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

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.

Local Differences in the Toxin Amount and Composition of Tetrodotoxin and Related Compounds in Pufferfish (Chelonodon patoca) and Toxic Goby (Yongeichthys criniger) Juveniles

“…The transferring of TTX from prey organisms has been reported in the pufferfish genus Takifugu, such as T. rubripes and T. alboplumbeus [13,14,19,40]. Alternatively, although the proposed composition of TTX and biosynthetic intermediates has been investigated in various species [41][42][43][44][45], there have been no studies on how these substances in the prey are ingested and transferred to predators. Therefore, it was speculated that various TTX-related compounds in the prey would be directly ingested and accumulated in the body of predators, such as ribbon worms and newts [46][47][48][49].…”

The road not taken: Evolution of tetrodotoxin resistance in the Sierra garter snake (Thamnophis couchii) by a path less travelled

“…Table 1). This mutation appears to be unique among snakes (Feldman et al, 2012;McGlothlin et al, 2016), but is seen in several other TTX-resistant taxa (Geffeney et al, 2019;Gendreau et al, 2021;Jost et al, 2008;Vaelli et al, 2020;Vlasenko et al, 2021) and confers a roughly 15-fold reduction in TTX ligation to the pore (Jost et al, 2008). Additionally, we found that all Th.…”

“…couchii contain the same mutation in the skeletal muscle channel gene, SCN4A (encoding Na v 1.4): a single amino acid substitution (M1267T) in the DIII P‐loop of Na v 1.4 at a critical TTX binding site (detailed in Feldman et al, 2009) (Figure 3; Table 1). This mutation appears to be unique among snakes (Feldman et al, 2012; McGlothlin et al, 2016), but is seen in several other TTX‐resistant taxa (Geffeney et al, 2019; Gendreau et al, 2021; Jost et al, 2008; Vaelli et al, 2020; Vlasenko et al, 2021) and confers a roughly 15‐fold reduction in TTX ligation to the pore (Jost et al, 2008). Additionally, we found that all Th.…”

“…The molecular underpinning of TTX resistance is thought to be functional variation in the P‐loops of voltage‐gated sodium channels (Na v loci), which show convergent replacements across the many species of metazoans that have evolved TTX resistance (e.g., Feldman et al, 2012; Geffeney et al, 2019; Gendreau et al, 2021; Jost et al, 2008; Vlasenko et al, 2021). We first screened variation in SCN4A , the gene that encodes the sodium channel expressed in skeletal muscle (Na v 1.4), as variation at this locus appears to explain whole‐animal phenotypes in Th.…”

“…Substitutions at this site alter TTX ligation to the outer pore of the channel (Terlau et al, 1991;Du et al, 2009), and when this exact mutation was constructed in rat Na v 1.4, it led to a 15-fold increase in TTX resistance (Jost et al, 2008). This same replacement has evolved & Gilly, 2015;Vaelli et al, 2020;Gendreau et al, 2021), pufferfishes (Jost et al, 2008), and even invertebrates such as blue-ringed octopus (Geffeney et al, 2019) and some marine ribbon worms (Vlasenko et al, 2021). Oddly, however, large differences in TTX resistance at both the whole-animal and muscle level in Th.…”