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The CAP protein superfamily (Cysteine-rich secretory proteins (CRISPs), Antigen 5 (Ag5), and Pathogenesis-related 1 (PR-1) proteins) is widely distributed, but for toxinologists, snake venom CRISPs are the most familiar members. Although CRISPs are found in the majority of venoms, very few of these proteins have been functionally characterized, but those that have been exhibit diverse activities. Snake venom CRISPs (svCRISPs) inhibit ion channels and the growth of new blood vessels (angiogenesis). They also increase vascular permeability and promote inflammatory responses (leukocyte and neutrophil infiltration). Interestingly, CRISPs in lamprey buccal gland secretions also manifest some of these activities, suggesting an evolutionarily conserved function. As we strive to better understand the functions that CRISPs serve in venoms, it is worth considering the broad range of CRISP physiological activities throughout the animal kingdom. In this review, we summarize those activities, known crystal structures and sequence alignments, and we discuss predicted functional sites. CRISPs may not be lethal or major components of venoms, but given their almost ubiquitous occurrence in venoms and the accelerated evolution of svCRISP genes, these venom proteins are likely to have functions worth investigating.

Venom proteins evolve rapidly, and as a trophic adaptation are excellent models for predator–prey evolutionary studies. The key to a deeper understanding of venom evolution is an integrated approach, combining prey assays with analysis of venom gene expression and venom phenotype. Here, we use such an approach to study venom evolution in the Amazon puffing snake, Spilotes sulphureus , a generalist feeder. We identify two novel three-finger toxins: sulditoxin and sulmotoxin 1. These new toxins are not only two of the most abundant venom proteins, but are also functionally intriguing, displaying distinct prey-specific toxicities. Sulditoxin is highly toxic towards lizard prey, but is non-toxic towards mammalian prey, even at greater than 22-fold higher dosage. By contrast, sulmotoxin 1 exhibits the reverse trend. Furthermore, evolutionary analysis and structural modelling show highest sequence variability in the central loop of these proteins, probably driving taxon-specific toxicity. This is, to our knowledge, the first case in which a bimodal and contrasting pattern of toxicity has been shown for proteins in the venom of a single snake in relation to diet. Our study is an example of how toxin gene neofunctionalization can result in a venom system dominated by one protein superfamily and still exhibit flexibility in prey capture efficacy.

Arbovirus infection of Aedes aegypti salivary glands (SGs) determines transmission. However, there is a dearth of knowledge on SG immunity. Here, we characterized SG immune response to dengue, Zika and chikungunya viruses using high-throughput transcriptomics. We also describe a transcriptomic response associated to apoptosis, blood-feeding and lipid metabolism. The three viruses differentially regulate components of Toll, Immune deficiency (IMD) and c-Jun N- terminal Kinase (JNK) pathways. However, silencing of the Toll and IMD pathway components showed variable effects on SG infection by each virus. In contrast, regulation of the JNK pathway produced consistent responses in both SGs and midgut. Infection by the three viruses increased with depletion of the activator Kayak and decreased with depletion of the negative regulator Puckered. Virus-induced JNK pathway regulates the complement factor, Thioester containing protein-20 (TEP20), and the apoptosis activator, Dronc, in SGs. Individual and co-silencing of these genes demonstrate their antiviral effects and that both may function together. Co-silencing either TEP20 or Dronc with Puckered annihilates JNK pathway antiviral effect. Upon infection in SGs, TEP20 induces antimicrobial peptides (AMPs), while Dronc is required for apoptosis independently of TEP20. In conclusion, we revealed the broad antiviral function of JNK pathway in SGs and showed that it is mediated by a TEP20 complement and Dronc-induced apoptosis response. These results expand our understanding of the immune arsenal that blocks arbovirus transmission.

