toxins Article Molecular Identification and Toxin Analysis of Alexandrium spp. in the Beibu Gulf: First Report of Toxic A. tamiyavanichii in Chinese Coastal Waters Yixiao Xu 1,2,* , Xilin He 1, Huiling Li 1, Teng Zhang 1, Fu Lei 3, Haifeng Gu 4 and Donald M. Anderson 5 1 Key Laboratory of Environment Change and Resources Use in Beibu Gulf, Ministry of Education, Nanning Normal University, Nanning 530001, China; [email protected] (X.H.); [email protected] (H.L.); [email protected] (T.Z.) 2 Guangxi Laboratory on the Study of Coral Reefs in the South China Sea, Guangxi University, Nanning 530004, China 3 Guangxi Key Laboratory of Marine Environmental Science, Beibu Gulf Marine Research Center, Guangxi Academy of Sciences, Nanning 530007, China; [email protected] 4 Third Institute of Oceanography, Ministry of Natural Resources, Xiamen 361005, China; [email protected] 5 Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA; [email protected] * Correspondence: [email protected] Abstract: The frequency of harmful algal blooms (HABs) has increased in China in recent years. Information about harmful dinoflagellates and paralytic shellfish toxins (PSTs) is still limited in China, especially in the Beibu Gulf, where PSTs in shellfish have exceeded food safety guidelines on multiple occasions. To explore the nature of the threat from PSTs in the region, eight Alexandrium strains were isolated from waters of the Beibu Gulf and examined using phylogenetic analyses of large subunit (LSU) rDNA, small subunit (SSU) rDNA, and internal transcribed spacer (ITS) sequences. Their toxin composition profiles were also determined using liquid chromatography-tandem mass spectrometry Citation: Xu, Y.; He, X.; Li, H.; (LC-MS/MS). All eight strains clustered in the phylogenetic tree with A. pseudogonyaulax, A. affine, Zhang, T.; Lei, F.; Gu, H.; Anderson, and A. tamiyavanichii from other locations, forming three well-resolved groups. The intraspecific D.M. Molecular Identification and genetic distances of the three Alexandrium species were significantly smaller than interspecific genetic Toxin Analysis of Alexandrium spp. in distances for Alexandrium species. Beibu Gulf isolates were therefore classified as A. pseudogonyaulax, the Beibu Gulf: First Report of Toxic A. tamiyavanichii in Chinese Coastal A. affine, and A. tamiyavanichii. No PSTs were identified in A. pseudogonyaulax, but low levels Waters. Toxins 2021, 13, 161. https:// of gonyautoxins (GTXs) 1 to 5, and saxitoxin (STX) were detected in A. tamiyavanichii (a total of doi.org/10.3390/toxins13020161 4.60 fmol/cell). The extremely low level of toxicity is inconsistent with PST detection above regulatory levels on multiple occasions within the Beibu Gulf, suggesting that higher toxicity strains may occur Received: 25 January 2021 in those waters, but were unsampled. Other explanations including biotransformation of PSTs in Accepted: 16 February 2021 shellfish and the presence of other PST-producing algae are also suggested. Understanding the Published: 18 February 2021 toxicity and phylogeny of Alexandrium species provides foundational data for the protection of public health in the Beibu Gulf region and the mitigation of HAB events. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: Alexandrium tamiyavanichii; paralytic shellfish poisoning; molecular identification; toxicity; published maps and institutional affil- harmful algal blooms; Beibu Gulf iations. Key Contribution: The first record of Alexandrium affine, A. pseudogonyaulax and A. tamiyavanichii from the seawaters of Beibu Gulf, and the first report of toxicity and phylogeny of A. tamiyavanichii from Chinese coastal waters. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and 1. Introduction conditions of the Creative Commons The genus Alexandrium is an important toxic and harmful algal blooms (HABs) di- Attribution (CC BY) license (https:// noflagellate distributed in coastal waters worldwide [1]. This genus was first described by creativecommons.org/licenses/by/ Halim [2] and it has since become one of the most studied dinoflagellate genera. Among 4.0/). Toxins 2021, 13, 161. https://doi.org/10.3390/toxins13020161 https://www.mdpi.com/journal/toxins ToxinsToxinsToxinsToxins 2021 2021 2021 2021, 13, 13, ,,13 x13, x,FOR , x FORx FOR FOR PEER PEER PEER PEER REVIEW REVIEW REVIEW REVIEW 2 2of 2of2 17of of 17 17 17 1.1. 1.Introduction1. Introduction IntroductionIntroduction Toxins 2021, 13, 161 TheTheTheThe genus genus genusgenus Alexandrium Alexandrium AlexandriumAlexandrium is is an isis an an animportant important importantimportant toxic toxic toxictoxic and and andand harmful harmful harmfulharmful algal algal algalalgal blooms blooms bloomsblooms (HABs) (HABs) (HABs)(HABs) dino- dino- dino-dino-2 of 16 flagellateflagellateflagellateflagellate distributed distributed distributeddistributed in in incoastalin coastal coastalcoastal waters waters waterswaters worl worl worlworldwidedwidedwidedwide [1]. [1]. [1].