Establishing superfine nanofibrils for robust polyelectrolyte artificial spider silk and powerful artificial muscles.

Spider silk exhibits an excellent combination of high strength and toughness, which originates from the hierarchical self-assembled structure of spidroin during fiber spinning. In this work, superfine nanofibrils are established in polyelectrolyte artificial spider silk by optimizing the flexibility of polymer chains, which exhibits combination of breaking strength and toughness ranging from 1.83 GPa and 238 MJ m-3 to 0.53 GPa and 700 MJ m-3, respectively. This is achieved by introducing ions to control the dissociation of polymer chains and evaporation-induced self-assembly under external stress. In addition, the artificial spider silk possesses thermally-driven supercontraction ability. This work provides inspiration for the design of high-performance fiber materials.

A review on complete silk gene sequencing and de novo assembly of artificial silk.

Silk, especially spider and insect silk, is a highly versatile biomaterial with potential applications in biomedicine, materials science, and biomimetic engineering. The primary structure of silk proteins is the basis for the mechanical properties of silk fibers. Biotechnologies such as single-molecule sequencing have facilitated an increasing number of reports on new silk genes and assembled silk proteins. Therefore, this review aims to provide a comprehensive overview of the recent advances in representative spider and insect silk proteins, focusing on identification methods, sequence characteristics, and de novo design and assembly. The review discusses three identification methods for silk genes: polymerase chain reaction (PCR)-based sequencing, PCR-free cloning and sequencing, and whole-genome sequencing. Moreover, it reveals the main spider and insect silk proteins and their sequences. Subsequent de novo assembly of artificial silk is covered and future research directions in the field of silk proteins, including new silk genes, customizable artificial silk, and the expansion of silk production and applications are discussed. This review provides a basis for the genetic aspects of silk production and the potential applications of artificial silk in material science and biomedical engineering.

Role of ErbB and IL-1 signaling pathways in the dermonecrotic lesion induced by Loxosceles sphingomyelinases D.

Sphingomyelinase D (SMase D), the main toxic component of Loxosceles venom, has a well-documented role on dermonecrotic lesion triggered by envenomation with these species; however, the intracellular mechanisms involved in this event are still poorly known. Through differential transcriptomics of human keratinocytes treated with L. laeta or L. intermedia SMases D, we identified 323 DEGs, common to both treatments, as well as upregulation of molecules involved in the IL-1 and ErbB signaling. Since these pathways are related to inflammation and wound healing, respectively, we investigated the relative expression of some molecules related to these pathways by RT-qPCR and observed different expression profiles over time. Although, after 24 h of treatment, both SMases D induced similar modulation of these pathways in keratinocytes, L. intermedia SMase D induced earlier modulation compared to L. laeta SMase D treatment. Positive expression correlations of the molecules involved in the IL-1 signaling were also observed after SMases D treatment, confirming their inflammatory action. In addition, we detected higher relative expression of the inhibitor of the ErbB signaling pathway, ERRFI1, and positive correlations between this molecule and pro-inflammatory mediators after SMases D treatment. Thus, herein, we describe the cell pathways related to the exacerbation of inflammation and to the failure of the wound healing, highlighting the contribution of the IL-1 signaling pathway and the ERRFI1 for the development of cutaneous loxoscelism.

Identification of a common epitope in knottins and phospholipases D present in Loxosceles sp venom by a monoclonal antibody.

In the Americas and specially in Brazil, the Loxosceles intermedia, Loxosceles gaucho and Loxosceles laeta are the three most medically relevant brown spider species, and whose bites can lead to the condition known as loxoscelism. Here, we report the development of a tool capable of identifying a common epitope amongst Loxosceles sp. venom's toxins. A murine monoclonal antibody (LmAb12) and its recombinant fragments (scFv12P and diabody12P) have been produced and characterized. This antibody and its recombinant constructs were able to recognize proteins of Loxosceles spider venoms with specificity. The scFv12P variant was also able to detect low concentrations of Loxosceles venom in a competitive ELISA assay, displaying potential as a venom identification tool. The primary antigenic target of LmAb12 is a knottin, a venom neurotoxin, that has a shared identity of 100 % between the L. intermedia and L. gaucho species and high similarity to L. laeta. Furthermore, we observed LmAb12 was able to partially inhibit in vitro hemolysis, a cellular event typically induced by the Loxosceles sp. venoms. Such behavior might be due to LmAb12 cross-reactivity between the antigenic target of LmAb12 and the venom's dermonecrotic toxins, the PLDs, or even the existence of synergism between these two toxins.