Arboviruses such as dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses infect close to half a billion people per year, and are primarily transmitted through Aedes aegypti bites. Infection-induced changes in mosquito salivary glands (SG) influence transmission by inducing antiviral immunity, which restricts virus replication in the vector, and by altering saliva composition, which influences skin infection. Here, we profiled SG proteome responses to DENV serotype 2 (DENV2), ZIKV and CHIKV infections by using high-resolution isobaric-tagged quantitative proteomics. We identified 218 proteins with putative functions in immunity, blood-feeding or related to the cellular machinery. We observed that 58, 27 and 29 proteins were regulated by DENV2, ZIKV and CHIKV infections, respectively. While the regulation patterns were mostly virus-specific, we separately depleted four uncharacterized proteins that were upregulated by all three viral infections to determine their effects on these viral infections. Our study suggests that gamma-interferon responsive lysosomal thiol-like (GILT-like) has an anti-ZIKV effect, adenosine deaminase (ADA) has an anti-CHIKV effect, salivary gland surface protein 1 (SGS1) has a pro-ZIKV effect and salivary gland broad-spectrum antiviral protein (SGBAP) has an antiviral effect against all three viruses. The comprehensive description of SG responses to three global pathogenic viruses and the identification of new restriction factors improves our understanding of the molecular mechanisms influencing transmission.

Background Venom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulatory investigations in elapid snakes have been limited. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis ), in addition to comparative genomics, to identify cis- and trans- acting regulation of venom production in an elapid in comparison to viperids ( Crotalus viridis and C. tigris ). Results Although there is conservation in high-level mechanistic pathways regulating venom production (unfolded protein response, Notch signaling and cholesterol homeostasis), there are differences in the regulation of histone methylation enzymes, transcription factors, and microRNAs in venom glands from these two snake families. Histone methyltransferases and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland in comparison to the viperids, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis- regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. The presence of Sp1 binding sites across multiple elapid toxin gene promoter regions that have been experimentally determined to regulate expression, in addition to upregulation of Sp1 after venom milking, suggests this transcription factor is involved in elapid toxin expression. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target a diversity of toxin transcripts in the elapid P. textilis venom gland, but only snake venom metalloproteinase transcripts in the viperid C. viridis venom gland. These results suggest differences in toxin gene posttranscriptional regulation between the elapid P. textilis and viperid C. viridis. Conclusions Our comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin regulation between these two snake families, demonstrating multiple different regulatory mechanisms underpin a venomous phenotype.

Dengue (DENV) and chikungunya (CHIKV) viruses are among the most preponderant arboviruses. Although primarily transmitted through the bite of Aedes aegypti mosquitoes, Aedes albopictus and Aedes malayensis are competent vectors and have an impact on arbovirus epidemiology. Here, to fill the gap in our understanding of the molecular interactions between secondary vectors and arboviruses, we used transcriptomics to profile the whole-genome responses of A. albopictus to CHIKV and of A. malayensis to CHIKV and DENV at 1 and 4 days post-infection (dpi) in midguts. In A. albopictus , 1793 and 339 genes were significantly regulated by CHIKV at 1 and 4 dpi, respectively. In A. malayensis , 943 and 222 genes upon CHIKV infection, and 74 and 69 genes upon DENV infection were significantly regulated at 1 and 4 dpi, respectively. We reported 81 genes that were consistently differentially regulated in all the CHIKV-infected conditions, identifying a CHIKV-induced signature. We identified expressed immune genes in both mosquito species, using a de novo assembled midgut transcriptome for A. malayensis , and described the immune architectures. We found the JNK pathway activated in all conditions, generalizing its antiviral function to Aedines. Our comprehensive study provides insight into arbovirus transmission by multiple Aedes vectors.

