Are We Underestimating Benthic Cyanotoxins? Extensive Sampling Results from Spain

Article Are We Underestimating Benthic Cyanotoxins? Extensive Sampling Results from Spain

Enrique A. Cantoral Uriza 1, Antonia D. Asencio 2 and Marina Aboal 3,*

1 Unidad Multidisciplinaria de Docencia e Investigación (UMDI), Facultad de Ciencias, Universidad Nacional Autónoma de México, Campus Juriquilla, C.P. Querétaro 76230, Mexico; [email protected] 2 Departamento de Biología Aplicada (Botánica), Facultad de Ciencias Experimentales, Universidad Miguel Hernández, Campus de Elche, E-03202 Alicante, Spain; [email protected] 3 Laboratorio de Algología, Departamento de Biología Vegetal, Facultad de Biología, Universidad de Murcia, Campus de Espinardo, E-30100 Murcia, Spain * Correspondence: [email protected]; Tel.: +34-868-884-940

Academic Editor: Luis M. Botana Received: 29 August 2017; Accepted: 23 November 2017; Published: 28 November 2017

Abstract: Microcystins (MCs) are potent hepatotoxins, and their presence in water bodies poses a threat to wildlife and human populations. Most of the available information refers to plankton, and much less is known about microcystins in other habitats. To broaden our understanding of the presence and environmental distribution of this group of toxins, we conducted extensive sampling throughout Spain, under a range of conditions and in distinct aquatic and terrestrial habitats. More than half of the tested strains were toxic; concentrations of the hepatotoxin were low compared with planktic communities, and the number of toxic variants identified in each sample of the Spanish strains ranged from 1–3. The presence of microcystins LF and LY (MC-LF and MC-LY) in the tested samples was significant, and ranged from 21.4% to 100% of the total microcystins per strain. These strains were only detected in cyanobacteria Oscillatoriales and Nostocales. We can report, for the first time, seven new species of microcystin producers in high mountain rivers and chasmoendolithic communities. This is the first report of these species in Geitlerinema and the confirmation of Anatoxin-a in Phormidium uncinatum. Our findings show that microcystins are widespread in all habitat types, including both aerophytic and endolithic peat bogs and that it is necessary to identify all the variants of microcystins in aquatic bodies as the commonest toxins sometimes represent a very low proportion of the total.

Keywords: Anatoxin-a; aquatic and aerophytic habitats; cyanobacteria; microcystins; MC-LF; MC-LR; MC-LY; MC-RR; MC-YR; Spain

1. Introduction Benthic cyanobacteria that produce microcystins (MCs) were ﬁrst detected and characterised in communities in alpine lakes [1]. Since then, their presence in benthic communities in Spain [2,3], California [4], Egypt [5], Morocco [6,7], New Zealand [8], and other countries [9] has been reported. More recently, the presence of benthic cyanobacteria has been demonstrated in 30% of fordable rivers in California, and the number of potentially toxic genera has increased [10]. Foliaceus lichens of the genera Nephroma, Sticta, Lobaria, and Peltigera produce microcystins, including some new variants [11,12]. All these lichens contain the cyanobacteria Nostoc as a phycosymbiont. Some Nostocs found outside of lichens can also produce MCs [13], and their capacity to do so has been connected to stress conditions. However, no toxins have been detected in Nostoc

Toxins 2017, 9, 385; doi:10.3390/toxins9120385 www.mdpi.com/journal/toxins Toxins 2017, 9, 385 2 of 13 Toxins 2017, 9, 385 2 of 13 communecommune var.var. flagelliformeflagelliforme BornetBornet andand Flahault,Flahault, whichwhich is commoncommon in desert regions, andand is frequentlyfrequently consumedconsumed asas foodfood inin countriescountries suchsuch asas ChinaChina [[14].14]. VanVan ApeldoornApeldoorn etet al.al. [[15]15] reportedreported aa totaltotal ofof 4040 toxictoxic generagenera ofof benthicbenthic cyanobacteria,cyanobacteria, butbut thethe numbernumber hashas increased increased since since that that report report [16 ].[16]. However, However, most most efforts efforts to study to thesestudy bacteria these bacteria have focused have onfocused studying on studying planktic communities,planktic communities, lakes, and lakes, reservoirs. and reservoirs. AsideAside from their toxicity toxicity to to humans, humans, the the actual actual role role of ofmicrocystins microcystins in the in theenvironment environment is not is notcompletely completely understood, understood, in either in either planktic planktic or benthic or benthic algal communities algal communities [17]. There [17]. is There a large is body a large of bodyliterature of literature about the about toxic theeffects toxic of effectsmicrocystins of microcystins on angiosperms, on angiosperms, fungi, bacteria, fungi, animals, bacteria, and animals, algae, andbut much algae, more but much has to more be done has to in be order done to in elucidate order to elucidatethis complex this complexissue. These issue. compound These compoundss interfere interferewith biological with biologicalprocesses that processes are necessary that are for necessary the survival for of the organisms. survival They of organisms. are potent inhibitors They are potentof certain inhibitors essential of certainprotein essential phosphatases protein (PP1 phosphatases and PP2) (PP1 for andall eukaryotes PP2) for all eukaryotes[18,19], and [ 18affect,19], andmicroalgae affect microalgae growth [20 growth]. However, [20]. However,the promotion the promotion or suppression or suppression of photosynthesis of photosynthesis has also been has alsoobserved been in observed several types in several of bent typeshic algae, of benthic including algae, diatoms including [21], diatoms which are [21 ],the which food arepreferred the food by preferredmany herbivores. by many herbivores.Microcystins Microcystins may also mayinhibit also the inhibit proteases the proteases employed employed in the in thedigestion digestion of ofheterotrophs heterotrophs [22 [],22 ],and and evidence evidence suggests suggests that that their their presence presence in in water water may causecause histologicalhistological degenerationdegeneration in macroinvertebrates,macroinvertebrates, although the survival ratios are relatively high belowbelow 55 ppbppb ofof MC-LRMC-LR andand MC-LWMC-LW [23 [23].]. Toxins Toxins may may also also accumulate accumulate in fishin fish tissue, tissue, and and pathological pathological effects effects have beenhave studiedbeen studied extensively extensively [24,25 [].24,25]. InIn recentrecent years,years, thethe frequencyfrequency ofof whatwhat have,have, untiluntil now,now, beenbeen consideredconsidered rarerare variantsvariants ofof microcystinsmicrocystins (Figure1 1)) has has beenbeen confirmedconfirmed inin severalseveral countries,countries, usingusing aa varietyvariety ofof methodsmethods [[2626–30]. MolecularMolecular toolstools havehave advancedadvanced considerablyconsiderably in thethe lastlast fewfew years,years, butbut quantitativequantitative methodsmethods areare stillstill notnot adequateadequate forfor detectingdetecting toxicity.toxicity. InIn mostmost cases,cases, the relationship betweenbetween gene expression andand toxintoxin productionproduction isis notnot yetyet sufficientlysufficiently understoodunderstood [[3131].]. MostMost environmentalenvironmental agenciesagencies recommendrecommend usingusing differentdifferent complementarycomplementary methodologiesmethodologies toto detectdetect andand quantifyquantify microcystinsmicrocystins [[1818].].

