Complete Genome Sequence of Spiroplasma helicoides TABS-2T (DSM 22551), a Bacterium Isolated from a Horsefly (Tabanus abactor).
ABSTRACT: Spiroplasma helicoides TABS-2T (DSM 22551) was isolated from the gut of a horsefly (Tabanus abactor) collected near Ardmore, Oklahoma, USA, in 1987. Here, we report the complete genome sequence of this bacterium to facilitate the investigation of its biology and the comparative genomics among Spiroplasma species.
Project description:Spiroplasma litorale TN-1(T) (DSM 21781) was isolated from the gut of a green-eyed horsefly (Tabanus nigrovittatus), collected at Ocracoke Island in North Carolina in 1983. Here, we report the complete genome sequence of this bacterium to facilitate the investigation of its biology.
Project description:Blood-feeding arthropods rely heavily on the pharmacological properties of their saliva to get a blood meal and suppress immune reactions of hosts. Little information is available on antihemostatic substances in horsefly salivary glands although their saliva has been thought to contain wide range of physiologically active molecules. In traditional Eastern medicine, horseflies are used as anti-thrombosis material for hundreds of years. By proteomics coupling transcriptome analysis with pharmacological testing, several families of proteins or peptides, which exert mainly on anti-thrombosis functions, were identified and characterized from 60,000 pairs of salivary glands of the horsefly Tabanus yao Macquart (Diptera, Tabanidae). They are: (I) ten fibrin(ogen)olytic enzymes, which hydrolyze specially alpha chain of fibrin(ogen) and are the first family of fibrin(ogen)olytic enzymes purified and characterized from arthropods; (II) another fibrin(ogen)olytic enzyme, which hydrolyzes both alpha and beta chain of fibrin(ogen); (III) ten Arg-Gly-Asp-motif containing proteins acting as platelet aggregation inhibitors; (IV) five thrombin inhibitor peptides; (V) three vasodilator peptides; (VI) one apyrase acting as platelet aggregation inhibitor; (VII) one peroxidase with both platelet aggregation inhibitory and vasodilator activities. The first three families are belonging to antigen five proteins, which show obvious similarity with insect allergens. They are the first members of the antigen 5 family found in salivary glands of blood sucking arthropods to have anti-thromobosis function. The current results imply a possible evolution from allergens of blood-sucking insects to anti-thrombosis agents. The extreme diversity of horsefly anti-thrombosis components also reveals the anti-thrombosis molecular mechanisms of the traditional Eastern medicine insect material.
Project description:Spiroplasma sp. TU-14 was isolated from a contaminated sample of Entomoplasma lucivorax PIPN-2T obtained from the International Organization for Mycoplasmology collection. Here, we report the complete genome sequence of this bacterium to facilitate the investigation of its biology and the comparative genomics among Spiroplasma spp.
Project description:A novel family of RGD-containing molecules (Tablysin-15) has been molecularly characterised from the salivary gland of the haematophagous horsefly Tabanus yao. Tablysin-15 does not share primary sequence homology to any disintegrin discovered so far, and displays an RGD motif in the N-terminus of the molecule. It is also distinct from disintegrins from Viperidae since its mature form is not released from a metalloproteinase precursor. Tablysin-15 exhibits high affinity binding for platelet ?IIb?3 and endothelial cell ?V?3 integrins, but not for ?5?1 or ?2?1. Accordingly, it blocks endothelial cell adhesion to vitronectin (IC50 ~1 nM) and marginally to fibronectin (IC50 ~1 ?M), but not to collagen. It also inhibits fibroblast growth factor (FGF)-induced endothelial cell proliferation, and attenuates tube formation in vitro. In platelets, Tablysin-15 inhibits aggregation induced by collagen, ADP and convulxin, and prevents static platelet adhesion to immobilised fibrinogen. In addition, solid-phase assays and flow cytometry demonstrates that ?IIb?3 binds to Tablysin-15. Moreover, immobilised Tablysin-15 supports platelet adhesion by a mechanism which was blocked by anti-integrin ?IIb?3 monoclonal antibody (e.g. abciximab) or by EDTA. Furthermore, Tablysin-15 dose-dependently attenuates thrombus formation to collagen under flow. Consistent with these findings, Tablysin-15 displays antithrombotic properties in vivo suggesting that it is a useful tool to block ?IIb?3, or as a prototype to develop antithrombotics. The RGD motif in the unique sequence of Tablysin-15 represents a novel template for studying the structure-function relationship of the disintegrin family of inhibitors.
Project description:The ventral compound eye of many insects contains polarization-sensitive photoreceptors, but little is known about how they are integrated into visual functions. In female horseflies, polarized reflections from animal fur are a key stimulus for host detection. To understand how polarization vision is mediated by the ventral compound eye, we investigated the band-eyed brown horsefly Tabanus bromius using anatomical, physiological, and behavioral approaches. Serial electron microscopic sectioning of the retina and single-cell recordings were used to determine the spectral and polarization sensitivity (PS) of photoreceptors. We found 2 stochastically distributed subtypes of ommatidia, analogous to pale and yellow of other flies. Importantly, the pale analog contains an orthogonal analyzer receptor pair with high PS, formed by an ultraviolet (UV)-sensitive R7 and a UV- and blue-sensitive R8, while the UV-sensitive R7 and green-sensitive R8 in the yellow analog always have low PS. We tested horsefly polarotaxis in the field, using lures with controlled spectral and polarization composition. Polarized reflections without UV and blue components rendered the lures unattractive, while reflections without the green component increased their attractiveness. This is consistent with polarotaxis being guided by a differential signal from polarization analyzers in the pale analogs, and with an inhibitory role of the yellow analogs. Our results reveal how stochastically distributed sensory units with modality-specific division of labor serve as separate and opposing input channels for visual guidance.
