toxins 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