Transcriptome analysis and characterization of genes associated to leaf tannin content in foxtail millet [Setaria italica (L.) P. Beauv.].

BACKGROUND: Chinese chestnut is an economically important tree species whose yield and quality are seriously affected by red spider attack. Tannins is one of the most important class secondary metabolites in plants, and is closely associated with plant defense mechanisms against insect and herbivory. In our previous studies, it was revealed that several low-tannin foxtail millet varieties growing under the Chinese chestnut trees could attract red spiders to feed on their leaves and protect the chestnut trees from the infestation of red spiders, meanwhile, the growth and yield of foxtail millet plants themselves were not greatly affected. RESULTS: To identify genes related to leaf tannin content and selection of foxtail millet germplasm resources with low tannin content for interplanting with Chinese chestnut and preventing the red spider attack, the leaves of 4 varieties with different levels of tannin content were harvested for comparative transcriptome analysis. In total, 335 differentially expressed genes (DEGs) were identified. For acquisition of gene functions and biological pathways they involved in, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were performed, and several DEGs were found to possibly participate in the tannins biosynthesis pathway and transport processes of precursors. In addition, according to the PlantTFDB database, some transcription factors were predicted among the DEGs, suggesting their role in regulation of tannins biosynthesis pathway. CONCLUSION: Our results provide valuable gene resources for understanding the biosynthesis and regulation mechanisms of tannins in foxtail millet, and pave the way for speeding up the breeding of low-tannin varieties through marker-assisted selection, which could be utilized for interplanting with Chinese chestnut trees to confer protection against red spider attack.

Australian funnel-web spiders evolved human-lethal δ-hexatoxins for defense against vertebrate predators.

Australian funnel-web spiders are infamous for causing human fatalities, which are induced by venom peptides known as δ-hexatoxins (δ-HXTXs). Humans and other primates did not feature in the prey or predator spectrum during evolution of these spiders, and consequently the primate lethality of δ-HXTXs remains enigmatic. Funnel-web envenomations are mostly inflicted by male spiders that wander from their burrow in search of females during the mating season, which suggests a role for δ-HXTXs in self-defense since male spiders rarely feed during this period. Although 35 species of Australian funnel-web spiders have been described, only nine δ-HXTXs from four species have been characterized, resulting in a lack of understanding of the ecological roles and molecular evolution of δ-HXTXs. Here, by profiling venom-gland transcriptomes of 10 funnel-web species, we report 22 δ-HXTXs. Phylogenetic and evolutionary assessments reveal a remarkable sequence conservation of δ-HXTXs despite their deep evolutionary origin within funnel-web spiders, consistent with a defensive role. We demonstrate that δ-HXTX-Ar1a, the lethal toxin from the Sydney funnel-web spider Atrax robustus, induces pain in mice by inhibiting inactivation of voltage-gated sodium (NaV) channels involved in nociceptive signaling. δ-HXTX-Ar1a also inhibited inactivation of cockroach NaV channels and was insecticidal to sheep blowflies. Considering their algogenic effects in mice, potent insecticidal effects, and high levels of sequence conservation, we propose that the δ-HXTXs were repurposed from an initial insecticidal predatory function to a role in defending against nonhuman vertebrate predators by male spiders, with their lethal effects on humans being an unfortunate evolutionary coincidence.

Acanthocytosis and brain damage in area postrema and choroid plexus: Description of novel signs of Loxosceles apachea envenomation in rats.

