Genomic organization and expression pattern of cytochrome P450 genes in the wolf spider Pardosa pseudoannulata.

As one of the dominant natural enemies for insect pests, the pond wolf spider, Pardosa pseudoannulata, plays important roles in pest control. Insecticide applications threaten P. pseudoannulata and consequently weaken its control effects. The roles of P450 monooxygenases in insecticide detoxifications have been richly reported in insects, but there are few reported in spiders. In this study, 120 transcripts encoding P. pseudoannulata P450s were identified based on whole genome sequencing. Compared to P450s of Aedes aegypti and Nilaparvata lugens, several novel P450 families were found, such as CYP3310. KEGG analysis of the CYP3310 family indicated that the family might be involved in the synthesis and metabolism of polyunsaturated fatty acids and hydrocarbons. The potential P450s involved in insecticide metabolism were obtained according to the high FPKM values in fat bodies based on transcriptome sequencing. However, none of the selected P450 genes was significantly upregulated by the treatments of deltamethrin or imidacloprid. The present study provides genomic and transcriptomic information of spider P450s, especially for their roles in the synthesis and metabolism of endogenous and exogenous compounds, such as polyunsaturated fatty acids, hydrocarbons and insecticides.

Sphingomyelinase D Activity in Sicarius tropicus Venom: Toxic Potential and Clues to the Evolution of SMases D in the Sicariidae Family.

The spider family Sicariidae includes three genera, Hexophthalma, Sicarius and Loxosceles. The three genera share a common characteristic in their venoms: the presence of Sphingomyelinases D (SMase D). SMases D are considered the toxins that cause the main pathological effects of the Loxosceles venom, that is, those responsible for the development of loxoscelism. Some studies have shown that Sicarius spiders have less or undetectable SMase D activity in their venoms, when compared to Hexophthalma. In contrast, our group has shown that Sicarius ornatus, a Brazilian species, has active SMase D and toxic potential to envenomation. However, few species of Sicarius have been characterized for their toxic potential. In order to contribute to a better understanding about the toxicity of Sicarius venoms, the aim of this study was to characterize the toxic properties of male and female venoms from Sicarius tropicus and compare them with that from Loxosceles laeta, one of the most toxic Loxosceles venoms. We show here that S. tropicus venom presents active SMases D. However, regarding hemolysis development, it seems that these toxins in this species present different molecular mechanisms of action than that described for Loxosceles venoms, whereas it is similar to those present in bacteria containing SMase D. Besides, our results also suggest that, in addition to the interspecific differences, intraspecific variations in the venoms' composition may play a role in the toxic potential of venoms from Sicarius species.

Comparative characterization of the hemocyanin-derived phenol oxidase activity from spiders inhabiting different thermal habitats.

Enzymes adapted to cold temperatures are commonly characterized for having higher Michaelis-Menten constants (KM) values and lower optimum and denaturation temperature, when compared to other meso or thermophilic enzymes. Phenoloxidase (PO) enzymes are ubiquitous in nature, however, they have not been reported in spiders. It is the oxygen carrier protein hemocyanin (Hc), found at high concentrations in their hemolymph, which displays an inducible PO activity. Hence, we hypothesize that Hc-derived PO activity could show features of cold adaptation in alpine species. We analyzed the Hc from two species of Theraphosidae from different thermal environments: Euathlus condorito (2400 m a.s.l.) and Grammostola rosea (500 m a.s.l.). Hc was purified from the hemolymph of both spiders and was characterized by identifying subunit composition and measuring the sodium dodecyl sulfate (SDS)-induced PO activity. The high-altitude spider Hc showed higher PO activity under all conditions and higher apparent Michaelis-Menten constant. Moreover, the optimum temperature for PO activity was lower for E. condorito Hc. These findings suggest a potential adaptation at the level of Hc-derived PO activity in Euathlus condorito, giving insights on possible mechanisms used by this mygalomorph spider to occupy extremes and variable thermal environments.

