Cloning and Immunosuppressive Properties of an Acyl-Activating Enzyme from the Venom Apparatus of Tetrastichus brontispae (Hymenoptera: Eulophidae).

Venom injected into the host plays vital roles in facilitating successful parasitization and development for parasitoid wasps, especially those devoid of polydnavirus, and the abundant venom proteins appear to be most likely involved in parasitization success. Previously, we found the four most abundant venom proteins, including 4-coumarate:CoA ligase-like 4 (4CL4-like), in the Tetrastichus brontispae (Hymenoptera: Eulophidae) venom apparatus. In this study, we cloned, expressed T. brontispae 4CL4-like (Tb4CL4-like) in Escherichia coli, and investigated its immunosuppressive properties. The deduced amino acid sequence for Tb4CL4-like shares high identity at conserved amino acids associated with the acyl-activating enzyme (AAE) consensus motif but shows only <40% identity with the members in the AAE superfamily. mRNA abundance analysis indicated that Tb4CL4-like was transcribed mainly in the venom apparatus. Recombinant Tb4CL4-like inhibited Octodonta nipae (Coleoptera: Chrysomelidae) pupal cellular encapsulation and spreading by targeting the hemocyte cytoskeleton and reduced the hemocyte-mediated phagocytosis of E. coli in vivo. Moreover, Tb4CL4-like exhibited greater affinity to palmitic acid and linolenic acid based on the molecular docking assay and is hypothesized to be involved in fatty acid metabolism. In conclusion, our results suggest that Tb4CL4-like may be an immunity-related AAE protein that is involved in the regulation of host immunity through fatty acid metabolism-derived signaling pathways.

Convergent recruitment of adamalysin-like metalloproteases in the venom of the red bark centipede (Scolopocryptops sexspinosus).

Many venom proteins have presumably been convergently recruited by taxa from diverse venomous lineages. These toxic proteins have characteristics that allow them to remain stable in solution and have a high propensity for toxic effects on prey and/or potential predators. Despite this well-established convergent toxin recruitment, some toxins seem to be lineage specific. To further investigate the toxic proteins found throughout venomous lineages, venom proteomics and venom-gland transcriptomics were performed on two individual red bark centipedes (Scolopocryptops sexspinosus). Combining the protein phenotype with the transcript genotype resulted in the first in-depth venom characterization of S. sexspinosus, including 72 venom components that were identified in both the transcriptome and proteome and 1468 nontoxin transcripts identified in the transcriptome. Ten different toxin families were represented in the venom and venom gland with the majority of the toxins belonging to metalloproteases, CAPS (cysteine-rich secretory protein, antigen 5, and pathogenesis-related 1 proteins), and ß-pore-forming toxins. Nine of these toxin families shared a similar proteomic structure to venom proteins previously identified from other centipedes. However, the most highly expressed toxin family, the adamalysin-like metalloproteases, has until now only been observed in the venom of snakes. We confirmed adamalysin-like metalloprotease activity by means of in vivo functional assays. The recruitment of an adamalysin-like metalloprotease into centipede venom represents a striking case of convergent evolution.

Insect venom phospholipases A1 and A2: Roles in the envenoming process and allergy.

Insect venom phospholipases have been identified in nearly all clinically relevant social Hymenoptera, including bees, wasps and ants. Among other biological roles, during the envenoming process these enzymes cause the disruption of cellular membranes and induce hypersensitive reactions, including life threatening anaphylaxis. While phospholipase A2 (PLA2) is a predominant component of bee venoms, phospholipase A1 (PLA1) is highly abundant in wasps and ants. The pronounced prevalence of IgE-mediated reactivity to these allergens in sensitized patients emphasizes their important role as major elicitors of Hymenoptera venom allergy (HVA). PLA1 and -A2 represent valuable marker allergens for differentiation of genuine sensitizations to bee and/or wasp venoms from cross-reactivity. Moreover, in massive attacks, insect venom phospholipases often cause several pathologies that can lead to fatalities. This review summarizes the available data related to structure, model of enzymatic activity and pathophysiological roles during envenoming process of insect venom phospholipases A1 and -A2.

Centipede venoms and their components: resources for potential therapeutic applications.

Venomous animals have evolved with sophisticated bio-chemical strategies to arrest prey and defend themselves from natural predators. In recent years, peptide toxins from venomous animals have drawn considerable attention from researchers due to their surprising chemical, biochemical, and pharmacological diversity. Similar to other venomous animals, centipedes are one of the crucial venomous arthropods that have been used in traditional medicine for hundreds of years in China. Despite signifying pharmacological importance, very little is known about the active components of centipede venoms. More than 500 peptide sequences have been reported in centipede venomous glands by transcriptome analysis, but only a small number of peptide toxins from centipede has been functionally described. Like other venomous animals such as snakes, scorpions, and spiders, the venom of centipedes could be an excellent source of peptides for developing drugs for treatments as well as bio-insecticides for agrochemical applications. Although centipede venoms are yet to be adequately studied, the venom of centipedes as well as their components described to date, should be compiled to help further research. Therefore, based on previous reports, this review focusses on findings and possible therapeutic applications of centipede venoms as well as their components.