Loxocelism is a neglected medical problem that depends on its severity, can cause a cutaneous or viscero-cutaneous syndrome. This syndrome is characterized by hemostatic effects and necrosis, and the severity of the loxoscelism depends on the amount of venom injected, the zone of inoculation, and the species. In the Chihuahuan desert, the most abundant species is L. apachea. Its venom and biological effects are understudied, including neurological effects. Thus, our aim is to explore the effect of this regional species of medical interest in the United States-Mexico border community, using rat blood and central nervous system (CNS), particularly, two brain structures involved in brain homeostasis, Area postrema (AP) and Choroid plexus (PC). L. apachea specimens were collected and venom was obtained. Different venom concentrations (0, 0.178 and 0.87 µg/g) were inoculated into Sprague-Dawley rats (intraperitoneal injection). Subsequently, blood was extracted and stained with Wright staining; coronal sections of AP were obtained and stained with Hematoxylin-Eosin (HE) staining and laminin γ immunolabelling, the same was done with CP sections. Blood, AP and CP were observed under the microscope and abnormalities in erythrocytes and fluctuation in leukocyte types were described and quantified in blood. Capillaries were also quantified in AP and damage was described in CP. L. apachea venom produced a segmented neutrophil increment (neutrophilia), lymphocyte diminishment (leukopenia) and erythrocytes presented membrane abnormalities (acanthocytosis). Extravasated erythrocytes were observed in HE stained sections from both, AP and CP, which suggest that near to this section a hemorrhage is present; through immunohistofluorescence, a diminishment of laminin γ was observed in AP endothelial cells and in CP ependymal cells when these structures were exposed to L. apachea venom. In conclusion, L. apachea venom produced leukopenia, netrophilia and acanthocytosis in rat peripheral blood, and also generated hemorrhages on AP and CP through degradation of laminin γ.

Bacterial and Arachnid Sphingomyelinases D: Comparison of Biophysical and Pathological Activities.

Sphingomyelinases D have only been identified in arachnid venoms, Corynebacteria, Arcanobacterium, Photobacterium and in the fungi Aspergillus and Coccidioides. The arachnid and bacterial enzymes share very low sequence identity and do not contain the HKD sequence motif characteristic of the phospholipase D superfamily, however, molecular modeling and circular dichroism of SMases D from Loxosceles intermedia and Corynebacterium pseudotuberculosis indicate similar folds. The phospholipase, hemolytic and necrotic activities and mice vessel permeabilities were compared and both enzymes possess the ability to hydrolyze phospholipids and also promote similar pathological reactions in the host suggesting the existence of a common underlying mechanism in tissue disruption. J. Cell. Biochem. 118:2053-2063, 2017. © 2016 Wiley Periodicals, Inc.

Peptidomics of Acanthoscurria gomesiana spider venom reveals new toxins with potential antimicrobial activity.

Acanthoscurria gomesiana is a Brazilian spider from the Theraphosidae family inhabiting regions of Southeastern Brazil. Potent antimicrobial peptides as gomesin and acanthoscurrin have been discovered from the spider hemolymph in previous works. Spider venoms are also recognized as sources of biologically active peptides, however the venom peptidome of A. gomesiana remained unexplored to date. In this work, a MS-based workflow was applied to the investigation of the spider venom peptidome. Data-independent and data-dependent LC-MS/MS acquisitions of intact peptides and of peptides submitted to multiple enzyme digestions, followed by automated chromatographic alignment, de novo analysis, database and homology searches with manual validations showed that the venom is composed by <165 features, with masses ranging from 0.4-15.8kDa. From digestions, 135 peptides were identified from 17 proteins, including three new mature peptides: U1-TRTX-Agm1a, U1-TRTX-Agm2a and U1-TRTX-Agm3a, containing 3, 4 and 3 disulfide bonds, respectively. The toxins U1-TRTX-Agm1a differed by only one amino acid from U1-TRTX-Ap1a from A. paulensis and U1-TRTX-Agm2a was derived from the genicutoxin-D1 precursor from A. geniculata. These toxins have potential applications as antimicrobial agents, as the peptide fraction of A. gomesiana showed activity against Escherichia coli, Enterobacter cloacae and Candida albicans strains. MS data are available via ProteomeXchange Consortium with identifier PXD003884. BIOLOGICAL SIGNIFICANCE: Biological fluids of the Acanthoscurria gomesiana spider are sources of active molecules, as is the case of antimicrobial peptides and acylpolyamines found in the hemolymphs. The venom is also a potential source of toxins with pharmacological and biotechnological applications. However, to our knowledge no A. gomesiana venom toxin structure has been determined to date. Using a combination of high resolution mass spectrometry, transcriptomics and bioinformatics, we employed a workflow to fully sequence, determine the number of disulfide bonds of mature peptides and we found new potential antimicrobial peptides. This workflow is suitable for complete peptide toxin sequencing when handling limited amount of venom samples and can accelerate the discovery of peptides with potential biotechnological applications.

