistence of these blooms are perceived to be increasing globally over the lastThe years occurrence, [8–13] and distribution, have been magnitude, related to and increasing persistence human of these activities blooms and are global perceived to be increasing warming [9–14]. However,globally over cyanobacterial the last years proliferations [8–13] and have are been intricate related phenomena to increasing and human their activities and global occurrence is the warming result of [9 –14 the]. However, juncture cyanobacterial of several factors, proliferations related are but intricate not exclusive phenomena to, and their occurrence increasing/reductionis theof nutrient result of inputs, the juncture increasing of several river factors, barriers, related increased but not water exclusive stratification to, increasing/reduction periods, changes inof the nutrient hydrological inputs, cycle, increasing increasing river global barriers, temperatures, increased and water CO stratification2 [9–16]. Whether periods, changes in the or not this is connectedhydrological to climate cycle, change increasing massive global cyanobacterial temperatures, blooms and CO are2 [no9–16 longer]. Whether seasonal, or not this is connected persisting during allto year, climate and change also, not massive restrict cyanobacterialto lentic water bloomssystems arewhere no longerrivers and seasonal, estuaries persisting during all have also been affectedyear, [8,9,17,18 and also,]. The not reports restrict of to freshwater lentic water cyanobacterial systems where blooms rivers reaching and the estuaries sea have also been are increasing and haffectedave been [8 ,most9,17,18 probably]. The reports underestimated of freshwater [19 cyanobacterial–22]. Furthermore, blooms the reaching effect of thethese sea are increasing and blooms in the marinehave ecosystem been most and probably the impact underestimated in human and [19 wildlife–22]. Furthermore, are largely theunknown effect of [19 these–22]. blooms in the marine The most commonlyecosystem found and freshwater the impact toxins in human—microcystins and wildlife—are are produced largely unknown by several [19 bloom–22]. The- most commonly forming cyanobacteriafound species, freshwater such toxins—microcystins—are as Microcystis, Planktothrix produced, Anabaena by several, Anabaenopsis bloom-forming, and cyanobacteria species, Aphanizomenon. Thesesuch genera as Microcystis—Microcystis, Planktothrix and ,PlanktothrixAnabaena, Anabaenopsis—are globally, and distributedAphanizomenon and. These form genera—Microcystis blooms in lakes andand reservoirsPlanktothrix all over—are the globally world. distributed In freshwater, and the form main blooms concern in lakes from and microcystin reservoirs all over the world. intoxication is throughIn freshwater, ingestion or the dialysis main concern of/with from contaminate microcystin drinking intoxication water, isrecreational through ingestion contact or dialysis of/with and animal poisoningcontaminate [6,23]. These drinking hepatotoxins water, recreational are known to contact be chemically and animal stable poisoning in both [ 6fresh,23]. and These hepatotoxins are marine water, to persistknown in sediments, to be chemically to accumulate stable in in both both fresh freshwater and marine and water,marine to filter persist-feeders in sediments, and to accumulate to transfer up the foodin bothchain freshwater [19–21,24–26 and]. In marine a recent filter-feeders review by Preece and to et transfer al. [22] the up authors the food describe chain [19–21,24–26]. In a freshwater blooms occurrencesrecent review in by estuarine Preece et and al. [coastal22] the waters authors in describe North and freshwater South America, blooms occurrences Europe, in estuarine and Africa, Australia, Turkey,coastal and waters Japan. in NorthIn some and of Souththe reports, America, microcystins Europe, Africa,were detected Australia, in the Turkey, water and Japan. In some and were accumulatingof the in reports, marine microcystinsshellfish with were animal detected deaths in implicated the water [22]. and Some were accumulatingcountries, like in marine shellfish Australia and USA,with have animal recently deaths defined implicated microcystins [22]. Someguidelines countries, for fish, like prawns Australia and and molluscs USA, haveto recently defined apply in some statesmicrocystins [26,27]. In general, guidelines however, for fish, governments, prawns and food molluscs standards to apply regulations in some and states legal [26,27]. In general, guidelines for seafoodhowever, safety governments,do not yet include food standardsfreshwater regulations phytoplankton and legal relat guidelinesed toxins [23,27 for seafood]. In safety do not yet the same way, theinclude Portuguese freshwater legislation phytoplankton only includes related the toxins obligation [23,27]. In of the monitoring same way, marine the Portuguese legislation phytoplankton relatedonly toxins includes in theseafood obligation through of monitoring the Law Decree marine n.