Envenomation of humans by snakes is a complex and continuously evolving medical emergency, and treatment is made that much more difficult by the diverse biochemical composition of many venoms. Venomous snakes and their venoms also provide models for the study of molecular evolutionary processes leading to adaptation and genotype-phenotype relationships. To compare venom complexity and protein sequences, venom gland transcriptomes are assembled, which usually requires the sacrifice of snakes for tissue. However, toxin transcripts are also present in venoms, offering the possibility of obtaining cDNA sequences directly from venom. This study provides evidence that unknown full-length venom protein transcripts can be obtained from the venoms of multiple species from all major venomous snake families. These unknown venom protein cDNAs are obtained by the use of primers designed from conserved signal peptide sequences within each venom protein superfamily. This technique was used to assemble a partial venom gland transcriptome for the Middle American Rattlesnake (Crotalus simus tzabcan) by amplifying sequences for phospholipases A2, serine proteases, C-lectins, and metalloproteinases from within venom. Phospholipase A2 sequences were also recovered from the venoms of several rattlesnakes and an elapid snake (Pseudechis porphyriacus), and three-finger toxin sequences were recovered from multiple rear-fanged snake species, demonstrating that the three major clades of advanced snakes (Elapidae, Viperidae, Colubridae) have stable mRNA present in their venoms. These cDNA sequences from venom were then used to explore potential activities derived from protein sequence similarities and evolutionary histories within these large multigene superfamilies. Venom-derived sequences can also be used to aid in characterizing venoms that lack proteomic profiles and identify sequence characteristics indicating specific envenomation profiles. This approach, requiring only venom, provides access to cDNA sequences in the absence of living specimens, even from commercial venom sources, to evaluate important regional differences in venom composition and to study snake venom protein evolution.

Snake venoms are complex mixtures of toxins that differ on interspecific (between species) and intraspecific (within species) levels. Whether venom variation within a group of closely related species is explained by the presence, absence and/or relative abundances of venom toxins remains largely unknown. Taipans (Oxyuranus spp.) and brown snakes (Pseudonaja spp.) represent medically relevant species of snakes across the Australasian region and provide an excellent model clade for studying interspecific and intraspecific venom variation. Using liquid chromatography with ultraviolet and mass spectrometry detection, we analyzed a total of 31 venoms covering all species of this monophyletic clade, including widespread localities. Our results reveal major interspecific and intraspecific venom variation in Oxyuranus and Pseudonaja species, partially corresponding with their geographical regions and phylogenetic relationships. This extensive venom variability is generated by a combination of the absence/presence and differential abundance of venom toxins. Our study highlights that venom systems can be highly dynamical on the interspecific and intraspecific levels and underscores that the rapid toxin evolvability potentially causes major impacts on neglected tropical snakebites.

Mosquitoes are responsible for the transmission of numerous pathogens, including Plasmodium parasites, arboviruses and filarial worms. They pose a significant risk to public health with over 200 million cases of malaria per annum and approximately 4 billion people at risk of arthropod-borne viruses (arboviruses). Mosquito populations are geographically expanding into temperate regions and their distribution is predicted to continue increasing. Mosquito symbionts, including fungi, bacteria and viruses, have desirable traits for mosquito disease control including spreading horizontally and vertically through mosquito populations and potentially colonising multiple important vector species. Paratransgenesis, genetic modification of mosquito symbionts with effectors to target the pathogen rather than the vector, is a promising strategy to prevent the spread of mosquito-borne diseases. A variety of effectors can be expressed but venom toxins are excellent effector candidates because they are target specific, potent and stable. However, the only toxins to be explored in mosquito paratransgenesis to date are scorpine and mutated phospholipase A2. To enhance the scope, effectiveness and durability of paratransgenesis, an expanded arsenal of effectors is required. This review discusses other potential toxin effectors for future paratransgenesis studies based on prior in vitro and in vivo antiparasitic and antiviral studies and highlights the need for further research and investment in this area. In terms of mosquito-borne diseases, paratransgenesis strategies have been developed to target Plasmodium . We postulate the potential to apply this principle to target arboviruses using antiviral toxin effectors. Graphical Abstract