Disabling spidroin N-terminal homologs' reverse reaction unveils why its intermolecular disulfide bonds have not evolved for 380 million years.

Spiders, ubiquitous predators known for their powerful silks, rely on spidroins that self-assemble from high-concentration solutions stored in silk glands, which are mediated by the NT and CT domains. CT homodimers containing intermolecular disulfide bonds enhance silk performance, promoting spider survival and reproduction. However, no NT capable of forming such disulfide bonds has been identified. Our study reveals that NT homodimers with sulfur substitution can form under alkaline conditions, shedding light on why spiders have not evolved intermolecular disulfide bonds in the NT module during their 380 million years of evolution. This discovery significantly advances our comprehension of spider evolution and silk spinning mechanisms, while also providing novel insights into protein storage, assembly, as well as the mechanisms and therapeutic strategies for neurodegenerative diseases associated with protein aggregation.

Recombinant Production, NMR Solution Structure, and Membrane Interaction of the Phα1ß Toxin, a TRPA1 Modulator from the Brazilian Armed Spider Phoneutria nigriventer.

Phα1ß (PnTx3-6) is a neurotoxin from the spider Phoneutria nigriventer venom, originally identified as an antagonist of two ion channels involved in nociception: N-type voltage-gated calcium channel (CaV2.2) and TRPA1. In animal models, Phα1ß administration reduces both acute and chronic pain. Here, we report the efficient bacterial expression system for the recombinant production of Phα1ß and its 15N-labeled analogue. Spatial structure and dynamics of Phα1ß were determined via NMR spectroscopy. The N-terminal domain (Ala1-Ala40) contains the inhibitor cystine knot (ICK or knottin) motif, which is common to spider neurotoxins. The C-terminal α-helix (Asn41-Cys52) stapled to ICK by two disulfides exhibits the µs-ms time-scale fluctuations. The Phα1ß structure with the disulfide bond patterns Cys1-5, Cys2-7, Cys3-12, Cys4-10, Cys6-11, Cys8-9 is the first spider knottin with six disulfide bridges in one ICK domain, and is a good reference to other toxins from the ctenitoxin family. Phα1ß has a large hydrophobic region on its surface and demonstrates a moderate affinity for partially anionic lipid vesicles at low salt conditions. Surprisingly, 10 µM Phα1ß significantly increases the amplitude of diclofenac-evoked currents and does not affect the allyl isothiocyanate (AITC)-evoked currents through the rat TRPA1 channel expressed in Xenopus oocytes. Targeting several unrelated ion channels, membrane binding, and the modulation of TRPA1 channel activity allow for considering Phα1ß as a gating modifier toxin, probably interacting with S1-S4 gating domains from a membrane-bound state.

Functional Profiling of the A-Family of Venom Peptides from the Wolf Spider Lycosa shansia.

The venoms of spiders from the RTA (retro-lateral tibia apophysis) clade contain diverse short linear peptides (SLPs) that offer a rich source of therapeutic candidates. Many of these peptides have insecticidal, antimicrobial and/or cytolytic activities, but their biological functions are unclear. Here, we explore the bioactivity of all known members of the A-family of SLPs previously identified in the venom of the Chinese wolf spider (Lycosa shansia). Our broad approach included an in silico analysis of physicochemical properties and bioactivity profiling for cytotoxic, antiviral, insecticidal and antibacterial activities. We found that most members of the A-family can form α-helices and resemble the antibacterial peptides found in frog poison. The peptides we tested showed no cytotoxic, antiviral or insecticidal activities but were able to reduce the growth of bacteria, including clinically relevant strains of Staphylococcus epidermidis and Listeria monocytogenes. The absence of insecticidal activity may suggest that these peptides have no role in prey capture, but their antibacterial activity may help to defend the venom gland against infection.

Biochemical mechanism involved in the enhancement of the Young's modulus of silk by the SpiCE protein.

