Proteomic analysis of the venom of the social wasp Apoica pallens (Hymenoptera: Vespidae)

Wasps are a diverse group of insects that possess a sting apparatus associated with a venom gland, which is used for predation and colony defense. The biochemistry of Hymenoptera venom has been evaluated in relation to allergy and immunology, and proteomics has been shown to be a powerful tool for the identification of compounds with pharmacological potential. Data on wasps venom the of genus Apoica are scarce, so the objective of the present work was to identify the venom proteins of the eusocial wasp Apoica pallens, as a first step towards further investigation of applied uses of the venom and its protein constituents. The venom proteins were separated by two-dimensional gel electrophoresis, followed by MALDI-TOF/TOF mass spectrometry. A total of 259 spots were detected, with molecular weights from 4.9 to 141 kDa. Thirty of these proteins were identified and classified into eight functional categories: allergen, enzyme, metabolism, structural, environmental response, proteoglycan, active in DNA and RNA, and unknown function. Due to the few available proteomic data for wasp venom, many proteins could not be identified, which makes studies with proteomic analysis of Hymenoptera venom even more important.     

The primary structure of the toxin Laticauda semifasciata III, a weak and reversibly acting neurotoxin from the venom of a sea snake, Laticauda semifasciata

A weak and reversibly acting neurotoxic protein of Laticauda semifasciata venom, Laticauda semifasciata III (component LsIII), was sequenced. Component LsIII consists of 66 amino acid residues and has five disulphide bridges, one of which was located between residues 26 and 30. The weak and reversible neurotoxicity of component LsIII is discussed in relation to its structure, which falls between those of the neuro- and cardiotoxins of sea snakes and Elapidae snakes isolated and sequenced so far.     

管氏肿腿蜂毒液器官超微结构观察

应用透射电镜技术,观察了管氏肿腿蜂Scleroderma guani毒液器官的超微结构.毒腺由基膜层、分泌细胞层、导管细胞层和内膜层构成,分泌细胞内含内质网、末端附器、分泌囊泡、分泌颗粒、液泡等细胞器,其内合成的毒液由末端附器输送至毒腺的腔体.毒囊由肌肉鞘层、上皮细胞层和内膜层组成,肌肉鞘内的肌纤丝规则排列不交错,上皮细胞层内细胞器稀少,内膜层呈波浪状均匀加厚.     

Characterization and biochemical analyses of venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

During parasitism, the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) induces a developmental arrest in host pupae that is sustained until the fly is either consumed by developing larvae or the onset of death. Bioassays using fluids collected from the female reproductive system (calyx, alkaline gland, acid gland, and venom reservoir) indicated that the venom gland and venom reservoir are the sources of the arrestant and inducer(s) of death. Infrared spectroscopic analyses revealed that crude venom is acidic and composed of amines, peptides, and proteins, which apparently are not glycosylated. Reversed phase high performance liquid chromatography (HPLC) and sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the proteinaceous nature of venom and that it is composed mostly of mid to high molecular weight proteins in the range of 13 to 200.5 kilodaltons (kDa). Ammonium sulfate precipitation and centrifugal size exclusion membranes were used to isolate venom proteins. SDS-PAGE protein profiles of the isolated venom fractions displaying biological activity suggest that multiple proteins contribute to arresting host development and eliciting death. Additionally, HPLC fractionation coupled with use of several internal standards implied that two of the low molecular weight proteins were apamin and histamine. However, in vitro assays using BTI-TN-5B1-4 cells contradict the presence of these agents.     