Cytotoxic and genotoxic effects on human keratinocytes triggered by sphingomyelinase D from Loxosceles venom.

The spiders of the Loxosceles genus (called brown or violin spiders) are of medical relevance in several countries due to the many human envenomation cases reported. The main component of Loxosceles venom is the enzyme sphingomyelinase D (SMase D), which is responsible for the local and systemic effects induced by the whole venom. Here, we investigated the cytotoxic and genotoxic effects caused by Loxosceles laeta venom and SMase D on human keratinocytes to better understand the dermonecrosis development mechanism. Our findings indicate that whole venom, as well as SMase D, increases intracellular superoxide levels, leading to DNA damage. These effects appear to be dependent on the binding of SMase D to the cell surface, although the complete pathway triggered as a result of the binding still needs to be elucidated. Moreover, after SMase D treatment, we observed the presence of histone γH2AX, suggesting that the cells are undergoing DNA repair. Moreover, when ATR kinase was inhibited, the cell viability of human keratinocytes was decreased. Together, our findings strongly suggest that L. laeta venom, as well as SMase D, increases intracellular superoxide levels, leading to DNA damage in human keratinocytes. Additionally, the induced DNA damage is repaired through the activation of an apparent ATR-mediated DNA-damage response. This knowledge may contribute to a better understanding of the behaviour of human keratinocytes during cutaneous loxoscelism, a condition that affects thousands of people around the world.

Antioxidant enzymes as biomarkers of Cu and Pb exposure in the ground spiders Lycosa terrestris and Pardosa birmanica.

Heavy metal exposure induces oxidative stress in terrestrial organisms, which they counteract via activation of antioxidant biomarkers. The present study investigated the effects of copper (Cu) and lead (Pb) on the total antioxidant capacity (TAC) and antioxidant enzymes such as Catalase (CAT), Glutathione reductase (GR), Superoxide dismutase (SOD) and Glutathione peroxidase (GPX) in two spider species, namely Lycosa terrestris and Pardosa birmanica. The spiders were exposed to Cu and Pb separately (10 ppm) or in combination (10 ppm each) via two different exposure routes (i.e. food and soil) for 10, 20 and 40 days. The results showed that metal accumulation and antioxidant biomarker responses in spiders were metal- and species-dependent. Also, the levels of all antioxidant biomarkers increased significantly with increasing exposure time and metal load in the bodies of spiders via both exposure routes. The significant inhibition of TAC and antioxidant enzyme activities was only observed in single Pb treatment through soil exposure. In L. terrestris, the activities of detoxification enzymes and TAC were significantly enhanced on single Cu exposure than Pb via both exposure routes. However, in P. birmanica consistent variation among antioxidant parameters were observed depending on the metal load and exposure routes. The combined metal exposure caused more pronounced increase in the level of antioxidants compared to single metal exposure in both species, mainly via food exposure. These results suggest that the antioxidant enzymes and TAC are sensitive to single and combined metal exposure via both uptake routes. These data show that antioxidant parameters can be used potential biomarkers of oxidative stress associated with metal exposure and for monitoring environmental health using spiders as bioindicators.

Identification, genomic organization and expression pattern of glutathione transferase in Pardosa pseudoannulata.

