The linear venom peptide Ammogarypin has recently been described from Ammogarypus lawrencei venom. We outsourced its synthesis via solid-phase synthesis to GenScript Biotech (Rijswijk, the Netherlands). Details on the peptide as provided by the vendor: 1) Molecular weight: 4,682.5 g/mol; 2) Length: 46AA; 3) HPLC purity: 84.2%; and 4) was supplied as a lyophilized powder.

The 46 amino acid sequence of Ammogarypin was taken from the original work in which it was described (Krämer et al., 2025). The hydrophobicity was evaluated using the heliQuest online analysis tool (Gautier et al., 2008). Molecular weight, Iso-electric point and the net charge at neutral pH were calculated using PepCalc.com (Innovagen AB, Sweden). Peptide structure was predicted using Alphafold3 (Abramson et al., 2024).

To determine whether the survival rates of insects injected with varying venom concentrations differed significantly, we performed one-way fixed-effects ANOVA (Lindman, 2012). We then carried out pairwise comparisons using Tukey’s Honestly Significant Difference (HSD) test (Abdi and Williams, 2010) to identify which specific treatment groups differed significantly. All analyses were performed in the R environment version 4.5.1 (R Core Team, 2025), using the packages car (Fox et al., 2013) and multcomp (Hothorn et al., 2008).

Myzus persicae (Julius-Kühn Institute, Braunschweig, Germany) was reared on healthy 3–4-week-old turnip plants (Brassica rapa L. Tonda A Colletto Viola) in insect rearing tents (BugDorm, MegaView Science, Taiwan) inside a closed, ventilated climate chamber at constant 20 °C with a 16:8 photoperiod and relative humidity of 60%–70%. Plants were replaced regularly. The experiments were conducted using 7-day old nymphs.

For injection assays, nymphs were immobilized using a membrane pump (ILMVAC GmbH, Germany) on a custom-fabricated device constructed from a 200 µL pipette tip (Eppendorf, Germany) and Parafilm (Bemis Company, WI, United States). Exactly 15 nL was administered via microinjection at a rate of 50 nL/s dorsoventrally between the third pair of legs. Following the injection of 20 aphids per treatment, they were transferred in pools of five to Petri dishes containing 2% agar medium and a fresh potato leaf. Mortality was monitored at 1, 3, and 24 h post-injection. Three biological repetitions were conducted.

Feeding assays were carried out using a previously established in vivo assay in which a Parafilm mimics the outer leaf or stem sheets including cuticle and epidermis, and the artificial diet the phloem sap (Jancovich et al., 1997; Kirfel et al., 2020). Assays were conducted on a 24-well plate (Sarstedt AG, Germany) that was filled with small glass vials (Agilent Technologies, CA, United States) closing off the wells completely. The peptide was dissolved in nuclease-free water to achieve final concentrations of 500, 250, and 50 ng/μL in the artificial diet respectively (Jancovich et al., 1997). The vials were sealed with two layers of Parafilm (Bemis Company, Inc., WIS, United States) surrounding 20 µL of artificial diet containing yeast extract and sucrose plus 10 µL of diluted peptide. Artificial diet mixed with nuclease-free water and Imidacloprid (4 ppm) were used as controls. The aphids were monitored daily for 4 days. Raw data for all assays performed on M. persicae are available as Supplementary Table S1.

Drosophila suzukii were tested for insecticidal effects caused by the analyzed toxin as described previously (Peng et al., 2025; Fischer et al., 2024). Briefly, flies were maintained in a ventilated climate chamber at constant 26 °C with a 12:12 photoperiod and a relative humidity of 65% on a diet prepared as described previously (Abdelhafiz et al., 2025). The experiments were carried out using synchronized 3–7-day old flies post-enclosure.

Injection assays were carried out using flies that were anesthetized using CO2 and sorted by sex. We injected in three biological repetitions containing 20 females each. The solubilized peptide with concentrations of 500 ng/μL, 250 ng/μL, 100 ng/μL and 50 ng/μL were injected (46 nL vol) using a free-hand nanoinjector (Drummond Scientific, Broomall, PA, United States). Tap-water and Spinosad (25 ppm) served as negative and positive controls, respectively. Mortality was monitored at 1, 3 and 24 h post-injection.

For feeding assays, 20 flies were starved for 6 h before being transferred to a 50 mL Drosophila vial containing twelve 3 µL drops of treatment solution with a concentration of 500 ng/μL on a piece of parafilm. After 6 h and visual confirmation of consumption, the flies were moved to a new vial containing the previously described diet and monitored daily for 4 days. Three biological repetitions were conducted. Raw data for all assays performed on D. suzukii are available as Supplementary Video S1, videos of effects following injection of Ammogarypin are presented in Supplementary Table S3.

