Many chemical insecticides are becoming less efficacious due to rising resistance in pest species, which has created much interest in the development of new, eco-friendly bioinsecticides. Since insects are the primary prey of most spiders, their venoms are a rich source of insect-active peptides that can be used as leads for new bioinsecticides or as tools to study molecular receptors that are insecticidal targets. In the present study, we isolated two insecticidal peptides, µ/ω-TRTX-Mb1a and -Mb1b, from venom of the African tarantula Monocentropus balfouri. Recombinant µ/ω-TRTX-Mb1a and -Mb1b paralyzed both Lucilia cuprina (Australian sheep blowfly) and Musca domestica (housefly), but neither peptide affected larvae of Helicoverpa armigera (cotton bollworms). Both peptides inhibited currents mediated by voltage-gated sodium (Na_V) and calcium channels in Periplaneta americana (American cockroach) dorsal unpaired median neurons, and they also inhibited the cloned Blattella germanica (German cockroach) Na_V channel (BgNa_V1). An additional effect seen only with Mb1a on BgNa_V1 was a delay in fast inactivation. Comparison of the Na_V channel sequences of the tested insect species revealed that variations in the S1–S2 loops in the voltage sensor domains might underlie the differences in activity between different phyla. Full article

Theraphosid spiders (tarantulas) are venomous arthropods found in most tropical and subtropical regions of the world. Tarantula venoms are a complex cocktail of toxins with potential use as pharmacological tools, drugs and bioinsecticides. Although numerous toxins have been isolated from tarantula venoms, little research has been carried out on the venom of Australian tarantulas. We therefore investigated the venom profile of the Australian theraphosid spider Phlogius crassipes and examined whether there are ontogenetic changes in venom composition. Spiders were divided into four ontogenic groups according to cephalothorax length, then the venom composition of each group was examined using gel electrophoresis and mass spectrometry. We found that the venom of P. crassipes changes continuously during development and throughout adulthood. Our data highlight the need to investigate the venom of organisms over the course of their lives to uncover and understand the changing functions of venom and the full range of toxins expressed. This in turn should lead to a deeper understanding of the organism’s ecology and enhance the potential for biodiscovery. Full article

The piercing-sucking mouthparts of the true bugs (Insecta: Hemiptera: Heteroptera) have allowed diversification from a plant-feeding ancestor into a wide range of trophic strategies that include predation and blood-feeding. Crucial to the success of each of these strategies is the injection of venom. Here we review the current state of knowledge with regard to heteropteran venoms. Predaceous species produce venoms that induce rapid paralysis and liquefaction. These venoms are powerfully insecticidal, and may cause paralysis or death when injected into vertebrates. Disulfide-rich peptides, bioactive phospholipids, small molecules such as N,N-dimethylaniline and 1,2,5-trithiepane, and toxic enzymes such as phospholipase A₂, have been reported in predatory venoms. However, the detailed composition and molecular targets of predatory venoms are largely unknown. In contrast, recent research into blood-feeding heteropterans has revealed the structure and function of many protein and non-protein components that facilitate acquisition of blood meals. Blood-feeding venoms lack paralytic or liquefying activity but instead are cocktails of pharmacological modulators that disable the host haemostatic systems simultaneously at multiple points. The multiple ways venom is used by heteropterans suggests that further study will reveal heteropteran venom components with a wide range of bioactivities that may be recruited for use as bioinsecticides, human therapeutics, and pharmacological tools. Full article

The inhibitor cystine knot (ICK) is an unusual three-disulfide architecture in which one of the disulfide bonds bisects a loop formed by the two other disulfide bridges and the intervening sections of the protein backbone. Peptides containing an ICK motif are frequently considered to have high levels of thermal, chemical and enzymatic stability due to cross-bracing provided by the disulfide bonds. Experimental studies supporting this contention are rare, in particular for spider-venom toxins, which represent the largest diversity of ICK peptides. We used ω-hexatoxin-Hv1a (Hv1a), an insecticidal toxin from the deadly Australian funnel-web spider, as a model system to examine the contribution of the cystine knot to the stability of ICK peptides. We show that Hv1a is highly stable when subjected to temperatures up to 75 °C, pH values as low as 1, and various organic solvents. Moreover, Hv1a was highly resistant to digestion by proteinase K and when incubated in insect hemolymph and human plasma. We demonstrate that the ICK motif is essential for the remarkable stability of Hv1a, with the peptide’s stability being dramatically reduced when the disulfide bonds are eliminated. Thus, this study demonstrates that the ICK motif significantly enhances the chemical and thermal stability of spider-venom peptides and provides them with a high level of protease resistance. This study also provides guidance to the conditions under which Hv1a could be stored and deployed as a bioinsecticide. Full article

Voltage-gated sodium (Na_V) channels are responsible for propagating action potentials in excitable cells. Na_V1.7 plays a crucial role in the human pain signalling pathway and it is an important therapeutic target for treatment of chronic pain. Numerous spider venom peptides have been shown to modulate the activity of Na_V channels and these peptides represent a rich source of research tools and therapeutic lead molecules. The aim of this study was to determine the diversity of Na_V1.7-active peptides in the venom of an Australian Phlogius sp. tarantula and to characterise their potency and subtype selectivity. We isolated three novel peptides, μ-TRTX-Phlo1a, -Phlo1b and -Phlo2a, that inhibit human Na_V1.7 (hNa_V1.7). Phlo1a and Phlo1b are 35-residue peptides that differ by one amino acid and belong in NaSpTx family 2. The partial sequence of Phlo2a revealed extensive similarity with ProTx-II from NaSpTx family 3. Phlo1a and Phlo1b inhibit hNa_V1.7 with IC₅₀ values of 459 and 360 nM, respectively, with only minor inhibitory activity on rat Na_V1.2 and hNa_V1.5. Although similarly potent at hNa_V1.7 (IC₅₀ 333 nM), Phlo2a was less selective, as it also potently inhibited rNa_V1.2 and hNa_V1.5. All three peptides cause a depolarising shift in the voltage-dependence of hNa_V1.7 activation. Full article

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. Full article

Although known for their potent venom and ability to prey upon both invertebrate and vertebrate species, the Barychelidae spider family has been entirely neglected by toxinologists. In striking contrast, the sister family Theraphosidae (commonly known as tarantulas), which last shared a most recent common ancestor with Barychelidae over 200 million years ago, has received much attention, accounting for 25% of all the described spider toxins while representing only 2% of all spider species. In this study, we evaluated for the first time the venom arsenal of a barychelid spider, Trittame loki, using transcriptomic, proteomic, and bioinformatic methods. The venom was revealed to be dominated by extremely diverse inhibitor cystine knot (ICK)/knottin peptides, accounting for 42 of the 46 full-length toxin precursors recovered in the transcriptomic sequencing. In addition to documenting differential rates of evolution adopted by different ICK/knottin toxin lineages, we discovered homologues with completely novel cysteine skeletal architecture. Moreover, acetylcholinesterase and neprilysin were revealed for the first time as part of the spider-venom arsenal and CAP (CRiSP/Allergen/PR-1) were identified for the first time in mygalomorph spider venoms. These results not only highlight the extent of venom diversification in this neglected ancient spider lineage, but also reinforce the idea that unique venomous lineages are rich pools of novel biomolecules that may have significant applied uses as therapeutics and/or insecticides. Full article