The pond wolf spider, Pardosa pseudoannulata, is one of the dominant natural enemies in farmlands and plays important roles in controlling a range of insect pests. The spider is less sensitive to many insecticides than the target pests such as the brown planthopper, Nilaparvata lugens. The different sensitivity to a certain insecticide between species is mostly attributed to the differences in both molecular targets and detoxification enzymes. As one of the most important detoxification enzymes, glutathione transferases (GSTs) play a key role as phase II enzyme in the enzymic detoxification in organisms. Until now, there are few studies on spiders' GSTs, limiting the understanding of insecticide selectivity between insect pests and natural enemy spiders. In this study, based on the transcriptome and genome sequencing of P. pseudoannulata, thirteen full-length transcripts encoding GSTs were identified and analyzed. Interestingly, Delta family, which is thought to be specific to the Insecta, was identified in P. pseudoannulata. Further, vertebrate/mammalian-specific Mu family was also identified in P. pseudoannulata. The mRNA expression levels of cytosolic GSTs in different tissues were determined, and most GST genes were abundant in the gut and the fat body. To investigate GST candidates involving in insecticide detoxification, the mRNA levels of cytosolic GSTs were tested after spiders' exposure to either imidacloprid or deltamethrin. The results showed that PpGSTD3 and PpGSTT1 responded to at least one of these two insecticides. The present study helped understand the function of GSTs in P. pseudoannulata and enriched the genetic information of natural enemy spiders.

Effect of Pesticides on Biological Control Potential of Neoscona theisi (Araneae: Araneidae).

The present study was designed to record the effect of λ-cyhalothrin, Bifenthrin, and Glyphosate on the mortality, avoidance behavior, foraging activity, and activity of Acetylcholine esterase (AChE) and Carboxylesterase (CarE) in Neoscona theisi (Walckenaer, 1841). Highest mortality (70%) in N. theisi was recorded against λ-cyhalothrin. However, Glyphosate was found to be least toxic. Spider spent less time on insecticides/herbicide-treated surfaces. Insecticides/herbicide-treated N. theisi consumed less prey than untreated control spiders. Similarly, when N. theisi were offered insecticide/herbicide-treated prey, they consumed significantly less. Increased AChE and CarE activities were recorded in insecticides/herbicide-treated spiders as compared to control group. Total protein contents were less in insecticides/herbicide-treated spiders than control group. The results revealed that λ-cyhalothrin is more harmful to spiders as compared to Bifenthrin and Glyphosate. It is suggested that the effect of all pesticides used in agro-ecosystem on beneficial insects should be evaluated before using them in the fields.

Biochemical and structural characterization of a protein complex containing a hyaluronidase and a CRISP-like protein isolated from the venom of the spider Acanthoscurria natalensis.

Spider venoms are composed of a complex mixture of bioactive molecules. The structural and functional characterization of these molecules in the venom of the Brazilian spider Acanthoscurria natalensis, has been little explored. The venom was fractionated using reversed-phase liquid chromatography. The fraction with hyaluronidase activity was named AnHyal. The partial sequencing of AnHyal revealed the presence of a CRISP-like protein, in addition to hyaluronidase, comprising 67% coverage for hyaluronidase from Brachypelma vagans and 82% for CRISP-like protein from Grammostola rosea. 1D BN-PAGE zymogram assays of AnHyal confirmed the presence of enzymatically active 53 kDa monomer and 124 and 178 kDa oligomers. The decomposition of the complexes by 2D BN/SDS-PAGE zymogram assays showed two subunits, 53 (AnHyalH) and 44 kDa (AnHyalC), with sequence similarity to hyaluronidase and CRISP proteins, respectively. The secondary structure of AnHyal is composed by 36% of α-helix. AnHyal presented maximum activity at pH between 4.0 and 6.0 and 30 and 60 °C, showed specificity to hyaluronic acid substrate and presented a KM of 617.9 µg/mL. Our results showed that hyaluronidase and CRISP proteins can form a complex and the CRISP protein may contribute to the enzymatic activity of AnHyalH.

Methamidophos Influences Midgut Proteinase Activity and Subcellular Structures in the Wolf Spider Pardosa pseudoamulata (Araneae: Lycosidae).