(a) (b)

Figure 1. Structure of microcystins (a) LF (MC-LF) and (b) LY (MC-LY). Figure 1. Structure of microcystins (a) LF (MC-LF) and (b) LY (MC-LY).

Benthic toxicity was initially related to Oscillatoriales and Anatoxin-a [32,33]. However, it seems that theseBenthic toxins toxicity are not was as initially prevalent related as originally to Oscillatoriales thought, and at least Anatoxin-a in some [ 32countries,33]. However, or geographical it seems thatareas these [34]. toxins Some arespecies not asof prevalentcyanobacteria as originally may produ thought,ce both at microcystins least in some and countries Anatoxin or- geographicala at the same areastime [35 [34,].36 Some], although species most of cyanobacteria studies focus may on only produce one type both of microcystins toxin at a time. and Anatoxin-aAnatoxin-a at(ANT the same-a) is timea neurotoxic [35,36], althoughalkaloid, mostwhich studies is an agonist focus onat neuronal only one typenicotinic of toxin acetylcholine at a time. receptors Anatoxin-a [37 (ANT-a),38]. is a neurotoxicIn this study, alkaloid, extensive whichis sampling an agonist was at neuronalperformed nicotinic throughout acetylcholine Spain, along receptors an environmental [37,38]. gradientIn this that study, focused extensive especially sampling on benthic was performed aquatic systems, throughout including Spain, aerophytic along an environmentaland endolithic gradienthabitats, in that an focusedattempt especiallyto broaden on the benthic perception aquatic of the systems, environmental including presenc aerophytice of cyanotoxins. and endolithic habitats, in an attempt to broaden the perception of the environmental presence of cyanotoxins. 2. Results 2. Results Different habitats were sampled throughout Spain, from both Atlantic and Mediterranean Different habitats were sampled throughout Spain, from both Atlantic and Mediterranean regions. regions. These habitats were high mountain streams or creeks of the Pyrenees and Sierra Nevada These habitats were high mountain streams or creeks of the Pyrenees and Sierra Nevada mountains, mountains, peat bogs from the Pyrenees, middle mountain streams from northeast and southeast peat bogs from the Pyrenees, middle mountain streams from northeast and southeast Spain and the Spain and the Canary Islands, saline and freshwater lagoons from northwest Spain, saline and freshwater springs from Mediterranean marshes, and a cave and building located in the southeast (Figure 2, Table 1).

Toxins 2017, 9, 385 3 of 13

ToxinsCanary 2017 Islands,, 9, 385 saline and freshwater lagoons from northwest Spain, saline and freshwater springs3 of 13 from Mediterranean marshes, and a cave and building located in the southeast (Figure2, Table1). Most areas were granitic, but others were were calcareous. calcareous. Most Most of of the the samples samples contained contained freshwater, freshwater, but some had saline water. The range of altitudes varied from 5 m to 2500 m above sea level. Rainfall ranged between <200 mm mm and and 1843 mm. The The mean mean air air temperature temperature was was between between 5 5 and and 22.5 22.5 °C.◦C. Conductivity varied between 61.6 and 2850 µμScm −11, ,with with pH pH between between 7 7 and and 8.5 8.5 (Figure (Figure 3).3).

Figure 2. MapMap of of the the study study area area with with sampled sampled localities localities.. Grey Grey areas areas indicate indicate mountain mountain systems whose altitude isis higherhigher thanthan 1500 1500 m m above above sea sea level level (asl). (asl). The The insert insert represents represents the Canarythe Canary Islands. Islands. Scale Scale bars barsrepresent represent 100 km. 100 km.

Table 1.1. ListList of of the sampled localities, indicating habitat type, lithology, altitudes, geographic coordinates, mean rainfall, and mean temperature.