º phytoplankton 293/98 (1998) related [28] transposed toxins in seafood through the from the EuropeanLaw Directive Decree n.º 293/9891/492/CEE (1998) (19 [2891)] transposed [29]. The from Portuguese the European legislation Directive concerning91/492/CEE (1991) [29]. cyanobacteria and microcystinsThe Portuguese is only legislation related concerning to drinking cyanobacteria water safety and(Law microcystins Decree nº 306/2007 is only related [30] to drinking water transposed from thesafety European (Law Decree Drinking306/2007 Water Directive [30] transposed [31]) and from the the legislation European concerning Drinking Water the Directive [31]) and quality of bathing waterthe legislation [32,33], that concerning was transposed the quality from of the bathing European water Bathing [32,33], Water that was Directive transposed [34], from the European poorly addresses thisBathing question. Water In the Directive light of [what34], poorly appears addresses to be an additional this question. and increasing In the light concern, of what appears to be an it is important to reportadditional freshwater and increasing cyanobacteria concern, blooms it is in important coastal waters to report and freshwater to access cyanobacteriarecreational blooms in coastal and seafood safety.waters In the present and to access work, recreationalwe describe and the seafoodoccurrence safety. of a In freshwater the present cyanobacterium work, we describe the occurrence bloom in marine waterof a freshwaterand land-sea cyanobacterium interface at a bloomshellfish in marineharvesting water and and bathing land-sea area interface located at on a shellfish harvesting the W coast of Portugal.and bathing We went area further located testing on the cell W coastconcentration of Portugal. and We microcystin went further content testing in the cell concentration and water and shellfish,microcystin cyanobacteria content strain in viability the water in anda marine shellfish, environment cyanobacteria with contrasting strain viability salinities in a marine environment and the presence ofwith microscystin contrasting related salinities genes and to evaluate the presence potential of microscystin toxic impact related in the genesecosystem. to evaluate potential toxic impact in the ecosystem. 2. Results 2. Results In November of 2016, during the regular monitoring program for the screening of harmful marine phytoplankton, In a high November concentration of 2016, of during the filamentous the regular cyanobacteria monitoring programPlanktotrix for agardhii the screening of harmful (4,960,608 cells·L−1) wasmarine observed phytoplankton, in a marine a sample high concentration at the northwest of thePortuguese filamentous coast cyanobacteria (geographical Planktotrix agardhii − coordinates: 39°21′00.4(4,960,608″ N 9°2 cells2′19.4·L″ W).1) was The observed area is a in shellfish a marine harvesting sample at site the northwestand a recreational Portuguese sea coast (geographical ◦ 0 ◦ 0 beach used for bathingcoordinates: and surfing. 39 21 00.4” N 9 22 19.4” W). The area is a shellfish harvesting site and a recreational sea The observed cyanobacteriabeach used for are bathing from the and group surfing. Oscillatoriales with no heterocytes nor akinetes, the filaments were blueThe-green, observed solitary cyanobacteria and without are from sheaths. the group The Oscillatorialesfilaments were with continually no heterocytes nor akinetes, attenuated at one extremitythe filaments and werestraight blue-green, at the other, solitary the andcells without were 3.54 sheaths. ± 0.365 The µm filaments wide and were 2.65 continually ± attenuated 0.467 µm long with atno one constrictions extremity at and cross straight-cell walls, at the the other, apical the cell cells was were narrowed 3.54 ± 0.365at oneµ sidem wide of the and 2.65 ± 0.467 µm filament with calyptralong with and no rounded constrictions at the at cross-cell other side walls, (Figure the apical 1a,f). cell These was narrowed morphological at one side of the filament characteristics werewith in congruence calyptra and with rounded the description at the other of the side cyano (Figurebacterium1a,f). These Planktothrix morphological agardhii characteristicsa were typically freshwater organism known to produce the hepatotoxins Microcystins.

