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Aphid control often relies on synthetic pesticides, but their overuse has raised concerns about resistance development and negative impact on wildlife and human health. Consequently, the search for new biopesticide agents has gained significant attention. Nemertide alpha-1, a peptide toxin from the marine nemertean worm Lineus longissimus (Gunnerus, 1770), is known for its pesticide activity but has less documented biological safety. This study investigates the aphid feeding deterrence and biological safety of the experimental biopesticide nemertide alpha-1. Nemertide alpha-1 demonstrated a clear dose-dependent repellent effect on the penetration behaviour of the green peach aphid (Myzus persicae, Sulzer). It also demonstrates bacteriostatic and bactericidal effects in an MIC (Minimum Inhibitory Concentration) assay, respectively, on E. coli (MIC: 112.5 µM) and S. aureus (MIC: 28.4 µM). In a bacterial liposome leakage assay, nemertide alpha-1 exhibits a less pronounced effect than the melittin control (20% maximum leakage at 100 µM), strengthening the hypothesis on the specificity of its neurotoxic mode of action. It is not toxic to mammalian cell U-937 GTB with only a slight decline in the percentage of survival at the highest concentration tested (80 µM). Finally, nemertide alpha-1 displays thermal stability over time for four weeks in three different conditions: cold (6 °C), room temperature (20–24 °C), and physiological temperature (37 °C). Nemertide alpha-1 deters green peach aphid feeding in the low micromolar range and exhibits low antimicrobial properties and very low toxicity to human cells. Its potential utility is further underscored by thermal stability over time.

Peptide toxins from marine invertebrates have found use as drugs and in biotechnological applications. Many marine habitats, however, remain underexplored for natural products, and the Southern Ocean is among them. Here, we report toxins from one of the top predators in Antarctic waters: the nemertean worm Parborlasia corrugatus (McIntosh, 1876). Transcriptome mining revealed a total of ten putative toxins with a cysteine pattern similar to that of alpha nemertides, four nemertide-beta-type sequences, and two novel full-length parborlysins. Nemertean worms express toxins in the epidermal mucus. Here, the expression was determined by liquid chromatography combined with mass spectrometry. The findings include a new type of nemertide, 8750 Da, containing eight cysteines. In addition, we report the presence of six cysteine-containing peptides. The toxicity of tissue extracts and mucus fractions was tested in an Artemia assay. Notably, significant activity was observed both in tissue and the high-molecular-weight mucus fraction, as well as in a parborlysin fraction. Membrane permeabilization experiments display the membranolytic activity of some peptides, most prominently the parborlysin fraction, with an estimated EC₅₀ of 70 nM.

During a research program to identify new cholinesterase inhibitors of natural origin, two new 7,8-didehy-droprotoberberine alkaloids (1 and 2) and nine known compounds (3-11) were isolated from the capsules of the common ornamental poppy, Papaver setiferum (previously P. pseudo-orientale). Despite their reported instability, the 7,8-didehydroprotoberberines isolated herein appeared relatively stable, particularly as their trifluoroacetic acid salts. The spatial distributions of the isolated alkaloids were also analyzed using desorption electrospray ionization imaging mass spectrometry. The alkaloids were localized predominantly within the walls and vascular bundles of the capsules, with the highest relative abundances occurring in the lower half of the capsules toward the peduncle. The relative abundances of the alkaloids were also compared across plant development stages. Although most alkaloids did not show clear patterns in their concentration across development stages, the concentration of suspected oxidation products clearly spiked upon plant death. Finally, all isolated natural products were screened for inhibitory activities against a panel of cholinesterases, from both human and animal sources. These studies identified several competitive inhibitors of cholinesterases with potency in the low micromolar range (1-4, 6, 7), offering new lead compounds for the development of cholinesterase inhibitory drugs.

Peptide toxins find use in medicine, biotechnology, and agriculture. They are exploited as pharmaceutical tools, particularly for the investigation of ion channels. Here, we report the synthesis and activity of a novel family of peptide toxins: the cystine-knotted α nemertides. Following the prototypic α-1 and -2 (1 and 2), six more nemertides were discovered by mining of available nemertean transcriptomes. Here, we describe their synthesis using solid phase peptide chemistry and their oxidative folding by using an improved protocol. Nemertides α-2 to α-7 (2–7) were produced to characterize their effect on voltage-gated sodium channels (Blatella germanica BgNaV1 and mammalian NaVs1.1–1.8). In addition, ion channel activities were matched to in vivo tests using an Artemia microwell assay. Although nemertides demonstrate high sequence similarity, they display variability in activity on the tested NaVs. The nemertides are all highly toxic to Artemia, with EC50 values in the sub-low micromolar range, and all manifest preference for the insect BgNaV1 channel. Structure–activity relationship analysis revealed key residues for NaV-subtype selectivity. Combined with low EC50 values (e.g., NaV1.1: 7.9 nM (α-6); NaV1.3: 9.4 nM (α-5); NaV1.4: 14.6 nM (α-4)) this underscores the potential utility of α-nemertides for rational optimization to improve selectivity.