A piezoelectric quartz crystal impedance (PQCI) sensor was used to investigate influences of the insecticide methamidophos on proteinase activity in midguts of the wolf spider, Pardosa pseudoamulata (Araneae: Lycosidae). Results from PQCI indicated that low-concentration dose methamidophos (0.008%) can activate the proteinase but high-concentration dose methamidophos (0.016-0.032%) can inhibit the enzyme activity. The changes in subcellular structure of spider midgut cells were also observed. Electron micrographs of spider midgut epithelial cells showed that the low-dose methamidophos did not visibly impact the structure of these cells. Conversely, high-concentration dose methamidophos led to severe changes in the cell structure, including the karyotheca dissolved, the nucleolus, and the endoplasmic reticulum disappeared. These may contribute to changes in proteinase activity of spider. This work documents a feasible method for rapid and reliable detection of proteinase activity.

Identity and role of the non-conserved acid/base catalytic residue in the GH29 fucosidase from the spider Nephilingis cruentata.

α-l-Fucosidases are widely occurring enzymes that remove fucose residues from N- and O-fucosylated glycoproteins. Comparison of amino acid sequences of fucosidases reveals that although the nucleophile is conserved among all α-l-fucosidases, the position of the acid/base residue is quite variable. Although several site-directed mutation studies have previously been performed on bacterial fucosidases, the only eukaryotic fucosidase so studied was the human fucosidase. Recent alignments indicate that human and Arthropoda α-l-fucosidases share at least 50% identity and the acid/base residue seems to be conserved among them suggesting a common acid/base residue in Metazoa. Here we describe the cloning and expression in Pichia pastoris of a very active α-l-fucosidase from the spider Nephilingis cruentata (NcFuc) with a Km value for pNPFuc of 0.4 mM. NcFuc hydrolyzed fucoidan, 2´fucosyllactose and also lacto-N-difucohexaose II. Mutants modified at the conserved residues D214N, E209A, E59A were expressed and characterized. The 500-fold lower kcat of D214N than the wild type was consistent with a role in catalysis, as was the 8000-fold lower kcat value of E59A. This was supported by the 57-fold increase in the kcat of E59A upon addition of azide. A complex pH/rate profile was seen for the wild-type and mutant forms of NcFuc, similar to those measured previously for the Sulfolobus fucosidase. The non-conservative catalytic structure and distinct active site organization reinforce the necessity of structural studies of new fucosidases.

First characterization of fucosidases in spiders.

l-fucose is a constituent of glycoconjugates in different organisms. Fucosidases catalyze the removal of fucose residues, and have been correlated to different physiological and pathological processes, such as fertilization, cancer, fucosidosis, and digestion in molluscs and ticks. An α-l-fucosidase sequence was identified from the transcriptome and proteome from the midgut diverticula of the synanthropic spider Nephilingis cruentata. In this article, we describe the isolation of this α-l-fucosidase and the characterization of its activity using substrates and inhibitors demonstrating different specificities among fucosidases. The enzyme had a Km of 32 and 400 µM for 4-methylumbelliferyl α-l-fucopyranoside and 4-nitrophenyl α-l-fucopyranoside, respectively; and was unable to hydrolyze fucoidan. Nephilingis cruentata α-l-fucosidase was inhibited competitively by fucose and fuconojyrimycin. The fucosidase had two distinct pH optima even in the isolated form, due to oligomerization dependent on pH, as previously described to other fucosidases. Alignment and molecular homology modeling of the protein sequence with other fucosidases indicated that the active sites and catalytic residues were different, including residues involved in acid/base catalysis. Phylogenetic analysis showed, for the first time, gene-duplication events for fucosidases in Arachnida species. All these data reveal that studies on fucosidases in organisms distinct from bacteria, fungi, and humans are important.

Spider's venom phospholipases D: A structural review.

Spider venoms are complex mixtures of proteins, peptides and small organic and inorganic molecules. Among the proteins, phospholipases D (PLDs) present the major portion, and till now they are the most studied enzymes in spider venom. These PLDs have been divided into two classes, I and II, based on their primary and tertiary structure. Currently, crystal structures of both classes of these enzymes are available in the Protein Data Bank (PDB). Their three-dimensional structure is composed of eight α-helices and eight ß-strands forming the ubiquitous fold called triosephosphate isomerase (TIM) barrel. These enzymes use general acid-base catalysis to hydrolyzes their substrate. In this review, we have described the structural features, structure-based mechanisms of catalysis, maturation, and inhibition of these enzymes using the synthetic inhibitor.

