Whole transcriptome sequencing reveals the activity of the PLA2 family members in Androctonus crassicauda (Scorpionida: Buthidae) venom gland.

Phospholipase A2 is the most abundant venom gland enzyme, whose activity leads to the activation of the inflammatory response by accumulating lipid mediators. This study aimed to identify, classify, and investigate the properties of venom PLA2 isoforms. Then, the present findings were confirmed by chemically measuring the activity of PLA2. The sequences representing PLA2 annotation were extracted from the Androctonus crassicauda transcriptome dataset using BLAS searches against the local PLA2 database. We found several cDNA sequences of PLA2 classified and named by conducting multiple searches as platelet-activating factor acetylhydrolases, calcium-dependent PLA2s, calcium-independent PLA2s, and secreted PLA2s. The largest and smallest isoforms of these proteins range between approximately 70.34 kDa (iPLA2) and 17.75 kDa (cPLA2). Among sPLA2 isoforms, sPLA2GXIIA and sPLA2G3 with ORF encoding 169 and 299 amino acids are the smallest and largest secreted PLA2, respectively. These results collectively suggested that A. crassicauda venom has PLA2 activity, and the members of this protein family may have important biological roles in lipid metabolism. This study also revealed the interaction between members of PLA2s in the PPI network. The results of this study would greatly help with the classification, evolutionary relationships, and interactions between PLA2 family proteins in the gene network.

Divergence in toxin antigenicity and venom enzymes in Tityus melici, a medically important scorpion, despite transcriptomic and phylogenetic affinities with problematic Brazilian species.

The Brazilian scorpion Tityus melici, native to Minas Gerais and Bahia, is morphologically related to Tityus serrulatus, the most medically significant species in Brazil. Despite inhabiting scorpion-envenomation endemic regions, T. melici venom remains unexplored. This work evaluates T. melici venom composition and function using transcriptomics, enzymatic activities, and in vivo and in vitro immunological analyses. Next-Generation Sequencing unveiled 86 components putatively involved in venom toxicity: 39 toxins, 28 metalloproteases, seven disulfide isomerases, six hyaluronidases, three phospholipases and three amidating enzymes. T. serrulatus showed the highest number of toxin matches with 80-100 % sequence similarity. T. melici is of medical importance as it has a venom LD50 of 0.85 mg/kg in mice. We demonstrated venom phospholipase A2 activity, and elevated hyaluronidase and metalloprotease activities compared to T. serrulatus, paralleling our transcriptomic findings. Comparison of transcriptional levels for T. serrulatus and T. melici venom metalloenzymes suggests species-specific expression patterns in Tityus. Despite close phylogenetic association with T. serrulatus inferred from COI sequences and toxin similarities, partial neutralization of T. melici venom toxicity was achieved when using the anti-T. serrulatus antivenom, implying antigenic divergence among their toxins. We suggest that the Brazilian therapeutic scorpion antivenom could be improved to effectively neutralize T. melici venom.

Genome-wide identification, structural homology analysis, and evolutionary diversification of the phospholipase D gene family in the venom gland of three scorpion species.

BACKGROUND: Venom phospholipase D (PLDs), dermonecrotic toxins like, are the major molecules in the crude venom of scorpions, which are mainly responsible for lethality and dermonecrotic lesions during scorpion envenoming. The purpose of this study was fivefold: First, to identify transcripts coding for venom PLDs by transcriptomic analysis of the venom glands from Androctonus crassicauda, Hottentotta saulcyi, and Hemiscorpius lepturus; second, to classify them by sequence similarity to known PLDs and motif extraction method; third, to characterize scorpion PLDs; fourth to structural homology analysis with known dermonecrotic toxins; and fifth to investigate phylogenetic relationships of the PLD proteins. RESULTS: We found that the venom gland of scorpions encodes two PLD isoforms: PLD1 ScoTox-beta and PLD2 ScoTox-alpha I. Two highly conserved regions shared by all PLD1s beta are GAN and HPCDC (HX2PCDC), and the most important conserved regions shared by all PLD2s alpha are two copies of the HKDG (HxKx4Dx6G) motif. We found that PLD1 beta is a 31-43 kDa acidic protein containing signal sequences, and PLD2 alpha is a 128 kDa basic protein without known signal sequences. The gene structures of PLD1 beta and PLD2 alpha contain 6 and 21 exons, respectively. Significant structural homology and similarities were found between the modeled PLD1 ScoTox-beta and the crystal structure of dermonecrotic toxins from Loxosceles intermedia. CONCLUSIONS: This is the first report on identifying PLDs from A. crassicauda and H. saulcyi venom glands. Our work provides valuable insights into the diversity of scorpion PLD genes and could be helpful in future studies on recombinant antivenoms production.