Altitude Rainfall Mean Temperature Localities Habitat Lithology Altitude Coordinates Rainfall Mean Temperature Localities Habitat Lithology (m) Coordinates (mm) (°C) (m) (mm) (◦C) Prado Redondo, Prado Redondo, 37°0537◦05′023.023.0”″ N Sierra Nevada, CreekCreek GraniticGranitic 21002100 13221322 7.8 Sierra Nevada, Granada 3°243◦24′056.856.8”″ W GranadaSan Juan, Sierra 37◦05016.7” N Creek Granitic 2500 1322 7.8 SanNevada, Juan, Sierra Granada 37°053◦220′18.5”16.7″W N Creek Granitic 2500 1322 7.8 Nevada,Poqueira, Granada Sierra 3°2236◦59′18.5026.8”″ W N Creek Granitic 1540 ◦ 0 935 11.6 Poqueira,Nevada, Sierra Granada 36°593 21 ′00.2”26.8″W N Vall de Mulleres, Creek Granitic 1540 42◦37040.6” N 935 11.6 Nevada, Granada Peat bog Granitic 1609 3°21′00.2″ W 1843 8.5 Vielha, Lleida 0◦45034.9” E Vall deRiu Mulleres, Escrita, 42°3742◦34′40.6038”″ N N PeatStream bog Granitic Granitic 1700 1609 18431100 8.5 5.0 Vielha, LleidaLleida 0°450◦56′34.9052”″E E Fonts Lac S. Maurici, 4242°34◦32028.9”′38″ N N Riu Escrita, Lleida StreamSprings Granitic Granitic 1910 1700 ◦ 0 11001100 5.0 Lleida 90°5601 21.6”′52″ E W Riu Llebreta, 42◦34038” N Fonts Lac S. Maurici, Stream Granitic 1999 42°32′28.9″ N 1100 5.0 Lleida Springs Granitic 1910 0◦56052” E 1100 5.0 LleidaRiu Ter, 9°0142◦19′21.6059”″ NW River Granitic 1067 933 9.8 Villalonga, Girona 42°342◦180′47”38″ EN Riu Llebreta, Lleida Stream Granitic 1999 1100 5.0 Lagoa Carregal, 420°56◦330′002”52″ E N Lagoon Granitic 5 ◦ 0 933 9.8 RiuCorrubedo, Ter, Villalonga, A Coruña 42°199 02 00”′59″ WN Lagoa Vixán, River Granitic 1067 42◦330002”N 933 9.8 Girona Lagoon Granitic 5 2°18′47″ E 1014 14.8 Corrubedo, A Coruña 9◦02000”W LagoaGuayadeque, Carregal, 27◦56000.7” N Stream Volcanic 1273 42°33′002″ N 175 22.5 Corrubedo,Gran Canaria A Lagoon Granitic 5 15◦28057.7” W 933 9.8 9°02′00″ W PalacioCoruña Guevara, 37◦40029.82” N Building Marble 353 ◦ 0 232 17.6 LagoaLorca, Vixán, Murcia 1 41 51.54” W Río Alhárabe, 42°3338◦12′000250”″ N Corrubedo, A LagoonStream CalcareousGranitic 900 5 1014522 14.8 15.7 Moratalla, Murcia 19°02◦570′46”00″W W CoruñaAzud de Ojós, 38◦80 52” N Reservoir Calcareous 132 306 17.4 Guayadeque,Ojós, Murcia Gran 27°561◦20′000.732” W″ N Stream Volcanic 1273 175 22.5 Canaria 15°28′57.7″ W Palacio Guevara, 37°40′29.82″ N Building Marble 353 232 17.6 Lorca, Murcia 1°41′51.54″ W Río Alhárabe, 38°12′50″ N Stream Calcareous 900 522 15.7 Moratalla, Murcia 1°57′46″ W

Toxins 20172017,,9 9,, 385385 4 of 13 Toxins 2017, 9, 385 4 of 13

Azud de Ojós, Ojós, 38°8′ 52″ N Azud de Ojós, Ojós, Reservoir Calcareous 132 38°8′ 52″ N 306 17.4 Murcia Reservoir Calcareous Table 132 1. Cont. 1°20′32″ W 306 17.4 Murcia 1°20′32″ W Cueva de los Grajos, 38°14′21″ N Cueva de los Grajos, Cave Calcareous 250 38°14′21″ N 307 17.2 Cieza, Murcia Cave Calcareous Altitude250 1°25′08″ W Rainfall307 Mean Temperature17.2 Cieza,Localities Murcia Habitat Lithology Coordinates1°25′08″ W ◦ Río Chícamo, (m) 38º24′97″ N (mm) ( C) Río Chícamo, Stream Calcareous 290 38º24◦ 0′97″ N <200 10.0 Abanilla,Cueva de Murcia los Grajos, Stream Calcareous 290 381º0014′1821”″ W N <200 10.0 Abanilla, Murcia Cave Calcareous 250 1º00◦ 0′18″ W 307 17.2 MarjalCieza, Almenara, Murcia Freshwater 39°441 25′54.108” W″ N MarjalR íAlmenara,o Chícamo, Freshwater Calcareous 26 39°4438º24′54.1097”″ N N 467 17.5 Almenara, Castellón SpringStream CalcareousCalcareous 29026 0°11′17.30 ″ W 467<200 10.017.5 Almenara,Abanilla, Castellón Murcia Spring 0°111º00′17.318”″ W W MarjalMarjal de Almenara,Pego- FreshwaterSaline 38°39◦ 44520′54.1”08.2″ NN Marjal de Pego- Saline CalcareousCalcareous 26 5 38° 52′08.2″ N 637467 17.517.8 Oliva,Almenara, Valencia Castell ón SpringSpring Calcareous 5 0°020◦11′57.92017.3”″W W 637 17.8 Oliva, Valencia Spring 0°02◦ ′57.920 ″ W RíoMarjal Amadorio, de Pego-Oliva, Saline 3838°3052 08.2”′19″ N N Río Amadorio, Stream CalcareousCalcareous 5 32 38°30◦ 0 ′19″ N 300637 17.818.0 Vilajoyosa,Valencia Alicante StreamSpring Calcareous 32 00°1302 57.92”′58″ W W 300 18.0 Vilajoyosa,Río Amadorio, Alicante 380°13◦30′58019”″ W N Río Algar, Callosa, Stream Calcareous 32 38°39◦ ′33.670 ″ N 300 18.0 RíoVilajoyosa, Algar, Callosa, Alicante Stream Calcareous 247 38°390 13′33.6758” W″ N 519 16.9 AlicanteRío Algar, Stream Calcareous 247 380°5◦39′45.58033.67”″ W N 519 16.9 Alicante Stream Calcareous 247 0°5′45.58″ W 519 16.9 Callosa, Alicante 0◦5045.58” W