Toxins 2017, 9, 391 3 of 13 in congruence with the description of the cyanobacterium Planktothrix agardhii a typically freshwater organismToxins known 2017, to9, 12 produce the hepatotoxins Microcystins. 3 of 13

Toxins 2017, 9, 12 3 of 13

Figure 1. Light microscopy photographs of Planktothrix agardhii strains observed in this study. (a,f)— Figure 1. Light microscopy photographs of Planktothrix agardhii strains observed in this study. P. agardhii filaments observed in marine water from the sea pier; (b,c)—P. agardhii filaments observed (a,f)—P. agardhiiin freshwaterﬁlaments from S.observed Domingos reservoir; in marine (d,e water)—P. agardhii from strain the seaIPMA2; pier; (g,h (b)—,cP.)— agardhiiP. agardhii strain ﬁlaments observed inIPMA5; freshwater (i,j)—P. agardhii from S. strain Domingos IPMA3. The reservoir; arrow indicates (d,e)— theP. calyptra. agardhii Scalestrain bar IPMA2;5 µm. (g,h)—P. agardhii strain IPMA5;Figure (1.i ,Lightj)—P. microscopy agardhii strain photographs IPMA3. of Planktothrix The arrow agardhii indicates strains the observed calyptra. in this Scale study. bar (a 5,f)µ—m. P.Following agardhii filaments this observation, observed in the marin sitee was water monitored from the seaclosely. pier; (Samplesb,c)—P. agardhiiwere collected filaments at observed three sites: at thein freshwatersea pier (salinities from S. fromDomingos 35 to reservoir;37), beach ( d(salinities,e)—P. agardhii from 7strain to 13) IPMA2; and river (g,h outfall)—P. agardhii (salinities strain from Following3 toIPMA5; 5).this One (i observation,,j sample)—P. agardhii was takenstrain the IPMA3. in site the was freshwateThe monitoredarrowr indicates reservoir closely. the located calyptra. Samples inland Scale bar atwere the5 µm. same collected water line at three sites: at the sea(salinity pier (salinities 0). from 35 to 37), beach (salinities from 7 to 13) and river outfall (salinities Cell concentrations in water were high at all the locations and present during all sampling period from 3 to 5).Following One sample this observation, was taken the in site the was freshwater monitored reservoir closely. Samples located were inland collected at theat three same sites: water line atbut the with sea pier very (salinities different fromorders 35 of to magnitude, 37), beach (salinitiesdecreasing from from 7 theto 13) river and outfall river tooutfall the sea (salinities (Figure from2). (salinity 0). 3 At to the 5). Onesea pier, sample cell concentrations was taken in theshowed freshwate high oscillationsr reservoir weekly located (Figure inland 2 ata) theand samewere waterbetween line −1 −1 Cell(salinity concentrations4,960,608 0). cells·L inand water 174,244 were cells·L high (Figure at all 2a the). At locations the beach andthe cell present concentrations during were allsampling higher period than the sea pier, ranging from 296.229 × 106 cells·L−1 to 124.571 × 106 cells·L−1 (Figure 2b). At the river but with veryCell different concentrations orders in water of magnitude, were high at decreasingall the locations from and present the river during outfall all sampling to the period sea (Figure 2). outfall, P. agardhii concentrations were the highest recorded, between 6810.3 × 106 cells·L−1 and 2328 × but with very different orders of magnitude, decreasing from the river outfall to the sea (Figure 2). At the sea10 pier,6 cells·L cell−1 ( concentrationsFigure 2c) with orders showed of magnitude high oscillations similar to the weekly freshwater (Figure reservoir2a) (8935. and7 were× 106 between At the sea− 1pier, cell concentrations− showed1 high oscillations weekly (Figure 2a) and were between 4,960,608 cellscells·L·L−1). and 174,244 cells·L (Figure2a). At the beach the cell concentrations were higher 4,960,608 cells·L−1 and 174,244 cells·L−1 (Figure 2a). At the beach the cell concentrations were higher than the sea pier, ranging from 296.229 × 106 cells·L−1 to 124.571 × 106 cells·L−1 (Figure2b). At the than the sea pier, ranging from 296.229 × 106 cells·L−1 to 124.571 × 106 cells·L−1 (Figure 2b). At the river 6 −1 river outfall,outfall,P. P. agardhii agardhii concentrationsconcentrations were were the highest the highest recorded, recorded, between 6810. between3 × 106 cells·L 6810.3−1 and× 232108 ×cells ·L − and 232810×6 cells·L106 cells−1 (Figure·L 1 2(Figurec) with orders2c) with of magnitude orders of similar magnitude to the freshwater similar toreservoir the freshwater (8935.7 × 10 reservoir6 (8935.7 ×cells·L106 cells−1). ·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. 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 of the Portugueserunoff at the National beach betwee Shellﬁshn the sea Monitoring and the river; System (c)—Sampling at the point sea pier; 3: river (b outfall)—Sampling near the beach. point 2: River runoff atThe the arrow beach indicates between the thefirst seaobserved and theoccurrence; river; ((*)c)—Sampling—No sampling. point 3: river outfall near the beach.