Silk fibers are known for their superior mechanical properties, with the strongest possessing over seven times the toughness of kevlar. Recently, low molecular weight non-spidroin protein, spider-silk constituting element (SpiCE), has been reported to enhance the mechanical properties of silk; however, its specific action mechanism has not yet been elucidated. Here, we explored the mechanism by which SpiCE strengthened the mechanical properties of major ampullate spidroin 2 (MaSp2) silk through hydrogen bonds and salt bridges of the silk structure via all-atom molecular dynamics simulations. Tensile pulling simulation on silk fiber with SpiCE protein revealed that the SpiCE protein enhanced the Young's modulus by up to 40% more than that of the wild type. Bond characteristic analysis revealed that SpiCE and MaSp2 formed more hydrogen bonds and salt bridges than the MaSp2 wild-type model. Sequence analysis of MaSp2 silk fiber and SpiCE protein revealed that SpiCE protein contained more amino acids that could act as hydrogen bond acceptors/donors and salt bridge partners. Our results provide insights into the mechanism by which non-spidroin proteins strengthen the properties of silk fibers and lay the groundwork for the development of material selection criteria for the design of de novo artificial silk fibers.

Reference genome of the long-jawed orb-weaver, Tetragnatha versicolor (Araneae: Tetragnathidae).

Climate-driven changes in hydrological regimes are of global importance and are particularly significant in riparian ecosystems. Riparian ecosystems in California provide refuge to many native and vulnerable species within a xeric landscape. California Tetragnatha spiders play a key role in riparian ecosystems, serving as a link between terrestrial and aquatic elements. Their tight reliance on water paired with the widespread distributions of many species make them ideal candidates to better understand the relative role of waterways versus geographic distance in shaping the population structure of riparian species. To assist in better understanding population structure, we constructed a reference genome assembly for Tetragnatha versicolor using long-read sequencing, scaffolded with proximity ligation Omni-C data. The near-chromosome-level assembly is comprised of 174 scaffolds spanning 1.06 Gb pairs, with a scaffold N50 of 64.1 Mb pairs and BUSCO completeness of 97.6%. This reference genome will facilitate future study of T. versicolor population structure associated with the rapidly changing environment of California.

Subsidy Quality Affects Common Riparian Web-Building Spiders: Consequences of Aquatic Contamination and Food Resource.

Anthropogenic stressors can affect the emergence of aquatic insects. These insects link aquatic and adjacent terrestrial food webs, serving as high-quality subsidy to terrestrial consumers, such as spiders. While previous studies have demonstrated that changes in the emergence biomass and timing may propagate across ecosystem boundaries, the physiological consequences of altered subsidy quality for spiders are largely unknown. We used a model food chain to study the potential effects of subsidy quality: Tetragnatha spp. were exclusively fed with emergent Chironomus riparius cultured in the absence or presence of either copper (Cu), Bacillus thuringiensis var. israelensis (Bti), or a mixture of synthetic pesticides paired with two basal resources (Spirulina vs. TetraMin®) of differing quality in terms of fatty acid (FA) composition. Basal resources shaped the FA profile of chironomids, whereas their effect on the FA profile of spiders decreased, presumably due to the capacity of both chironomids and spiders to modify (dietary) FA. In contrast, aquatic contaminants had negligible effects on prey FA profiles but reduced the content of physiologically important polyunsaturated FAs, such as 20:4n-6 (arachidonic acid) and 20:5n-3 (eicosapentaenoic acid), in spiders by approximately 30% in Cu and Bti treatments. This may have contributed to the statistically significant decline (40%-50%) in spider growth. The observed effects in spiders are likely related to prey nutritional quality because biomass consumption by spiders was, because of our experimental design, constant. Analyses of additional parameters that describe the nutritional quality for consumers such as proteins, carbohydrates, and the retention of contaminants may shed further light on the underlying mechanisms. Our results highlight that aquatic contaminants can affect the physiology of riparian spiders, likely by altering subsidy quality, with potential implications for terrestrial food webs. Environ Toxicol Chem 2023;42:1346-1358. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Methods for Silk Property Analyses across Structural Hierarchies and Scales.

Silk from silkworms and spiders is an exceptionally important natural material, inspiring a range of new products and applications due to its high strength, elasticity, and toughness at low density, as well as its unique conductive and optical properties. Transgenic and recombinant technologies offer great promise for the scaled-up production of new silkworm- and spider-silk-inspired fibres. However, despite considerable effort, producing an artificial silk that recaptures the physico-chemical properties of naturally spun silk has thus far proven elusive. The mechanical, biochemical, and other properties of pre-and post-development fibres accordingly should be determined across scales and structural hierarchies whenever feasible. We have herein reviewed and made recommendations on some of those practices for measuring the bulk fibre properties; skin-core structures; and the primary, secondary, and tertiary structures of silk proteins and the properties of dopes and their proteins. We thereupon examine emerging methodologies and make assessments on how they might be utilized to realize the goal of developing high quality bio-inspired fibres.