丽蝇蛹集金小蜂Pacifastin蛋白酶抑制剂基因nvpp-1和nvpp-2的功能研究

Pacifastin蛋白酶抑制剂在昆虫免疫与发育中起着重要作用。为了明确其在寄生蜂中的相关功能, 本研究分别克隆获得编码丽蝇蛹集金小蜂Pacifastin蛋白酶抑制剂开放阅读框的cDNA序列nvpp-1和nvpp-2, 序列长度分别为723和888 bp, 分别编码240和295个氨基酸残基。预测结果表明, nvpp-1和nvpp-2推导氨基酸序列N端均含一个长度为17个氨基酸残基的信号肽序列。序列分析和进化树构建结果表明, NVPP-1和NVPP-2分别含有5个和4个典型的Pacifastin保守结构域, 并与疑黑瘤姬蜂Pimpla hypochondriaca毒液蛋白CVP4 聚为一类。实时荧光定量RT-PCR结果表明, nvpp-1和nvpp-2于该蜂雌蜂各组织中均发生转录, 且在胸、 腹部残体（解剖后腹部剩余部分）和毒器官中的转录水平较高; 于毒器官中, 其在羽化初期（0和1 d）转录水平较高, 其转录水平显著降低。Western blot结果表明, NVPP-1和NVPP-2均只在毒液中被大量检出, 在其他待测组织中均未被检出, 而刚羽化时（0 d）其在毒液中含量较低。利用pET-28a (+) 载体分别对nvpp-1和nvpp-2进行了原核表达, 并对重组表达产物进行纯化。分别测定重组NVPP-1和NVPP-2对4种不同丝氨酸蛋白酶（胰蛋白酶、 糜蛋白酶、 蛋白酶K和弹性蛋白酶）的抑制效果, 结果表明, 重组NVPP-1和NVPP-2分别能显著抑制糜蛋白酶和胰蛋白酶活性。同时还分别测定了两种重组蛋白对寄主家蝇蛹血淋巴自身的酚氧化酶活性及原酚氧化酶激活反应的影响, 结果表明, 重组蛋白对家蝇蛹血淋巴原酚氧化酶激活反应亦有抑制效果, 但其均不能显著影响血淋巴自身的酚氧化酶活性。综上所述, 丽蝇蛹集金小蜂毒液中含有Pacifastin蛋白酶抑制剂NVPP-1和NVPP-2, 分别为糜蛋白酶抑制剂和胰蛋白酶抑制剂家族成员, 均能显著影响寄主家蝇蛹血淋巴原酚氧化酶激活反应, 从而削弱寄主体液免疫水平。本研究所获结果加深了我们对昆虫尤其是寄生蜂Pacifastin蛋白酶抑制剂作用的认识。     

Evidence of alarm pheromones in the venom of Polistes dominulus workers (Hymenoptera: Vespidae)

Abstract.  The active and coordinating capacity of defending the nest is a key feature of social insects. The present study investigates the presence of alarm pheromones in the venom of workers of the social wasp, Polistes dominulus . Laboratory experiments were performed with caged colonies of P. dominulus using a wind tunnel apparatus to test the behavioural response of workers to venom released by other workers and to venom extracts. Contrary to that previously reported for European paper wasps, the present results show that the venom is the source of alarm pheromones. Field experiments combining a visual (black target) and a chemical stimulus (venom extract) were performed to test the effect of the venom on the reaction of colonies. Wasps leave the nest, land on the visual target and attack the target significantly more once exposed to venom extract plus target than to solvent plus target. This work shows that the venom of P. dominulus workers elicits an alarm response, reduces the threshold for attack and acts as an attractant on targets. These results using P. dominulus indicate that, in both American and European species, colony defence is based on the same features, suggesting that chemical alarm is a widespread trait in the genus Polistes .     

Tarantula (Eurypelma californicum) venom, a multicomponent system

The venom of the tarantula Eurypelma californicum was analysed biochemically, the components were isolated and characterized. The pH value of the crude venom is 5.3 +/- 0.3. After dilution with distilled water, UV-absorption spectra showed a single maximum at 258 nm (pH ca. 7.0). A second maximum at 328 nm emerged above pH 8.0. Protein concentration of the venom is ca. 65 mg/ml. After Coomassie staining SDS-PAGE patterns show three major bands with apparent molecular masses around 40 kDa, 4.3 kDa and 1.3 kDa besides some weak high molecular protein bands. The following low-molecular mass constituents were determined in the crude venom: ATP, ADP, AMP, glutamic acid, aspartic acid, gamma-aminobutyric acid, glucose and the ions potassium, sodium, calcium, magnesium and chloride; the osmolality was 361 micro0smol/ml. The LD50 value for female cockroaches was 0.15 microliters venom per g body weight and for male cockroaches 0.4 microliters venom per g body weight. Separation of the crude venom by gel chromatography yielded four elution peaks. Peak I contains the enzyme hyaluronidase. The activity is 200-900 U/microliters. Peak II contains a mixture of toxic peptides. Peak III contains the 1.3-kDa components of SDS-PAGE and peak IV mainly contains ATP. Venom proteins including the enzyme hyaluronidase were precipitated by 5% trichloroacetic acid. The supernatant was separated by HPLC into 13 fractions. Fraction 1 contains glutamic acid, aspartic acid, gamma-aminobutyric acid and ATP; fraction 2 contains ATP, ADP and AMP as well as a component 2' visible in SDS-PAGE as 1.3-kDa band and consisting of spermine and tryptophan; fraction 3 contains ATP and an unknown component 3'; fractions 4-6 also show a 1.3-kDa band in SDS-PAGE, fraction 4 being tyrosylspermine and fractions 5 and 6 containing compounds of spermine and aromatic molecules; fraction 7 contains a peptide which lacks aromatic amino acids, it was sequenced from the N-terminus; fractions 8-13 contain very similar toxic peptides. The peptides in fractions 11 and 12, labeled ESTX for Eurypelma spider toxin, were cleaved with different enzymes and sequenced. They differ in one amino acid in position 26. Homologies with scorpion toxins and with a toxin of the spider Segestria florentina were found.     