Varying Modes of Selection Among Toxin Families in the Venoms of the Giant Desert Hairy Scorpions (Hadrurus).

Venoms are primarily believed to evolve under strong diversifying selection resulting from persistent coevolution between predator and prey. Recent research has challenged this hypothesis, proposing that venoms from younger venomous lineages (e.g., snakes and cone snails) are governed predominantly by diversifying selection, while venoms from older venomous lineages (e.g., centipedes, scorpions, and spiders) are under stronger purifying selection. However, most research in older lineages has tested selection at more diverse phylogenetic scales. Although these tests are important for evaluating broad macroevolutionary trends underlying venom evolution, they are less equipped to detect species-level evolutionary trends, which likely have large impacts on venom variation seen at more diverse phylogenetic scales. To test for selection among closely related species from an older venomous lineage, we generated high-throughput venom-gland transcriptomes and venom proteomes for four populations of Giant Desert Hairy Scorpions (Hadrurus), including three Hadrurus arizonensis populations and one Hadrurus spadix population. We detected significant episodic and pervasive diversifying selection across a highly abundant toxin family that likely has a major role in venom function ([Formula: see text]KTxs), providing a contrast to the stronger purifying selection identified from other studies on scorpion venoms. Conversely, we detected weak episodic diversifying and/or stronger purifying selection in four toxin families (non-disulfide bridged peptides, phospholipase A2s, scorpine-like antimicrobial peptides, and serine proteases), most of which were less abundant and likely have ancillary functional roles. Finally, although we detected several major toxin families at disproportionate transcriptomic and/or proteomic abundances, we did not identify significant sex-based variation in Hadrurus venoms.

Identification, classification, and characterization of alpha and beta subunits of LVP1 protein from the venom gland of four Iranian scorpion species.

Scorpion venoms contain bioactive peptides and proteins. Some, can be used for pharmaceutical purposes. So, identification of venom proteins matters because, in addition to determining the function of the toxins can also be an excellent guide to developing new drugs. Here, we got transcriptome of venom glands from four Iranian scorpion species, including Hemsicorpius lepturus, Mesobuthus eupeus, Andructunus crassicuada, and Hottentotta saulcyi using cDNA library synthesis and high-throughput transcriptomic analysis of the venom glands. In a comparative way, we identified the cDNA encoding isoforms of subunits (alpha and beta) of BotLVP1/BmLVP1-like protein in the venom gland of three species except for H. lepturus. Characterization and structure determination of the LVP1_like proteins combined with gene map analysis provided evidence of the existence of some isoforms of LVP1_like proteins, encoded by genes with two exons and one intron, which can be classified in CSαß superfamily in the venom gland of three Iranian scorpion species. According to the high similarity with BotLVP1 and BmLVP1, these proteins could also be potent to mediate cholesterol homeostasis. However, further research is needed to prove it, and this study just may lay the foundation lead to light up this way.

Unveiling the Protein Components of the Secretory-Venom Gland and Venom of the Scorpion Centruroides possanii (Buthidae) through Omic Technologies.