Age-Related Modulations of AQP4 and Caveolin-1 in the Hippocampus Predispose the Toxic Effect of Phoneutria nigriventer Spider Venom.

We have previously demonstrated that Phoneutria nigriventer venom (PNV) causes blood-brain barrier (BBB) breakdown, swelling of astrocytes end-feet and fluid permeation into brain interstitium in rats. Caveolae and water channels respond to BBB alterations by co-participation in shear stress response and edema formation/resolution. Herein, we showed post-natal developmental-related changes of two BBB-associated transporter proteins: the endothelial caveolin-1 (Cav-1), the major scaffolding protein from caveolae frame, and the astroglial aquaporin-4 (AQP4), the main water channel protein expressed in astrocytic peri-vascular end-feet processes, in the hippocampus of rats intraperitoneally-administered PNV. Western blotting protein levels; immunohistochemistry (IHC) protein distribution in CA1, CA2, and CA3 subfields; and gene expression by Real Time-Polymerase Chain Reaction (qPCR) were assessed in post-natal Day 14 (P14) and 8-10-week-old rats over critical periods of envenomation. The intensity and duration of the toxic manifestations indicate P14 neonate rats more vulnerable to PNV than adults. Histologically, the capillaries of P14 and 8-10-week-old rats treated with PNV showed perivascular edema, while controls did not. The intensity of the toxic manifestations in P14 decreases temporally (2 > 5 > 24 h), while inversely the expression of AQP4 and Cav-1 peaked at 24 h when clinically PNV-treated animals do not differ from saline controls. IHC of AQP4 revealed that hippocampal CA1 showed the least expression at 2 h when toxic manifestation was maximal. Subfield IHC quantification revealed that in P14 rats Cav-1 peaked at 24 h when toxic manifestations were absent, whereas in 8-10-week-old rats Cav-1 peaked at 2 h when toxic signs were highest, and progressively attenuated such increases until 24 h, remaining though significantly above baseline. Considering astrocyte-endothelial physical and functional interactions, we hypothesize that age-related modulations of AQP4 and Cav-1 might be linked both to changes in functional properties of astrocytes during post-natal development and in the BBB breakdown induced by the venom of P. nigriventer.

Cry1Ab-expressing rice did not influence expression of fecundity-related genes in the wolf spider Pardosa pseudoannulata.

The impact of Bacillus thuringiensis (Bt) toxin proteins on non-target predatory arthropods is not well understood at the cellular and molecular levels. Here, we investigated the potential effects of Cry1Ab expressing rice on fecundity of the wolf spider, Pardosa pseudoannulata, and some of the underlying molecular mechanisms. The results indicated that brown planthoppers (BPHs) reared on Cry1Ab-expressing rice accumulated the Cry toxin and that reproductive parameters (pre-oviposition period, post-oviposition stage, number of eggs, and egg hatching rate) of the spiders that consumed BPHs reared on Bt rice were not different from those that consumed BPHs reared on the non-Bt control rice. The accumulated Cry1Ab did not influence several vitellin (Vt) parameters, including stored energy and amino acid composition, during one generation. We considered the possibility that the Cry toxins exert their influence on beneficial predators via more subtle effects detectable at the molecular level in terms of gene expression. This led us to transcriptome analysis to detect differentially expressed genes in the ovaries of spiders exposed to dietary Cry1Ab and their counterpart control spiders. Eight genes, associated with vitellogenesis, vitellogenin receptor activity, and vitellin membrane formation were not differentially expressed between ovaries from the treated and control spiders, confirmed by qPCR analysis. We infer that dietary Cry1Ab expressing rice does not influence fecundity, nor expression levels of Vt-associated genes in P. pseudoannulata.

