Transfer and bioaccumulation of pesticides in terrestrial arthropods and food webs: State of knowledge and perspectives for research.

Arthropods represent an entry point for pesticide transfers in terrestrial food webs, and pesticide accumulation in upper chain organisms, such as predators can have cascading consequences on ecosystems. However, the mechanisms driving pesticide transfer and bioaccumulation in food webs remain poorly understood. Here we review the literature on pesticide transfers mediated by terrestrial arthropods in food webs. The transfer of pesticides and their potential for bioaccumulation and biomagnification are related to the chemical properties and toxicokinetic of the substances, the resistance and detoxification abilities of the contaminated organisms, as well as by their effects on organisms' life history traits. We further identify four critical areas in which knowledge gain would improve future predictions of pesticides impacts on terrestrial food webs. First, efforts should be made regarding the effects of co-formulants and pesticides mixtures that are currently understudied. Second, progress in the sensitivity of analytical methods would allow the detection of low concentrations of pesticides in small individual arthropods. Quantifying pesticides in arthropods preys, their predators, and arthropods or vertebrates at higher trophic level would bring crucial insights into the bioaccumulation and biomagnification potential of pesticides in real-world terrestrial food webs. Finally, quantifying the influence of the trophic structure and complexity of communities on the transfer of pesticides could address several important sources of variability in bioaccumulation and biomagnification across species and food webs. This narrative review will inspire future studies aiming to quantify pesticide transfers in terrestrial food webs to better capture their ecological consequences in natural and cultivated landscapes.

Biotic Ligand Modeling for the Effect of Major Cations on the Uptake of Cadmium in Folsomia candida Exposed in a Sand-Solution Medium.

Biotic ligand modeling (BLM) approaches are already applied to predict the bioavailability and possible risk of metals in surface water, but need further development for soils. The present study investigated the effect of major cations (Ca2+, Mg2+, Na+, K+, and H+) on cadmium bioaccumulation in the springtail Folsomia candida. To avoid the complexity of real soils and enable control of elemental speciation in the exposure medium, the animals were exposed to different cadmium concentrations in an inert quartz sand-solution medium. Accumulation of cadmium in the animals was measured after 7 days exposure at different cation concentrations. Among the cations, only Ca2+ significantly affected the uptake of cadmium in the springtails. Mg2+ also had higher effects compared with other selected cations. Using a BLM approach, the uptake of cadmium in the animals predicted by taking into account both Ca2+ and Mg2+ activities correlated well with the measured values (R2 = 0.68). The final estimated conditional binding constants for cadmium (log KCd-BL), Ca (log KCa-BL), and Mg (log KMg-BL) of 1.06, 2.14, and 1.23 L/mol, respectively, were in agreement with previously reported values. The match between predicted and measured uptake data confirms the applicability and usefulness of the BLM for predicting the bioavailability of cadmium to springtails and opens the way for its application in soil. Environ Toxicol Chem 2024;43:1090-1096. © 2024 SETAC.

Polyproline type II helical antifreeze proteins are widespread in Collembola and likely originated over 400 million years ago in the Ordovician Period.

Antifreeze proteins (AFPs) bind to ice crystals to prevent organisms from freezing. A diversity of AFP folds has been found in fish and insects, including alpha helices, globular proteins, and several different beta solenoids. But the variety of AFPs in flightless arthropods, like Collembola, has not yet been adequately assessed. Here, antifreeze activity was shown to be present in 18 of the 22 species of Collembola from cold or temperate zones. Several methods were used to characterize these AFPs, including isolation by ice affinity purification, MALDI mass spectrometry, amino acid composition analysis, tandem mass spectrometry sequencing, transcriptome sequencing, and bioinformatic investigations of sequence databases. All of these AFPs had a high glycine content and were predicted to have the same polyproline type II helical bundle fold, a fold unique to Collembola. These Hexapods arose in the Ordovician Period with the two orders known to produce AFPs diverging around 400 million years ago during the Andean-Saharan Ice Age. Therefore, it is likely that the AFP arose then and persisted in many lineages through the following two ice ages and intervening warm periods, unlike the AFPs of fish which arose independently during the Cenozoic Ice Age beginning ~ 30 million years ago.

Harnessing the functional diversity of plant cystatins to design inhibitor variants highly active against herbivorous arthropod digestive proteases.