Centruroides possanii is a recently discovered species of "striped scorpion" found in Mexico. Certain species of Centruroides are known to be toxic to mammals, leading to numerous cases of human intoxications in the country. Venom components are thought to possess therapeutic potential and/or biotechnological applications. Hence, obtaining and analyzing the secretory gland transcriptome and venom proteome of C. possanii is relevant, and that is what is described in this communication. Since this is a newly described species, first, its LD50 to mice was determined and estimated to be 659 ng/g mouse weight. Using RNA extracted from this species and preparing their corresponding cDNA fragments, a transcriptome analysis was obtained on a Genome Analyzer (Illumina) using the 76-base pair-end sequencing protocol. Via high-throughput sequencing, 19,158,736 reads were obtained and ensembled in 835,204 sequences. Of them, 28,399 transcripts were annotated with Pfam. A total of 244 complete transcripts were identified in the transcriptome of C. possanii. Of these, 109 sequences showed identity to toxins that act on ion channels, 47 enzymes, 17 protease inhibitors (PINs), 11 defense peptides (HDPs), and 60 in other components. In addition, a sample of the soluble venom obtained from this scorpion was analyzed using an Orbitrap Velos apparatus, which allowed for identification by liquid chromatography followed by mass spectrometry (LC-MS/MS) of 70 peptides and proteins: 23 toxins, 27 enzymes, 6 PINs, 3 HDPs, and 11 other components. Until now, this work has the highest number of scorpion venom components identified through omics technologies. The main novel findings described here were analyzed in comparison with the known data from the literature, and this process permitted some new insights in this field.

Congruence between ultraconserved element-based matrices and phylotranscriptomic datasets in the scorpion Tree of Life.

Scorpions are ancient and historically renowned for their potent venom. Traditionally, the systematics of this group of arthropods was supported by morphological characters, until recent phylogenomic analyses (using RNAseq data) revealed most of the higher-level taxa to be non-monophyletic. While these phylogenomic hypotheses are stable for almost all lineages, some nodes have been hard to resolve due to minimal taxonomic sampling (e.g. family Chactidae). In the same line, it has been shown that some nodes in the Arachnid Tree of Life show disagreement between hypotheses generated using transcritptomes and other genomic sources such as the ultraconserved elements (UCEs). Here, we compared the phylogenetic signal of transcriptomes vs. UCEs by retrieving UCEs from new and previously published scorpion transcriptomes and genomes, and reconstructed phylogenies using both datasets independently. We reexamined the monophyly and phylogenetic placement of Chactidae, sampling an additional chactid species using both datasets. Our results showed that both sets of genome-scale datasets recovered highly similar topologies, with Chactidae rendered paraphyletic owing to the placement of Nullibrotheas allenii. As a first step toward redressing the systematics of Chactidae, we establish the family Anuroctonidae (new family) to accommodate the genus Anuroctonus.

The Impact of Whole Genome Duplication on the Evolution of the Arachnids.

The proliferation of genomic resources for Chelicerata in the past 10 years has revealed that the evolution of chelicerate genomes is more dynamic than previously thought, with multiple waves of ancient whole genome duplications affecting separate lineages. Such duplication events are fascinating from the perspective of evolutionary history because the burst of new gene copies associated with genome duplications facilitates the acquisition of new gene functions (neofunctionalization), which may in turn lead to morphological novelties and spur net diversification. While neofunctionalization has been invoked in several contexts with respect to the success and diversity of spiders, the overall impact of whole genome duplications on chelicerate evolution and development remains imperfectly understood. The purpose of this review is to examine critically the role of whole genome duplication on the diversification of the extant arachnid orders, as well as assess functional datasets for evidence of subfunctionalization or neofunctionalization in chelicerates. This examination focuses on functional data from two focal model taxa: the spider Parasteatoda tepidariorum, which exhibits evidence for an ancient duplication, and the harvestman Phalangium opilio, which exhibits an unduplicated genome. I show that there is no evidence that taxa with genome duplications are more successful than taxa with unduplicated genomes. I contend that evidence for sub- or neofunctionalization of duplicated developmental patterning genes in spiders is indirect or fragmentary at present, despite the appeal of this postulate for explaining the success of groups like spiders. Available expression data suggest that the condition of duplicated Hox modules may have played a role in promoting body plan disparity in the posterior tagma of some orders, such as spiders and scorpions, but functional data substantiating this postulate are critically missing. Spatiotemporal dynamics of duplicated transcription factors in spiders may represent cases of developmental system drift, rather than neofunctionalization. Developmental system drift may represent an important, but overlooked, null hypothesis for studies of paralogs in chelicerate developmental biology. To distinguish between subfunctionalization, neofunctionalization, and developmental system drift, concomitant establishment of comparative functional datasets from taxa exhibiting the genome duplication, as well as those that lack the paralogy, is sorely needed.

