Wolbachia neither induces nor suppresses transcripts encoding antimicrobial peptides

Wolbachia are intracellular maternally inherited microorganisms that are associated with reproductive abnormalities such as cytoplasmic incompatibility (CI), feminization and parthenogenesis in the various arthropod species they infect. Surveys indicate that these bacteria infect more than 16% of all insect species as well as isopods, mites and nematodes, making Wolbachia one of the most ubiquitous parasites yet described. However, nothing is known about the interactions of this bacterium with the host's immune system. We studied the expression of inducible antimicrobial markers in the adults of two Wolbachia infected insect species, Drosophila simulans and Aedes albopictus. The lack of available immune markers in the mosquito species led us to clone part of the defensin gene from this species, which was found to be very similar to the other mosquito defensins cloned from Anopheles gambiae and Aedes aegypti. Comparisons of the expression pattern of the antibacterial markers between Wolbachia-infected and cured lines, and also between bacteria-challenged and unchallenged adults indicated that Wolbachia does not either constitutively induce or suppress the transcription of these antibacterial genes. In addition, no difference in the transcription of these genes was found between double and single Wolbachia-infected strains or between strains in which Wolbachia has different tissue tropisms.

Expressed sequenced tags and new genes from the cattle tick, Boophilus microplus

We used the expressed sequenced tags (ESTs) approach to study the genome of the cattle tick Boophilus microplus. One hundred and forty-two of our 234 unique ESTs were from genes not previously identified from ticks, mites or any other arachnids. The largest class of identified ESTs (29%) was from genes involved in transcription and translation. Ninety-one ESTs (39% of all ESTs) did not match any sequences in international databases; some of these may be specific to ticks. Thirteen percent of our ESTs were from ribosomal proteins and two ESTs were for genes implicated in resistance to pesticides. (C) 1998 Chapman & Hall Ltd.

Phylogeny of the hard ticks (Ixodidae) inferred from 18S rRNA indicates that the genus Aponomma is paraphyletic

We examined the phylogeny of ticks (Acari:Parasitiformes:Ixodida) and their closest known mite relatives (Acari:Parasitiformes:Mesostigmata and Holothyrida) using 18S rRNA sequences. In our analyses, we included sequences from 36 taxa. Sequences for 13 hard ticks (Family Ixodidae), 5 soft ticks (Family Argasidae), and 2 mesostigmatid mites were obtained from the GenBank database and we generated sequences for 15 hard ticks and 1 holothyrid mite. Ten of these tick species were endemic to Australia. Our analyses indicated that the suborder Holothyrida is more closely related to Ixodida than to Mesostigmata, the group used as outgroup in earlier molecular studies. This finding is consistent with Lehtinen's (1991) hypothesis that the Holothyrida rather than the Mesostigmata is the sister-group to the Ixodida. Within the hard ticks the genus Aponomma and thus the family Amblyomminae were paraphyletic. Taxonomic revision of these taxa is needed. The genus Amblyomma was paraphyletic without the inclusion of typical Aponomma species (Ap. latum and Ap. fimbriatum). There was a basal divergence between endemic Australian and other species in both the Metastriata and the Prostriata divisions of the hard ticks. (C) 1999 Academic Press.

Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge

We have isolated a family of insect-selective neurotoxins from the venom of the Australian funnel-web spider that appear to be good candidates for biopesticide engineering. These peptides, which we have named the Janus-faced atracotoxins (J-ACTXs), each contain 36 or 37 residues, with four disulfide bridges, and they show no homology to any sequences in the protein/DNA databases. The three-dimensional structure of one of these toxins reveals an extremely rare vicinal disulfide bridge that we demonstrate to be critical for insecticidal activity. We propose that J-ACTX comprises an ancestral protein fold that we refer to as the disulfide-directed beta-hairpin.

Why fear snakes and spiders? Evidence for a learning-based account of fear-relevant stimulus-processing

Fear-relevant stimuli, such as snakes, spiders and heights, preferentially capture attention as compared to nonfear-relevant stimuli. This is said to reflect an encapsulated mechanism whereby attention is captured by the simple perceptual features of stimuli that have evolutionary significance. Research, using pictures of snakes and spiders, has found some support for this account; however, participants may have had prior fear of snakes and spiders that influenced results. The current research compared responses of snake and spider experts who had little fear of snakes and spiders, and control participants across a series of affective priming and visual search tasks. Experts discriminated between dangerous and nondangerous snakes and spiders, and expert responses to pictures of nondangerous snakes and spiders differed from those of control participants. The current results dispute that stimulus fear relevance is based purely on perceptual features, and provides support for the role of learning and experience.

