Insecticidal peptides from the theraposid spider Brachypelma albiceps: An NMR-based model of Ba2

Soluble venom and purified fractions of the theraposid spider Brachypelma albiceps were screened for insecticidal peptides based on toxicity to crickets. Two insecticidal peptides, named Ba1 and Ba2, were obtained after the soluble venom was separated by high performance liquid chromatography and cation exchange chromatography. The two insecticidal peptides contain 39 amino acid residues and three disulfide bonds, and based on their amino acid sequence, they are highly identical to the insecticidal peptides from the theraposid spiders Aphonopelma sp. from the USA and Haplopelma huwenum from China indicating a relationship among these genera. Although Ba1 and Ba2 were not able to modify currents in insect and vertebrate cloned voltage-gated sodium ion channels, they have noteworthy insecticidal activities compared to classical arachnid insecticidal toxins indicating that they might target unknown receptors in insect species. The most abundant insecticidal peptide Ba2 was submitted to NMR spectroscopy to determine its 3-D structure; a remarkable characteristic of Ba2 is a cluster of basic residues, which might be important for receptor recognition.     

Oxyopinins, large amphipathic peptides isolated from the venom of the wolf spider Oxyopes kitabensis with cytolytic properties and positive insecticidal cooperativity with spider neurotoxins

Five amphipathic peptides with antimicrobial, hemolytic, and insecticidal activity were isolated from the crude venom of the wolf spider Oxyopes kitabensis. The peptides, named oxyopinins, are the largest linear cationic amphipathic peptides from the venom of a spider that have been chemically characterized at present. According to their primary structure Oxyopinin 1 is composed of 48 amino acid residues showing extended sequence similarity to the ant insecticidal peptide ponericinL2 and to the frog antimicrobial peptide dermaseptin. Oxyopinins 2a, 2b, 2c, and 2d have highly similar sequences. At least 27 out of 37 amino acid residues are conserved. They also show a segment of sequence similar to ponericinL2. Circular dichroism analyses showed that the secondary structure of the five peptides is essentially alpha-helical. Oxyopinins showed disrupting activities toward both biological membranes and artificial vesicles, particularly to those rich in phosphatidylcholine. Electrophysiological recordings performed on insect cells (Sf9) showed that the oxyopinins produce a drastic reduction of cell membrane resistance by opening non-selective ion channels. Additionally, a new paralytic neurotoxin named Oxytoxin 1 was purified from the same spider venom. It contains 69 amino acid residue cross-linked by five disulfide bridges. Application of mixtures containing oxyopinins and Oxytoxin 1 to insect larvae showed a potentiation phenomenon, by which an increase lethality effect is observed. These results suggest that the linear amphipathic peptides in spider venoms and neuropeptides cooperate to capture insects efficiently.     

Novel insecticidal peptides from Tegenaria agrestis spider venom may have a direct effect on the insect central nervous system

Fractionation of venom from an agelenid spider, Tegenaria agrestis, resulted in the isolation of a family of three peptides with potent insecticidal activity. These peptide toxins, TaITX-1, -2, and -3, whose sequences were revealed from cloned cDNAs, each consist of 50 amino acid residues, six of which are cysteines. They appear to be amidated at their C-termini and exhibit greater than 90% sequence identity. Unlike other reported spider toxins, the TaI toxins are processed from precursors containing no propeptide sequences. In lepidopteran larvae and corn rootworm beetles, the insecticidal Tegenaria toxins cause an unusual excitatory symptomatology with 50% paralytic doses ranging from 0.23 to 2.6 nmol/g. In a series of electrophysiological experiments performed in house fly larvae, these toxins caused an elevated rate of firing from central nervous system neurons. No significant effects were found when any peripheral sensory or motor systems were examined. Thus, it appears that the TaI toxins may act in a fashion not previously reported for insecticidal peptide toxins; they may act directly on the insect central nervous system. Arch. Insect Biochem. Physiol. 38:19–31, 1998. © 1998 Wiley-Liss, Inc.     