Centipede venom: recent discoveries and current state of knowledge.

Centipedes are among the oldest extant venomous predators on the planet. Armed with a pair of modified, venom-bearing limbs, they are an important group of predatory arthropods and are infamous for their ability to deliver painful stings. Despite this, very little is known about centipede venom and its composition. Advances in analytical tools, however, have recently provided the first detailed insights into the composition and evolution of centipede venoms. This has revealed that centipede venom proteins are highly diverse, with 61 phylogenetically distinct venom protein and peptide families. A number of these have been convergently recruited into the venoms of other animals, providing valuable information on potential underlying causes of the occasionally serious complications arising from human centipede envenomations. However, the majority of venom protein and peptide families bear no resemblance to any characterised protein or peptide family, highlighting the novelty of centipede venoms. This review highlights recent discoveries and summarises the current state of knowledge on the fascinating venom system of centipedes.

Immunotherapy with commercial venoms is efficacious for anaphylactic reactions to Vespa orientalis stings.

BACKGROUND: Vespa orientalis (VO) stings occasionally induce anaphylaxis. In the absence of commercial VO venom, allergists use commercial venoms for immunotherapy, despite having no indication regarding efficacy. We attempted to examine the effectiveness of immunotherapy with commercial venoms in patients with VO allergy and to identify the venom accountable for this effect. METHODS: Patients who unequivocally identified VO as the culprit insect were administered venom immunotherapy with the commercial venoms available in Israel to which they had positive skin tests. Patients were also skin tested with VO venom sac extracts and, after reaching the maintenance dose, were sting challenged by a live insect. The allergenic components in the venom were determined by immunoblotting. RESULTS: Twelve patients were recruited and, based on their skin test results, all were treated with yellow jacket (YJ) venom, either alone or combined with other venoms. All 8 patients who were sting challenged by VO demonstrated positive skin test responses to VO venom. Six of the stung patients tolerated the sting challenge uneventfully. Two patients developed minimal transient symptoms that resolved spontaneously. SDS-PAGE with patient sera suggested cross-reactivity between VO and YJ venoms at molecular weights of 39-42 kDa. Using phospholipases, antigen 5 and hyaluronidase derived from several Vespa, Dolichovespula and Vespula species, hyaluronidase is possibly accountable for inducing the allergic reaction. CONCLUSION: In the absence of commercial VO venom the practice of treating patients allergic to this insect with available commercial venoms seems to be efficacious and YJ venom is probably responsible for this effect.

Venom from the centipede Scolopendra viridis Say: purification, gene cloning and phylogenetic analysis of a phospholipase A2.

Venom components from the centipede Scolopendra viridis Say were studied, using both the soluble venom and a cDNA library prepared from mRNA of the venomous glands. Separation of the soluble venom by high performance liquid chromatography (HPLC) permitted to obtain at least 54 different fractions. The fraction eluting at 46.24 min showed phospholipase activity. The enzyme was purified to homogeneity and the first 25 amino acid residues were identified by Edman degradation. From the cDNA library several genes were cloned, one of which codes for a protein with identical amino acid sequence as the one experimentally determined. The cloned gene codes for a signal peptide of 28 amino acids and a mature peptide of 119 residues. The molecular weight of the enzyme was estimated by mass spectrometry and shown to be 13,752 Da, which matches exactly with the molecular mass expected from the deduced amino acid sequence of the gene. Phylogenetic analysis of this sequence, in comparison with other known from venomous animals, showed that it is more similar to snake phospholipases than to insect or arachnid sequences, suggesting that it has been submitted to convergent evolution. To the best of our knowledge this is the first time that a phospholipase from this species of animal is fully characterized. We have named it Scol/Pla.

Proteases from Lonomia obliqua venomous secretions: comparison of procoagulant, fibrin(ogen)olytic and amidolytic activities.

The hemorrhagic syndrome caused by Lonomia obliqua caterpillars is an increasing problem in Southern Brazil. The clinical profile is characterized by both hemorrhagic and pro-coagulant symptoms, constituting a paradoxical action of the venom. The effects upon blood coagulation and fibrin(ogen)olysis have been shown to result from the combined action of several active principles found mostly in the bristle extract. The present study reports quali-quantitative differences among L. obliqua secretions: Cryosecretion, hemolymph, bristle extract and tegument extract. Cryosecretion and hemolymph displayed strong amidolytic activity upon several substrates, presented moderated procoagulant activity and high fibrinogen degrading ability. Bristle and tegument extracts presented low amidolytic activity, but bristle extract showed the most potent procoagulant activity and both extracts presented low fibrinogen degrading ability. The differential involvement of these secretions during the accidents with L. obliqua can elucidate the different symptoms presented after envenomation.

Identification and partial characterisation of hyaluronidases in Lonomia obliqua venom.