Figure 3. Ranges of the main ecological variables in the sampled habitats. Figure 3. Ranges of the main ecological variables in the sampled habitats.

Toxic species were detected in all the sampled habitats, including both aerophytic and endolithic Toxic speciesspecies werewere detecteddetected inin all the sampled habitats,habitats, including both aerophytic and endolithic species (Figure 4). The proportion of toxic strains varied between 28% and 100% of all the strains species (Figure4 4).). TheThe proportionproportion ofof toxictoxic strainsstrains variedvaried betweenbetween 28%28% andand 100%100% ofof allall thethe strainsstrains studied. Table 2 below lists all the isolated and extracted species. studied. Table2 2 below below lists lists all all the the isolated isolated and and extracted extracted species. species.

8 8

7 7

6 6

5 Non-toxic 5 Non-toxic

4 Toxic 4 Toxic 3 3 Number of strains of Number Number of strains of Number 2 2

1 1

0 0 Endolithic Lagoons River/ Streams Cave Springs Peat-bog Endolithic Lagoons River/ Streams Cave Springs Peat-bog

Figure 4. Distribution of the toxic strains in each habitat. Figure 4. Distribution of the toxic strains in each habitat.

Toxins 2017, 9, 385 5 of 13

Table 2. Isolated and extracted strains. Taxonomic order, locality and habitat are listed. Toxicity is Toxinshighlighted 2017, 9, 385 in bold (dark grey). 5 of 13

Table 2. IsolatedTaxa and extracted strains. Order Taxonomic order, Habitat locality and habitat are listed. Locality Toxicity is highlightedHyella balani inLehman bold (dark grey). Chroococcales Euendolithic, spring Marjal Oliva-Pego, Valencia

