Abstract The slipper lobster ( Thenus australiensis ) is rapidly emerging as a high-potential species for commercial aquaculture. Because females exhibit superior growth characteristics due to less frequent moulting after sexual maturity, developing monosex breeding strategies is highly desirable for industry profitability. However, the lack of genomic resources and early sex-identification tools has hindered this development. Here, we report the first draft male genome assembly for T. australiensis , generated using a combination of whole-genome shotgun sequencing, DArT-seq, and multi-tissue transcriptomics. The curated assembly spans 0.913 Gbp with high functional completeness (93.0% BUSCO), providing a robust repertoire of 30,100 protein-coding genes. Through k-mer subtraction and population-level DArT-seq genotyping, we provide definitive evidence that T. australiensis utilizes an XX/XY sex-determination system. Crucially, by identifying male-specific structural variations within a neo-Y locus, we developed a diagnostic PCR assay targeting a male-exclusive sequence. This 171 bp marker achieved 100% accuracy in phenotypic sex identification across wild-caught populations. Ultimately, these foundational genomic resources, combined with a highly reliable molecular sexing tool, provide the critical framework necessary for early sex sorting, broodstock management, and the commercial advancement of monosex slipper lobster farming. Highlights First draft genome of slipper lobster (0.913 Gbp) assembled. Multi-omics approach confirms an XX/XY sex-determination system. Developed a 171 bp diagnostic PCR sex marker with 100% accuracy. Facilitates early sex sorting for profitable monosex aquaculture.

IntroductionScorpion venoms are natural sources of neurotoxins that primarily modulate the properties of neuronal ion channels, particularly voltage-gated sodium and potassium channels. However, they also contain a variety of small bioactive molecules that have been largely neglected in the omics era of toxinological research.MethodsIn the present study, we employed a high-throughput FLIPR screen of arthropod venoms against nicotinic acetylcholine receptors (nAChRs) expressed in the human SH-SY5Y neuroblastoma cell line. ResultsThe venom of the Malaysian forest scorpion Heterometrus spinifer (family: Scorpionidae) proved to be an agonist of nAChRs, and the small choline ester senecioylcholine was identified as being responsible for the observed effect. Synthetic senecioylcholine and its isomers tigloylcholine and angeloylcholine were not insecticidal when injected into sheep blowflies. DiscussionBased on previous reports of senecioylcholine being toxic to vertebrates along with the agonistic effect we observed on human nAChRs, we postulate that senecioylcholine in scorpion venom is likely to assist with vertebrate prey capture and defense against vertebrate predators. Since senecioylcholine has only been previously reported in marine gastropods and moths, its presence in scorpion venom seems to represent an unusual case of convergent evolution.

Abstract Global food supply strongly depends on honeybee pollination services, which are threatened by insecticides and pests such as parasitic Varroa destructor mites. Chemical varroacides/acaricides are hampered by resistance development, necessitating the development of sustainable and environmentally friendly alternatives, with arthropod venom peptides being considered promising sources of acaricidal toxins. With only a few acaricidal venom peptides being reported, we performed a systematic topical screening of 50 arthropod venoms against V. destructor, with 78% of the venoms causing 100% mortality after 24 h. Deconvolution of the venoms from the Tasmanian cave spider Hickmania troglodytes and the Giant Japanese funnel-web spider Gigathele gigas led to identification of the varroacidal peptides Ht1a and Gg1a. Topical application of Ht1a and Gg1a reduced varroa mite but not honeybee survival, despite Ht1a inhibiting voltage-gated sodium channels from varroa and honeybee with equal potency. Ht1a and Gg1a were inactive against human skeletal muscle (hNaV1.4), cardiac (NaV1.5), neuronal NaV channel isoforms, and human voltage-gated calcium channel CaV2.2. At human α3β2/4 nicotinic acetylcholine receptors, Gg1a was inactive while 10 µM of Ht1a partially blocked nicotine-mediated Ca2+ influx. Our data reveal Ht1a and Gg1a as promising candidates for the development of novel varroa mite treatments of honeybee hives.

Scorpion venoms are likewise a medical burden as well as a source of novel bioresources. Despite their important dual role, the venoms of most scorpion species remain under- or unstudied. Among these is the venom of the Black Judaicus scorpion, Hottentotta judaicus (Simon, 1872), a common yet neglected species native to the Middle East. Here, we employ venom gland transcriptomics to investigate its toxin-encoding precursor profile to gain insight into its toxin repertoire. The venom was found to be composed primarily of various short scorpion toxins, long scorpion toxins from the 3 C-C as well as the 4 C-C type, and enzymatic components. Minor components include, defensins and putative antimicrobial peptides. Several identified toxins show similarity to known neurotoxins from lethal buthids or to toxins with translational value in biomedicine, agriculture, and industrial production, thus rendering H. judaicus both, a potential health concern and source of novel bioresources. Our work provides an extended perspective on the venom profile of this species and represents a basis for future follow-up studies. ### Competing Interest Statement The authors have declared no competing interest.