The overall aims of the projects included in this thesis were to discover, synthesize and characterize disulphide-stabilized peptides from marine worms (Nemertea sp.) and plants (Viola sp.). 

One of the main outcomes of this thesis is the discovery of a new family of highly active cysteine-rich toxins, alpha nemertides, from nemertean worms (paper II). Functional characterization and production routes of nemertides were further explored (papers II-III). In addition, 12 new cyclotides from the bog violet were discovered (paper I). Finally, transcriptomes and mucus of the Antarctic nemertean Parborlasia corrugatus were investigated for toxin content (paper IV).

 In paper I wild-type leaf and callus tissue of the endangered bog violet, V. uliginosa, were analyzed using transcriptomics and LC-MS, resulting in the discovery of 12 new cyclotides (i.e. cysteine-rich cyclic peptides). In addition, cyclotide expression under different cell-growth conditions was monitored.

In paper II  the discovery and initial characterization of a new family of highly active peptides, the alpha nemertides, from the epidermal mucus of the world’s longest animal; Lineus longissimus is described. The most abundant alpha nemertide, alpha-1, was extracted in minute amounts, prompting the use solid phase peptide synthesis (SPPS) for further characterization. The tertiary structure of alpha-1 was elucidated and revealed an inhibitory cystine knot (ICK) framework. The knotted core-structure is similar to the cyclic cystine knot (CCK) motif, found in the cyclotides described in paper I.

In manuscript III, the production route established in paper II was used to produce nemertides alpha 1-7. These were tested in vivo in an Artemia microwell assay as well as on an extended panel of voltage-gated sodium channels (Na_V1.1 – 1.8 and BgNa_V1). All seven alpha nemertides were highly active in the in vivo Artemia assay with EC₅₀ values in the sub to low µM range. The alpha nemertides were also active in the Na_Vs tested. However, differences in the activity profiles were observed, indicating an opportunity for future optimization of alpha nemertides to reach higher specificity to certain Na_V subtypes.

In manuscript IV, the exploration of nemertide toxins was extended to include the Antarctic P. corrugatus. Resulting findings include a set of cysteine-rich peptides, some similar to the nemertides previously discovered in paper II. Two purified peptides and one fraction were evaluated for their membranolytic activity.

Most ribbon worms (phylum: Nemertea) are found in marine environments, where they act as predators and scavengers. They are characterized by an eversible proboscis that is used to hunt for prey and thick mucus covering their skin. Both proboscis and epidermal mucus mediate toxicity to predators and preys. Research into the chemical nature of the substances that render toxicity has not been extensive, but it has nevertheless led to the identification of several compounds of potential medicinal use or for application in biotechnology. This review provides a complete account of the current status of research into nemertean toxins.

Polypeptides from animal venoms have found important uses as drugs, pharmacological tools, and within biotechnological and agricultural applications. We here report a novel family of cystine knot peptides from nemertean worms, with potent activity on voltage-gated sodium channels. These toxins, named the alpha-nemertides, were discovered in the epidermal mucus of Lineus longissimus, the 'bootlace worm' known as the longest animal on earth. The most abundant peptide, the 31-residue long alpha-1, was isolated, synthesized, and its 3D NMR structure determined. Transcriptome analysis including 17 species revealed eight alpha-nemertides, mainly distributed in the genus Lineus. alpha-1 caused paralysis and death in green crabs (Carcinus maenas) at 1 mu g/kg (similar to 300 pmol/kg). It showed profound effect on invertebrate voltage-gated sodium channels (e.g. Blattella germanica Na(v)1) at low nanomolar concentrations. Strong selectivity for insect over human sodium channels indicates that a-nemertides can be promising candidates for development of bioinsecticidal agents.

Cyclotides are a family of plant proteins that are characterized by a cyclic backbone and a knotted disulfide topology. Their cyclic cystine knot (CCK) motif makes them exceptionally resistant to thermal, chemical, and enzymatic degradation. By disrupting cell membranes, the cyclotides function as host defense peptides by exhibiting insecticidal, anthelmintic, antifouling, and molluscicidal activities. In this work, we provide the first insight into the evolution of this family of plant proteins by studying the Violaceae, in particular species of the genus Viola. We discovered 157 novel precursor sequences by the transcriptomic analysis of six Viola species: V. albida var. takahashii, V. mandshurica, V. orientalis, V. verecunda, V. acuminata, and V. canadensis. By combining these precursor sequences with the phylogenetic classification of Viola, we infer the distribution of cyclotides across 63% of the species in the genus (i.e., ~380 species). Using full precursor sequences from transcriptomes, we show an evolutionary link to the structural diversity of the cyclotides, and further classify the cyclotides by sequence signatures from the non-cyclotide domain. Also, transcriptomes were compared to cyclotide expression on a peptide level determined using liquid chromatography-mass spectrometry. Furthermore, the novel cyclotides discovered were associated with the emergence of new biological functions.