New Invasive Nemertean Species (Cephalothrix Simula) in England with High Levels of Tetrodotoxin and a Microbiome Linked to Toxin Metabolism

Abstract

The marine nemertean Cephalothrix simula originates from the Pacific Ocean but in recent years has been discovered in northern Europe. The species has been associated with high levels of the marine neurotoxin Tetrodotoxin, traditionally associated with Pufferfish Poisoning. This study reports the first discovery of two organisms of C. simula in the UK, showing the geographical extent of this species is wider than originally described. Species identification was initially conducted morphologically, with confirmation by Cox 1 DNA sequencing. 16S gene sequencing enabled the taxonomic assignment of the microbiome, showing the prevalence of a large number of bacterial genera previously associated with TTX production including Alteromonas, Vibrio and Pseudomonas. LC-MS/MS analysis of the nemertean tissue revealed the presence of multiple analogues of TTX, dominated by the parent TTX, with a total toxin concentration quantified at 54 µg TTX per g of tissue. Pseudomonas luteola isolated from C. simula, together with Vibrio alginolyticus from the native nemertean Tubulanus annulatus, were cultured at low temperature and both found to contain TTX. Overall, this paper confirms the high toxicity of a newly discovered invasive nemertean species with links to toxin-producing marine bacteria and the potential risk to human safety. Further work is required to assess the geographical extent and toxicity range of C. simula along the UK coast in order to properly gauge the potential impacts on the environment and human safety.

Stable Tetrodotoxin Production by Bacillus sp. Strain 1839

“…For example, early research of Alteromonas tetraodonis noted the phosphate concentration in medium to affect the TTX production [8]. Recent works showed better TTX yield because of cultivation at lower temperatures in a range of 22-25 • C [9,10]. In a number of studies, TTX production depended on the growth phase of the bacteria and time of cultivation, respectively [8,11].…”

The Microbial Community of Tetrodotoxin-Bearing and Non-Tetrodotoxin-Bearing Ribbon Worms (Nemertea) from the Sea of Japan

“…According to Yotsu-Yamashita et al [15], the late stages of biosynthesis and metabolism of TTX and its analogues may involve oxidation routes of 5,6,11-trideoxyTTX. TTX analogues including 5-deoxyTTX, 11-deoxyTTX, and 5,6,11-trideoxyTTX itself, presumably involved in at least one oxidation route were found in C. simula [12,13] and K. manchenkoi [13]. As no conversion between TTX and its non-equilibrium analogues was detected in animals, the above mentioned oxidation reactions were supposed to proceed in TTX-producing microorganisms [16,17].…”

“…In this study, by using 16S rRNA gene amplicon sequencing, we characterized and compared the microbiome composition of four TTX-bearing and four non-TTX-bearing species of marine ribbon worms. The data on the microbiome of TTX-bearing animals is extremely scarce, with only one study characterizing the microbial community of two C. simula samples [12]. We are not aware of other studies addressing the microbiome of TTX-bearing animals by using metagenomic approach.…”

“…TTX and its analogues were found in Cephalothrix linearis [7,8], Cephalothrix sp. [9][10][11], and Cephalothrix simula [12,13]. Screenings for TTXs in other ribbon worms showed that the paleonemertean T. punctatus, hoplonemerteans Quasitetrastemma stimpsoni, Collarenemertes bimaculata, and Nipponnemertes punctatula, as well as the pilidiophores Kulikovia alborostrata (=Lineus alborostratus), Kulikovia manchenkoi, and Yininemertes pratensis contained TTXs but in much smaller concentrations than the Cephalothrix species [11][12][13][14].…”

“…These were found in cultivable microflora of nemerteans. Members of the genera Vibrio, Pseudomonas, Pseudoalteromonas, and Bacillus isolated from both TTX-bearing and non-TTX-bearing nemerteans were claimed to synthesize TTX [12,[18][19][20][21].…”