Identification of novel non-autonomous CemaT transposable elements and evidence of their mobility within the C. elegans genome

We describe here two new transposable elements, CemaT4 and CemaT5, that were identified within the sequenced genome of Caenorhabditis elegans using homology based searches. Five variants of CemaT4 were found, all non-autonomous and sharing 26 bp inverted terminal repeats (ITRs) and segments (152-367 bp) of sequence with similarity to the CemaT1 transposon of C. elegans. Sixteen copies of a short, 30 bp repetitive sequence, comprised entirely of an inverted repeat of the first 15 bp of CemaT4's ITR, were also found, each flanked by TA dinucleotide duplications, which are hallmarks of target site duplications of mariner-Tc transposon transpositions. The CemaT5 transposable element had no similarity to maT elements, except for sharing identical ITR sequences with CemaT3. We provide evidence that CemaT5 and CemaT3 are capable of excising from the C. elegans genome, despite neither transposon being capable of encoding a functional transposase enzyme. Presumably, these two transposons are cross-mobilised by an autonomous transposon that recognises their shared ITRs. The excisions of these and other non-autonomous elements may provide opportunities for abortive gap repair to create internal deletions and/or insert novel sequence within these transposons. The influence of non-autonomous element mobility and structural diversity on genome variation is discussed.

The structure of versutoxin (delta-atracotoxin-Hv1) provides insights into the binding of site 3 neurotoxins to the voltage-gated sodium channel

Background: Versutoxin (delta-ACTX-Hv1) is the major component of the venom of the Australian Blue Mountains funnel web spider, Hadronyche versuta. delta-ACTX-Hv1 produces potentially fatal neurotoxic symptoms in primates by slowing the inactivation of voltage-gated sodium channels; delta-ACTX-Hv1 is therefore a useful tool for studying sodium channel function. We have determined the three-dimensional structure of delta ACTX-Hv1 as the first step towards understanding the molecular basis of its interaction with these channels. Results: The solution structure of delta-ACTX-Hv1, determined using NMR spectroscopy, comprises a core beta region containing a triple-stranded antiparallel beta sheet, a thumb-like extension protruding from the beta region and a C-terminal 3(10) helix that is appended to the beta domain by virtue of a disulphide bond. The beta region contains a cystine knot motif similar to that seen in other neurotoxic polypeptides. The structure shows homology with mu-agatoxin-l, a spider toxin that also modifies the inactivation kinetics of vertebrate voltage-gated sodium channels. More surprisingly, delta-ACTX-Hv1 shows both sequence and structural homology with gurmarin, a plant polypeptide. This similarity leads us to suggest that the sweet-taste suppression elicited by gurmarin may result from an interaction with one of the downstream ion channels involved in sweet-taste transduction. Conclusions: delta-ACTX-Hv1 shows no structural homology with either sea anemone or alpha-scorpion toxins, both of which also modify the inactivation kinetics of voltage-gated sodium channels by interacting with channel recognition site 3. However, we have shown that delta-ACTX-Hv1 contains charged residues that are topologically related to those implicated in the binding of sea anemone and alpha-scorpion toxins to mammalian voltage-gated sodium channels, suggesting similarities in their mode of interaction with these channels.

Discovery and structure of a potent and highly specific blockerof insect calcium channels

We have isolated a novel family of insect-selective neurotoxins that appear to be the most potent blockers of insect voltage-gated calcium channels reported to date. These toxins display exceptional phylogenetic specificity, with at least a 10,000-fold preference for insect versus vertebrate calcium channels. The structure of one of the toxins reveals a highly structured, disulfide-rich core and a structurally disordered C-terminal extension that is essential for channel blocking activity. Weak structural/functional homology with omega -agatoxin-IVA/B, the prototypic inhibitor of vertebrate P-type calcium channels, suggests that these two toxin families might share a similar mechanism of action despite their vastly different phylogenetic specificities.

The cystine knot motif in toxins and implications for drug design

The cystine knot structural motif is present in peptides and proteins from a variety of species, including fungi, plants, marine molluscs. insects and spiders. It comprises an embedded ring formed by two disulfide bonds and their connecting backbone segments which is threaded by a third disulfide bond. It is invariably associated with nearby beta-sheet structure and appears to be a highly efficient motif for structure stabilization. Because of this stability it makes an ideal framework for molecular engineering applications. In this review we summarize the main structural features of the cystine knot motif, focussing on toxin molecules containing either the inhibitor cystine knot or the cyclic cystine knot. Peptides containing these motifs are 26-48 residues long and include ion channel blockers, haemolytic agents, as well as molecules having antiviral and antibacterial activities. The stability of peptide toxins containing the cystine knot motif, their range of bioactivities and their unique structural scaffold can be harnessed for molecular engineering applications and in drug design. Applications of cystine knot molecules for the treatment of pain. and their potential use in antiviral and antibacterial applications are described. (C) 2000 Elsevier Science Ltd. All rights reserved.