Structural features, intrinsic disorder, and modularity of a pyriform spidroin 1 core repetitive domain.

Orb-weaving spiders produce up to seven silk types, each with distinct biological roles, protein compositions, and mechanics. Pyriform (or piriform) silk is composed of pyriform spidroin 1 (PySp1) and is the fibrillar component of attachment discs that attach webs to substrates and to each other. Here, we characterize the 234-residue repeat unit (the "Py unit") from the core repetitive domain of Argiope argentata PySp1. Solution-state nuclear magnetic resonance (NMR) spectroscopy-based backbone chemical shift and dynamics analysis demonstrate a structured core flanked by disordered tails, structuring that is maintained in a tandem protein of two connected Py units, indicative of structural modularity of the Py unit in the context of the repetitive domain. Notably, AlphaFold2 predicts the Py unit structure with low confidence, echoing low confidence and poor agreement to the NMR-derived structure for the Argiope trifasciata aciniform spidroin (AcSp1) repeat unit. Rational truncation, validated through NMR spectroscopy, provided a 144-residue construct retaining the Py unit core fold, enabling near-complete backbone and side chain 1H, 13C, and 15N resonance assignment. A six α-helix globular core is inferred, flanked by regions of intrinsic disorder that would link helical bundles in tandem repeat proteins in a beads-on-a-string architecture.

Mesoscale Confinement in Bagworm Silk: A Hidden Structural Organization.

While silk fibers produced by silkworms and spiders are frequently described as a network of amorphous protein chains reinforced by crystalline ß-sheet nanodomains, the importance of higher-order, self-assembled structures has been recognized for advanced modeling of mechanical properties. General acceptance of hierarchical structural models is, however, currently limited by lack of experimental results. Indeed, X-ray scattering studies of spider's dragline-type fibers have been particularly limited by low crystallinities. Here we are reporting on probing the local structure of exceptionally crystalline bagworm silk fibers by X-ray nanobeam scattering. Probing the comparable thickness of cross sections with an X-ray nanobeam allows removing the strong scattering background from the outer sericin layer and reveals a hidden structural organization due to a radial gradient in diameters of mesoscale nanofibrillar bundles in the fibroin phase. Our results provide direct support for lateral interactions between nanofibrils.

Biomolecular Click Reactions Using a Minimal pH-Activated Catcher/Tag Pair for Producing Native-Sized Spider-Silk Proteins.

A type of protein/peptide pair known as Catcher/Tag pair spontaneously forms an intermolecular isopeptide bond which can be applied for biomolecular click reactions. Covalent protein conjugation using Catcher/Tag pairs has turned out to be a valuable tool in biotechnology and biomedicines, but it is essential to increase the current toolbox of orthogonal Catcher/Tag pairs to expand the range of applications further, for example, for controlled multiple-fragment ligation. We report here the engineering of novel Catcher/Tag pairs for protein ligation, aided by a crystal structure of a minimal CnaB domain from Lactobacillus plantarum. We show that a newly engineered pair, called SilkCatcher/Tag enables efficient pH-inducible protein ligation in addition to being compatible with the widely used SpyCatcher/Tag pair. Finally, we demonstrate the use of the SilkCatcher/Tag pair in the production of native-sized highly repetitive spider-silk-like proteins with >90 % purity, which is not possible by traditional recombinant production methods.

A secretory system for extracellular production of spider neurotoxin huwentoxin-I in Escherichia coli.