Protein engineering approaches have been proposed to improve the inhibitory properties of plant cystatins against herbivorous arthropod digestive proteases, generally involving the site-directed mutagenesis of functionally relevant amino acids or the selection of improved inhibitor variants by phage display approaches. Here, we propose a novel approach where the function-related structural elements of a cystatin are substituted by the corresponding elements of an alternative cystatin. Inhibitory assays were first performed with 20 representative plant cystatins and model Cys proteases, including arthropod proteases, to appreciate the extent of functional variability among the plant cystatin family. The most, and less, potent of these cystatins were then used as 'donors' of structural elements to create hybrids of tomato cystatin SlCYS8 used as a model 'recipient' inhibitor. In brief, inhibitory activities against Cys proteases strongly differed from one plant cystatin to another, with Ki (papain) values diverging by more than 30-fold and inhibitory rates against arthropod proteases varying by up to 50-fold depending on the enzymes assessed. In line with theoretical assumptions from docking models generated for different Cys protease-cystatin combinations, structural element substitutions had a strong impact on the activity of recipient cystatin SlCYS8, positive or negative depending on the basic inhibitory potency of the donor cystatin. Our data confirm the wide variety of cystatin inhibitory profiles among plant taxa. They also demonstrate the usefulness of these proteins as a pool of discrete structural elements for the design of cystatin variants with improved potency against herbivorous pest digestive Cys proteases.

Beauvericin potentiates the activity of pesticides by neutralizing the ATP-binding cassette transporters in arthropods.

Multi-drug resistance is posing major challenges in suppressing the population of pests. Many herbivores develop resistance, causing a prolonged survival after exposure to a previously effective pesticide. Consequently, resistant pests reduce the yield of agricultural production, causing significant economic losses and reducing food security. Therefore, overpowering resistance acquisition of crop pests is a must. The ATP binding cassette transporters (ABC transporters) are considered as the main participants to the pesticide efflux and their neutralization will greatly contribute to potentiate failed treatments. Real-Time PCR analysis of 19 ABC transporter genes belonging to the ABCB, ABCC, ABCG, and ABCH revealed that a broad range of efflux pumps is activated in response to the exposure to pesticides. In this study, we used beauvericin (BEA), a known ABC transporters modulator, to resensitize different strains of Tetranychus urticae after artificial selection for resistance to cyflumetofen, bifenazate, and abamectin. Our results showed that the combinatorial treatment of pesticide (manufacturer's recommended doses) + BEA (sublethal doses: 0.15 mg/L) significantly suppressed the resistant populations of T. urticae when compared to single-drug treatments. Moreover, after selective pressure for 40 generations, the LC50 values were significantly reduced from 36.5, 44.7, and 94.5 (pesticide) to 8.3, 12.5, and 23.4 (pesticide + BEA) for cyflumetofen, bifenazate, and abamectin, respectively. While the downstream targets for BEA are still elusive, we demonstrated hereby that it synergizes with sub-lethal doses of different pesticides and increases their effect by inhibiting ABC transporters. This is the first report to document such combinatorial activity of BEA against higher invertebrates paving the way for its usage in treating refractory cases of resistance to pesticides. Moreover, we demonstrated, for the first time, using in silico techniques, the higher affinity of BEA to ABC transformers subfamilies when compared to xenobiotics; thus, elucidating the pathway of the mycotoxin.

Initial defensive secretory compounds emitted from the live millipede and the induction of apoptotic cell death.

The initial defensive secretory compounds emitted from a live millipede have not yet been clarified. This study focused on elucidating the initial secretory compounds emitted from a live millipede. Pre-concentration of the defensive secretory volatile organic compounds (VOC) from the live Polidesmida millipedes, Chamberlinius hualienensis and Oxidus gracilis, was performed using a three-stage VOC concentration technique by an on-line GC/MS system. As a result, the monoterpenes derived from the plant metabolite; i.e., α-pinene, α-thujene, ß-pinene, 3-carene, ß-myrcene, ß-phellandrene, γ-terpinene, o,m,p-cymenes, limonene and camphene were first detected as the initial secretory substances. It was elucidated that some plant monoterpenes have a repellent effect and antifungal and antibacterial actions which are used as defensive substances. In addition, this study also confirmed that these monoterpenes induced apoptotic cell death involved in the induction of the caspase 3/7 activity. The millipede feeds on fallen or withered leaves containing the monoterpenes. Thus, the millipede accumulates the plant defensive secretions in the exocrine defense glands of the body somites, which would be used as against predators.

Crystal waters on the nine polyproline type II helical bundle springtail antifreeze protein from Granisotoma rainieri match the ice lattice.

