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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.

The overall aims of the projects included in this thesis were to discover, functionally characterise, explore biological safety and evaluate potential utility of cysteine-rich peptides, named alpha-nemertides, from marine worms (Nemertea sp.). 

One of the main outcomes of this thesis is the discovery of a new subfamily of nemertean peptide toxins, from the epidermal mucus of an unexplored marine worm (Paper I). Structure-activity relationships of alpha-nemertides was investigated via alanine scanning on the archetype nemertide alpha-1 (Paper II). Finally, a preliminary investigation of nemertide alpha-1 feeding repellence and acute toxicity was perform in vivo on pest insects (Paper III-IV) alongside with the analysis of its human and biological safety (Paper III). 

In Manuscript I, the discovery and initial characterisation of a new subfamily of nemertean peptide toxins from Lineus viridis is presented. The new subfamily was named delta-nemertide and the archetype, nemertide delta-1, was successfully isolated and described. Furthermore, transcriptomic analysis led to the addition of seven delta-nemertide putative sequences. Nemertide delta-1 share many similarities with the previously described nemertide alpha-1 except for a 30-fold higher potency on measured insect voltage-gated sodium channels. 

In Manuscript II, the study focused on determining the importance for selectivity and acute toxicity of each amino acid from nemertide alpha-1 sequence. The mutants’ toxicity was assessed in vitro on a selection of voltage-gated sodium channels and in vivo on Artemia salina. Five positions crucial for overall selectivity were highlighted and one mutant named S12A demonstrated specific selectivity to insect voltage-gated sodium channels. 

In Manuscript III, nemertide alpha-1 displayed feeding deterrence and feeding behaviour changes at the low molecular level on green peach aphids (Myzus persicae), a known pest insect. In parallel, the peptide demonstrated attractive environmental features by having very low lytic activity on liposomes and low antimicrobial effect on gram positive and negative bacteria. It also showed very low cytotoxicity on human cell lines and high temperature stability on a short period of time. 

In Manuscript IV, oral toxicity of nemertide alpha-1 was investigated on two mosquito’s species responsible of being diseases vectors: Anopheles coluzzii and Aedes aegypti. The peptide did not display significant feeding repellence on mosquitos. It also generated fast paralysis symptoms within 10-15 min post ingestion and eventually led to death after 24h. Additional physiological effects were observed via the presence of a swollen abdomen and air bubbles in the crop. 

Natural product discovery by isolation and structure elucidation is a laborious task often requiring ample quantities of biological starting material and frequently resulting in the rediscovery of previously known compounds. However, peptides are a compound class amenable to an alternative genomic, transcriptomic, and in silico discovery route by similarity searches of known peptide sequences against sequencing data. Based on the sequences of barrettides A and B, we identified five new barrettide sequences (barrettides C-G) predicted from the North Atlantic deep-sea demosponge Geodia barretti (Geodiidae). We synthesized, folded, and investigated one of the newly described barrettides, barrettide C (NVVPCFCVEDETSGAKTCIPDNCDASRGTNP, disulfide connectivity I-IV, II-III). Co-elution experiments of synthetic and sponge-derived barrettide C confirmed its native conformation. NMR spectroscopy and the anti-biofouling activity on larval settlement of the bay barnacle Amphibalanus improvisus (IC₅₀ 0.64 μM) show that barrettide C is highly similar to barrettides A and B in both structure and function. Several lines of evidence suggest that barrettides are produced by the sponge itself and not one of its microbial symbionts.

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.