Pseudocapsa dubia ErcegovicTaxa Chroococcales Order Chasmoendolithic Habitat Palacio Guevara, Locality Lorca, Murcia Gloeotrichia natans (Hedwig) Euendolithic, Hyella balani Lehman Nostocales Chroococcales Epiphytic, lagoon LagoaMarjal Vixá n,Oliva-Pego, Corrubedo, Valencia A Coruña Rabenhorst ex Bornet et Flahault spring Nostochopsis lobata Palacio Guevara, Lorca, Pseudocapsa dubia Ercegovic NostocalesChroococcales Epiphytic, Chasmoendolithic lagoon Lagoa Vixán, Corrubedo, A Coruña Wood ex Bornet & Flahault Murcia GloeotrichiaRivularia natans biasolettiana (Hedwig) Rabenhorst ex RíoLagoa Alhárabe, Vixán, Moratalla, Corrubedo, Murcia A NostocalesNostocales Epilithic,Epiphytic, stream lagoon (Meneghini exBornet Bornet et & Flahault Flahault Río Chícamo,Coruña Abanilla, Murcia Scytonema drilosiphon Lagoa Vixán, Corrubedo, A Nostochopsis lobata Wood ex Bornet & FlahaultNostocales Nostocales Epilithic, Epiphytic, cave lagoon Cueva Grajos, Cieza, Murcia Elenkin & V. I. Polyansky Coruña Geitlerinema splendidum Río Alhárabe, Moratalla, Rivularia biasolettiana (Meneghini ex BornetOscillatoriales & Epilithic, spring Ullal Almenara, Castellón (Greville ex Gomont) Anagnostidis Nostocales Epilithic, stream Murcia Flahault Homoeothrix juliana Río Chícamo, Abanilla, Murcia Oscillatoriales Epilithic, stream Río Amadorio, Alicante (BornetScytonema & Flahault drilosiphon ex Gomont) Elenkin Kirchner & V. I. Polyansky Nostocales Epilithic, cave Cueva Grajos, Cieza, Murcia GeitlerinemaLeptolyngbya splendidum subtilis (Greville ex Gomont) OscillatorialesOscillatoriales Epilithic, Epilithic, spring spring Ullal Almenara,Almenara, Castell Castellónón (West) AnagnostidisAnagnostidis LeptolygnbyaHomoeothrix truncata juliana(Lemmermann) (Bornet & Flahault ex OscillatorialesOscillatoriales Epilithic, Epilithic, stream stream RRíoío Amadorio,Amadorio, Alicante Alicante AnagnostidisGomont) & Kom Kirchnerárek Leptolyngbya subtilis (West) Anagnostidis Oscillatoriales Epilithic, spring Ullal Almenara, Castellón Oscillatoria margaritifera Leptolygnbya truncata (Lemmermann)Oscillatoriales Epilithic, spring Ullal Almenara, Castellón Kützing ex Gomont Oscillatoriales Epilithic, stream Río Amadorio, Alicante Anagnostidis & Komárek Oscillatoria sancta Kützing ex Gomont Oscillatoriales Epilithic, stream Río Ter, Vilallonga, Lérida Oscillatoria margaritifera Kützing ex Gomont Oscillatoriales Epilithic, spring Ullal Almenara, Castellón Geitlerinema carotinum Oscillatoria sancta Kützing ex GomontOscillatoriales Oscillatoriales Epipelic, Epilithi reservoirc, stream Río Azud Ter, Oj Vilallonga,ós, Ojós, Murcia Lérida (Geitler) Anagnostidis Geitlerinema carotinum (Geitler) Anagnostidis Oscillatoriales Epipelic, reservoir Azud Ojós, Ojós, Murcia Phormidium autumnale (Agardh) Phormidium autumnale (Agardh) TrevisanOscillatoriales ex Epilithic, stream Río AlhRíoá Alhárabe,rabe, Moratalla, Moratalla, Murcia Trevisan ex Gomont Oscillatoriales Epilithic, stream Gomont Murcia Pseudanabaena frigida (Fritsch) Pseudanabaena frigida (Fritsch) AnagnostidisOscillatoriales Oscillatoriales Epipelic, Epipe peatlic, bog peat bog Vall dede Molleres,Molleres, Viella, Viella, L éLéridarida Anagnostidis Río Alhárabe, Moratalla, Schizothrix rivularianum Voronichin Oscillatoriales Epipelic, stream Schizothrix rivularianum Voronichin Oscillatoriales Epipelic, stream Río Alhárabe,Murcia Moratalla, Murcia Phormidium uncinatum Barranco Guayadeque, Gran Phormidium uncinatum Gomont ex GomontOscillatoriales Oscillatoriales Epipelic, Epipelic, stream stream Barranco Guayadeque, Gran Canaria Gomont ex Gomont Canaria

Eleven of of the the 24 24 analysed analysed strains strains contained contained microc microcystin.ystin. The Theproportion proportion of toxic of toxicstrains strains to non- to toxicnon-toxic strains strains was wassimilar similar for fordifferent different taxonomic taxonomic orders orders of ofcyanobacteria, cyanobacteria, and and varied varied between between 50% 50% and 70%: 50% in Chroococcales, 60% in Oscillatoriales and 67% in Nostocales (Figure5 5).).

8 Non-toxic 6 Toxic

Strain number 4

0 Chroococcales Oscillatoriales Nostocales

FigureFigure 5. 5. DistributionDistribution of of the the toxic toxic strains strains in currently recognised taxonomic orders.

The number of microcystin variants detected per strain varied between one and three (Table 3). MC-RR was the most common microcystin in all of the studied strains (39%), followed by MC-LY (28%) and MC-LF (17%).The least common microcystins were MC-LR (11%) and MC-YR (5%) (Figure 6).

Toxins 2017, 9, 385 6 of 13

The number of microcystin variants detected per strain varied between one and three (Table3). MC-RR was the most common microcystin in all of the studied strains (39%), followed by MC-LY (28%) and MC-LF (17%).The least common microcystins were MC-LR (11%) and MC-YR (5%) (Figure6).

Toxins 2017Table, 9, 385 3. Types of toxins present in the different isolated strains (nd = not detected). 6 of 13

Toxins 2017, 9, 385 6 of 13 TaxaTable 3. Types of toxins present Locality in the different isolated Microcystinsstrains (nd = not detected). Anatoxins