Identification of a precursor processing protease from the spider Cupiennius salei essential for venom neurotoxin maturation.

Spider venom neurotoxins and cytolytic peptides are expressed as elongated precursor peptides, which are post-translationally processed by proteases to yield the active mature peptides. The recognition motifs for these processing proteases, first published more than 10 years ago, include the processing quadruplet motif (PQM) and the inverted processing quadruplet motif (iPQM). However, the identification of the relevant proteases was still pending. Here we describe the purification of a neurotoxin precursor processing protease from the venom of the spider Cupiennius salei The chymotrypsin-like serine protease is a 28-kDa heterodimer with optimum activity at venom's pH of 6.0. We designed multiple synthetic peptides mimicking the predicted cleavage sites of neurotoxin precursors. Using these peptides as substrates, we confirm the biochemical activity of the protease in propeptide removal from neurotoxin precursors by cleavage C-terminal of the PQM. Furthermore, the PQM protease also cleaves the iPQM relevant for heterodimerization of a subgroup of neurotoxins. An involvement in the maturing of cytolytic peptides is very likely, due to high similarity of present protease recognition motifs. Finally, bioinformatics analysis, identifying sequences of homolog proteins from 18 spiders of 9 families, demonstrate the wide distribution and importance of the isolated enzyme for spiders. In summary, we establish the first example of a PQM protease, essential for maturing of spider venom neurotoxins. In the future, the here described protease may be established as a powerful tool for production strategies of recombinant toxic peptides, adapted to the maturing of spider venom toxins.

Characterisation of protein families in spider digestive fluids and their role in extra-oral digestion.

BACKGROUND: Spiders are predaceous arthropods that are capable of subduing and consuming relatively large prey items compared to their own body size. For this purpose, spiders have evolved potent venoms to immobilise prey and digestive fluids that break down nutrients inside the prey's body by means of extra-oral digestion (EOD). Both secretions contain an array of active proteins, and an overlap of some components has been anecdotally reported, but not quantified. We systematically investigated the extent of such protein overlap. As venom injection and EOD succeed each other, we further infer functional explanations, and, by comparing two spider species belonging to different clades, assess its adaptive significance for spider EOD in general. RESULTS: We describe the protein composition of the digestive fluids of the mygalomorph Acanthoscurria geniculata and the araneomorph Stegodyphus mimosarum, in comparison with previously published data on a third spider species. We found a number of similar hydrolases being highly abundant in all three species. Among them, members of the family of astacin-like metalloproteases were particularly abundant. While the importance of these proteases in spider venom and digestive fluid was previously noted, we now highlight their widespread use across different spider taxa. Finally, we found species specific differences in the protein overlap between venom and digestive fluid, with the difference being significantly greater in S. mimosarum compared to A. geniculata. CONCLUSIONS: The injection of venom precedes the injection with digestive fluid, and the overlap of proteins between venom and digestive fluid suggests an early involvement in EOD. Species specific differences in the overlap may reflect differences in ecology between our two study species. The protein composition of the digestive fluid of all the three species we compared is highly similar, suggesting that the cocktail of enzymes is highly conserved and adapted to spider EOD.

Characterization of the Fifth Putative Acetylcholinesterase in the Wolf Spider, Pardosa pseudoannulata.