Genetic Diversity of Hottentotta sp. Scorpions (Scorpionidae: Buthidae) in Khuzestan, Iran.

The genus of Hottentotta sp. scorpion is one of the few medically important scorpions in Iran. This study assessed the genetic relationship analysis of the cytochrome c oxidase subunit I (COXI) and 12sRNA genes and morphometric parameters among the population of Hottentotta sp in Khuzestan. Morphological analysis using the ANOVA T-test with a significance level of P-value less than 0.05 showed differences between the Hottetotta saulcyi and Hottetotta zagrosensis. However, this method was not able to distinguish between members of the same species. The amplification of gene fragments was done on 12srRNA (374 bp), and cytochrome c oxidase subunit I (COXI) (624 bp) from Hottentotta sp. collected from Khuzestan by PCR. Based on sequence 12srRNA, all H. saulcyi specimens (HS4, HS6 and HS7) except HS5 were included in cluster B. While two specimens of H. Zagrosensis (HZ6 and HZ1) with 99% bootstrap value were placed in cluster A. By using 12srRNA sequences, the highest genetic distance between the Khuzestan specimens was related to HS5 and HS7, which was calculated to be 16.7%. However, the amount of amino acid difference between HS5 and HS7 using the COXI sequence was 9.2%. The genetic distances of HS7 and HS5 with the only scorpion reference sequence, H. saulcyi, were 11.8% and 9.2%, respectively. Morphological data showed the separation of the two species, consistent with molecular phylogenetic trees. On the other hand, the genetic distance of specimens HS7 and HS5 with other members of the group as well as the scorpion reference sequence using the COXI gene, confirmed the possibility of an intraspecies difference that could not be proved by the morphological data alone.

Dual Functions of labial Resolve the Hox Logic of Chelicerate Head Segments.

Despite an abundance of gene expression surveys, comparatively little is known about Hox gene function in Chelicerata. Previous investigations of paralogs of labial (lab) and Deformed (Dfd) in a spider have shown that these play a role in tissue maintenance of the pedipalp segment (lab-1) and in patterning the first walking leg identity (Dfd-1), respectively. However, extrapolations of these data across chelicerates are hindered by the existence of duplicated Hox genes in arachnopulmonates (e.g., spiders and scorpions), which have resulted from an ancient whole genome duplication (WGD) event. Here, we investigated the function of the single-copy ortholog of lab in the harvestman Phalangium opilio, an exemplar of a lineage that was not subject to this WGD. Embryonic RNA interference against lab resulted in two classes of phenotypes: homeotic transformations of pedipalps to chelicerae, as well as reduction and fusion of the pedipalp and leg 1 segments. To test for combinatorial function, we performed a double knockdown of lab and Dfd, which resulted in a homeotic transformation of both pedipalps and the first walking legs into cheliceral identity, whereas the second walking leg is transformed into a pedipalpal identity. Taken together, these results elucidate a model for the Hox logic of head segments in Chelicerata. To substantiate the validity of this model, we performed expression surveys for lab and Dfd paralogs in scorpions and horseshoe crabs. We show that repetition of morphologically similar appendages is correlated with uniform expression levels of the Hox genes lab and Dfd, irrespective of the number of gene copies.

A first molecular characterization of the scorpion telson microbiota of Hadrurus arizonensis and Smeringurus mesaensis.

Scorpions represent an ancient lineage of arachnids that have radiated across the globe and are incredibly resilient-since some thrive in harsh environments and can exist on minimal and intermittent feedings. Given the emerging importance of microbiomes to an organism's health, it is intriguing to suggest that the long-term success of the scorpion bauplan may be linked to the microbiome. Little is known about scorpion microbiomes, and what is known, concentrates on the gut. The microbiome is not limited to the gut, rather it can be found within tissues, fluids and on external surfaces. We tested whether the scorpion telson, the venom-producing organ, of two species, Smeringurus mesaensis and Hadrurus arizonensis, contain bacteria. We isolated telson DNA from each species, amplified bacterial 16S rRNA genes, and identified the collection of bacteria present within each scorpion species. Our results show for the first time that telsons of non-buthid scorpion species do indeed contain bacteria. Interestingly, each scorpion species has a phylogenetically unique telson microbiome including Mollicutes symbionts. This study may change how we view scorpion biology and their venoms.