[1]. This This ThisThis genus genus genusgenus was was waswas first first firstfirst described described describeddescribed by by by by HalimHalimHalimHalim [2] [2] [2] [2]and and andand it it has it ithas hashas since since sincesince become become becomebecome one one oneone of of ofthof th e thth emoste emost mostmost studied studied studiedstudied dinoflagellate dinoflagellate dinoflagellatedinoflagellate genera. genera. genera.genera. Among Among AmongAmong thethethethe over over overover 30 30 3030species species speciesspecies of of ofAlexandriumof Alexandrium AlexandriumAlexandrium reported reported reportedreported to to todate,to date, date,date, at at atleastat least leastleast half half halfhalf can can cancan cause cause causecause paralytic paralytic paralyticparalytic shell- shell- shell-shell- the over 30 species of Alexandrium reported to date, at least half can cause paralytic shellfish fishfishfishfish poisoning poisoning poisoningpoisoning (PSP) (PSP) (PSP)(PSP) [3,4]. [3,4]. [3,4].[3,4]. Paralytic Paralytic ParalyticParalytic shellfish shellfish shellfishshellfish toxins toxins toxinstoxins (PSTs) (PSTs) (PSTs)(PSTs) accumulate accumulate accumulateaccumulate in in inshellfish,in shellfish, shellfish,shellfish, fish, fish, fish,fish, poisoning (PSP) [3,4]. Paralytic shellfish toxins (PSTs) accumulate in shellfish, fish, and andandandand other other otherother organisms organisms organismsorganisms through through throughthrough the the thethe food food foodfood web. web. web.web. Humans Humans HumansHumans who who whowho consume consume consumeconsume sh sh shellfishshellfishellfishellfish containing containing containingcontaining andother other organisms organisms through through the the food food web. web. Humans Humans who who consume consume shellfish shellfish containing containing PSTs PSTsPSTs can can become become poisoned; poisoned; PSTs PSTs thus thus re representspresents a amajor major health health hazard hazard [1,5]. [1,5]. Alexandrium Alexandrium PSTsPSTscan cancan become becomebecome poisoned; poisoned;poisoned; PSTs PSTsPSTs thus thusthus represents rerepresentspresents aa majormajor health healthhealth hazard hazard [1,5].[1,5]. [1,5]. AlexandriumAlexandriumAlexandrium is isis aisis a common a acommon acommoncommon common species species speciesspecies species in in inthein the in thethe coastal thecoastal coastalcoastal coastal waters waters waterswaters waters of of ofChina.of China. ofChina.China. China. Over Over OverOver Overthe the thethe past past the pastpast 15 past15 1515years, years, years, 15years, years, 24 24 2424HABs HABs HABsHABs 24HABs due due duedue due toto toAlexandriumto Alexandrium AlexandriumAlexandriumto Alexandrium spp. spp. spp.spp. have havespp. havehave occurred haveoccurred occurredoccurred occurred in in inthein the thethe inBohai, Bohai, Bohai, theBohai, Bohai,Yellow, Yellow, Yellow,Yellow, Yellow, and and andand South South and SouthSouth SouthChina China ChinaChina ChinaSeas Seas SeasSeas [5], Seas[5], [5],[5], with with [withwith5], with disastrousdisastrousdisastrousdisastrousdisastrous consequences consequences consequencesconsequences consequences for for forfor local local local forlocal marine localmarine marinemarine marine fi fisheries, fisheries,fisheries,sheries, fisheries, ecosystems, ecosystems, ecosystems,ecosystems, ecosystems, an an andand coastald dcoastal and coastalcoastal coastal landscapes. landscapes. landscapes.landscapes. landscapes. TheTheTheThe morphological morphological The morphologicalmorphological morphological identification identification identificationidentification identification of of of Alexandriumof Alexandrium AlexandriumAlexandrium of Alexandrium is is based isis based basedbasedis basedprimarily primarily primarilyprimarily primarily on on on onthe the thethe onfine fine fine thefine struc- struc- fine struc-struc- struc- tureturetureture of ofture ofofthe the thethe ofapical apical theapicalapical apicalpore pore porepore comp porecomp compcomplex, complex,lex,lex,lex, the the thethe first first thefirstfirst apical apical first apicalapical apicaland and andand sixt sixt and sixtsixthh precingularh sixthhprecingular precingularprecingular precingular plates, plates, plates,plates, plates, the the thethe pres- pres- pres- thepres- pres- ence/absenceence/absenceence/absenceence/absenceence/absence of of ofofa a ventral a aventral ofventralventral a ventral pore