Spiders in dermatology.

Spider bites represent an unusual and potentially over-represented clinical diagnosis. Despite a common fear of spiders, known as arachnophobia, current knowledge suggests that only a small number of families within the order Araneae are medically relevant. Moreover, most cutaneous spider reactions, including both evenomations and physical trauma, produce mild, local symptoms which may be managed with supportive care alone. The differential diagnosis for spider bites may be broad, especially if the offending arachnid is not seen or found. We describe a series of spiders relevant to the dermatologist in the United States.

Tratamento cirúrgico da úlcera loxoscélica: solução prática para um problema de difícil resolução / Surgical treatment of ulcer loxoscélica: practical solution to a difficult problem to solve

Contexto: O manuscrito descreve a reparação cirúrgica de uma úlcera crônica resultante da picada de uma aranhamarrom,um problema de difícil resolução, o qual habitualmente demora meses até a cicatrização.Descrição do caso: A vítima foi picada na coxa direita e desenvolveu fases de envenenamento típicas da picada deLoxosceles, incluindo a placa mármore, a escara e a úlcera profunda e de bordos emoldurados, características doacidente. Devido ao processo cicatricial lento, cuidados intensivos com curativos, dificuldade na deambulação, além dofator estético, optou-se por cirurgia excisional da lesão e sutura direta das bordas. O tratamento cirúrgico proporcionoucicatrização total da lesão dentro de 15 dias.Conclusões: A úlcera loxoscélica apresenta difícil cicatrização, evoluindo para a cronicidade. A cirurgia com excisãocompleta e síntese primária constitui técnica adequada para a cura da doença.

Sponge implant in Swiss mice as a model for studying loxoscelism.

Envenomation by Loxosceles spider bite leads to a set of signs and symptoms, called loxoscelism, which in most cases manifests through the dermonecrotic frame. The development of a smaller size animal model, of easy handling and maintenance, and lower cost is needed to study the loxoscelism pathogenesis. The inflammatory effects of the Loxosceles similis crude venom was evaluated considering neutrophil and macrophage activation, vasodilatation, hyperhaemia, edema and hemorrhage and TNF-α and VEGF production using the murine sponge implant model. Thirty two male Swiss mice (6-8 weeks old) were implanted subcutaneously with polyether-polyurethane sponge discs. Fourteen days post implantation, animals were separated into two groups: (1) control group--16 mice received 30 µL of saline intra-implant; (2) treated group-sixteen mice injected with 0.5 µg/30 µL of L. similis crude venom intra-implant. The animals were euthanized with xylazine/ketamine after 1 and 4 h post- injection. Microscopically, implants of the treated groups presented an acute inflammation characterized by: neutrophilic infiltrate, edema, vasodilatation hyperhaemia, and severe hemorrhage. Some vessels presented ruptured walls. Under morphometric analysis, vessel area was bigger in the treated groups compared with the control ones. The biochemical parameters, hemoglobin content, inflammatory enzyme activities (myeloperoxidase and n-acethyl-ß-D glucosaminidase) and levels of the cytokines, TNF-α and VEGF, were also significantly higher in the venom-treated groups. The effects of Loxosceles venom in the granulation tissue of the implant in mice were similar to those observed in cutaneous loxoscelism in other species (human and rabbits). Consequently, the murine sponge implant model provides a new method to investigate cellular/molecular mechanisms associated with cutaneous loxoscelism.

Loxoscelismo cutáneo. A propósito de un caso clínico / Skin loxoscelism. A clinical case report

Loxosceles laeta es una araña de distribución mundial y la necrosis cutánea caracteriza a su mordedura. Las formas sistémicas son de baja frecuencia pero alta mortalidad. El diagnóstico es de sospecha ante una lesión intensamente dolorosa o por la identificación del arácnido. La recomendación para la utilización de suero antiloxosceles no es uniforme. En este artículo reportamos un caso de loxoscelismo a forma cutáneo necrótica que requirió cirugía de debridamiento temprano y con buena evolución. El objetivo es concientizar a los pediatras de la existencia de esta entidad que sólo será diagnosticada tempranamente si pensamos en ella, lo que permitirá el oportuno y adecuado abordaje del paciente.