A springtail (Collembola) identified as Granisotoma rainieri was collected from snow in Hokkaido, Japan, in late winter when nighttime temperatures were below zero. Extracts of these arthropods showed antifreeze activity by shaping ice crystals and stopping their growth. The glycine-rich proteins responsible for this freezing point depression were isolated by ice-affinity purification and had principal masses of ~ 6.9 and 9.6 kDa. We identified a transcript for a 9.6-kDa component and produced it as a His-tagged recombinant protein for structural analysis. Its crystal structure was solved to a resolution of 1.21 Å and revealed a polyproline type II helical bundle, similar to the six-helix Hypogastrura harveyi AFP, but with nine helices organized into two layers held together by an extensive network of hydrogen bonds. One of the layers is flat, regular, and hydrophobic and likely serves as the ice-binding side. Although this surface makes close protein-protein contacts with its symmetry mate in the crystal, it has bound chains of waters present that resemble those on the basal and primary prism planes of ice. Molecular dynamic simulations indicate most of these crystal waters would preferentially occupy these sites if exposed to bulk solvent in the absence of the symmetry mate. These prepositioned waters lend further support to the ice-binding mechanism in which AFPs organize ice-like waters on one surface to adsorb to ice. DATABASES: Structural data are available in the Protein Data Bank under the accession number 7JJV. Transcript data are available in GenBank under accession numbers MT780727, MT780728, MT780729, MT780730, MT780731 and MT985982.

First characterization of multixenobiotic activity in Collembola: An approach on cadmium-induced response.

ATP-binding cassette (ABC) efflux pumps mediate the activity of the Multixenobiotic Resistance (MXR) mechanism and have been proposed as a biomarker of environmental pollution mainly in aquatic invertebrates. MXR activity was never investigated in Collembola and represents a potential tool for soil biomonitoring. This study aimed to characterize for the first time the activity of ABC efflux pumps in the gut of collembolan species, and investigate its responsiveness to cadmium (Cd), a common stressor found in polluted soils. We performed in vitro rhodamine-B accumulation assays in the presence of model inhibitors of ABC efflux pumps: verapamil hydrochloride as P-gp (P-glycoprotein) inhibitor, and MK571, as MRPs (multidrug resistance-related proteins) inhibitor. We also performed rhodamine-B accumulation assays under Cd-exposure (209 µg/L;1 µM). Our results showed that all species presented basal (noninduced) level of MXR activity in their gut. Efflux pumps P-gp and/or MRPs activity were confirmed in Cyphoderus innominatus, Cyphoderus similis, and Folsomia candida, the standard species. The rhodamine-B accumulation assays performed with Cd, applied as soil pollutant, showed that the gut of non-standard species C. similis and Trogolaphysa sp. presented an increase of MXR activity for both P-gp and MRP transporters, indicating the potential of these species as test organisms for soil ecotoxicology studies in Neotropical region. Our findings suggest a functional role of ABC transporters in the collembolan gut and their cellular involvement in Cd defense response, corroborating that MXR phenotype in Collembola can be a promising tool for bioindication of soil contamination.

Chitin and Chitosan Derivatives as Biomaterial Resources for Biological and Biomedical Applications.

Chitin is a long-chain polymer of N-acetyl-glucosamine, which is regularly found in the exoskeleton of arthropods including insects, shellfish and the cell wall of fungi. It has been known that chitin can be used for biological and biomedical applications, especially as a biomaterial for tissue repairing, encapsulating drug for drug delivery. However, chitin has been postulated as an inducer of proinflammatory cytokines and certain diseases including asthma. Likewise, chitosan, a long-chain polymer of N-acetyl-glucosamine and d-glucosamine derived from chitin deacetylation, and chitosan oligosaccharide, a short chain polymer, have been known for their potential therapeutic effects, including anti-inflammatory, antioxidant, antidiarrheal, and anti-Alzheimer effects. This review summarizes potential utilization and limitation of chitin, chitosan and chitosan oligosaccharide in a variety of diseases. Furthermore, future direction of research and development of chitin, chitosan, and chitosan oligosaccharide for biomedical applications is discussed.

Zinc modulation of proton currents in a new voltage-gated proton channel suggests a mechanism of inhibition.