The neurotoxins of the sea snake Laticauda schistorhynchus.

Erabutoxins a and b, the major neurotoxins in the venom of the sea snake Laticauda semifasciata, were detected in the venom of Laticauda schistorhynchus. The identity of the toxins was confirmed on the basis of elution position on CM-cellulose column chromatography, disc electrophoretic mobility, amino acid analysis and toxicity measurement.     

Neutralization of some hematological and hemostatic alterations induced by neuwiedase, a metalloproteinase isolated from Bothrops neuwiedi pauloensis snake venom, by the aqueous extract from Casearia mariquitensis (Flacourtiaceae)

The aqueous extract from the leaves of Casearia mariquitensis (C. m.), a plant found in Brazilian open pastures, was assayed for its ability to inhibit some hematological and hemostatic effects induced by neuwiedase, a 22 kDa class P-I metalloproteinase from the venom of the South American pit viper Bothrops neuwiedi pauloensis. The aqueous extract from C. m. was able to neutralize the hematological alterations induced by the crude venom (C.V.) upon erythrocytes when the venom was incubated at a ratio of 1:10 (w/w, venom/extract), but it did not neutralize the platelet decreasing ability of C.V. The plasma fibrinogen concentration decreased approximately 36% and 83% when 0.6 LD(50) of the C.V. or neuwiedase, respectively, were injected by i.p. route in mice, and the aqueous extract from C. m. was able to inhibit this effect. The Bbeta fibrinogen chain was protected against degradation caused by crude venom and neuwiedase when the venom or toxin were incubated with C. m. extract. We also observed that this extract exerted a very slight effect on the clotting time, prolonging it only to a little extent. The pulmonary hemorrhage induced by neuwiedase when injected intravenously with 0.6 LD(50) was completely inhibited when this toxin was incubated with the extract at a ratio of 1:10 (w/w, toxin/extract). It is concluded that C. m. displays components able to inhibit some hematological and systemic alterations induced by C.V.     

腰带长体茧蜂毒液器官和卵巢的形态学及其超微结构

应用超薄切片和电镜技术,观察内寄生蜂腰带长体茧蜂Macrocentrus cingulum Brischke毒液器官和卵巢的形态结构。腰带长体茧蜂毒液器官由1个毒囊和2条毒腺组成,毒腺接于毒囊的顶端。毒腺由单层分泌细胞、退化的外胚层细胞和环腔的内膜构成,分泌细胞主要由1个明显的细胞核和1个较大囊状细胞器构成,囊状细胞器的功能是分泌毒液。毒囊由肌肉鞘和扁平细胞层构成,但没有分泌细胞。腰带长体茧蜂卵巢1对,每个卵巢由10条左右卵巢小管组成,与侧输卵管相接处略微膨大形成卵巢萼区。2条侧输卵管在产卵管基部会合形成1条总输卵管与产卵管相接。毒液器官通过毒囊的毒液导管附着在总输卵管上。对寄生蜂毒液器官的生物学、细胞学及在分类进化上的意义进行研究。     

A case of hermaphroditism in Scyliorhinus stellaris (Linné, 1758) (Pisces, Scyliorhinidae): morphological and histological study

The authors describe an hermaphrodite Scyliorhinus stellaris with both complete but juvenile male and female genital apparatus.     