Background: Acetylcholinesterase (AChE) is an important neurotransmitter hydrolase in invertebrate and vertebrate nervous systems. The number of AChEs is various among invertebrate species, with different functions including the 'classical' role in terminating synaptic transmission and other 'non-classical' roles. Methods: Using rapid amplification of cDNA ends (RACE) technology, a new putative AChE-encoding gene was cloned from Pardosa pseudoannulata, an important predatory natural enemy. Sequence analysis and in vitro expression were employed to determine the structural features and biochemical properties of this putative AChE. Results: The cloned AChE contained the most conserved motifs of AChEs family and was clearly clustered with Arachnida AChEs. Determination of biochemical properties revealed that the recombinant enzyme had the obvious preference for the substrate ATC (acetylthiocholine iodide) versus BTC (butyrylthiocholine iodide). The AChE was highly sensitive to AChE-specific inhibitor BW284C51, but not butyrylcholinesterase-specific inhibitor tetraisopropyl pyrophosphoramide (ISO-OMPA). Based on these results, we concluded that a new AChE was identified from P. pseudoannulata and denoted as PpAChE5. Conclusion: Here we report the identification of a new AChE from P. pseudoannulata and increased the AChE number to five in this species. Although PpAChE5 had the biggest Vmax value among five identified AChEs, it showed relatively low affinity with ATC. Similar sensitivity to test insecticides indicated that this AChE might serve as the target for both organophosphorus and carbamate insecticides.

Recombinant Phospholipase D from Loxosceles gaucho Binds to Platelets and Promotes Phosphatidylserine Exposure.

Spider envenomation, from the genus Loxosceles, is frequently reported as a cause of necrotic lesions in humans around the world. Among the many components found in the venom of Loxosceles genus, phospholipases D (PLDs) are the most investigated, since they can cause a massive inflammatory response, dermonecrosis, hemolysis and platelet aggregation, among other effects. Even though the PLDs induce strong platelet aggregation, there are no studies showing how the PLDs interact with platelets to promote this effect. Since many agonists must interact with specific receptors on the platelet membrane to induce aggregation, it is reasonable to expect that the PLDs may, in some way, also interact with platelets, to induce this activity. Therefore, to address this possibility, in this work, a recombinant PLD, called LgRec1, from L. gaucho was fused to enhanced green fluorescent protein (EGFP) and used as a probe to detect the interaction of LgRec1 to platelets, by fluorescence-activated cell sorter (FACS) and confocal microscopy. The preservation of biological activities of this chimera toxin was also analyzed. As a first, the results show that LgRec1 does not require plasma components to bind to platelets, although these components are necessary to LgRec1 to induce platelet aggregation. Also, the attachment of LgRec1 to human platelets' cell membranes suggests that the exposure of phosphatidylserine (PS) may act as a scaffold for coagulation factors. Therefore, the results add new information about the binding of Loxosceles PLDs to platelets, which may help unravel how these toxins promote platelet aggregation.

Efficacy of DNA barcoding for the species identification of spiders from Western Ghats of India.

DNA barcoding has emerged as an additional tool for taxonomy and as an aid to taxonomic impediments. Due to their extensive morphological variation, spiders are taxonomically challenging. Therefore, all over the world, attempts are being made to DNA barcode species of spiders. Till now no attempts were made to DNA barcode Indian spiders despite their rich diversity. We have generated DNA barcodes for 60 species (n = 112) of spiders for the first time from India. Although only 17 species were correctly identified at the species level, DNA barcoding correctly discriminated 99% of the species studied here. We have also found high intraspecies nucleotide divergence in Plexippus paykulli suggesting cryptic diversity that needs to be studied in detail. Our study also showed non-specific amplification of the Cytochrome Oxidase I (COI) gene of endosymbiont bacteria Wolbachia. However, these cases are very rare and could be resolved by the use of modified or group specific primers.

Mitochondrial DNA phylogeny of camel spiders (Arachnida: Solifugae) from Iran.