The arrow indicates the ﬁrst observed occurrence; (*)—No sampling. Toxins 2017, 9, 391 4 of 13

Toxins 2017, 9, 12 4 of 13 Filaments were isolated from the water to freshwater Z8 culture media. Cultures were established with successFilaments indicating were that isolatedP. agardhii from is the preferably water to a freshwater freshwater Z8 species. culture Cultures media. Cultures were also were obtained fromestablished all the locations with success including indicating the sea that pier, P. indicatingagardhii is preferably that the cells a freshwater were viable species. at the Cultures sea water were with a salinityalso of obtained 35. The from list ofall P.the agardhii locationscultures including established the sea pier, and indicating used in that this the study cells is were displayed viable at in the Table 1. sea water with a salinity of 35. The list of P. agardhii cultures established and used in this study is displayedTable 1. List in Table of Planktothrix 1. 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 at samplingTable 1. pointList of 4. 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 at sampling point 4. Strain Number Origin Date of Isolation StrainIPMA1 Number RiverOrigin outfall Date 29 November of Isolation 2016 IPMA2IPMA 1 River Sea outfall Pier 29 8Nov Novemberember 2016 2016 IPMA3IPMA 2 RiverSea outfallPier 08 29 November November 2016 2016 IPMA4IPMA 3 River Sea outfall Pier 29 8November November 2016 2016 IPMA5IPMA 4 S. DomingosSea Pier reservoir 08 15 November November 2016 2016 IPMA6IPMA 5 S. S. Domingos Domingos reservoir 15 15 November November 2016 2016 IPMA 6 S. Domingos reservoir 15 November 2016 Figure1 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. TheThe morphometry morphometry of of the the strains strains was was betweenbetween 3.44 ±± 0.4640.464 and and 3.64 3.64 ± 0.601± 0.601 for cell for width cell width and and betweenbetween 2.45 2.45± 0.492 ± 0.492 and and 2.57 2.57± ± 0.4660.466 for the cell cell length length (Figure (Figure 3).3 ).No No significant signiﬁcant differences differences were were obtainedobtained between between strains strains for for cell cell widthwidth (ANOVA; F5, F 6005, 600= 1.682;= 1.682; p ˃ 0.05)p > and 0.05) cell and length cell (ANOVA; length (ANOVA; F5, F5, 600600= = 0.845;0.845; pp ˃> 0.05). 0.05).

