Proteinaceous Toxins in the Mucus and Proboscis of the Ribbon Worm Cephalothrix cf. simula (Palaeonemertea: Nemertea)

<i>Cephalothrix</i> cf. <i>simula</i> is a highly toxic ribbon worm of the class Palaeonemertea, known for its high concentrations of tetrodotoxin. Recent transcriptomic and proteomic studies across Nemertea have revealed that species from all classes possess a diverse array of protein and peptide toxins, which are associated with unicellular glands of the proboscis and the integument epithelium. Previous studies have identified a large number of putative toxins in the transcriptome of <i>C</i>. cf. <i>simula</i>; however, corresponding proteomic data have so far been lacking. This study presents the first comprehensive analysis of the mucus and proboscis proteome of <i>C</i>. cf. <i>simula</i> using high-performance liquid chromatography-tandem mass spectrometry. We identified three putative toxins in the proboscis and three in the mucus. Additionally, four cysteine-rich peptides with putative toxic activity were identified in the mucus and one in the proboscis. The expression of the corresponding genes in both tissues was quantified using quantitative real-time PCR. The toxin compositions of the proboscis and mucus showed clear signs of functional specialization, with no overlapping toxins and tissue-specific patterns of gene expression. Feeding experiments combined with transmission electron microscopy confirmed the involvement of specialized proboscis structures, pseudocnidae, in delivering toxins into the prey.