In the present study, the mitochondrial DNA phylogeny of five solifuge families of Iran is presented using phylogenetic analysis of mitochondrial cytochrome c oxidase, subunit 1 (COI) sequence data. Moreover, we included available representatives from seven families from GenBank to examine the genetic distance between Old and New World taxa and test the phylogenetic relationships among more solifuge families. Phylogenetic relationships were reconstructed based on the two most probabilistic methods, Maximum Likelihood (ML) and Bayesian inference (BI) approaches. Resulting topologies demonstrated the monophyly of the families Daesiidae, Eremobatidae, Galeodidae, Karschiidae and Rhagodidae, whereas the monophyly of the families Ammotrechidae and Gylippidae was not supported. Also, within the family Eremobatidae, the subfamilies Eremobatinae and Therobatinae and the genus Hemerotrecha were paraphyletic or polyphyletic. According to the resulted topologies, the taxonomic placements of Trichotoma michaelseni (Gylippidae) and Nothopuga sp. 1 (Ammotrechidae) are still remain under question and their revision might be appropriate. According to the results of this study, within the family Galeodidae, the validity of the genus Galeodopsis is supported, while the validity of the genus Paragaleodes still remains uncertain. Moreover, our results revealed that the species Galeodes bacillatus, and Rhagodes melanochaetus are junior synonyms of G. caspius, and R. eylandti, respectively.

Bacterial and Arachnid Sphingomyelinases D: Comparison of Biophysical and Pathological Activities.

Sphingomyelinases D have only been identified in arachnid venoms, Corynebacteria, Arcanobacterium, Photobacterium and in the fungi Aspergillus and Coccidioides. The arachnid and bacterial enzymes share very low sequence identity and do not contain the HKD sequence motif characteristic of the phospholipase D superfamily, however, molecular modeling and circular dichroism of SMases D from Loxosceles intermedia and Corynebacterium pseudotuberculosis indicate similar folds. The phospholipase, hemolytic and necrotic activities and mice vessel permeabilities were compared and both enzymes possess the ability to hydrolyze phospholipids and also promote similar pathological reactions in the host suggesting the existence of a common underlying mechanism in tissue disruption. J. Cell. Biochem. 118:2053-2063, 2017. © 2016 Wiley Periodicals, Inc.

Identification and Biochemical Properties of Two New Acetylcholinesterases in the Pond Wolf Spider (Pardosa pseudoannulata).

Acetylcholinesterase (AChE), an important neurotransmitter hydrolase in both invertebrates and vertebrates, is targeted by organophosphorus and carbamate insecticides. In this study, two new AChEs were identified in the pond wolf spider Pardosa pseudoannulata, an important predatory natural enemy of several insect pests. In total, four AChEs were found in P. pseudoannulata (including two AChEs previously identified in our laboratory). The new putative AChEs PpAChE3 and PpAChE4 contain most of the common features of the AChE family, including cysteine residues, choline binding sites, the conserved sequence 'FGESAG' and conserved aromatic residues but with a catalytic triad of 'SDH' rather than 'SEH'. Recombinant enzymes expressed in Sf9 cells showed significant differences in biochemical properties compared to other AChEs, such as the optimal pH, substrate specificity, and catalytic efficiency. Among three test substrates, PpAChE1, PpAChE3 and PpAChE4 showed the highest catalytic efficiency (Vmax/KM) for ATC (acetylthiocholine iodide), with PpAChE3 exhibiting a clear preference for ATC based on the VmaxATC/VmaxBTC ratio. In addition, the four PpAChEs were more sensitive to the AChE-specific inhibitor BW284C51, which acts against ATC hydrolysis, than to the BChE-specific inhibitor ISO-OMPA, which acts against BTC hydrolysis, with at least a 8.5-fold difference in IC50 values for each PpAChE. PpAChE3, PpAChE4, and PpAChE1 were more sensitive than PpAChE2 to the tested Carb insecticides, and PpAChE3 was more sensitive than the other three AChEs to the tested OP insecticides. Based on all the results, two new functional AChEs were identified from P. pseudoannulata. The differences in AChE sequence between this spider and insects enrich our knowledge of invertebrate AChE diversity, and our findings will be helpful for understanding the selectivity of insecticides between insects and natural enemy spiders.