For antibacterial activity, we carried out assays as disclosed earlier (Hurka et al., 2022). Single colonies from Staphylococcus aureus DSMZ 2569 and Escherichia coli DSMZ 102053 were picked, transferred to a 15 mL cultivation tube containing 5 mL Tryptic Soy Broth (TSB) media and grown in an incubator for 24 h at 180 rpm and 37 °C. Around 2 mL of the overnight culture was transferred into a new cultivation tube containing 5 mL TSB media and grown for 3–4 h at 180 rpm and 37 °C. After incubation OD₆₀₀ was measured using a BioTek Eon microplate reader and the strains were diluted to 0.00125 for S. aureus and 0.000312 for E. coli. We seed 96-multiwell plates, in triplicate, exposing the bacteria to 200 μmol/L peptide in 100 µL medium, followed by OD₆₀₀ measurements every 20 min for 48 h following peptide exposure. The growth was normalized to the control cultures in DMSO, and the blank medium. Raw data of the antibacterial activity assay are presented in Supplementary Table S4.

Cytotoxicity was determined as previously described (Krämer et al., 2022). Peptide and ionomycin (Cayman Chemical, Ann Arbor, MI, United States) were dissolved in DMSO to create 10 mM stock solutions. MDCK II (Madin-Darby canine kidney) cells were seeded and cultured to 90% confluence in 96-well plates and treated with the compounds (at 100 µM) or DMSO for 48 h at 37 °C in a 5% CO2 atmosphere. Cell viability was determined using CellTiterGlo Luminescent Cell Viability Assay (Promega, Walldorf, Germany). Luminescence was measured in black 96-well plates in a Synergy H4 microplate reader (Biotek, Waldbronn, Germany). Relative light units (RLU) were normalized to the DMSO control set at 100%. Duplicate measurements were used to calculate the means and standard deviations. Raw data of the cytotoxicity assay are presented in Supplementary Table S5.

Hemolytic activity was assessed as described previously (Avella et al., 2024). Briefly, horse blood erythrocytes were purified by adding 900 µL DPBS (Dubelco’s Phosphate-Buffered Saline) to 100 µL of defibrinated horse blood (Thermo Fisher Scientific, Waltham, MA, United States). The cells were centrifuged for 5 min at 804 rcf and 4 °C, the supernatant was discarded and the pellet was resuspended in 1 mL of DPBS. We repeated this process 3 times until the supernatant was clear. A 1% (w/v) erythrocyte suspension in DPBS was used for further analysis. We mixed 50 µL of a 400 μmol/L solution of Ammogarypin in a 96-multiwell v-plate and incubated for 2 h at 37 °C and 130 rpm. Afterwards, the plate was centrifuged for 5 min at 804 rcf and 4 °C, and 50 µL supernatant was transferred into a new plate. We measured the hemolysis photometrically via OD₄₀₅ (Optical Density at 405 nm) in a BioTek Eon microplate reader (BioTek, Winooski, VT, United States of America). A 1% (v/v) Triton X-100 solution served as positive controls, Apis mellifera crude venom (50 μg/μL) served as biological controls and DMSO was used as negative control. All measurements were carried out in triplicates. Raw data of the hemolytic assay are presented in Supplementary Table S6.

As a first step towards the functional assessment of Ammogarypin, we employed in silico approaches to predict its physicochemical properties and structure. We used the primary structure of the mature peptide (SPVADPEAGILDTIKNVIGKVKGVITDPKVLDAVKAAIAAIKDSLK-CONH2) and subjected it to various tools in order to predict its properties. Our analysis revealed, that Ammogarypin has a hydrophobicity of 35% across its entire sequence and features a molecular weight of 4,682.5 g/mol. At neutral pH, it is a cationic peptide with a net charge of +2 and a calculated iso-electric point (pI) of 9.95. To further gather insights into its structure, we employed AI-based structural predictions via Alphafold3. This revealed that Ammogarypin is indeed a linear peptide that contains two adjacent amphipathic alpha-helical domains (Figure 1).

Under natural conditions, pseudoscorpions employ their venoms to facilitate the capture of prey which inter alia comprises small insects (Krämer et al., 2019). Therefore, to unveil the biological role of Ammogarypin, we first set out to test its activity on insect models. We selected injection assays into the dipteran Drosophila suzukii and the aphid Myzus persicae as primary assays to determine the insecticidal activity of Ammogarypin as this application is the best approximation to the natural deployment of pseudoscorpion toxins. Following injection, insects were monitored and regularly checked for a total of 24 h post-injection to capture immediate and long-term effects caused by the peptide.