Eight new species of the spider genera Raveniola and Sinopesa from China and Vietnam (Araneae,Nemesiidae)

Eight new species, seven Raveniola Zonstein, 1987 and one Sinopesa Raven & Schwendinger, 1995 from China and Vietnam are described: Raveniolaalpinasp. n., Raveniolabellulasp. n., Raveniolachayisp. n., Raveniolagracilissp. n., Raveniolarugosasp. n., Raveniolaspirulasp. n. and Raveniolayajiangensissp. n. and Sinopesaninhbinhensissp. n. Keys to all East-Asian congeners, diagnoses of the new species, and new distribution data of Raveniolamontana Zonstein & Marusik, 2012, with a first record for Sichuan, China, are provided.     

Purification and characterization of huwentoxin-II, a neurotoxic peptide from the venom of the Chinese bird spider Selenocosmia huwena.

A neurotoxic peptide, huwentoxin-II (HWTX-II), was purified from the venom of the Chinese bird spider Selenocosmia huwena by ion exchange chromatography and reversed phase HPLC. The toxin can reversibly paralyse cockroaches for several hours, with an ED50 of 127 +/- 54 microg/g. HWTX-II blocks neuromuscular transmission in an isolated mouse phrenic nerve diaphragm preparation and acts cooperatively to potentiate the activity of huwentoxin-I. The complete amino sequence of HWTX-II was determined and found to consist of 37 amino acid residues, including six Cys residues. There is microheterogeneity (Ile/Gln) in position 10, and mass spectrometry indicated that the two isoproteins have a tendency to dimerize. It was determined by mass spectrometry that the six Cys residues are involved in three disulphide bonds. The sequence of HWTX-II is highly homologous with ESTX, a toxin from the tarantula Eurypefina californicum.     

An analysis of the secondary structure of spider spidroins I and II belonging to different species

We have analyzed the secondary structure of spidroin proteins of I and II types, related to spiders of different species. We used standard methods of secondary structure prediction NNPREDICT and JPRED and also analyzed the occurrences of oligopeptides with a preferred secondary structure with the help of the OLIGON program. We have demonstrated that local segments of the polypeptide chain can adopt alpha- and beta-conformations as well as the left-handed helix of polyproline II type. Periodical patterns found in the amino acid distribution indicate that there is a possibility of development of a macroscopic order accompanied by local conformational transitions.     

Comparative insecticidal activity of different plant materials from six common plant species against Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) is one of the major insect pests of stored grains. Due to export legislation and zero-tolerance for live insect in trade commodities, extensive use of synthetic insecticides is in practice in order to eliminate pest infestations from the lots. Currently, the one and only acceptable chemical to be used in stored grain is phosphine but due to its excessive usage the stored grain pests are becoming resistant against it. Hence discovery of alternative compounds is much needed. In this study we have compared insecticidal efficacy of different plant materials from six commonly grown plants of Pakistan, viz. Allium sativum (Alliaceae), Zingiber officinale (Zingiberaceae), Cymbopogon citratus (Poaceae), Eucalyptus globulus (Myrtaceae), Nicotiana tabacum (Solanaceae), and Azadirachta indica (Meliaceae) against T. castaneum infesting stored wheat, rice, corn and gram pulse. Various plant parts were dried, powdered, and used as admixtures to the stored commodities in the experiments. The results have suggested that A. sativum (garlic) and Z. officinale (ginger) were more effective resulting into 15 times higher adult mortality and 4 to 5 times reduction in grain weight losses when mixed with rice grains. Similarly, A. indica when admixture with wheat checked the population growth in the resources resulting into 3.5 times less adult production compared to controls. A subsequent experiment was conducted to study the dose response of neem seed powder against the beetle pest infesting milled products. Surprisingly, better control was observed either at lowest (1% w/w) or the highest doses (5% w/w). This finding is of great interest to understand the underlying phenomenon which we assume is the ability of T. castaneum to feed selectively in flour mediums, however, further research on this aspect is required to be investigated. The results of this study support the use of botanicals for stored product pest management.     