Project description:A diverse group of physiologically active peptides/proteins are present in the salivary glands of horsefly Tabanus yao (Diptera, Tabanidae) that facilitate acquisition of blood meal. However, their roles in the regulation of local inflammation remains poorly understood.Induction expression profiles of immune-related molecules in the salivary glands of T. yao was analyzed by quantitative PCR (qPCR) after bacterial feeding. A significantly up-regulated molecule (cecropin-TY1) was selected for anti-inflammatory assay in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages. The transcription levels of inducible NO synthase (iNOS) and pro-inflammatory cytokines were quantified by qPCR. Nitric oxide (NO) production was determined by Griess reagent. Pro-inflammatory cytokine production was determined by an enzyme-linked immunosorbent assay (ELISA). The inflammatory signals were assayed by Western blotting analysis. The secondary structure of cecropin-TY1 was measured by Circular dichroism (CD) spectroscopy. Interaction of cecropin-TY1 with LPS was evaluated by the dissociation of fluorescein isothiocyanate (FITC)-conjugated LPS aggregates and neutralization of LPS determined by a quantitative Chromogenic End-point Tachypleus amebocyte lysate (TAL) assay kit. Homology modeled structure analysis and mutation of key residues/structures were performed to understand its structure-activity relationship.Cecropin-TY1 was demonstrated to possess high anti-inflammatory activity and low cytotoxicity toward mouse macrophages. In LPS-stimulated mouse peritoneal macrophage, addition of cecropin-TY1 significantly inhibited the production of nitric oxide (NO) and pro-inflammatory cytokines. Further study revealed that cecropin-TY1 inhibited inflammatory cytokine production by blocking activation of mitogen-activated protein kinases (MAPKs) and transcriptional nuclear factor-κB (NF-κB) signals. Cecropin-TY1 even interacted with LPS and neutralized LPS. The secondary structure analysis revealed that cecropin-TY1 adopted unordered structures in hydrophobic environment but converted to α-helical confirmation in membrane mimetic environments. Homology modeled structure analysis demonstrated that cecropin-TY1 adopted two α-helices (Leu3-Thr24, Ile27-Leu38) linked by a hinge (Leu25-Pro26) and the structure surface was partly positively charged. Structure-activity relationship analysis indicated that several key residues/structures are crucial for its anti-inflammatory activity including α-helices, aromatic residue Trp2, positively charged residues Lys and Arg, hinge residue Pro26 and N-terminal amidation.We found a novel anti-inflammatory function of horsefly-derived cecropin-TY1 peptide, laying groundwork for better understanding the ectoparasite-host interaction of horsefly with host and highlighting its potency in anti-inflammatory therapy for sepsis and endotoxin shock caused by Gram-negative bacterial infections.
Project description:BACKGROUND:More than 170 species of tabanids are known in Europe, with many occurring only in limited areas or having become very rare in the last decades. They continue to spread various diseases in animals and are responsible for livestock losses in developing countries. The current monitoring and recording of horseflies is mainly conducted throughout central Europe, with varying degrees of frequency depending on the country. To the detriment of tabanid research, little cooperation exists between western European and Eurasian countries. METHODS:For these reasons, we have compiled available sources in order to generate as complete a dataset as possible of six horsefly species common in Europe. We chose Haematopota pluvialis, Chrysops relictus, C. caecutiens, Tabanus bromius, T. bovinus and T. sudeticus as ubiquitous and abundant species within Europe. The aim of this study is to estimate the distribution, land cover usage and niches of these species. We used a surface-range envelope (SRE) model in accordance with our hypothesis of an underestimated distribution based on Eurocentric monitoring regimes. RESULTS:Our results show that all six species have a wide range in Eurasia, have a broad climatic niche and can therefore be considered as widespread generalists. Areas with modelled habitat suitability cover the observed distribution and go far beyond these. This supports our assumption that the current state of tabanid monitoring and the recorded distribution significantly underestimates the actual distribution. Our results show that the species can withstand extreme weather and climatic conditions and can be found in areas with only a few frost-free months per year. Additionally, our results reveal that species prefer certain land-cover environments and avoid other land-cover types. CONCLUSIONS:The SRE model is an effective tool to calculate the distribution of species that are well monitored in some areas but poorly in others. Our results support the hypothesis that the available distribution data underestimate the actual distribution of the surveyed species.
Project description:Due to poor diagnostic facilities and a lack of medical alertness, allergy to Vespa wasps may be underestimated. Few allergens have been identified from Vespa wasps.Possible native allergen proteins were purified from the wasp venoms (WV) (Vespa magnifica Smith) by gel filtration, ion exchange chromatography, respectively. Their sequences were determined by Edman degradation and cDNA cloning. Their allergenicities were assayed by enzyme-linked immunosorbent assay inhibition tests (ELISA-IT), immunoblots, and skin prick tests (SPTs). Their cross allergencities with Tab y 2 and Tab y 5 purified from the horsefly (Tabanus yao Macquart) were also determined. Two native allergens were identified from the WV, respectively. They are a 25-KDa antigen 5 protein (Ag5) (Vesp ma 5) and a 35-KDa hyaluronidase (Vesp ma 2). They represented major allergens in Vespa magnifica by immunoblots and SPTs. ELISA inhibition of pooled sera IgE reactivity to both the WV and the horsefly salivary gland extracts (HSGE) using four purified allergens (Vesp ma 2, Vesp ma 5 and previously purified Tab y 2 and Tab y 5) was significant. Their cross allergenicities were confirmed by ELISA-IT, immunoblots, and SPTs. They represented the cross reactive allergens from wasp and horsefly and proved the so called wasp-horsefly syndrome.