Due to their advantages in structural stability and versatility, cysteine-rich peptides, which are secreted from the venom glands of venomous animals, constitute a naturally occurring pharmaceutical arsenal. However, the correct folding of disulfide bonds is a challenging task in the prokaryotic expression system like Escherichia coli due to the reducing environment. Here, a secretory expression plasmid pSE-G1M5-SUMO-HWTX-I for the spider neurotoxin huwentoxin-I (HWTX-I) with three disulfides as a model of cysteine-rich peptides was constructed. By utilizing the signal peptide G1M5, the fusion protein 6 × His-SUMO-HWTX-I was successfully secreted into extracellular medium of BL21(DE3). After enrichment using cation-exchange chromatography and purification utilizing the Ni-NTA column, 6 × His-SUMO-HWTX-I was digested via Ulp1 kinase to release recombinant HWTX-I (rHWTX-I), which was further purified utilizing RP-HPLC. Finally, both impurities with low and high molecular weights were completely removed. The molecular mass of rHWTX-I was identified as being 3750.8 Da, which was identical to natural HWTX-I with three disulfide bridges. Furthermore, by utilizing whole-cell patch clamp, the sodium currents of hNav1.7 could be inhibited by rHWTX-I and the IC50 value was 419 nmol/L.

Riparian Spiders: Sentinels of Polychlorinated Dibenzo-p-dioxin and Dibenzofuran-Contaminated Sediment.

Polychlorinated dibenzo-p-dioxin and polychlorinated dibenzofuran (PCDD/F) are persistent, toxic, and bioaccumulative. Currently, PCDD/F monitoring programs primarily use fish and birds with potentially large home ranges to monitor temporal trends over broad spatial scales; sentinel organisms that provide targeted sediment contaminant information across small geographic areas have yet to be developed. Riparian orb-weaving spiders, which typically have small home ranges and consume primarily adult aquatic insects, are potential PCDD/F sentinels. Recent studies have demonstrated that spider tissue concentrations indicate the source and magnitude of dioxin-like chlorinated compounds in contaminated sediments, including polychlorinated biphenyls (PCBs). Our aim in the present study was to assess the utility of riparian spiders as sentinels for PCDD/F-contaminated sediments. We measured PCDD/F (total [Σ] and homologs) in surface sediments and spiders collected from three sites within the St. Louis River basin (Minnesota and Wisconsin, USA). We then compared (1) patterns in ΣPCDD/F concentrations between sediment and spiders, (2) the distribution of homologs within sediments and spiders when pooled across sites, and (3) the relationship between sediment and spider concentrations of PCDD/F homologs across 13 stations sampled across the three sites. The ΣPCDD/F concentrations in sediment (mean ± standard error 286 591 ± 97 614 pg/g) were significantly higher than those in riparian spiders (2463 ± 977 pg/g, p < 0.001), but the relative abundance of homologs in sediment and spiders were not significantly different. Spider homolog concentrations were significantly and positively correlated with sediment concentrations across a gradient of sediment PCDD/F contamination (R2 = 0.47, p < 0.001). Our results indicate that, as has been shown for other legacy organic chemicals like PCBs, riparian spiders are suitable sentinels of PCDD/F in contaminated sediment. Environ Toxicol Chem 2023;42:414-420. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Complete gene sequence and mechanical property of the fourth type of major ampullate silk protein.

Spiders spin a great diversity of silk types for daily survival and reproduction. Of the six orb-weaver silk types, the dragline silk forming orb web frame attracts the most attention because of its extremely high tensile strength and toughness. So far, four types of major ampullate silk proteins (MaSp1-4) that make up dragline silk have been identified. These MaSp types have diversified amino acid motifs that underlie the impressive mechanical property of dragline silk by forming particular structures. Existing knowledge of MaSp4 proteins is fragmented, making it difficult to illuminate the structure and function of MaSp4. Here, we report the full-length MaSp4 gene with 11,334 bp from the orb-weaving spider Araneus ventricosus. Removing the only intron, the spliced complete transcript of MaSp4 gene is 6897 bp and encodes 2298 amino acids. Analysis of the primary structure of A. ventricosus MaSp4 protein reveals the repetitive region lacks poly-A and GGX motifs but has the unique GPGPQ motifs. Quantitative real-time PCR analyses show high levels of MaSp4 mRNA were detected in major ampullate gland. Structural characterization using CD- and FTIR sepctroscopy reveals a mainly α-helical solution conformation and a very high ß-turn content within fibers. Collectively, our new findings provide complete template for recombinant silk protein with specific properties and support that the GPGPQ motif found in MaSp4 could increase flexibility in dragline silk by packing in more ß-turns, expanding the repertoire of sequences known to form ß-turn that is available for artificial chimeric silk fibers. STATEMENT OF SIGNIFICANCE: Dragline silk forming orb web frame attracts the most attention because of its extremely high tensile strength and toughness. So far, four types of major ampullate silk proteins (MaSp1-4) that make up dragline silk have been identified. Existing knowledge of MaSp4 proteins is fragmented, making it difficult to illuminate the structure and function of MaSp4. Here, we report the full-length MaSp4 gene from the orb-weaving spider Araneus ventricosus. We further identify the sequence, structure, and mechanical property of MaSp4 protein, providing a new insight into the structure-funtion relationships associated with MaSp4. Collectively, our new findings provide complete template for recombinant silk protein with specific properties and support that the GPGPQ motif found in MaSp4 could increase flexibility in dragline silk by packing in more ß-turns, expanding the repertoire of sequences known to form ß-turn that is available for artificial chimeric silk fibers.