Conspecific male to female envenomation, though rare, has been documented across venomous taxa. While traditionally interpreted as a coercive mating strategy to enhance male reproductive success and to avoid cannibalism, this explanation may not fully account for the behaviour in scorpions, which exhibit minor sexual size dimorphism and complex courtship rituals. This review explores the possibly multifaceted roles of sexual stinging in scorpions. We highlight potential adaptive strategies, such as venom metering and compositional plasticity, that allow males to subdue females without causing lethal harm. We discuss hypotheses on the evolution of sexual stings, ranging from sexual coercion to chemical seduction and cooperative signalling. Finally, we propose future research directions, including comparative venomics, behavioural assays, and ecological studies, to shed light on the selective pressures that shape this enigmatic behaviour. By integrating insights from physiology, ecology, and evolution, this review advances our understanding of sexual envenomation as a dynamic interplay between conflict and cooperation in scorpion reproductive strategies.

Scorpion venoms are likewise a medical burden as well as a source of novel bioresources. Despite their important dual role, the venoms of most scorpion species remain under- or unstudied. Among these is the venom of the Black Judaicus scorpion, Hottentotta judaicus (Simon, 1872), a common yet neglected species native to the Middle East. Here, we employ venom gland transcriptomics to investigate its toxin-encoding precursor profile to gain insight into its toxin repertoire. The venom was found to be composed primarily of various short scorpion toxins, long scorpion toxins from the 3 C-C as well as the 4 C-C type, and enzymatic components. Minor components include, defensins and putative antimicrobial peptides. Several identified toxins show similarity to known neurotoxins from lethal buthids or to toxins with translational value in biomedicine, agriculture, and industrial production, thus rendering H. judaicus both, a potential health concern but possibly also as source of novel bioresources. Our work provides an extended perspective on the venom profile of this species and represents a basis for future follow-up studies.

Most spiders deploy paralytic venom for prey capture, but adults of the yellow sac spider (Cheiracanthium punctorium) instead produce a predominantly defensive venom to safeguard their offspring. Here, we characterized the molecular repertoire of C. punctorium venom to shed light on its evolutionary history. Unlike venom in other spiders, C. punctorium venom mostly comprises neurotoxic double domain neurotoxin 19 family (CSTX) peptides and enzymes, such as phospholipase A2 (PLA2). Comparative venomics in four spiders representing two infraorders showed that CSTXs arose following the mygalomorph-araneomorph split ca. 300 mya by means of ancestral gene duplication and functional specialization. A gene fusion event then merged CSTXs from two distinct clades to form the double domain toxin. PLA2 proteins were convergently recruited to C. punctorium to fulfil a defensive function and are strikingly similar to proalgesic PLA2 proteins in bee venom. These complex, multimodal molecular innovations in venom systems highlight natures tendency to use the same molecular solutions for similar ecological challenges across diverse animal lineages. ### Competing Interest Statement The authors have declared no competing interest.

Peptide toxins from spider venoms are being increasingly hailed as environmentally friendly alternatives to market-dominating small-molecule chemical insecticides. While the stability of knotted spider-venom peptides towards enzymatic degradation, temperature changes and varying pH conditions has already been examined, their susceptibility to sunlight remains unclear. Field applications of insecticides demand that the insecticidal component is active for at least a few days to ensure sufficient eradication of the targeted insect pests. We therefore exposed four insecticidal spider-venom peptides (ω-Hv1a, ω/κ-Hv1a, Ta1a and Dc1a) to continuous artificial sunlight for up to 7 days. After certain incubation periods, we quantified the percentage of intact peptide and identified sites of peptide cleavage. We found that after 3 days of continuous exposure (=6 days of 12 h/d sunlight), the amount of remaining intact peptide was 16 % (Ta1a), 21 % (Dc1a), 55 % (ω-Hv1a), and 67 % (ω/κ-Hv1a), whereas bovine serum albumin was completely degraded. Even after 7 days (=14 days of 12 h/d sunlight) exposure, more than 50 % of ω/κ-Hv1a and ω-Hv1a remained intact. Peptides with lower molecular mass tended to be less susceptible to sunlight, while cleavage of peptide bonds involving proline or cysteine were most susceptible to photochemical degradation. The photochemical changes detected by mass spectrometry mainly comprised oxidations, deamidations, and cysteine-targeted modifications.

