Tetrodotoxins Secretion and Voltage-Gated Sodium Channel Adaptation in the Ribbon Worm Kulikovia alborostrata (Takakura, 1898) (Nemertea).

Anna E Vlasenko,Alexandra O Pereverzeva,Konstantin V Yakovlev,Vasiliy G Kuznetsov,Peter V Velansky,Timur Yu Magarlamov,Grigorii V Malykin

doi:10.3390/toxins13090606

Abstract

Nemertea is a phylum of marine worms whose members bear various toxins, including tetrodotoxin (TTX) and its analogues. Despite the more than 30 years of studying TTXs in nemerteans, many questions regarding their functions and the mechanisms ensuring their accumulation and usage remain unclear. In the nemertean Kulikovia alborostrata, we studied TTX and 5,6,11-trideoxyTTX concentrations in body extracts and in released mucus, as well as various aspects of the TTX-positive-cell excretion system and voltage-gated sodium (Nav1) channel subtype 1 mutations contributing to the toxins’ accumulation. For TTX detection, an immunohistological study with an anti-TTX antibody and HPLC-MS/MS were conducted. For Nav1 mutation searching, PCR amplification with specific primers, followed by Sanger sequencing, was used. The investigation revealed that, in response to an external stimulus, subepidermal TTX-positive cells released secretions actively to the body surface. The post-release toxin recovery in these cells was low for TTX and high for 5,6,11-trideoxyTTX in captivity. According to the data obtained, there is low probability of the targeted usage of TTX as a repellent, and targeted 5,6,11-trideoxyTTX secretion by TTX-bearing nemerteans was suggested as a possibility. The Sanger sequencing revealed identical sequences of the P-loop regions of Nav1 domains I–IV in all 17 studied individuals. Mutations comprising amino acid substitutions, probably contributing to nemertean channel resistance to TTX, were shown.

Highlights

Nemertea is a phylum of marine worms, consisting of more than 1300 species, most of which are active predators [1]
The TTX and its analogues (TTXs) content in the secretions and body extracts of nemertean K. alborostrata was studied with high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS)
The ribbon worms were kept for 30 days, and, after this time, measurements of the TTX concentrations in their secretions showed an increase in 16 out of 17 worms, while the TTX concentration in the remaining specimen was higher than the limit of quantification (LoQ) (0.6 ng/mL of the extract) (Worm 4); it could be clearly quantified

Summary

Introduction

Nemertea is a phylum of marine worms, consisting of more than 1300 species, most of which are active predators [1]. Most studies have focused on toxin detection in nemerteans [4,5,6,7,8] and the search for toxin sources [9,10,11], while a small number of studies included physiological experiments concerning the transportation and function of TTX and its analogues (TTXs) in ribbon worms. The physiological experiments dedicated to the study of the secretion of the highly toxic nemertean Cephalothrix simula (=C. linearis) showed the presence of TTX in mucus secreted upon stimulation of the animal [12,13]. For many TTX-secreting animals, the function of the toxin as a predator deterrent was suggested. The realization of this function in ribbon worms supposes the recovery of TTX in secreting cells through migration from the tissues of the internal environment. In the current research, using 17 specimens of the ribbon worm Kulikovia alborostrata, we studied the dynamics of TTX concentration in the secretion produced at different time intervals, and four specimens were used for the investigation of toxin localization at different stages of the excretion process

Methods

Results

Discussion

Conclusion