Loxoscelismo: epidemiología y clínica de una patología endémica en el país / Loxoscelism: epidemiology and clinical aspects of an endemic patology in the country

Loxosceles es una araña de distribución mundial con características sinantrópicas. Su picadura se caracteriza por necrosis cutánea que, en un bajo porcentaje de casos, se acompaña de manifestaciones sistémicas. En los cuadros sistémicos lamortalidad puede ser elevada. No existen métodos de laboratorio para el diagnóstico asistencial, que se realiza por los signos clínicos y la identificaciónde los ejemplares. El tratamiento específico es la aplicación del antiveneno. El objetivo de esta presentación es brindar una actualización sobre las características de este envenenamiento, su diagnóstico y tratamiento dirigidos hacia el paciente pediátrico.

Loxosceles is a global distribution spider with synanthropic characteristics. It is responsible for skin necrosis that in a low percentage is accompanied by systemic manifestations. Mortality can be high when systemic manifestationsare present. There is no laboratory methods available for diagnostic. Clinical signs and identification of individuals lead to it. The applicationof antivenom is the specific treatment.The objetive of this presentation is to update on the characteristics of this poisoning, its diagnosis and treatment directed toward the pediatric patient.

Loxoscelismo: epidemiología y clínica de una patología endémica en el país / Loxoscelism: epidemiology and clinical aspects of an endemic patology in the country

Loxosceles es una araña de distribución mundial con características sinantrópicas. Su picadura se caracteriza por necrosis cutánea que, en un bajo porcentaje de casos, se acompaña de manifestaciones sistémicas. En los cuadros sistémicos lamortalidad puede ser elevada. No existen métodos de laboratorio para el diagnóstico asistencial, que se realiza por los signos clínicos y la identificaciónde los ejemplares. El tratamiento específico es la aplicación del antiveneno. El objetivo de esta presentación es brindar una actualización sobre las características de este envenenamiento, su diagnóstico y tratamiento dirigidos hacia el paciente pediátrico.(AU)

Loxosceles is a global distribution spider with synanthropic characteristics. It is responsible for skin necrosis that in a low percentage is accompanied by systemic manifestations. Mortality can be high when systemic manifestationsare present. There is no laboratory methods available for diagnostic. Clinical signs and identification of individuals lead to it. The applicationof antivenom is the specific treatment.The objetive of this presentation is to update on the characteristics of this poisoning, its diagnosis and treatment directed toward the pediatric patient.(AU)

Transcriptome analysis of Loxosceles laeta (Araneae, Sicariidae) spider venomous gland using expressed sequence tags.

BACKGROUND: The bite of spiders belonging to the genus Loxosceles can induce a variety of clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, haemolysis, and persistent inflammation. In order to examine the transcripts expressed in venom gland of Loxosceles laeta spider and to unveil the potential of its products on cellular structure and functional aspects, we generated 3,008 expressed sequence tags (ESTs) from a cDNA library. RESULTS: All ESTs were clustered into 1,357 clusters, of which 16.4% of the total ESTs belong to recognized toxin-coding sequences, being the Sphingomyelinases D the most abundant transcript; 14.5% include "possible toxins", whose transcripts correspond to metalloproteinases, serinoproteinases, hyaluronidases, lipases, C-lectins, cystein peptidases and inhibitors. Thirty three percent of the ESTs are similar to cellular transcripts, being the major part represented by molecules involved in gene and protein expression, reflecting the specialization of this tissue for protein synthesis. In addition, a considerable number of sequences, 25%, has no significant similarity to any known sequence. CONCLUSION: This study provides a first global view of the gene expression scenario of the venom gland of L. laeta described so far, indicating the molecular bases of its venom composition.