Lepidopterans are the most economically significant crop pests. They are mainly controlled with chemical insecticides that often suffer from insect resistance and adverse effects on the environment and human health. Insect-specific spider-venom peptides are considered safer, eco-friendly alternatives to chemical insecticides, and two insecticidal spider-venom derived peptides have already been commercialised by Vestaron Corporation, while more candidates are being progressed in global discovery pipelines. This study focusses on two insecticidal spider-venom peptides—Dc1a and Ta1a— and assesses their suitability in targeting five species of lepidopteran pests by injection and oral application, using Vestaron’s commercial ω/κ-Hv1a as a reference. In addition, the potential of bacterial Bt Cry toxins in providing synergistic insecticidal activities with these spider-venom peptides is evaluated. We found that when applied by injection, all peptides caused paralysis and mortality against all tested lepidopteran species within a narrow dose range. In contrast, the differences in insecticidal activity were more pronounced when orally applied. Helicoverpa consistently was the genus most susceptible to the spider-venom peptides, independent of the route of application, which we presume being related to its particular food preferences. Furthermore, we found synergistic activities for co-application of each of the spider-venom peptides with sublethal amounts of Bt Cry toxins in H. armigera. Overall, our results indicate that Dc1a and Ta1a are suitable bioinsecticide candidates for targeting certain lepidopteran pests, with co-application of Bt Cry toxins considered a viable strategy for increasing their efficacy as foliar sprays.

Microbes play vital roles in ecological systems, yet their presence and functions within venom environments of venomous organisms remain understudied. Despite the prevalent belief in the sterility of venoms, recent findings reveal diverse microbial communities within venom systems. This review aims to explore the relationships between venoms and microbes, highlighting their potential roles in evolutionary processes, ecological interactions, and therapeutic advancements. Venoms, composed of toxins utilized in hunting or defense, represent a rich source of natural products with applications in drug discovery and therapy, exemplified by FDA-approved venom toxin-derived drugs. Understanding microbial resistance mechanisms against antimicrobial peptides can illuminate coevolutionary processes and guide therapeutic development. Integrating hologenomic evolution and microbial ecology frameworks will facilitate comprehensive research on venom-microbiome interactions, and reveal the evolutionary drivers of venom diversification. Investigating and investing in these relationships promises advancements in understanding evolution, ecology, and biotechnology, with implications for human health and ecological conservation. This review synthesizes existing knowledge, identifies many gaps in literature, and investigates critical unanswered questions in the field of venom microbiology, encouraging ongoing and future collaborative research.

Spider venoms are primarily composed of small neurotoxic peptides. However, recent studies suggested a hitherto overlooked diversity of spider venom enzymes, although their functional space still remains largely unexplored. We tested 10 spider venoms for enzymatic activities covering six enzyme classes and found that all tested enzymatic activities can be detected in at least some of the venoms and that hyaluronidases exhibit particularly high enzymatic activities. With this, our study provides functional evidence for the proposed biological significance of enzymes in spider venoms, but more detailed investigations are required.

Most spiders deploy paralytic venom for prey capture, but adults of the Nurse´s thorn finger (Cheiracanthium punctorium) instead produce a predominantly defensive venom to safeguard their offspring. Here, we characterize the molecular repertoire of C. punctorium venom to shed light on its evolutionary history. Unlike venom in other spiders, C. punctorium venom mostly comprises neurotoxic double-domain neurotoxin 19 family (CSTX) peptides and enzymes, such as phospholipase A2 (PLA2). Comparative venomics in four spiders representing two infraorders shows that CSTXs arise following the mygalomorph-araneomorph split ~300 mya by means of ancestral gene duplication and functional specialization. A gene fusion event then appeared to have merged CSTXs from two distinct clades to form the double-domain toxin. PLA2 proteins are convergently recruited to C. punctorium to fulfil a defensive function and are strikingly similar to proalgesic PLA2 proteins in bee venom. These complex, multimodal molecular innovations in venom systems highlight nature's tendency to use the same molecular solutions for similar ecological challenges across diverse animal lineages.