The HV 1 voltage-gated proton (HV 1) channel is a key component of the cellular proton extrusion machinery and is pivotal for charge compensation during the respiratory burst of phagocytes. The best-described physiological inhibitor of HV 1 is Zn2+ . Externally applied ZnCl2 drastically reduces proton currents reportedly recorded in Homo sapiens, Rattus norvegicus, Mus musculus, Oryctolagus cuniculus, Rana esculenta, Helix aspersa, Ciona intestinalis, Coccolithus pelagicus, Emiliania huxleyi, Danio rerio, Helisoma trivolvis, and Lingulodinium polyedrum, but with considerable species variability. Here, we report the effects of Zn2+ and Cd2+ on HV 1 from Nicoletia phytophila, NpHV 1. We introduced mutations at potential Zn2+ coordination sites and measured Zn2+ inhibition in different extracellular pH, with Zn2+ concentrations up to 1000 µm. Zn2+ inhibition in NpHV 1 was quantified by the slowing of the activation time constant and a positive shift of the conductance-voltage curve. Replacing aspartate in the S3-S4 loop with histidine (D145H) enhanced both the slowing of activation kinetics and the shift in the voltage-conductance curve, such that Zn2+ inhibition closely resembled that of the human channel. Histidine is much more effective than aspartate in coordinating Zn2+ in the S3-S4 linker. A simple Hodgkin Huxley model of NpHV 1 suggests a decrease in the opening rate if it is inhibited by zinc or cadmium. Limiting slope measurements and high-resolution clear native gel electrophoresis (hrCNE) confirmed that NpHV 1 functions as a dimer. The data support the hypothesis that zinc is coordinated in between the dimer instead of the monomer. Zinc coordination sites may be potential targets for drug development.

Antiviral peptides as promising therapeutic drugs.

While scientific advances have led to large-scale production and widespread distribution of vaccines and antiviral drugs, viruses still remain a major cause of human diseases today. The ever-increasing reports of viral resistance and the emergence and re-emergence of viral epidemics pressure the health and scientific community to constantly find novel molecules with antiviral potential. This search involves numerous different approaches, and the use of antimicrobial peptides has presented itself as an interesting alternative. Even though the number of antimicrobial peptides with antiviral activity is still low, they already show immense potential to become pharmaceutically available antiviral drugs. Such peptides can originate from natural sources, such as those isolated from mammals and from animal venoms, or from artificial sources, when bioinformatics tools are used. This review aims to shed some light on antimicrobial peptides with antiviral activities against human viruses and update the data about the already well-known peptides that are still undergoing studies, emphasizing the most promising ones that may become medicines for clinical use.

A Centipede Toxin Family Defines an Ancient Class of CSαß Defensins.

Disulfide-rich peptides (DRPs) play diverse physiological roles and have emerged as attractive sources of pharmacological tools and drug leads. Here we describe the 3D structure of a centipede venom peptide, U-SLPTX15-Sm2a, whose family defines a unique class of one of the most widespread DRP folds known, the cystine-stabilized α/ß fold (CSαß). This class, which we have named the two-disulfide CSαß fold (2ds-CSαß), contains only two internal disulfide bonds as opposed to at least three in all other confirmed CSαß peptides, and constitutes one of the major neurotoxic peptide families in centipede venoms. We show the 2ds-CSαß is widely distributed outside centipedes and is likely an ancient fold predating the split between prokaryotes and eukaryotes. Our results provide insights into the ancient evolutionary history of a widespread DRP fold and highlight the usefulness of 3D structures as evolutionary tools.

High levels of microplastic pollution in the sediments and benthic organisms of the South Yellow Sea, China.

Microplastics, emerging contaminants in the ocean, are thought to sink and accumulate in sediments, and thus may pose a potential ecological risk to benthic communities. In this study, abundances and characteristics of microplastics in sediments and benthic organisms from the South Yellow Sea were investigated. First, we optimized the sediment sampling for microplastic analysis and found that the top layer (0-5 cm) had the highest abundance, and microplastic abundances decreased significantly with increase in sediment depth. The abundance of microplastics was 560-4205 n/kg dry weight in the surface sediments (the topmost 3 cm) of 14 sites and 1.7-47.0 n/g wet weight in the tissues of benthic organisms. Moreover, microplastic abundances in sediments and benthic organisms were both positively correlated with water depth. Fibers, transparent microplastics, and small microplastics (<0.5 mm) were the most dominant types in sediments and organisms. FTIR analysis showed that polypropylene (PP, 31%), polyester (PE, 24%), nylon (19%), and polystyrene (PS, 15%) were the most abundant polymers in sediments. The results of SEM showed rough surfaces and obvious cracks on the microplastics isolated from sediments. In addition, characteristics of microplastics in Ophiura sarsii, Crangon affinis, and Acila mirabilis were compared. Our results demonstrate that a comprehensive investigation of microplastics in sediments and benthic communities will help to fully understand the ecological risk of microplastic pollution.