Host plant specificity in several species of generalist mite predators

1. Species in the genus Neoseiulus are considered to be generalist predators. with some species used in biological control programmes against phytophagous mites and insects. 2. A general survey of Neoseiulus species in inland Australia indicated that different species are associated with particular tree species. This pattern of host plant use was investigated for four Neoseiulus species (N. buxeus, N. cappari, N. brigarinus, N. eremitus) by means of a sampling programme through time and across space. 3. Each species of Neoseiulus was collected entirely or mostly from one species of tree: little or no overlap was detected despite the tree species growing in well-mixed stands. Host plant specificity thus appears to be strong in this genus. 4. Species in two other genera (Pholaseius and Australiseiulus), also considered to be predatory, showed a similar association with particular tree species. 5. The implications for the use of these predators in biological control are considerable. In particular, phytoseiid species with specific needs in terms of host plants may not be suitable for use as general purpose predators. Meeting the needs of phytoseiids through the modification of host plant attributes may be a step towards enhancing their efficacy as biological control agents.

Argyrodes: phylogeny, sociality and interspecific interactions - a report on the Argyrodes symposium, Badplaas 2001

Argyrodes Simon 1864 is a large, cosmopolitan theridiid genus whose members exhibit a wide range of foraging techniques which usually involve exploiting other spiders, either by using their webs, stealing their food, or preying on them directly. We held a symposium on this genus at the 15th International Congress of Arachnology, Badplaas, South Africa in order to obtain a clearer perspective on the relationship between the phylogeny of the genus and the different foraging techniques. We concluded that Argyrodes forms a monophyletic group within the Theridiidae, and that there are clear monophyletic clades within the genus (already identified as species groups) that appear to share behavioral characteristics. We found no clear indication that foraging behaviors such as kleptoparasitism (stealing food) evolved from araneophagy (eating spiders) or vice versa. However, it appears that species that specialize in either kleptoparasitism or araneophagy use additional techniques in comparison to species that readily use both foraging modes. During our examination of Argyrodes/host interactions we noted the importance of Nephila species as hosts of Argyrodes species around the world and the impact of Argyrodes on Nephila. We also noted the fluid nature of the relationship between Argyrodes and the spiders with which they interact. For example, an Argyrodes/host relationship can change to an Argyrodes/prey relationship, and the type of kleptoparasitic behavior employed by an Argyrodes can change when it changes host species. The importance of eating silk was also noted and identified as an area for further research. We concluded that more work involving international collaboration is needed to fully understand the phylogeny of the genus and the relationships between the different types of foraging behaviors.

The mitochondrial 12S gene is a suitable marker of populations of Sarcoptes scabiei from wombats, dogs and humans in Australia

We sequenced part of the mitochondrial 12S ribosomal RNA gene of 23 specimens of Sarcoptes scabiei from eight wombats, one dog and three humans. Twelve of the 326 nucleotide positions varied among these mites and there were nine haplotypes (sequences) that differed by 1-8 nucleotides. Phylogenetic analyses indicated that these mites were from two lineages: (1) mites from wombats from Victoria, Australia, and mites from the humans and dog from the Northern Territory, Australia (haplotypes 1-4, 9); and (2) mites from the humans and dog from the Northern Territory (haplotypes 5-8). Mites from the three different hosts (wombats, a dog and humans) had not diverged phylogenetically; rather, these mites had similar 12S sequences. Thus, we conclude that these mites from wombats, humans and a dog are closely related, and that they diverged from a common ancestor relatively recently. This conclusion is consistent with the argument that people and/or their dogs introduced to Australia the S. scabiei mites that infect wombats Australia. So, S. scabiei, which has been blamed for the extinction of populations of wombats in Australia, may be a parasitic mite that was introduced to Australia with people and/or their dogs. These data show that the mitochondrial 12S rRNA gene may be a suitable population marker of S. scabiei from wombats, dogs and humans in Australia.

Solution Structures of the cis- and trans-Pro30 Isomers of a Novel 38-Residue Toxin from the Venom of Hadronyche Infensa sp. that Contains a Cystine-Knot Motif within Its Four Disulfide Bonds

The primary sequence and three-dimensional structure of a novel peptide toxin isolated from the Australian funnel-web spider Hadronyche infensa sp. is reported. ACTX-HI:OB4219 contains 38 amino acids, including eight-cysteine residues that form four disulfide bonds. The connectivities of these disulfide bonds were previously unknown but have been unambiguously determined in this study. Three of these disulfide bonds are arranged in an inhibitor cystine-knot (ICK) motif, which is observed in a range of other disulfide-rich peptide toxins. The motif incorporates an embedded ring in the structure formed by two of the disulfides and their connecting backbone segments penetrated by a third disulfide bond. Using NMR spectroscopy, we determined that despite the isolation of a single native homologous product by RP-HPLC, ACTX-HI:OB4219 possesses two equally populated conformers in solution. These two conformers were determined to arise from cis/trans isomerization of the bond preceding Pro30. Full assignment of the NMR spectra for both conformers allowed for the calculation of their structures, revealing, the presence of a triple-stranded antiparallel sheet consistent with the inhibitor cystine-knot (ICK) motif.