New fan worm genera and species (Polychaeta, Sabellidae, Fabriciinae) from the western Pacific, and cladistic relationships among genera

Five new species are described from among the genera Fabriciola Friedrich, 1939 (one species from Okinawa), Novafabricia Fitzhugh, 1990 (two species from Australia and Papua New Guinea, respectively), and two new monotypic genera, Brifacia gen. n. (Australia), and Pseudoaugeneriella gen. n. (Okinawa). All species are known only from the intertidal. Fabriciola rubra sp. n. is a member of the species complex with red peristomial and pygidial eyes, inferior thoracic flagellate setae, and abdominal pin-head setae. Novafabricia labrus sp. n., and N. exiguus sp. n., are distinguished from other species based on the distribution of inferior thoracic pseudospatulate setae. Brifacia metastellaris sp. n., resembles Fabricia stellaris (Müller, 1774) with regard to anterior peristomial ring collar construction and distribution of pseudospatulate setae, but differs in having abdominal uncini with relatively short manubria. Pseudoaugeneriella unirama sp. n., most closely resembles species of Augeneriella Banse, 1956 in general form, but has unbranched vascularized ventral filamentous appendages. Revised keys to fabriciin genera and selected species are provided. Cladistic relationships of these new genera and species to other Fabriciinae are examined and the status of some characters are reviewed. Whereas previous analyses have supported a monophyletic Augeneriella on the basis of branched, vascularized ventral filamentous appendages, present results show Augeneriella paraphyletic relative to Fabricia Blainville, 1828, Novafabricia, Fabricinuda Fitzhugh, 1990 Parafabricia Fitzhugh, 1992 and Brifacia. Similarly, some topologies show Novafabricia, which has only been defined by the presence of poorly developed dorsal lips, to be paraphyletic relative to Fabricia, Fabricinuda, Parafabricia, and Brifacia. The cladistic analyses do, however, support recognition of Brifacia and Pseudoaugeneriella. With the addition of Brifacia, the presence of unbranched, vascularized appendages is plesiomorphic for the Fabriciinae, which has not been previously hypothesized.     

Solution structure of agelenin, an insecticidal peptide isolated from the spider Agelena opulenta, and its structural similarities to insect-specific calcium channel inhibitors

Agelenin, isolated from the Agelenidae spider Agelena opulenta, is a peptide composed of 35 amino acids. We determined the three-dimensional structure of agelenin using two-dimensional NMR spectroscopy. The structure is composed of a short antiparallel beta-sheet and four beta-turns, which are stabilized by three disulfide bonds. Agelenin has characteristic residues, Phe9, Ser28 and Arg33, which are arranged similarly to the pharmacophore of the insect channel inhibitor, omega-atracotoxin-Hv1a. These observations suggest that agelenin and omega-atracotoxin-Hv1a bind to insect calcium channels in a similar manner. We also suggest that another mode of action may operate in the channel inhibition by omega-agatoxin-IVA and omega-atracotoxin-Hv2a.     

Astacin-like metalloproteases are a gene family of toxins present in the venom of different species of the brown spider (genus Loxosceles)

Brown spiders have a worldwide distribution, and their venom has a complex composition containing many different molecules. Herein, we report the existence of a family of astacin-like metalloprotease toxins in Loxosceles intermedia venom, as well as in the venom of different species of Loxosceles. Using a cDNA library from the L. intermedia venom gland, we cloned two novel cDNAs encoding astacin-like metalloprotease toxins, LALP2 and LALP3. Using an anti-serum against the previously described astacin-like toxin in L. intermedia venom (LALP1), we detected the presence of immunologically-related toxins in the venoms of L. intermedia, Loxosceles laeta, and Loxosceles gaucho. Zymographic experiments showed gelatinolytic activity of crude venoms of L. intermedia, L. laeta, and L. gaucho (which could be inhibited by the divalent metal chelator 1,10-phenanthroline) at electrophoretic mobilities identical to those reported for immunological cross-reactivity. Moreover, mRNAs extracted from L. laeta and L. gaucho venom glands were screened for astacin-like metalloproteases, and cDNAs obtained using LALP1-specific primers were sequenced, and their deduced amino acid sequences confirmed they were members of the astacin family with the family signatures (HEXXHXXGXXHE and MXY), LALP4 and LALP5, respectively. Sequence comparison of deduced amino acid sequences revealed that LALP2, LALP3, LALP4, and LALP5 are related to the astacin family. This study identified the existence of gene family of astacin-like toxins in the venoms of brown spiders and raises the possibility that these molecules are involved in the deleterious effects triggered by the venom.     

cDNA cloning, sequence analysis and molecular modeling of a new peptide from the scorpion Buthotus saulcyi venom