Gloeotrichia natansTableTaxa 3. Types of toxinsCorrubedo, presentLocality in Galicia the different isolated MC-LF, strains Microcystins (nd MC-RR = not detected). Anatoxins nd GeitlerinemaGloeotrichia carotinum natans OjCorrubedo,ós, Murcia Galicia MC-LF, MC-LY MC-RR nd ANT-a Taxa Locality Microcystins Anatoxins GeitlerinemaGeitlerinema splendidum carotinum Almenara,Ojós, Valencia Murcia MC-LF, MC-LY MC-RR ANT-a ANT-a Gloeotrichia natans Corrubedo, Galicia MC-LF, MC-RR nd Nostoc cf.Geitlerinemacommune splendidumSierra Nevada,Almenara, Granada Valencia MC-LF,MC-LF, MC-RR MC-LY ANT-a nd Geitlerinema carotinum Ojós, Murcia MC-LY ANT-a Oscillatoria margaritiferaNostoc cf. commune Almenara,Sierra Nevada, Valencia Granada MC-LF, MC-LF, MC-LY, MC-LY MC-RR nd nd OscillatoriaGeitlerinema margaritifera splendidum Almenara, Valencia MC-LF,MC-LF, MC-LY, MC-RR MC-RR ANT-a nd Phormidium autumnale Moratalla, Murcia MC-LR nd PhormidiumNostoc cf. commune autumnale SierraMoratalla, Nevada, Murcia Granada MC-LF, MC-LR MC-LY nd Phormidium uncinatum Guayadeque, Gran Canaria MC-LF, MC-LY ANT-a OscillatoriaPhormidium margaritifera uncinatum Guayadeque,Almenara, Gran Valencia Canaria MC-LF, MC-LF, MC-LY, MC-LY MC-RR ANT-a nd Phormidium sp. Sierra Nevada, Granada MC-LF, MC-LY nd PhormidiumPhormidium autumnale sp. SierraMoratalla, Nevada, Murcia Granada MC-LF, MC-LR MC-LY nd Pseudoanabaena frigida Vall de Mulleres, Lérida MC-RR nd PhormidiumPseudoanabaena uncinatum frigida Guayadeque,Vall de Mulleres, Gran LéridaCanaria MC-LF, MC-RR MC-LY ANT-a nd PseudocapsaPseudocapsaPhormidium dubia dubia sp. Palacio PalacioSierra de Guevara, deNevada, Guevara, MurciaGranada Murcia MC-RR, MC-RR,MC-LF, MC-LYMC-YR MC-YR nd nd Rivularia biasolettianaPseudoanabaenaRivularia biasolettiana frigida Río ChVallRíoícamo, de Chícamo, Mulleres, Murcia Murcia Lérida MC-LR, MC-LR, MC-RRMC-LY, MC-RR MC-RR nd nd SchizothrixSchizothrix rivularianumPseudocapsa rivularianum dubia R íoPalacio AlhRíoá rabe, Alhárabe,de Guevara, Murcia Murcia Murcia MC-LY, MC-RR,MC-LY, MC-YRMC-RR nd nd ScytonemaRivulariaScytonema drilosiphon biasolettiana drilosiphonCueva Cueva deRío los deChícamo, Grajos, los Grajos, Murcia Murcia MC-LR, MC-LYMC-LY, MC-RR nd nd Schizothrix rivularianum Río Alhárabe, Murcia MC-LY, MC-RR nd Scytonema drilosiphon Cueva de los Grajos, Murcia MC-LY nd

MC-LF 17% MC-RR 39% MC-LF MC-RR MC-LY 17% 39% 28% MC-LY MC-LR 28% 11% MC-LR 11% MC-YR 5% MC-YR 5% FigureFigure 6. Frequency 6. Frequency of of each each ofof the detected detected microcystin microcystin variants. variants.

MC-LR reached Figurethe highest 6. Frequency concentration of each of per the dry detected weight microcystin (1.87 μg/g), variants. followed by MC-LY (1.72 MC-LRμg/g), while reached MC-RR the (1.36 highest μg/g), concentrationMC-YR (0.78 μg/g), per and dry MC-LF weight (0.37 (1.87 μg/g)µ g/g),were less followed concentrated. by MC-LY (1.72 µAnatoxin-ag/g),MC-LR while reached reached MC-RR the the highest highest (1.36 µ concentrationconcentrationg/g), MC-YR ofper all (0.78dry the weight toxinsµg/g), (1.87(2.63 and μμg/g),g/g), MC-LF followedand was (0.37 bypresent MC-LYµg/g) in three(1.72 were less μg/g), while MC-RR (1.36 μg/g), MC-YR (0.78 μg/g), and MC-LF (0.37 μg/g) were less concentrated. concentrated.strains from Anatoxin-a Oscillatoriales reached (Table the 3, Figure highest 7). concentration of all the toxins (2.63 µg/g), and was Anatoxin-a reached the highest concentration of all the toxins (2.63 μg/g), and was present in three presentstrains in three from strains Oscillatoriales from Oscillatoriales (Table 3, Figure (Table 7). 3, Figure7). 3

2.5 3 2 2.5 1.5

g g-1 2 μ 1 1.5 g g-1 μ 0.5 1 0 0.5

Figure 7. Mean and range of the concentrations of each of the identified variants. Figure 7. Mean and range of the concentrations of each of the identified variants. Figure 7. Mean and range of the concentrations of each of the identiﬁed variants.

Toxins 2017, 9, 385 7 of 13

3. Discussion Toxins 2017, 9, 385 7 of 13 The ability to synthesise microcystins appeared very early in the evolution of cyanobacteria [39], but has been lost in certain phylogenetic branches over time. Most toxin screening efforts have been 3.made Discussion in freshwater aquatic habitats, mainly lagoons and reservoirs. Running water, salt water, high mountains, peat bogs and aerophytic habitats (except for lichens) have been very rarely sampled The ability to synthesise microcystins appeared very early in the evolution of cyanobacteria [39], [9,40]. but has been lost in certain phylogenetic branches over time. Most toxin screening efforts have been In this work, microcystins were detected for the first time in seven species, found in high made in freshwater aquatic habitats, mainly lagoons and reservoirs. Running water, salt water, mountain streams, caves and endolithic habitats: Pseudocapsa dubia, Gloeotrichia natans, Scytonema high mountains, peat bogs and aerophytic habitats (except for lichens) have been very rarely drilosiphon, Geitlerinema carotinum, Oscillatoria margaritifera, Pseudanabaena frigida, and Schizothrix sampled [9,40]. rivularianum (Figure 8). Anatoxin-a production was confirmed in Phormidium uncinatum, and was In this work, microcystins were detected for the first time in seven species, found in high mountain detected for the first time in Geitlerinema carotinum and G. splendidum. streams, caves and endolithic habitats: Pseudocapsa dubia, Gloeotrichia natans, Scytonema drilosiphon, The focus on freshwater environments is justified by the potential human health risks that the Geitlerinema carotinum, Oscillatoria margaritifera, Pseudanabaena frigida, and Schizothrix rivularianum presence of microcystins may present for local populations [41]. However, since neither the factors (Figure8). Anatoxin-a production was confirmed in Phormidium uncinatum, and was detected for the that trigger the synthesis of these compounds nor their ecological function are known, significant first time in Geitlerinema carotinum and G. splendidum. further research must be conducted in all habitats and phylogenetic branches [17,42].