FigureFigure 3. Morphometry 3. Morphometry of ofPlanktothrix Planktothrix agardhiiagardhii strainsstrains isolated isolated in inthis this study. study. Narrow Narrow lines lines indicate indicate the the 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 rpoSinceC1 gene morphology was performed of Planktothrix and confirmedis very the similar identificati betweenon of species the cultures the molecularalso as P. agardhii analysis by of the rpoC1BLAST gene was search. performed The phylogenetic and confirmed analysis the identification showed that of our the culturesstrains form also as a P. well agardhii-supportedby BLAST search.monophyletic The phylogenetic clade in one analysis of the showedunresolved that groups our strains of the P. form agardhii a well-supported/P. rubescens complex monophyletic (Figure 4). clade in oneP. agardhii of the unresolvedand P. rubesc groupsens are difficult of the P.to agardhiidistinguish/P. molecularly, rubescens complex differing (Figuremorphologically4). P. agardhii by its and P. rubescenscolor, oneare is difficult green and to the distinguish other is red molecularly, [9,35]. differing morphologically by its color, one is green and the otherP. agardhii is red growth, [9,35]. exposed to a range of salinities was evaluated using optical density 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 measurementsoutfall, sampling over point a 216 3 h— periodFigure 5b (Figure and the5). freshwater The growth reservoir, pattern sampling for all point the strains 4—Figure tested 5c). (IPMA2;The 3 andstrains 5) was grew similar well in regardless salinities between the origin 0 and of 10. the Growth strain (seainhibition pier, was sampling visible pointat salinities 1—Figure 20 and5 a;30 river outfall,for all sampling the strains point tested 3—Figure (Figure 5).5 b and the freshwater reservoir, sampling point 4—Figure5c). The strains grew well in salinities between 0 and 10. Growth inhibition was visible at salinities 20 and 30 for all the strains tested (Figure5).

Toxins 2017, 9, 391 5 of 13 Toxins 2017, 9, 12 5 of 13 Toxins 2017, 9, 12 5 of 13

Figure 4. FigurePhylogenetic 4. Phylogenetic tree of treeP. agardhii of P. agardhiistrains strains and closelyand closely related relatedPlanktothrix Planktothrixretrieved retrieved from from GenBank,FigureGenBank, inferred 4. Phylogenetic byinferred using tree by the using of MaximumP. the agardhii Maximum strains Likelihood Likelihood and closely method method related based based Planktothrix on on the the Kimura Kimuraretrieved 2-parameter 2 - fromparameter modelGenBank, of themodelrpo inferred ofC1 the gene rpo by C1 usingsequences. gene the sequences. Maximum The percentageThe Likelihood percentage bootstrap method bootst rap based values values on of theof1000 1000 Kimura replicates 2-parameter are are given given at each node. GenBank accession numbers are indicated after the species designation. The tree is at eachmodel node. of the GenBank rpoC1 gene accession sequences. numbers The percentage are indicated bootstrap after values the speciesof 1000 replicates designation. are given The at tree is unrooted and drawn to scale, with branch lengths measured in the number of substitutions per site. unrootedeach and node. drawn GenBank to scale, accession with numbers branch lengths are indicated measured after in the the species number designation. of substitutions The treeper is site. unrootedThe andanalysis drawn involve to scale,d 23 with nucleotide branch representativelengths measured sequences. in the number There were of substitutions a total of 452 per positions site. in The analysis involved 23 nucleotide representative sequences. There were a total of 452 positions The analysisthe final involve dataset.d 23The nucleotide grey box indicatesrepresentative the origin sequences. and strain There number were aof total the culturesof 452 positions obtained in in this in the ﬁnal dataset. The grey box indicates the origin and strain number of the cultures obtained the finalstudy. dataset. Accession The grey numbers: box indicates IPMA1 —theMG452723; origin and IPMA2 strain number—MG452724; of the IPMA3 cultures— obtainedMG45272 in5; IPMA4this — in thisstudy. study.MG452726; Accession Accession numbers:IPMA5 numbers:— MG452727;IPMA1 IPMA1—MG452723;—MG452723; IPMA6—MG452728. IPMA2—MG452724; IPMA2—MG452724; IPMA3—MG45272 IPMA3—MG452725;5; IPMA4— IPMA4—MG452726;MG452726; IPMA5 IPMA5—MG452727;—MG452727; IPMA6— IPMA6—MG452728.MG452728.