Comparative analysis of mitochondrial genomes reveals family-specific architectures and molecular features in scorpions (Arthropoda: Arachnida: Scorpiones).

Scorpions are a group of arachnids with great evolutionary success that comprise more than 2,000 described species. Mitochondrial genomes have been little studied in this clade. We describe and compare different scorpion mitochondrial genomes and analyze their architecture and molecular characteristics. We assembled eight new scorpion mitochondrial genomes from transcriptomic datasets, annotated them, predicted the secondary structures of tRNAs, and compared the nucleotide composition, codon usage, and relative synonymous codon usage of 16 complete scorpion mitochondrial genomes. Lastly, we provided a phylogeny based on all mitochondrial protein coding genes. We characterized the mitogenomes in detail and reported particularities such as dissimilar synteny in the family Buthidae compared to other scorpions, unusual tRNA secondary structures, and unconventional start and stop codons in all scorpions. Our comparative analysis revealed that scorpion mitochondrial genomes exhibit different architectures and features depending on taxonomic identity. We highlight the parvorder Buthida, particularly the family Buthidae, as it invariably exhibited different mitogenome features such as synteny, codon usage, and AT-skew compared to the parvorder Iurida that included the rest of the scorpion families we analyzed in this study. Our results provide a better understanding of the evolution of mitogenome features and phylogenetic relationships in scorpions.

An overview of Tityus cisandinus scorpion venom: Transcriptome and mass fingerprinting reveal conserved toxin homologs across the Amazon region and novel lipolytic components.

Tityus cisandinus, a neglected medically important scorpion in Ecuadorian and Peruvian Amazonia, belongs to a complex of species related to the eastern Amazon endemic Tityus obscurus, spanning a distribution of ca. 4000 km. Despite high morbidity and mortality rates, no effective scorpion antivenom is currently available in the Amazon region. Knowledge of the structural/functional relationships between T. cisandinus venom components and those from related Amazonian species is crucial for designing region-specific therapeutic antivenoms. In this work, we carried out the first venom gland transcriptomic study of an Amazonian scorpion outside Brazil, T. cisandinus. We also fingerprinted its total venom through MALDI-TOF MS, which supported our transcriptomic findings. We identified and calculated the expression level of 94 components: 60 toxins, 25 metalloproteases, five disulfide isomerases, three amidating enzymes, one hyaluronidase, and also uncovered transcripts encoding novel lipolytic beta subunits produced by New World buthid scorpions. This study demonstrates the high similarity between T. cisandinus and T. obscurus venoms, reinforcing the existence of a neglected complex of genetically and toxinologically related Amazonian scorpions of medical importance. Finally, we demonstrated the low recognition of currently available therapeutic sera against T. cisandinus and T. obscurus venoms, and concluded that these should be improved to protect against envenomation by Amazonian Tityus spp.

Genetic Diversity within Scorpio Genus (Scorpion: Scorpionidae) from Iran: Preliminary Evidence based on 16srRNA Sequence.

The yellow digger scorpion, Scorpio maurus, is a medically important scorpion for which little is known about its genetic diversity. Polymerase chain reaction products of 16srRNA gene fragments were generated from scorpion specimens named SmKh1 and SmKh2. These sequences showed high similarity with the only partial sequence of S. maurus isolate SCA1 large subunit ribosomal RNA gene available in the Genbank database. The drawing of the phylogeny tree showed two clusters, A and B. The two specimens (SmKh1 and SmKh2), which are placed in sub-cluster A2, were provided from Behbahan, Iran, and they have the closest relationship with the only sequence of S. maurus (MW281771), which is also collected from Behbahan. It is noteworthy that the two sequences obtained from S. maurus scorpions recorded from Miandoab (MK170444) and Mahabad (KU705354), which are in sub-cluster A1, are more similar to the scorpions isolated from the Mediterranean basin than those collected from Behbahan. This issue is probably due to the fact that patterns of genetic diversity are a reflection of variation in gene flow, which is also influenced by factors such as territorial barriers and geographical distances. We conclude that the scorpions of this study accompanied by similar scorpions in the Mediterranean basin, belong to the same species despite the insignificant differences.

