Localization and Bioreactivity of Cysteine-Rich Secretions in the Marine Gastropod Nucella lapillus

Abstract

Marine biodiversity has been yielding promising novel bioproducts from venomous animals. Despite the auspices of conotoxins, which originated the paradigmatic painkiller Prialt, the biotechnological potential of gastropod venoms remains to be explored. Marine bioprospecting is expanding towards temperate species like the dogwhelk Nucella lapillus, which is suspected to secrete immobilizing agents through its salivary glands with a relaxing effect on the musculature of its preferential prey, Mytilus sp. This work focused on detecting, localizing, and testing the bioreactivity of cysteine-rich proteins and peptides, whose presence is a signature of animal venoms and poisons. The highest content of thiols was found in crude protein extracts from the digestive gland, which is associated with digestion, followed by the peribuccal mass, where the salivary glands are located. Conversely, the foot and siphon (which the gastropod uses for feeding) are not the main organs involved in toxin secretion. Ex vivo bioassays with Mytilus gill tissue disclosed the differential bioreactivity of crude protein extracts. Secretions from the digestive gland and peribuccal mass caused the most significant molecular damage, with evidence for the induction of apoptosis. These early findings indicate that salivary glands are a promising target for the extraction and characterization of bioactive cysteine-rich proteinaceous toxins from the species.

Keywords: Gastropoda; bioreactives; dogwhelk; marine biotechnology; thiols; toxicity.

Figure 1

Concentration and distribution of thiols in N. lapillus. The results are expressed as nmol cysteine equivalents per mg total protein (mean + standard deviation) in different organs: foot (reference organ), siphon, (peribuccal) glandular mass, and digestive gland. Different letters indicate significant differences (Dunn’s test; p < 0.05).

Figure 2

Histological sections (paraffin) of multiple organs of the dogwhelk N. lapillus stained with a tetrachrome stain. (a) Cross section of the foot, showing the multi-directional muscle fibers (mf) and the epithelium (ep) where the mucous-secreting cells (mc) are stained greenish-blue. (b) Siphon section localized close to the digestive gland. Note the existence of an epithelium (ep), adjacent muscle fibers (mu), and the lumen (lm). Inset: detail of the duct with a single layer of epithelial cells. (c) Section across a salivary gland of the peribuccal glandular mass, showing the two types of secretory cells: protein-secreting cells (sc) with brownish cytoplasm (from Weigert’s hematoxylin) due to high number of ribosomes and mucous-secreting cells (mc), which are Alcian blue-positive (indicating acidic mucins). (d) Typical tubular-structure of the digestive gland. The digestive gland ducts are formed by a large number of small digestive vesicles (inset). Scale bars: (a,c,d) 50 µm; (b) 200 µm.

Figure 3

Comet assay results expressed as %DNA in tail from the gills of mussels exposed ex vivo to crude protein extracts of two dilutions from different organs—foot, peribuccal glandular mass (gland), siphon, and digestive gland of N. lapillus. Results are expressed as means + standard deviation, and they correspond to the nominal dilutions of 100% (D1) and 50% (D2). * indicates significant differences to the reference organ, the foot (Student’s t-test p < 0.05).

Figure 4

Representative histological gill sections of Mytilus sp. treated with the most concentrated (D1) extract from the N. lapillus peribuccal glandular mass (H&E). (a) Transversal cut of a control gill (treated with PBS only). Fz: frontal zone; az: abfrontal zone; sc: supporting cartilage. (b) Transversal cut of gill exposed to extract. The presence of hemocytes (hm), hemolytic infiltration (hi), and apoptotic cells (ap) was verified in both sections, but the frequency was higher in exposed gills. Inset: detail of an edematous (fluid-retaining) area with apoptotic cells (white arrow) and normal nuclei (nu) observed on the same section. Scale bars: 50 µm.