Figure 5. Growth curves of the Planktothrix agardhii strains exposed to several salinities. (a) P. agardhii FigureIPMA2 5. Growth collected curves at of the the sea Planktothrix pier; (b) agardhiiP. agardhii strains IPMA3 exposed collected to several at the salinities. river outfall; (a) P. ( agardhiic) P. agardhii Figure 5. Growth curves of the Planktothrix agardhii strains exposed to several salinities. (a) P. agardhii IPMA2IPMA5 collected collected at the at sea the pier;freshwater (b) P. reservoir.agardhii IPMA3 Range collectedof salinities at thetested: river (∆) outfall; 0; (○) 2.5; (c) (□)P. a5;gardhii (▲) 10; (●) IPMA2 collected at the sea pier; (b) P. agardhii IPMA3 collected at the river outfall; (c) P. agardhii IPMA5 IPMA520; collected (■) 30. at the freshwater reservoir. Range of salinities tested: (∆) 0; (○) 2.5; (□) 5; (▲) 10; (●) collected20; (■ at) 30. the freshwater reservoir. Range of salinities tested: (∆) 0; ( ) 2.5; () 5; (N) 10; ( ) 20; () 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. Toxins 2017, 9, 391 6 of 13

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 Toxins 2017, 9, 391 7 of 13 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 Figure6. 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 (Figure6). Between November and December of 2016, the samples were taken every week at three different locations at the site (Figure6). 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 (Figure6). The freshwater reservoir locates 5 km upstream in the river was sampled once to investigate the presence of cyanobacteria (Figure6). 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. Toxins 2017, 9, 12 8 of 13 Toxins 2017, 9, 391 8 of 13

FigureFigure 6.6. SchematicSchematic representationrepresentation atat scalescale ofof thethe samplingsampling sitesite atat thethe northwestnorthwest ofof thethe PortuguesePortuguese coast.coast. ((●1)1)—Monitoring—Monitoring stationstation ofof thethe PortuguesePortuguese NationalNational ShellﬁshShellfish MonitoringMonitoring SystemSystem atat thethe seasea pierpier (39(39°2◦211′000.4”00.4″ NN 99°2◦222′019.4”19.4″ W), ((●2)2)—Sampling—Sampling point 2, riverriver run-offrun-off atat thethe beachbeach betweenbetween thethe seasea andand thethe river river (39 (39°2◦211′003.0”03.0″ NN 99°2◦222′004.7”04.7″ W), ((●3)3)—Sampling—Sampling point 33 atat thethe riverriver outfalloutfall nearnear thethe beachbeach ◦ 0 ◦ 0 (39(39°221104.6”′04.6″ NN 99°2211′58.6”58.6″ W), (●4) ( 4)—Sampling—Sampling point 44 near the water damdam dischargeddischarged atat thethe freshwaterfreshwater ◦ 0 ◦ 0 reservoirreservoir located located 5 5 km km upstream upstream of of the the river river S. S. Domingos Domingos (39 (39°119 58.1”9′58.1 N″ N 9 9°119 9′07.5”07.5″ W), W), (− (−)—Sea)—Sea PiersPiers andand waterwater dam,dam, grey grey lines lines represent represent water water lines. lines.