The evolutionary history of the relict scorpion family Iuridae of the eastern Mediterranean.

Iuridae is a family of scorpions that exhibits a highly complex biogeographic and taxonomic history. Iuridae taxa are mainly found in Turkey and Greece, whereas a single species is found in northern Iraq. Several taxonomic revisions have been conducted on this family that initially comprised two genera. The latest taxonomic review, based on morphological and anatomical features, raised the number of Iuridae genera to four, and the number of species to 14. Sequence data from three molecular markers (COX1, 16S rDNA, ITS1) originating from numerous Iuridae taxa were analyzed within a phylogenetic framework. Divergence time-estimate analyses, species delimitation approaches and estimation of ancestral areas were implemented in order to: (1) reconstruct the phylogenetic relationships of the Iuridae taxa, (2) evaluate the morphological classifications, and (3) obtain insights into the biogeographic history of the family in the East Mediterranean. The multi-locus phylogeny clearly confirms an ancient division into two clades, Calchinae and Iurinae. Ancient patterns of isolation and dispersal are revealed. Both subfamilies are largely confined to the Anatolian peninsula and its few coastal islands; only the most derived genus Iurus has dispersed westward to Crete and Peloponnese. Based on our findings, three new genera of Iurinae (Metaiurus, Anatoliurus, and Letoiurus) are established. The genus Neocalchas emerges as one of the most ancient scorpion clades, with divergence time about 27 mya.

Differential venom gland gene expression analysis of juvenile and adult scorpions Androctonus crassicauda.

BACKGROUND: The Androctonus crassicauda, belonging to the genus Androctonus of the family Buthidae, is the most venomous scorpion in Middle East countries. However, the venom gland transcriptome profile of A. crassicauda scorpion has not yet been studied. In this study, we elucidated and compared the venom gland gene expression profiles of adult and juvenile male scorpion A. crassicauda using high-throughput transcriptome sequencing. This is the first report of transcriptional analysis of the venom glands of scorpions in different growth stages, with insights into the identification of the key genes during venom gland development. RESULTS: A total of 209,951 mRNA transcripts were identified from total RNA-seq data, of which 963 transcripts were differentially expressed (DE) in adult and juvenile scorpions (p < 0.01). Overall, we identified 558 up-regulated and 405 down-regulated transcripts in the adult compared to the juvenile scorpions, of which 397 and 269 unique unigenes were annotated, respectively. GO and KEGG enrichment analyses indicated that the metabolic, thermogenesis, cytoskeleton, estrogen signaling, GnRH signaling, growth hormone signaling, and melanogenesis pathways were affected by two different growth conditions and the results suggested that the DE genes related to those pathways are important genes associated with scorpion venom gland development, in which they may be important in future studies, including Chs, Elovl, MYH, RDX, ACTN, VCL, PIP5K, PP1C, FGFR, GNAS, EGFR, CREB, CoA, PLCB, CALM, CACNA, PKA and CAMK genes. CONCLUSIONS: These findings broadened our knowledge of the differences between adult and juvenile scorpion venom and opened new perspectives on the application of comparative transcriptome analysis to identify the special key genes.

Transcriptome Sequencing and Comparison of Venom Glands Revealed Intraspecific Differentiation and Expression Characteristics of Toxin and Defensin Genes in Mesobuthus martensii Populations.