Cuticular gas exchange by Antarctic sea spiders.

Many marine organisms and life stages lack specialized respiratory structures, like gills, and rely instead on cutaneous respiration, which they facilitate by having thin integuments. This respiratory mode may limit body size, especially if the integument also functions in support or locomotion. Pycnogonids, or sea spiders, are marine arthropods that lack gills and rely on cutaneous respiration but still grow to large sizes. Their cuticle contains pores, which may play a role in gas exchange. Here, we examined alternative paths of gas exchange in sea spiders: (1) oxygen diffuses across pores in the cuticle, a common mechanism in terrestrial eggshells, (2) oxygen diffuses directly across the cuticle, a common mechanism in small aquatic insects, or (3) oxygen diffuses across both pores and cuticle. We examined these possibilities by modeling diffusive oxygen fluxes across all pores in the body of sea spiders and asking whether those fluxes differed from measured metabolic rates. We estimated fluxes across pores using Fick's law parameterized with measurements of pore morphology and oxygen gradients. Modeled oxygen fluxes through pores closely matched oxygen consumption across a range of body sizes, which means the pores facilitate oxygen diffusion. Furthermore, pore volume scaled hypermetrically with body size, which helps larger species facilitate greater diffusive oxygen fluxes across their cuticle. This likely presents a functional trade-off between gas exchange and structural support, in which the cuticle must be thick enough to prevent buckling due to external forces but porous enough to allow sufficient gas exchange.

Human rickettsial pathogen modulates arthropod organic anion transporting polypeptide and tryptophan pathway for its survival in ticks.

The black-legged tick Ixodes scapularis transmits the human anaplasmosis agent, Anaplasma phagocytophilum. In this study, we show that A. phagocytophilum specifically up-regulates I. scapularis organic anion transporting polypeptide, isoatp4056 and kynurenine amino transferase (kat), a gene involved in the production of tryptophan metabolite xanthurenic acid (XA), for its survival in ticks. RNAi analysis revealed that knockdown of isoatp4056 expression had no effect on A. phagocytophilum acquisition from the murine host but affected the bacterial survival in tick cells. Knockdown of the expression of kat mRNA alone or in combination with isoatp4056 mRNA significantly affected A. phagocytophilum survival and isoatp4056 expression in tick cells. Exogenous addition of XA induces isoatp4056 expression and A. phagocytophilum burden in both tick salivary glands and tick cells. Electrophoretic mobility shift assays provide further evidence that A. phagocytophilum and XA influences isoatp4056 expression. Collectively, this study provides important novel information in understanding the interplay between molecular pathways manipulated by a rickettsial pathogen to survive in its arthropod vector.

Gene expression reveals evidence for EGFR-dependent proximal-distal limb patterning in a myriapod.

Evolution of segmented limbs is one of the key innovations of Arthropoda, allowing development of functionally specific specialized head and trunk appendages, a major factor behind their unmatched evolutionary success. Proximodistal limb patterning is controlled by two regulatory networks in the vinegar fly Drosophila melanogaster, and other insects. The first is represented by the function of the morphogens Wingless (Wg) and Decapentaplegic (Dpp); the second by the EGFR-signaling cascade. While the role of Wg and Dpp has been studied in a wide range of arthropods representing all main branches, that is, Pancrustacea (= Hexapoda + Crustacea), Myriapoda and Chelicerata, investigation of the potential role of EGFR-signaling is restricted to insects (Hexapoda). Gene expression analysis of Egfr, its potential ligands, and putative downstream factors in the pill millipede Glomeris marginata (Myriapoda: Diplopoda), reveals that-in at least mandibulate arthropods-EGFR-signaling is likely a conserved regulatory mechanism in proximodistal limb patterning.

Infection-derived lipids elicit an immune deficiency circuit in arthropods.