4.2.4.2. FilamentFilament IsolationIsolation andand CultureCulture EstablishmentEstablishment

CyanobacteriaCyanobacteria ﬁlaments filaments were were isolated isolated with with a a micropipettemicropipette under under the the invertedinverted microscopemicroscope Leica Leica®® DMi8DMi8 from from water water fresh fresh samples samples concentrated concentrated with with a a 10 10µ µmm net. net. Cultures Cultures were were established established by by transferringtransferring the the isolated isolated ﬁlaments filaments into into Z8 Z8 culture culture medium medium [52 [52]] at at salinities salinities 35 35 and and 0. 0. Successful Successful ◦ − − culturescultures were were maintained maintained at at 19 19± ±1 1 C°C with with a a light light intensity intensity of of 5 µ5mol μmol·photons·photons·m·m2−·2s·s−11 and aa 12:1212:12 hh light:darklight: dark cycle. cycle.

4.3.4.3. CyanobacteriaCyanobacteria IdentificationIdentification andand QuantificationQuantification ForFor thethe quantificationquantification ofof cyanobacteria,cyanobacteria, thethe samplessamples werewere preservedpreserved withwith neutralizedneutralized Lugol’sLugol’s iodineiodine solution,solution, settledsettled downdown inin sedimentationsedimentation chamberschambers (25(25 mL)mL) andand countedcounted usingusing anan invertedinverted ® microscopemicroscope LeicaLeica® DMi8DMi8 atat 400400×× magnificationmagnification following the procedureprocedure ofof thethe UtermöhlUtermöhl techniquetechnique describeddescribed in in thethe EuropeanEuropean StandardStandard EN15204 EN15204 [ 53[53].]. TheThe numbernumber ofof cellscells inin oscillatorialesoscillatoriales cyanobacteriacyanobacteria waswas calculatedcalculated by by dividing dividing the the measured measured filament filament length length by by the the mean mean cell cell length. length. ® ForFor thethe morphologicalmorphological characterization,characterization, thethe cyanobacteriacyanobacteria werewere analyzedanalyzed usingusing aa Leica Leica® DMi8 DMi8 invertedinverted microscope microscope at at 1000 1000×× magnification.magnification. The The morphological morphological characters characters evaluated evaluated were: were: cell cell dimensionsdimensions (length(length andand width),width), filamentfilament colorcolor andand shape,shape, constrictionsconstrictions atat thethe cellcell wall,wall, presencepresence ofof sheath,sheath, shape shape of of apical apical cell, cell, presence/absence presence/absence of calyptra of calyptra and andnecredia necredia [35,46 [35,46,54,55].,54,55]. Photographs Photographs were ® ® takenwere with taken a Leica with a DFC550 Leica® DFC55 digital0 camera. digital The camera. cell measurements The cell measurements were performed were using performed Leica usingLasx software.Leica® Lasx At software. least 50 At measurements least 50 measurements were done were for calculating done for calculating the size of the size cells of and the filaments. cells and Differencesfilaments. Differences in cell dimensions in cell dimensions (length and (length width) and were width) tested were with tested a one-way with a Analysis one-way ofAnalysis Variance of (ANOVA)Variance (ANOVA) for p < 0.05. for p < 0.05. ForFor thethe molecularmolecular identification,identification, thethe DNADNA waswas extractedextracted fromfrom thethe culturescultures usingusing thethe DNeasyDNeasy ® PlantPlant Mini Mini Kit, Kit, Quiagen Quiagen®.. The The total total DNA DNA concentration concentration was was quantified quantified using the Qubit Qubit™™ ® FluorometricFluorometric Quantitation, Thermo Thermo Fisher Fisher Sci Scientificentific® . A. DNA A DNA fragment fragment of 608 of bp 608 within bp within the rpo theC1 0 0 rpogeneC1 genewas amplified was amplified with with the primersthe primers RPOF RPOF (5′-TGGTCAAGTGGTTGGAGA (5 -TGGTCAAGTGGTTGGAGA-3-3′) and) and RPOR RPOR (5′-

Toxins 2017, 9, 391 9 of 13

(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) H2O + 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 Toxins 2017, 9, 391 10 of 13

(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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