Four novel insecticidal toxins were isolated from the venom of the spider Paracoelotes luctuosus (Araneae: Amaurobiidae) and named d-palutoxins IT1 to IT4. The four toxins are homologous 36±37 amino acid peptides reticulated by four disulfide bridges and three have amidated C-terminal residues. The d-palutoxins are highly homologous with the previously described m-agatoxins and curtatoxins (77±97%). The four peptides demonstrated significant toxicity against larvae of the crop pest Spodoptera litura (Lepidoptera: Noctuidae) in a microinjection bioassay, with LD 50 values in the 9±50 mg per g of insect range. This level of toxicity is equivalent to that of several of the most active scorpion toxins used in the development of recombinant baculoviruses, and the d-palutoxins appear to be insect specific. Electrophysiological experiments demonstrated that d-palutoxin IT1, the most active toxin acts by affecting insect sodium channel inactivation, resulting in the appearance of a late-maintained sodium current, in a similar fashion to insecticidal scorpion a and a-like toxins and is thus likely to bind to channel receptor site 3. However, d-palutoxin IT1 was distinguished by its lack of effect on peak sodium conductance, on the early phase of sodium current inactivation and the absence of a shift in the activation voltage of the sodium channels. d-Palutoxins are thus proposed as new insecticidal toxins related to the a and a-like scorpion toxins. They will be useful both in the development of recombinant baculoviruses in agrochemical applications and also as molecular probes for the investigation of molecular mechanisms of insect selectivity and structure and function of sodium channels.Keywords: baculovirus; insecticidal toxins; scorpion toxins; sodium channel; spider toxins.Intensive use of synthetic chemicals as the sole strategy for insect control is being reconsidered as public tolerance of the negative effects of synthetic pesticides increases and pressure mounts against widespread and indiscriminate use of agrochemicals. Microbial insecticides, and in particular the group of insect-specific baculoviruses [1], have been proposed as an attractive alternative in integrated pest management programs or specific applications. In the past, substantial research efforts have been devoted to the development of recombinant baculoviruses expressing proteins deleterious to insects with the goal of producing selective, fast-acting pathogens to be used in combination with other pest-control agents [2,3]. One of the most successful approaches to increase the speed of kill in baculoviruses has been genetic engineering into the viruses of arthropod toxin genes with neurotoxic effects on the insect nervous system. Significant decreases in the time of the appearance of lethal effects and the reduction of crop damage have been obtained with recombinant viruses expressing toxins [1,4,5]. The AaIT scorpion toxin from Androctonus australis and other scorpion toxins affecting sodium channel kinetics such as LqhaIT from Leiurus quinques...

The increased interest in spider venom toxins has made precise identification of venom samples necessary for reproducibility of biochemical and pharmacological studies. In the family Theraphosidae (Tarantulas), identification of specimens may be difficult and a new approach involving multidimensional biochemical analysis of venoms is proposed. Combined HPLC, capillary electrophoresis and matrix-assisted laser desorption/ionization fingerprinting of venom peptides permits accurate and reproducible identification of venom samples, and can be correlated with morphological observations. This rapid and very accurate methodology can be applied to minute amounts of samples obtained from live animals, on a large scale, with high reproducibility. Application of the methodology to five separate examples demonstrates confirmation of sample identity, separation of closely related species on the basis of venom profiles, and solution of classification problems not easily solved by morphological examination. Discussion of sample variability and the use of a dual matrix system is presented. # 1998 John Wiley & Sons, Ltd. Received 21 May 1998; Revised 29 June 1998; Accepted 30 June 1998 In the past five years, peptide toxins from spider venoms have received increased attention from biochemists and pharmacologists, in the light of the discovery of several original ion channel ligands.1-5 Spider venoms represent a rich source of novel pharmacological tools for the study of receptors in excitable cells and possess a wide variety of pharmacological properties. [6][7][8][9][10][11] Proper species identification prior to venom investigation is thus of paramount importance, especially since venoms have often to be sourced from pools of animals obtained through the pet trade. The Theraphosidae (true tarantulas) are the largest family of the mygalomorph spiders. Their taxonomy often remains unclear and species identification based on morphological features can prove challenging, especially with live specimens, and remains a field with few specialists.12-14 The geographical origin of specimens from the pet trade can be uncertain, and physical characteristics such as coloration may vary considerably within a population or with the physiological state of the spider (pre-or post-molting in particular). Taxonomic identification is often based on the description of one sex or the other, generally unavailable at the same time, or on particular features such as the morphology and position of stridulating organs or morphology of sexual organs, whose study is impossible without sacrifice of the specimen.13 These problems and the requirements of venom studies emphasize the need for a rapid and accurate species determination method, while keeping the specimens alive. As direct genome products, venom polypeptides appear to represent good biomarkers for species identification. Several reports of this approach to the identification of bacterial species by MALDI-TOFMS, HPLC and capillary electrofocusing have recently been presented, [15][16][17][18][1...

Pholcid spiders (Araneae: Pholcidae), officially "cellar spiders" but popularly known as "daddy long-legs," are renown for the potential of deadly toxic venom, even though venom composition and potency has never formally been studied. Here we detail the venom composition of male Physocyclus mexicanus using proteomic analyses and venom-gland transcriptomes ("venomics"). We also analyze the venom's potency on insects, and assemble available evidence regarding mammalian toxicity. The majority of the venom (51% of tryptic polypeptides and 62% of unique tryptic peptides) consists of proteins homologous to known venom toxins including enzymes (astacin metalloproteases, serine proteases and metalloendopeptidases, particularly neprilysins) and venom peptide neurotoxins. We identify 17 new groups of peptides (U 1−17-PHTX) most of which are homologs of known venom peptides and are predicted to have an inhibitor cysteine knot fold; of these, 13 are confirmed in the proteome. Neprilysins (M13 peptidases), and astacins (M12 peptidases) are the most abundant venom proteins, respectively representing 15 and 11% of the individual proteins and 32 and 20% of the tryptic peptides detected in crude venom. Comparative evidence suggests that the neprilysin gene family is expressed in venoms across a range of spider taxa, but has undergone an expansion in the venoms of pholcids and may play a central functional role in these spiders. Bioassays of crude venoms on crickets resulted in an effective paralytic dose of 3.9 µg/g, which is comparable to that of crude venoms of Plectreurys tristis and other Synspermiata taxa. However, crickets exhibit flaccid paralysis and regions of darkening that are not observed after P. tristis envenomation. Documented bites on humans make clear that while these spiders can bite, the typical result is a mild sting with no long-lasting effects. Together, the evidence we present indicates pholcid venoms are a source of interesting new peptides and proteins, and effects of bites on humans and other mammals are inconsequential.

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