Mesobuthus martensii, a famous and important Traditional Chinese Medicine has a long medical history and unique functions. It is the first scorpion species whose whole genome was sequenced worldwide. In addition, it is the most widespread and infamous poisonous animal in northern China with complex habitats. It possesses several kinds of toxins that can regulate different ion channels and serve as crucial natural drug resources. Extensive and in-depth studies have been performed on the structures and functions of toxins of M. martensii. In this research, we compared the morphology of M. martensii populations from different localities and calculated the COI genetic distance to determine intraspecific variations. Transcriptome sequencing by RNA-sequencing of the venom glands of M. martensii from ten localities and M. eupeus from one locality was analyzed. The results revealed intraspecific variation in the expression of sodium channel toxin genes, potassium channel toxin genes, calcium channel toxin genes, chloride channel toxin genes, and defensin genes that could be related to the habitats in which these populations are distributed, except the genetic relationships. However, it is not the same in different toxin families. M. martensii and M. eupeus exhibit sexual dimorphism under the expression of toxin genes, which also vary in different toxin families. The following order was recorded in the difference of expression of sodium channel toxin genes: interspecific difference; differences among different populations of the same species; differences between sexes in the same population, whereas the order in the difference of expression of potassium channel toxin genes was interspecific difference; differences between both sexes of same populations; differences among the same sex in different populations of the same species. In addition, there existed fewer expressed genes of calcium channel toxins, chloride channel toxins, and defensins (no more than four members in each family), and their expression differences were not distinct. Interestingly, the expression of two calcium channel toxin genes showed a preference for males and certain populations. We found a difference in the expression of sodium channel toxin genes, potassium channel toxin genes, and chloride channel toxin genes between M. martensii and M. eupeus. In most cases, the expression of one member of the toxin gene clusters distributed in series on the genome were close in different populations and genders, and the members of most clusters expressed in same population and gender tended to be the different. Twenty-one toxin genes were found with the MS/MS identification evidence of M. martensii venom. Since scorpions were not subjected to electrical stimulation or other special treatments before conducting the transcriptome extraction experiment, the results suggested the presence of intraspecific variation and sexual dimorphism of toxin components which revealed the expression characteristics of toxin and defensin genes in M. martensii. We believe this study will promote further in-depth research and use of scorpions and their toxin resources, which in turn will be helpful in standardizing the identification and medical applications of Quanxie in traditional Chinese medicine.

The divergent genome of Scorpion Group 1 (SG1) intracellular bacterium from the venom glands of Vaejovis smithi (Scorpiones: Vaejovidae).

Scorpions were among the first animals on land around 430 million years ago. Like many arachnids, scorpions have evolved complex venoms used to paralyze their prey and for self-defense. Here we sequenced and analyzed the metagenomic DNA from venom glands from Vaejovis smithi scorpions. A metagenome-assembled genome (MAG) of 624,025 bp was obtained corresponding to the previously reported Scorpion Group 1 (SG1). The SG1 genome from venom glands had a low GC content (25.8%) characteristic of reduced genomes, many hypothetical genes and genes from the reported minimal set of bacterial genes. Phylogenomic reconstructions placed the uncultured SG1 distant from other reported bacteria constituting a taxonomic novelty. By PCR we detected SG1 in all tested venom glands from 30 independent individuals. Microscopically, we observed SG1 inside epithelial cells from the venom glands using FISH and its presence in scorpion embryos suggested that SG1 is transferred from mother to offspring.

Kinetic Activity of Chromosomes and Expression of Recombination Genes in Achiasmatic Meiosis of Tityus (Archaeotityus) Scorpions.

Several species of Tityus (Scorpiones, Buthidae) present multi-chromosomal meiotic associations and failures in the synaptic process, originated from reciprocal translocations. Holocentric chromosomes and achiasmatic meiosis in males are present in all members of this genus. In the present study, we investigated synapse dynamics, transcriptional silencing by γH2AX, and meiotic microtubule association in bivalents and a quadrivalent of the scorpion Tityus maranhensis. Additionally, we performed RT-PCR to verify the expression of mismatch repair enzymes involved in crossing-over formation in Tityus silvestris gonads. The quadrivalent association in T. maranhensis showed delay in the synaptic process and long asynaptic regions during pachytene. In this species, γH2AX was recorded only at the chromosome ends during early stages of prophase I; in metaphase I, bivalents and quadrivalents of T. maranhensis exhibited binding to microtubules along their entire length, while in metaphase II/anaphase II transition, spindle fibers interacted only with telomeric regions. Regarding T. silvestris, genes involved in the recombination process were transcribed in ovaries, testes and embryos, without significant difference between these tissues. The expression of these genes during T. silvestris achiasmatic meiosis is discussed in the present study. The absence of meiotic inactivation by γH2AX and holo/telokinetic behavior of the chromosomes are important factors for the maintenance of the quadrivalent in T. maranhensis and the normal continuation of the meiotic cycle in this species.