Generalized vasculitic exanthem following Loxosceles reclusa envenomation.

Brown recluse spider bites (BRSB) cause a myriad of reactions ranging from local necrosis to potentially lethal systemic involvement. Envenomation may induce a generalized exanthem known clinically but not described histologically. We report a 49-year-old female who developed a generalized exanthem 24 hours after BRSB. The histopathology demonstrated a necrotizing vasculitis similar to that seen at an envenomation site but without epidermal necrosis. Loxoscelism should be considered in patients residing in endemic areas who present with a vasculitic exanthem.

Epidemiological and clinical manifestations of patients hospitalized with brown recluse spider bite.

BACKGROUND: Necrotic arachnidism represents a common health problem and standard treatments are usually safe and effective. OBJECTIVE: The aim of this study was to review documented Loxosceles species spider envenomations and identify the natural history of affected patients. METHODS: A retrospective single-centre study included 52 patients with necrotic arachnidism hospitalized in the dermatology department between 1997 and 2004. We examined the relationship between the epidemiological, clinical and laboratory parameters and degree of lesion severity, length of hospitalization and time to complete healing. RESULTS: The bites occurred predominantly in rural areas, 67% between April and August. Only 35% of the patients sought medical care within 24 h post bite. Most bites were of the extremities (67%). Time to complete healing ranged from 14 days to more than 8 weeks (mean, 4.8 weeks). A marked relationship was found between age, comorbidities, lesion severity and time to complete healing (P < 0.01). Duration of hospitalization was significantly longer in patients with severe thigh lesions (P < 0.02). CONCLUSIONS: Loxosceles species spider bites frequently induce necrotic, slowly healing ulcers on the fatty areas of the body. Early, appropriate systemic therapy may provide clinical benefit.

Verified bites by yellow sac spiders (genus Cheiracanthium) in the United States and Australia: where is the necrosis?

Spiders of the genus Cheiracanthium are frequently reported in review articles and medical references to be a definitive cause of dermonecrosis or necrotic arachnidism in humans. We provide 20 cases of verified bites by Cheiracanthium spiders from the United States and Australia, none with necrosis. A review of the international literature on 39 verified Cheiracanthium bites found only one case of mild necrosis in the European species C. punctorium. The basis for the suggestion that this spider genus causes dermonecrosis seems to be mostly inference from venom experiments in rabbits and guinea pigs, circumstantial spider involvement in human skin lesions, and repetitive citation of non-definitive reports in the medical literature. We discuss factors that lead to the erroneous elevation of virtually innocuous spiders to that of significant medical concern, which is a recurring problem in the medical community.

Host selection by a kleptobiotic spider.

Why do kleptobiotic spiders of the genus Argyrodes seem to be associated with spiders of the genus Nephila worldwide? Observations following introduction of experimental insect prey of different sizes and weights on to host webs revealed that: (1) small prey are more effectively retained on the web of Nephila clavipes than on the web of another common host, Leucauge venusta. (2) N. clavipes did not consume small prey that accumulated on the web whereas larger, heavier prey were enveloped and stored. (3) We observed clear partitioning of prey items between N. clavipes and Argyrodes spp.; diet selection by Argyrodes did not overlap with that of N. clavipes but closely overlapped with that of L. venusta. (4) L. venusta responds very quickly to prey impact whereas N. clavipes is slower, offering a temporal window of opportunity for Argyrodes foraging. (5) The ability of L. venusta to detect and respond to small items also means that it acts aggressively to Argyrodes spp., whereas N. clavipes does not. Consequently, food-acquisition behaviours of Argyrodes were clearly less risky with N. clavipes compared with L. venusta. We conclude that when a kleptobiotic organism has a choice of various host species, it will opt for the least risky host that presents the highest rate of availability of food items. The fact that Nephila species present such characteristics explains the worldwide association with Argyrodes kleptobiotic spiders.