Composition and toxicity of venom produced by araneophagous white-tailed spiders (Lamponidae: Lampona sp.).

Prey-specialised spiders are adapted to capture specific prey items, including dangerous prey. The venoms of specialists are often prey-specific and less complex than those of generalists, but their venom composition has not been studied in detail. Here, we investigated the venom of the prey-specialised white-tailed spiders (Lamponidae: Lampona), which utilise specialised morphological and behavioural adaptations to capture spider prey. We analysed the venom composition using proteo-transcriptomics and taxon-specific toxicity using venom bioassays. Our analysis identified 208 putative toxin sequences, comprising 103 peptides < 10 kDa and 105 proteins > 10 kDa. Most peptides belonged to one of two families characterised by scaffolds containing eight or ten cysteine residues. Toxin-like proteins showed similarity to galectins, leucine-rich repeat proteins, trypsins and neprilysins. The venom of Lampona was shown to be more potent against the preferred spider prey than against alternative cricket prey. In contrast, the venom of a related generalist was similarly potent against both prey types. These data provide insights into the molecular adaptations of venoms produced by prey-specialised spiders.

Extended phenotypes can underlie trade-offs: a case of social spiders.

Extended phenotypes engineered by animals can potentially improve safety and/or foraging. Whether the well-known trade-off between safety and foraging applies for extended phenotypes, and if so, how it is resolved has not been determined. Spiders build elaborate silk structures that serve as traps for their insect prey and often attach silken retreats (nests) to their capture webs. These extended phenotypes of spiders are made of silk that is considered costly since it is made of protein. Using the Indian social spider, Stegodyphus sarasinorum, we examined how simple proximal factors, like colony hunger state and group size, shape trade-offs in collectively built extended phenotypes that offer shelter and food. We found that well-fed colonies showed greater investment in retreat silk than starved colonies. However, the two groups did not differ in their investment in capture webs. Hence, our findings validate the starvation-risk taking hypothesis in an extended phenotypic paradigm by showing that hungry colonies trade-off retreat size for capture web, irrespective of group size.

Evaluation of recombinant toxin JFTX-23, an oral-effective anti-insect peptide from the spider Selenocosmia jiafu venom gland proteome.

Excessive utilization of chemical pesticides for pest control can lead to adverse consequences for the health of humans and other organisms and may also cause irreversible ecological changes; therefore, the use of biologically derived insecticides can be a safe alternate strategy. Transcriptomic studies have shown JFTX- 23,a small peptide from the spider Selenocosmia jiafuis highly similar to U1-TRTX-Sp1, a well-characterized oral-effective insecticide toxin from the Australian tarantula Selenotypus plumipes. First, we evaluated the JFTX-23 peptide sequence using bioinformatics tools and modeling studies. Preliminary results showed a high similarity of JFTX-23 to JZTX-58 (91.67%) and U1-TRTX-Sp1 (86.11%). Superimposition of the α-carbons of the modeled JFTX-23 and U1-TRTX-Sp1 demonstrated a very high similarity of the 3-D structure of the two peptides (RMSD of 0.02 Å).The injection assay of JFTX-23 in Helicoverpa armigera indicated an LD50 of 0.077 and 0.423 nmol/insect after 24 and 120 h, respectively. JFTX-23 was toxic to H. armigera via oral administration with an LC50 of 1.16 nmol/g food after 5 days, which was comparable to the toxicity of the oral-effective toxin U1-TRTX-Sp1. Our studies have shown that JFTX-23 is a potent oral-effective toxin that can be considered an attractive candidate for the biological control of insect pests.