In this study, the cDNA of a new peptide from the venom of the scorpion, Buthotus saulcyi, was cloned and sequenced. It codes for a 64 residues peptide (Bsaul1) which shares high sequence similarity with depressant insect toxins of scorpions. The differences between them mainly appear in the loop1 which connects the beta-strand1 to the alpha-helix and seems to be functionally important in long chain scorpion neurotoxins. This loop is three amino acids longer in Bsaul1 compared to other depressant toxins. A comparative amino acid sequence analysis done on Bsaul1 and some of alpha-, beta-, excitatory and depressant toxins of scorpions showed that Bsaul1 contains all the residues which are highly conserved among long chain scorpion neurotoxins. Structural model of Bsaul1 was generated using Ts1 (a beta-toxin that competes with the depressant insect toxins for binding to Na(+) channels) as template. According to the molecular model of Bsaul1, the folding of the polypeptide chain is being composed of an anti-parallel three-stranded beta-sheet and a stretch of alpha- helix, tightly bound by a set of four disulfide bridges. A striking similarity in the spatial arrangement of some critical residues was shown by superposition of the backbone conformation of Bsaul1 and Ts1.     

Solution structure of Phrixotoxin 1, a specific peptide inhibitor of Kv4 potassium channels from the venom of the theraphosid spider Phrixotrichus auratus

Animal toxins block voltage-dependent potassium channels (Kv) either by occluding the conduction pore (pore blockers) or by modifying the channel gating properties (gating modifiers). Gating modifiers of Kv channels bind to four equivalent extracellular sites near the S3 and S4 segments, close to the voltage sensor. Phrixotoxins are gating modifiers that bind preferentially to the closed state of the channel and fold into the Inhibitory Cystine Knot structural motif. We have solved the solution structure of Phrixotoxin 1, a gating modifier of Kv4 potassium channels. Analysis of the molecular surface and the electrostatic anisotropy of Phrixotoxin 1 and of other toxins acting on voltage-dependent potassium channels allowed us to propose a toxin interacting surface that encompasses both the surface from which the dipole moment emerges and a neighboring hydrophobic surface rich in aromatic residues.     

A lysine rich C-terminal tail is directly involved in the toxicity of CSTX-1, a neurotoxic peptide from the venom of the spider Cupiennius salei

CSTX-1 (74 amino acids, 8,352.62 Da) is a potent neurotoxin from the venom of Cupiennius salei. With the monoclonal antibody 9H3 against CSTX-1, we identified two similar peptides by Western blot analysis. These two peptides were purified by RP-HPLC: CSTX-2a (61 amino acids, 6865.75 Da) and CSTX-2b (60 amino acids, 6709.57 Da). Using ESI-MS analysis and sequencing we verified that CSTX-2a is a truncated version of CSTX-1. CSTX-2b differs from CSTX-2a by the absence of Arg61. Toxicity of CSTX-1, CSTX-2a, and CSTX-2b to Drosophila melanogaster showed that the absence of the last 13 amino acids of CSTX-1 results in a seven-fold activity loss. CSTX-2b, which lacks Arg61 is 190-fold less toxic. We conclude that the C-terminal part of CSTX-1, especially Arg61, is essential for the expression of toxicity. CSTX-1 is degraded to CSTX-2a and CSTX-2b by proteases that are released from venom gland cells by apocrine secretion.     

Distinct primary structures of the major peptide toxins from the venom of the spider Macrothele gigas that bind to sites 3 and 4 in the sodium channel

Six peptide toxins (Magi 1-6) were isolated from the Hexathelidae spider Macrothele gigas. The amino acid sequences of Magi 1, 2, 5 and 6 have low similarities to the amino acid sequences of known spider toxins. The primary structure of Magi 3 is similar to the structure of the palmitoylated peptide named PlTx-II from the North American spider Plectreurys tristis (Plectreuridae). Moreover, the amino acid sequence of Magi 4, which was revealed by cloning of its cDNA, displays similarities to the Na+ channel modifier delta-atracotoxin from the Australian spider Atrax robustus (Hexathelidae). Competitive binding assays using several 125I-labelled peptide toxins clearly demonstrated the specific binding affinity of Magi 1-5 to site 3 of the insect sodium channel and also that of Magi 5 to site 4 of the rat sodium channel. Only Magi 6 did not compete with the scorpion toxin LqhalphaIT in binding to site 3 despite high toxicity on lepidoptera larvae of 3.1 nmol/g. The K(i)s of other toxins were between 50 pM for Magi 4 and 1747 nM for Magi 1. In addition, only Magi 5 binds to both site 3 in insects (K(i)=267 nM) and site 4 in rat brain synaptosomes (K(i)=1.2 nM), whereas it showed no affinities for either mammal binding site 3 or insect binding site 4. Magi 5 is the first spider toxin with binding affinity to site 4 of a mammalian sodium channel.     