In D. suzukii, we observed insecticidal activities when the peptide was administered in higher amounts. Concentrations of 500 ng/μL caused death in 27% (±8%) of flies 1 h post injection, 45% (±25%) after 3 h and 77% (±15%) after 24 h respectively (Figure 2). Lower concentrations caused little to no effects at 1 h and 3 h post injection. Only after 24 h, some marginal insecticidal activity was observed, e.g., at 50 ng/μL exhibiting 17% fatalities (±12%) (Figure 2). Besides counting living and dead insects, we monitored for effects on the insect movement to gather further clues on the function and mode of action of the peptides. Interestingly, across the tested concentrations flies exhibited signs of spastic paralysis when observed at 1 h and 3 h post injection (see Supplementary Table S3), yet in many cases recovered without succumbing to the toxic effects. Therefore, Ammogarypin appears to cause neurotoxic and sometimes fatal effects in Diptera. Next, we injected our peptide into M. persicae aphids. This experiment revealed no activity when examined after 1 h and 3 h, yet low insecticidal activity was detected after 24 h at various concentrations (Figure 2). For instance, concentrations of 500 ng/μL and 250 ng/μL caused death in 30% (±18%) and 20% (±7%) of injected aphids respectively (Figure 2). Hence, our data supports that Ammogarypin also has no detrimental effect on aphids, yet to a much lesser extent compared to the dipteran D. suzukii.

Previous works have shown that some linear pseudoscorpion toxins cause insecticidal effects when applied orally, thereby offering translational potential as insecticide (Krämer et al., 2022). To test whether Ammogarypin shows similar activity, we proceeded to further test it in feeding assays in both insect models. After feeding, D. suzukii and M. persicae were monitored for 4 days and their survival was compared to conspecifics reared on nuclease-free water or insecticides (Spinosad for D. suzukii and Imidacloprid for M. persicae) as controls. Interestingly, in D. suzukii no effects were observed even when high concentrations were administered (Figure 2). As for M. persicae, we likewise recovered only marginal effects and the calculated probability of survival was above 80% after 3 days (Figure 2). Overall, this suggests, that Ammogarypin exerts much lesser toxicity via oral uptake and, compared to known orally active pseudoscorpion toxins, these effects are much less pronounced.

The one-way fixed-effects ANOVAs revealed a significant effect of peptide concentration on fruit fly survival at 1 h post-injection (F3,8 = 4.98, p = 0.031) and at 24 h post-injection (F3,8 = 9.25, p = 0.005), but not at 3 h post-injection (F3,8 = 3.82, p = 0.057). Specifically, Tukey’s HSD test indicated a significant difference between the 500 and 100 ng/μL concentrations at 1 h post-injection (p = 0.036), and between the 500 ng/μL concentration and all other venom concentrations at 24 h post-injection (250 ng/μL: p = 0.01; 100 ng/μL: p = 0.009; 50 ng/μL: p = 0.016). Meanwhile, the one-way fixed-effects ANOVAs revealed no significant effect of peptide concentration on aphid survival at all measured time points. Details of the results of the performed statistical tests are provided in Supplementary Tables S7, S8.

In the past it has been shown, that some linear pseudoscorpion toxins exert potent antimicrobial effects against prokaryotes (Krämer et al., 2022; Erkoc et al., 2024). Therefore, antimicrobial screening is an important element in the bioactivity profiling of these peptides. In response, we carried out exploratory screenings to test the toxins effects against two selected bacterial strains, the gram-negative E. coli DSMZ 102053 and gram-positive S. aureus DSMZ 2569. Both bacterial strains were cultured in a medium containing Ammogarypin at 200 μmol/L and their growth was recorded photometrically using the OD₆₀₀ method. The peptide exhibited a weak effect on the growth of gram-negative E. coli (Figure 3A), and no observable effect on gram-positive S. aureus (Figure 3B). However, in contrast to other linear pseudoscorpion venom peptides, their activity seems to be of much lesser potency.

Linear pseudoscorpion venom peptides potentially exert their activities via interaction with lipid bilayers of biomembranes (Erkoc et al., 2024; Pukala et al., 2007; Jenssen et al., 2006). Typical consequences are cytotoxicity and/or hemolytic activity by disruption of membranes and can ultimately lead to cell death (Erkoc et al., 2024; Krämer et al., 2022; Pukala et al., 2007; Dersch et al., 2024). On one hand, these effects could be biologically relevant, e.g., by allowing the peptides to serve as spreading factors or defensive components. However, understanding these aspects is equally important from a translational perspective. Both represent pivotal and unwanted side effects to be explored in preclinical screening of venom peptides and previously other linear pseudoscorpion venom peptides were already shown to cause cytotoxicity and hemolysis. Hence, we next set out to study effects on mammalian cells caused by Ammogarypin. In order to assess the cytotoxic activity of Ammogarypin, a photometric CellTiterGlo Luminescent Cell Viability Assay was conducted on mammalian MDCKII cells (Figure 4A). Interestingly, at neither of the tested concentrations the peptide caused cytotoxicity. To further evaluate the peptides’ capability to damage mammalian cells, we tested it against equine erythrocytes. Reminiscent to our cytotoxicity screening, we did not observe effects against this cell type (Figure 4B). Therefore our in vitro assessment targeting cellular structures suggests that Ammogarypin does not exhibit lytic activity on vertebrate cells. With that, it differs profoundly from many other linear pseudoscorpion venom peptides.