Figure 8. Images of the toxic strains that were isolated during this study: 1. Pseudocapsa dubia. 2. Figure 8. Images of the toxic strains that were isolated during this study: 1. Pseudocapsa dubia. Scytonema drilosiphon. 3. Gloeotrichia natans. 4. Pseudanabaena frigida. 5. Phormidium uncinatum. 6. 2. Scytonema drilosiphon. 3. Gloeotrichia natans. 4. Pseudanabaena frigida. 5. Phormidium uncinatum. Oscillatoria margaritifera. 7. Phormidium sp. 8. Geitlerinema splendidum. 9. Phormidium favosum. 10. 6. Oscillatoria margaritifera. 7. Phormidium sp. 8. Geitlerinema splendidum. 9. Phormidium favosum. Geitlerinema carotinum. 11. Leptolyngbya rivularianum. The scale bar represents 20 μm. 10. Geitlerinema carotinum. 11. Leptolyngbya rivularianum. The scale bar represents 20 µm.

Toxins 2017, 9, 385 8 of 13

The focus on freshwater environments is justified by the potential human health risks that the presence of microcystins may present for local populations [41]. However, since neither the factors that trigger the synthesis of these compounds nor their ecological function are known, significant further research must be conducted in all habitats and phylogenetic branches [17,42]. During the present study, we detected toxic strains in all of the studied habitats, which implies the need for more extensive reviews of the data from both the taxonomic and ecologic points of view. Our data showed that the frequency of microcystin producers was higher in Nostocales, although we found a similar proportion in Chroococales and Oscillatoriales. These data are similar to those found in other studies published elsewhere [15]. In most strains, more than one microcystin congener was detected. Interestingly, some of the most abundant congeners were those not considered to be particularly common. In particular, MC-LF and MC-LY were detected fairly frequently, and at relatively high concentrations. Even though these supposedly rare variants have probably always been present, they have only recently been widely reported [10,27] perhaps due to improved extraction and detection methods. These cogeners should be included in monitoring programmes, especially taking into account that the toxic capacity of the more hydrophobic variants such as MC-LF and MC-LY is sometimes much higher than MC-LR [22], which is typically used for risks assessments. The use of solid-phase extraction (SPE), coupled with immunoaffinity columns (IACs), improves the efficiency of extraction and clean-up of microcystins, especially in complex matrices [15]. The effects of exposure to MC-LF and MC-LW on the degradation, growth and proliferation of human cells is much higher than on the cells exposed to MC-LR, but phosphatase inhibition is much lower [22]. The toxicity of microcystin variants is strongly associated with the hydrophobicity of their structural amino acids. MC-LF and MC-LW therefore induce more pronounced cytotoxic effects on Caco-2 cells compared with MC-LR, and are taken up much more rapidly, or with a higher affinity, by human embryonic kidney cells and human primary hepatocytes [43,44]. Interest in algae as a food supplement has considerably increased of late. A new market has opened up for these products, which has the potential for vast economic benefits. Aquaculture has also undergone similar or even stronger growth, with the global yearly production of thousands of tons of fish and shellfish. However, no clear regulation or legislation exists for these products in most countries. Several authors have reported the presence of microcystins in food supplement products [45], as well as in fishponds and fish [26], but very little information and very few studied effects on consumer health are known. Increasing efforts to improve research in this field are needed by companies that prepare these products, not to mention by environmental agencies, countries, and governments, in order to create international regulations for blue-green algae supplements (BGAS), given their strong potential impact on populations’ health. Above all, international regulation is necessary, because BGAS is now available and easily sold on the internet, which increases possible risks worldwide. Different types of quantification and detection kits are available, but the effectiveness for these “rare” variants is very low in some cases [38]. The use of several complementary identification or quantification methods should be compulsory, and the utilisation of biological tests based on phosphatase inhibition as the first step should be emphasised. These functional toxicity assays permit the detection of toxicants that may escape standard analytical detection [46–48]. Ward et al. [18] have already suggested that most strains usually produce more than one variant, and that the concentrations of some variants might drop below the level of detection for chemical methods, and could thus escape analytical control. Puddick et al. [49] provides an assessment of the microcystin congener diversity produced amongst cyanobacterial strains. Climate warming will probably increase problems with cyanotoxins [42,43]. Thus, much work has to be done, in order to know how to prevent toxic events, how to avoid the conditions that promote their synthesis, and how to control cyanobacteria populations [50]. This is particularly important, as more hydrophobic variants will increase their predominance, given the current and future predicted atmospheric and environmental conditions [50–52]. Toxins 2017, 9, 385 9 of 13

Our data show that microcystins are widespread, in a variety of continental habitats, including high mountain streams, peat bogs, and aerophytic and endolithic communities. The results from our study demonstrate how important it is that screening for variants be intensiﬁed and globally managed, in order to prevent sanitary and environmental problems. Cyanotoxin-monitoring programmes worldwide should be revised to include a wider range of toxin variants (as has been done in some countries), or to initially test toxins according to “in vitro” toxicity assays, such as protein phosphatase inhibition.

