Tetrodotoxins in Larval Development of Ribbon Worm Cephalothrix cf. simula (Palaeonemertea, Nemertea)

The toxic ribbon worm, Cephalothrix cf. simula (Palaeonemertea, Nemertea), possesses extremely high concentrations of tetrodotoxin (TTX). Although TTX has been found in the eggs of this species, the fate of the toxin in the ontogeny of the animal has not been explored. Here, using high performance liquid chromatography with tandem mass spectrometry and immunohistochemistry with anti-TTX antibodies, we examined levels, profile, and localization of TTX and its analogues (TTXs) in larvae of C. cf. simula throughout 41 days post-fertilization. A detailed investigation of cells in sites of TTX-accumulation was performed with light and electron microscopy. Newly hatched larvae possessed weak TTX-like immunoreactivity in all cells. With subsequent development, intensity of TTX-labeling in the ectodermal structures, mesodermal cells and apical cylinder of the apical gland increased. In the ectodermal structures, an intense TTX-labeling was observed in the multiciliated, type II granular, type I mucoid, and basal cells of the epidermis, and in the type III granular cells of the mouth gland. In the mesoderm, TTX was localized in the muscle and unigranular parenchyma-like cells. Eggs and larvae of C. cf. simula contained five TTXs, with two major toxins - TTX and 5,6,11-trideoxyTTX. Level and relative proportion of TTXs did not differ significantly among developmental stages, confirming that larvae obtained toxins from maternal eggs and were able to retain it. The results of this study provide insights into the formation of TTX-bearing apparatus of C. cf. simula through the larval development.