Voltage-gated sodium (NaVs) channels are pore-forming transmembrane proteins that regulate the influx of sodium ions across cell membranes. Spider venoms are a rich source of NaV-modulating peptides with high selectivity and potency, making them important tools for understanding NaV structure and function. NaV1.8 is tetrodotoxin-resistant, expressed in the peripheral nervous system and contributes to the propagation of action potentials in nociceptive neurons, making it a potential therapeutic target for pain. We identified Tl1a, a 36 amino acid residue peptide isolated from the crude venom of the Peruvian tarantula species Thrixopelma longicolli as a modulator of NaV1.8. Tl1a was synthesized using solid-phase peptide synthesis, and activity was assessed using automated whole-cell patch-clamp recordings. Tl1a inhibited NaV1.8 peak current (IC50 210 nM), delayed the kinetics of activation, inhibited fast inactivation, and caused a persistent current as well as a depolarising shift in the voltage dependence of activation (ΔV1/2 +11 mV). Tl1a inhibited peak current with similar potency at NaV1.5 (IC50 282 nM) and KV2.1 (IC50 156 nM) and was 8-fold selective over the tetrodotoxin-sensitive NaV1.4 (IC50 1769 nM), NaV1.1 (2201 nM) and 6-fold selective over NaV1.7 (IC50 1278 nM) channels. Tl1a analogues with an increased number of charged amino acids in loop 4 of the peptide lost activity at NaV1.8 due to altered interactions with the domain IV S3-S4 extracellular loop. The results of this work contribute to a better understanding of the structure-activity relationships at tetrodotoxin-resistant NaV channels and may be useful for the future rational design of selective NaV1.8 peptide modulators.

The naming of venom toxins was largely ad hoc until the introduction of a rational nomenclature with a structured framework for naming venom peptides conveying taxonomic, pharmacological and structural information. Unfortunately, the resulting toxin names are susceptible to frequent name changes, necessitating a more stable nomenclature extending beyond peptides for long-term storage of toxin records in online repositories. The proposed toxin ID nomenclature includes a class-level taxonomic identifier plus a unique number within each class.

Understanding the mechanisms of sexual development would pave the way for producing mono-sex populations to aid the aquaculture industry. This study investigates the functions of the Y-linked iDmrt1 paralogue (Po-iDMY) and insulin-like androgenic gland hormone (Po-IAG) in the process of sexual development in the tropical rock lobster, Panulirus ornatus (TRL). Previously, we identified that Po-iDMY, a male-specific heterogametic (Y-linked) paralogue of the autosomal Po-iDmrt1 found in TRL, is a second sex-linked iDmrt gene identified in invertebrates. Using 5' and 3' rapid amplification of cDNA ends and data from a draft male genome (with an assembly genome size of approximately 2.446 Gbp and 87% BUSCO completeness), we obtained the full-length Po-iDMY gene (encoding a protein of 312 amino acids). A 411 bp male-specific sequence located at the 3' untranslated region of Po-iDMY mRNA was used as a sex marker, which was reported for the first time in our draft genome. However, Po-iDMY is not a master sex-determining factor since it was not expressed across developmental stages of embryos, juveniles and adults. Instead, we silenced Po-IAG at an early juvenile stage, generating two potential neo-females, implying that sexual manipulation could be a promising technique in TRL.

Arachnid venom peptides receive increasing attention from researchers for possible applications as human therapeutics, as bioinsecticides in agriculture or for targeting vectors of human disease. One commonly perceived disadvantage of peptides in contrast to small molecule drugs is their inability of crossing biological membranes comprised of lipid bilayers, providing a major obstacle for the delivery of peptide-based drugs and bioinsecticides. However, some arachnid venom peptides were reported to cross biological membranes, including cellular membranes, the vertebrate and insect blood brain barrier (BBB) and the insect midgut epithelium. This review will focus on these membrane-permeating arachnid peptides and discuss the underlying mechanisms. Different physico-chemical properties of membrane-permeating arachnid peptides and their contribution to the ability of crossing biological membranes will also be examined. In addition, several methods that facilitate or enable peptides to cross biological membranes will be discussed, which can be employed on peptides with no inherent membrane-permeating capabilities.

Insecticides are vital for safeguarding agricultural crops against pests, albeit many lack selectivity towards pest species and are poorly bio-degradable. This leads to targeting of beneficial organisms like pollinators and widespread environmental contamination of soil and water. Exposure to insecticides such as neonicotinoids causes insect paralysis and mortality at higher doses, while sublethal doses can disrupt other functions that are crucial for survival such as learning and memory performance. Potent and selective arachnid venom peptides affecting a variety of molecular targets are being explored as bioinsecticide candidates. However, their effect on insect learning is poorly understood. We therefore established a sucrose-induced conditioned place preference (CPP) assay using Drosophila melanogaster fruit flies to provide a means of evaluating how various classes of insecticidal compounds interact with insect memory to assess their broader ecological consequences. Our results confirmed the adverse effect of a sublethal dose of the neonicotinoid insecticide imidacloprid (20 pg/fly) on fly CPP formation upon daily injection during the conditioning phase. However, imidacloprid did not affect CPP retrieval when applied after the conditioning phase. Sublethal doses of the two insecticidal spider venom peptides μ-DGTX-Dc1a (Dc1a; 70 pg/fly) and U1-AGTX-Ta1a (Ta1a; 125 pg/fly) had no effect on either CPP formation or retrieval, underlining their potential as novel and safe bioinsecticide candidates.