4. Materials and Methods

4.1. Study Area Extensive sampling was performed throughout Spain (including the Canary Islands), from high mountain streams, such as those in the Pyrenees and Sierra Nevada mountains, medium and low mountain streams in the southeast, freshwater and saltwater springs from Mediterranean marshes, lagoons within Atlantic marshes, caves, and buildings. Most strains lived epilithically, but some were epipelic, and others were endolithic (chasmo or euendolithic) (Table1).

4.2. Culturing All the collected samples were isolated in different culture media (Bold’s Basal Medium with soil extract, BG-11 for freshwater or aerophytic strains, and SWES for saltwater species) and maintained as monoclonal cultures in the Algology Laboratory at Murcia University. Strains were transferred to 500-mL ﬂasks, and were maintained with BG-11 in aeration at 20 ◦C and 65–70 µM m−2 s−1 (PAR), in a 16:8 light-dark photoperiod, until enough biomass was obtained for the later analyses (25–1000 mg). The incubation period varied from 2 weeks to 2 months, depending on growth rates. Biomass was collected by ﬁltration and was lyophilised, weighed, and kept frozen (−20 ◦C) until analysed [26]. The analysis was done 1 week after collection.

4.3. Taxonomic Identification The monoclonal cultures were identified taxonomically by standard algological techniques under an OLYMPUS BX60 light microscope (OLYMPUS IBERIA S.A.U., Barcelona, Spain), equipped with a digital camera (OLYMPUS IBERIA S.A.U., Barcelona, Spain), following [53–55]. Comparisons were made with the preserved field material whenever necessary.

4.4. Microcystin Extraction and Quantification Freeze-dried and weighed biomass was ground with ground-glass homogenizers. Microcystins were extracted by sonicating for 30 min with 75% methanol, following the protocols of [36,37,48,52,56]. Cycles were repeated three times. Extracts were kept frozen (−20 ◦C), and were then concentrated in a Büchi Vac® V-500 vacuum dryer (BÜCHI Labortechnik AG, Postfach, Switzerland) and re-suspended in methanol for three cycles. Dried extracts were re-suspended in 1 mL of HPLC grade methanol, transferred to a 1.5-mL vial, and centrifuged in a Spectrafuge 24D microcentrifuge by Labnet International Inc. (Edison, New York, NY, USA) at 16 g for 5 min [26]. The resultant extracts were filtered through a regenerated 0.45 µm cellulose filter (Filter-Lab®, Eaton Filtration LLC, Tinton Falls, 3 NJ, USA) and kept frozen until analysed [26]. Extracts were collected with a 4 4 inch-long hypodermic needle (B/Braun Sterican®, B Braun España, Barcelona, Spain) attached to a 1-mL polypropylene syringe (BD PlastipakTM, BD, Madrid, Spain) and placed inside 1.8 amber phials (Scharlab S.L., Barcelona, Spain). The identification and quantification of microcystins were performed by HPLC in a high-resolution photodiode array detection analysis, using a HPLC-VWR Hitachi equipped with an L-2130 Diode Array model L-2455, following the recommendations of [27,45,46,48]. Analytes were separated in a reverse-phase Agilent silicon Zorbax column C18 (4.6 × 250 mm × 5µm). The gradient Toxins 2017, 9, 385 10 of 13 mobile phase was composed of water and acetonitrile (ACN), and was acidified with trifluoroacetic acid (TFA) (0.5 µLL−1). The flow rate was 1 mL min−1 and the injection volume was 20 µL. The results were confirmed by HPLC/MS TOF (Agilent 6220, Agilent Technologies Spain S.L, Madrid, Spain). The Anatoxin-a was extracted following the recommendations of [35,36], and was identified and quantified by HPLC photodiode array detection (Hitachi, Barcelona, Spain) and HPLC/MS (Agilent Technologies Spain S.L, Madrid, Spain).

4.5. Chemicals All the reagents were HPLC-grade and purchased from Sigma-Aldrich (St. Louis, MO, USA). The ultrapure grade water (Milli-Q water) came from the Millipore Corporate (Bedford, MA, USA). The standards of microcystins (MC-LF, MC-LR, MC-LY, MC-RR, MC-YR) and Anatoxin-a were obtained from Enzo Life Sciences (Farmingdale, NY, USA).

Acknowledgments: The PASPA-DGAPA-UNAM programme supported the sabbatical stay of the first author. Thanks are due to the Department of Plant Biology of Murcia University for allowing us to use its facilities. We thank Mª Ángeles Caravaca (Departamento de Biología Vegetal-UM), Mª del Mar Santiago and Almudena Gutiérrez (Servicio de Experimentación Agroforestal, SEAF-UM), Mª Dolores Pardo and José Rodríguez (Servicio de Instrumentación Científica, SUIC-UM) and Juana García (Servicio de Cultivo de Tejidos, SCT-UM) for technical support in the different steps of this work. Mª Dolores Belando Torrentes helped with graphic work. This text has been proofread by Helen Warburton, (H & A), Valencia, Spain. Author Contributions: M.A. conceived and designed the sampling and the experiments; E.A.C. and A.D.A. performed the experiments; M.A., E.A.C. and A.D.A. analysed the data; M.A. wrote the paper. All the authors contributed to the general discussion, revision, and manuscript editing. Conflicts of Interest: The authors declare no conflict of interest with the research, authorship and/or publication of this article.

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