Biological characteristics of three co‐occurring species of armorhead from different genera vary markedly from previous results for the Pentacerotidae

Biological characteristics of Pentaceropsis recurvirostris, Paristiopterus gallipavo and Parazanclistius hutchinsi were determined from commercial gillnet samples from temperate south‐western Australian coastal waters. Growth zones in otoliths, with more than a few such zones, were readily detectable only after the otoliths had been sectioned. Visual analyses and modelling of the trends in marginal increments on sectioned otoliths demonstrate that these opaque zones are formed annually. Maximum ages of 55, 36 and 49 years, derived for P. recurvirostris, P. gallipavo and P. hutchinsi, respectively, reflect relatively low mortalities. These longevities greatly exceed those estimated, using otoliths, for Pentaceros wheeleri and Pentaceros richardsoni, which belong to the other pentacerotid subfamily. These differences may be due to the counts of ‘daily’ growth zones in sectioned otoliths of P. wheeleri not representing the complete age range of that species and the zones detected in whole otoliths of P. richardsoni not constituting the complete range of annually‐formed zones. Pentaceropsis recurvirostris, P. gallipavo and P. hutchinsi recruited into the fishery in the sampling area as 2–3 year‐old fishes. Pentaceropsis recurvirostris and P. hutchinsi exhibited little or no subsequent growth throughout the remainder of their protracted life, whereas, P. gallipavo continued to grow for c. 5 years and then underwent little further growth. Spawning of P. recurvirostris and P. hutchinsi peaked in the austral winter and autumn, respectively, but in the austral spring and summer with P. gallipavo, which is more typical of temperate species. Although the females of P. gallipavo and P. hutchinsi were mature, this did not apply to a few P. recurvirostris, some of which were >20 years old, implying that any given female of this species does not always spawn every year. Ovarian mass greatly exceeded testis mass, indicative of pair spawning, which is consistent with field observations. In contrast to P. recurvirostris and P. hutchinsi, the sex ratio was heavily biased towards males and the spawning period longer in P. gallipavo, suggesting that selection pressures for spawning success were greater for this latter species.     

A new species of spider fly in the genus Sabroskya Schlinger from Malawi, with a key to Acrocerinae world genera (Diptera, Acroceridae)

In this paper we diagnose the genus Sabroskya Schlinger, 1960 and describe Sabroskya schlingerisp. n. from Malawi. We also provide dichotomous keys to species of Sabroskya and to world genera of the subfamily Acrocerinae, both extant and extinct.     

Metagenomic analysis of the viromes of three North American bat species: viral diversity among different bat species that share a common habitat

Effective prediction of future viral zoonoses requires an in-depth understanding of the heterologous viral population in key animal species that will likely serve as reservoir hosts or intermediates during the next viral epidemic. The importance of bats as natural hosts for several important viral zoonoses, including Ebola, Marburg, Nipah, Hendra, and rabies viruses and severe acute respiratory syndrome-coronavirus (SARS-CoV), has been established; however, the large viral population diversity (virome) of bats has been partially determined for only a few of the ～1,200 bat species. To assess the virome of North American bats, we collected fecal, oral, urine, and tissue samples from individual bats captured at an abandoned railroad tunnel in Maryland that is cohabitated by 7 to 10 different bat species. Here, we present preliminary characterization of the virome of three common North American bat species, including big brown bats (Eptesicus fuscus), tricolored bats (Perimyotis subflavus), and little brown myotis (Myotis lucifugus). In samples derived from these bats, we identified viral sequences that were similar to at least three novel group 1 CoVs, large numbers of insect and plant virus sequences, and nearly full-length genomic sequences of two novel bacteriophages. These observations suggest that bats encounter and disseminate a large assortment of viruses capable of infecting many different animals, insects, and plants in nature.