Paralytic shellfish toxin producing Aphanizomenon gracile strains isolated from Lake Iznik, Turkey

Highlights

• Three cyanobacteria strains were isolated from Lake Iznik, Turkey in 2016.

• Strains were identified as Aphanizomenon gracile with a polyphasic approach.

• All three strains produced STX, NEO, dcSTX and dcNEO.

• First report of a PST producer in Turkish waters.

• First observation of dcNEO in an A. gracile culture.

Abstract

Aphanizomenon gracile is one of the most widespread Paralytic Shellfish Toxin (PST) producing cyanobacteria in freshwater bodies in the Northern Hemisphere. It has been shown to produce various PST congeners, including saxitoxin (STX), neosaxitoxin (NEO), decarbamoylsaxitoxin (dcSTX) and gonyautoxin 5 (GTX5) in Europe, North America and Asia. Three cyanobacteria strains were isolated in Lake Iznik in northwestern Turkey. Morphological characterization of these strains suggested all three strains conformed to classical taxonomic identification of A. gracile with some differences such as clumping of filaments, partially hyaline cells in some filaments and longer than usual vegetative cells. Sequences of 16S rRNA gene of these strains were placed within an A. gracile cluster including the majority of PST producing strains, confirming the identification of these strains as A. gracile. These new strains possessed saxitoxin biosynthesis genes sxtA, sxtG and their sequences clustered with those of other A. gracile. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis demonstrated the presence of NEO, STX, dcSTX and decarbamoylneosaxitoxin (dcNEO) in all strains. This is the first report of a PST producer in any water body in Turkey and first observation of dcNEO in an A. gracile culture.

Introduction

Saxitoxin and its analogues are a group of guanidinium containing alkaloids produced either by marine dinoflagellates or freshwater cyanobacteria (Wiese et al., 2010). These toxins exert their effect by blocking voltage gated sodium channels, calcium channels and by modifying the gating behavior of potassium channels in mammals (Cusick and Sayler, 2013 and references therein). They are usually transferred to humans and animals via vector organisms such as shellfish (Wiese et al., 2010). In cases of severe mammalian intoxications they cause the paralysis of respiratory muscles and therefore they are collectively called Paralytic Shellfish Toxins (PSTs) (Cusick and Sayler, 2013, Wiese et al., 2010). While in marine environments the main route of intoxication is through the food web (Wiese et al., 2010), in fresh water environments exposure may result both from the food web and drinking water (Batoréu et al., 2005).

PSTs are produced by strains of various cyanobacterial species in freshwater environments. These include Dolichospermum circinale in Australia (Humpage et al., 1994), Cylindrospermopsis raciborskii (Lagos et al., 1999), Raphidiopsis brookii (Yunes et al., 2009), Geitlerinema spp., Phormidium uncinatum and Cylindrospermum stagnale (Borges et al., 2015) in South America, Microseira wollei (Foss et al., 2012, Onodera et al., 1997) in North America, Cuspidothrix issatschenkoi (Pereira et al., 2000) in Europe, Scytonema sp. in New Zealand (Smith et al., 2011) and Aphanizomenon gracile in North America, Europe and Asia (Ballot et al., 2010, Cirés et al., 2014, Jung et al., 2003, Ledreux et al., 2010, Liu et al., 2006a, Mahmood and Carmichael, 1986, Pereira et al., 2004, Perez et al., 2008).

Aphanizomenon gracile, one of the most widespread PST producing cyanobacteria, is a species of the temperate regions (Komarek and Komarkova, 2006). The first report of PST production in A. gracile was from a strain isolated in Durham, NH, USA (strain NH-5) (Mahmood and Carmichael, 1986). Although initially identified as an atypical non-fasciculated A. flos-aquae, subsequent molecular work placed this strain within an A. gracile cluster (Casero et al., 2014, Li et al., 2003). Isolation of PST producing A. gracile in Europe was much later, first in Portugal (Pereira et al., 2004), followed by Germany (Ballot et al., 2010), France (Ledreux et al., 2010), Spain (Cirés et al., 2014) and Norway (Ballot et al., 2016). On the other hand, in Asia, two A. gracile strains are known producers of PSTs. Aphanizomenon gracile NIES-81 (A. flos-aquae f. gracile), isolated in Japan in 1978, was shown to produce saxitoxin in 2003 (Jung et al., 2003) and strain DC-1 from China produced various PSTs (Liu et al., 2006a, Liu et al., 2006b).

Aphanizomenon gracile strains isolated to date proved to produce certain PST analogues including saxitoxin (STX), neosaxitoxin (NEO), decarbamoylsaxitoxin (dcSTX) and gonyautoxin-5 (GTX5). Strain NH-5 from USA (Mahmood and Carmichael, 1986), LMECYA-40 from Portugal (Pereira et al., 2004), PMC 627.10 and 638.10 from France (Ledreux et al., 2010) produced only STX and NEO. While strain UAM 529 from Spain produced only STX and dcSTX, the other Spanish strain UAM 531 produced STX, NEO and dcSTX (Cirés et al., 2014). While Japanese strain NIES-81 was shown to produce STX (Jung et al., 2003), German strains from lakes Scharmützel and Melang produced more diverse PSTs that included STX, NEO, dcSTX and GTX5 (Ballot et al., 2010). Strain NIVA-CYA 851 from Norway was more similar to German strains producing STX, NEO and GTX5 (Ballot et al., 2016). Chinese strain DC-1 was different from the above and was reported to produce STX, NEO, GTX5 (Liu et al., 2006a) or dcSTX, dcGTX2, dcGTX3, and GTX4 (Liu et al., 2006b) or NEO, GTX1 and GTX5 (Zhang et al., 2015).

Previous phylogenetic analyses based on 16S rRNA gene demonstrated close genetic relatedness between A. gracile and Aphanizomenon flos-aquae (Cirés et al., 2014, Gugger et al., 2002, Rajaniemi et al., 2005a, Wu et al., 2010). While some studies suggested that A. gracile and A. flos-aquae could be separated into different clusters in phylogenetic trees based on 16S rRNA, rpoB, rbcLX, cpcBA-IGS and concatenated genes (Casero et al., 2014, Cirés et al., 2014, De Figueiredo et al., 2010, Rajaniemi et al., 2005a, Wu et al., 2010), other studies argued against it (Gugger et al., 2002, Stüken et al., 2009).

Three cyanobacteria strains, tentatively identified as A. gracile, were isolated from Lake Iznik in Turkey in 2016. To our knowledge, this species was not previously reported in Lake Iznik. Therefore, the goals of this study were to perform polyphasic identification of these isolates and the investigation of their PST production.