By studying Lonomia obliqua (caterpillar) venom we were able to detect a lytic activity on purified hyaluronic acid. The venom hydrolyses purified chondroitin sulphate, but was unable to degrade either heparan sulphate or dermatan sulphate. Moreover, through purified hyaluronic acid-degrading kinetic assays, we observed that this lytic activity was caused by a hydrolase rather than lyase enzyme. In addition, by using the Reissig colorimetric reaction, we detected this hyaluronic acid hydrolase action as a beta-endohexosaminidase enzyme originating terminal N-acetylglucosamine residues rather than beta-endoglucuronidase, which may originate glucuronic acid residues. Zymogram analysis of the venom detected 49 and 53 kDa molecules with hyaluronic acid lytic activity. An examination of these hyaluronic acid degrading activities as a function of pH showed that these hydrolases had no apparent activities at a pH below 5.0 and higher than 8.0 and displayed their optimal activities at pH ranging from 6.0 to 7.0. Finally, through a fluorescence reaction to hyaluronic acid and confocal microscopy, we confirmed this cleaving action upon hyaluronic acid organised on the extracellular matrix of the dermis of rabbit. The data provide experimental evidence of the presence of hyaluronidases in the L. obliqua venom, probably involved in the harmful effects of the venom.

Lonofibrase, a novel alpha-fibrinogenase from Lonomia obliqua caterpillars.

Envenomation caused by Lonomia obliqua caterpillars is an increasing problem in Southern Brazil. The clinical profile is characterized by a profound hemorrhagic disorder. In the present study, we describe the characterization of a fibrin(ogen)olytic factor (lonofibrase) isolated from a venomous secretion of the caterpillars. The crude extract showed a dose-dependent inhibitory effect in the rate of thrombin-induced fibrinogen clotting and produced fragmentation of fibrinogen. Isolation of the fibrin(ogen)olytic enzyme was achieved by combining ion exchange chromatography followed by gel filtration in a fast protein liquid chromatography (FPLC) system. A single 35-kDa band was identified and the isolated enzyme named lonofibrase. Lonofibrase rapidly degrades Aalpha and Bbeta chains of fibrinogen, also being able to cleave fibrin in a distinct way from that observed with plasmin. The presence of lonofibrase with both fibrinogenolytic and fibrinolytic activities in L. obliqua secretion is coherent with the severe hemorrhagic clinical profile resulting from envenomation caused by these insects.

Fluorometric assay using naphthylamide substrates for assessing novel venom peptidase activities.

In the present study we examined the feasibility of using the fluorometry of naphthylamine derivatives for revealing peptidase activities in venoms of the snakes Bothrops jararaca, Bothrops alternatus, Bothrops atrox, Bothrops moojeni, Bothrops insularis, Crotalus durissus terrificus and Bitis arietans, of the scorpions Tityus serrulatus and Tityus bahiensis, and of the spiders Phoneutria nigriventer and Loxosceles intermedia. Neutral aminopeptidase (APN) and prolyl-dipeptidyl aminopeptidase IV (DPP IV) activities were presented in all snake venoms, with the highest levels in B. alternatus. Although all examined peptidase activities showed relatively low levels in arthropod venoms, basic aminopeptidase (APB) activity from P. nigriventer venom was the exception. Compared to the other peptidase activities, relatively high levels of acid aminopeptidase (APA) activity were restricted to B. arietans venom. B. arietans also exhibited a prominent content of APB activity which was lower in other venoms. Relatively low prolyl endopeptidase and proline iminopeptidase activities were, respectively, detectable only in T. bahiensis and B. insularis. Pyroglutamate aminopeptidase activity was undetectable in all venoms. All examined peptidase activities were undetectable in T. serrulatus venom. In this study, the specificities of a diverse array of peptidase activities from representative venoms were demonstrated for the first time, with a description of their distribution which may contribute to guiding further investigations. The expressive difference between snake and arthropod venoms was indicated by APN and DPP IV activities while APA and APB activities distinguished the venom of B. arietans from those of Brazilian snakes. The data reflected the relatively uniform qualitative distribution of the peptidase activities investigated, together with their unequal quantitative distribution, indicating the evolutionary divergence in the processing of peptides in these different venoms and/or the different abilities of the venoms examined to hydrolyze different peptides during envenomation.

The venomous hair structure, venom and life cycle of Lagoa crispata, a puss caterpillar of Oklahoma.

The presence of a unique population of Lagoa crispata, puss caterpillar, in western Oklahoma is reported. A detailed microscopic examination shows the structure of the L. crispata spines resemble the type 4 spines described by [Kawamoto, F., Kumada, N., 1984. Biology and venoms of lepidoptera. In: Tu, A.T. (Ed.), Handbook of Natural Toxins, Insect Poisons, Allergens and other invertebrate venoms, vol. 2, pp. 291-332 (ch. 9)]. The major food source of L. crispata are the leaves of oak (shin oak). The high tannin content of this food source results in spine extracts high in oak tannins. These extracts have activity but enzyme and toxin activity is lost with time. The gel filtration protein fractions are colored from brown to yellow and are inactive as enzymes or toxins. No hyaluronidase, protease or phosphohydrolase activity is detected in these protein fractions. The life cycle shows these caterpillars have 6 instars. Characterizations and annual emerging times of each instar are included.