The insect immune deficiency (IMD) pathway resembles the tumour necrosis factor receptor network in mammals and senses diaminopimelic-type peptidoglycans present in Gram-negative bacteria. Whether unidentified chemical moieties activate the IMD signalling cascade remains unknown. Here, we show that infection-derived lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and 1-palmitoyl-2-oleoyl diacylglycerol (PODAG) stimulate the IMD pathway of ticks. The tick IMD network protects against colonization by three distinct bacteria, that is the Lyme disease spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale. Cell signalling ensues in the absence of transmembrane peptidoglycan recognition proteins and the adaptor molecules Fas-associated protein with a death domain (FADD) and IMD. Conversely, biochemical interactions occur between x-linked inhibitor of apoptosis protein (XIAP), an E3 ubiquitin ligase, and the E2 conjugating enzyme Bendless. We propose the existence of two functionally distinct IMD networks, one in insects and another in ticks.

The Influence of Soil Characteristics on the Toxicity of Oil Refinery Waste for the Springtail Folsomia candida (Collembola).

We determined the toxicity of oil refinery waste in three soils using the springtail Folsomia candida (Collembola) in bioassays. Sublethal exposure to a concentration series of API-sludge presented EC50's for reproduction of 210 mg/kg in site soil; 880 mg/kg in LUFA2.2- and 3260 mg/kg in OECD-soil. The sludge was the least toxic in the OECD-soil with the highest clay and organic matter content, the highest maximum water holding capacity, and the least amount of sand. It was the most toxic in the reference site soil with the lowest organic matter content and highest sand content. The results emphasized the important role of soil characteristics such as texture and organic matter content in influencing toxicity, possibly by affecting bioavailability of toxicants.

Transcriptional profiling of the soil invertebrate Folsomia candida in pentachlorophenol-contaminated soil.

Pentachlorophenol (PCP), a widely used pesticide, is considered to be an endocrine disruptor. The molecular effects of chemicals with endocrine-disrupting potential on soil invertebrates are largely unknown. In the present study, the authors explored the transcriptional expression changes of collembola (Folsomia candida) in response to PCP contamination. A total of 92 genes were significantly differentially expressed at all exposure times, and the majority of them were found to be downregulated. In addition to the transcripts encoding cytochrome P450s and transferase enzymes, chitin-binding protein was also identified in the list of common differentially expressed genes. Analyses of gene ontology annotation and enrichment revealed that cell cycle-related transcripts were significantly induced by PCP, indicating that PCP can stimulate cell proliferation in springtail, as has been reported in human breast cancer cells. Enrichment of functional terms related to steroid receptors was observed, particularly in 20 significant differentially expressed genes involved in chitin metabolism in response to PCP exposure. Combined with confirmation by quantitative polymerase chain reaction, the results indicate that the adverse effects on reproduction of springtails after exposure to PCP can be attributed to a chemical-induced delay in the molting cycle and that molting-associated genes may serve as possible biomarkers for assessing toxicological effects.

Peptidomics combined with cDNA library unravel the diversity of centipede venom.

Centipedes are one of the oldest venomous arthropods using toxin as their weapon to capture prey. But little attention was focused on them and only few centipede toxins were demonstrated with activity on ion channels. Therefore, more deep works are needed to understand the diversity of centipede venom. In the present study, we use peptidomics combined with cDNA library to uncover the diversity of centipede Scolopendra subspinipes mutilans L. Koch. 192 peptides were identified by LC-MS/MS and 79 precursors were deduced by cDNA library. Surprisingly, the signal peptides of centipede toxins were more complicated than any other animal toxins and even exhibited large differences in homologues. Meanwhile, a large number of variants generated by alternative cleavage sites were detected by mass spectra. Odd number of cystein (3, 5, 7) found in the mature peptides were seldom seen in peptide toxins. In additional, two novel cysteine frameworks (C-C-C-CCC, C-C-C-C-CC-CC) were identified from 16 different cysteine frameworks from centipede peptides. Only 29 precursors have clear targets, while others may provide a potential diversity function for centipede. These findings highlight the extensive diversity of centipede toxins and provide powerful tools to understand the capture and defense weapon of centipede. BIOLOGICAL SIGNIFICANCE: Peptide toxins from venomous animal have attracted increasing attentions due to their extraordinary chemical and pharmacological diversity. Centipedes are one of the most used Chinese traditional medicines, but little was known about the active components. The venom of Scolopendra subspinipes mutilans L. Koch is first deeply analyzed in this work and most of peptides were never discovered before. Interestingly, the number and arrangement of cysteine showed a larger different to known peptide toxins such spider or scorpion toxins. Moreover, only 29 peptides from this centipede venom were identified with known function. It suggested that our work not only important to understand the composition of centipede venom, but also provide many valuable peptides for potential biological functions.