Venom apparatus and toxicity of the centipede Ethmostigmus rubripes (Chilopoda, Scolopendridae)

The venom apparatus of Ethmostigmus rubripes, a generalized predator, consists of the telopodites of the postcephalic segment, the basal article of w which contains the venom gland. Within the gland, venom granules are concentrated in intracellular secretory granules, from which they are discharged into vacuoles in the cytoplasm of the secretory cells and thereafter by exocytosis into the lumen of the gland. A venom duct carries venom to the venom claw, which introduces it into prey via a subterminal pore on the outer curvature of the claw. Pits containing pegs, presumed to be sensory, are concentrated near grooves leading to a cutting ridge proximal to the point of the claw. The venom is toxic both to mammals and insects.     

管氏肿腿蜂毒液丝氨酸蛋白酶同源物SgSPH对寄主血淋巴酚氧化酶活性的影响

[目的]本研究旨在通过克隆表达管氏肿腿蜂Scleroderma guani毒液丝氨酸蛋白酶同源物(serine protease homologue,SPH)基因SgSPH,探索其编码的毒液蛋白对寄主血淋巴酚氧化酶活性的影响.[方法]利用RT-PCR技术克隆管氏肿腿蜂毒液SgSPH基因的开放阅读框(ORF),采用生物信...     

Study of a Stridulatory Apparatus in Pardosa fulvipes (Collett) (Araneae, Lycosidae) by Scanning Electron Microscopy

The adult Pardosa fulvipes male has a stridulatory apparatus in which the file is the hairless striated surface of the book lung opercula, and the scraper part is formed by several stout denticulated hairs re-trolaterally on the fourth coxae and trochanters. The microscuipture of th; opsrcula and specialized hairs in the adult male are studied with the aid of scanning electron microscope and compared with homologous structures in the adult female and immatures. The microscuipture of the book lung opercula is also compared with that of related Pardosa species. A gradual transformation of the opercular microscuipture occurs in both male and female P. fulvipes leading to the species-specific sculpture in the adults. The behavioural prerequisites for stridulation in this species are discussed. P. fulvipes is hitherto the only lycosid known to have a stridulatory apparatus.     

Ultrastructure of the venom gland of the scorpion,Centruroides sculpturatus (Ewing)

The venom apparatus of the scorpion, C. sculpturatus (Ewing) was studied with light and electron microscopy. Each of the paired glands is lined by secretory epithelium made up of a single layer of columnar cells. Extensive folding in the epithelial layer creates a primitive acinar gland. The secretory products are either membrane-bound or unbound vesicles with discrete morphologies and are observed in the extruded venom, within the lumen of the gland, and within single secretory cells. The venom apparatus, including connective tissues, nerve cells, and muscle tunic is described and correlations are made with observations in other Athropods.     

Regulation of wing formation and adult development in an aphid host, Aphis fabae, by the parasitoid Aphidius colemani

Nymphs of presumptive winged gynoparae of Aphis fabae (Hemiptera: Aphididae), were exposed to female parasitoids, Aphidius colemani (Hymenoptera: Aphidiidae) and stung once with the ovipositor. Wing development was inhibited and, when aphids were parasitised during the early stages, they did not reach the adult stage but mummies with rudimentary or no wingbuds are observed in the host's fourth-stadium. These and previous studies have suggested that wing development may be inhibited by factor(s) from the maternal parasitoid injected into the host at the time of oviposition. In an attempt to identify such factor(s), saline extracts of whole female parasitoids, abdomens, ovaries and venom glands were prepared. When a saline extract of venom glands was injected into late-second-stadium aphids, many develop to fourth-stadium nymphs with rudimentary wingbuds, indicating an effect on wing formation but also showed developmental arrest and often died when attempting to moult to the adult stage. It appears that host death may be related to physiological/biochemical interactions of parasitoid and host rather than just late stage parasitoid larvae ingesting the host's vital organs. Injections with extracts into later host stadia gave similar results with regard to development to the adult, although aphids injected in the late-fourth-stadium develop normally to the adult stage with no effect on wing formation. The results indicate that the earlier the injection before the final moult the greater the effect of the injected extract on preventing adult development.Extracts prepared from head + thorax do not affect aphid development and the results indicate that there is an active factor(s) - likely a protein - in the female parasitoid's venom that disrupts wing development and/or inhibits development to the adult stage. Surprisingly, injections of extracts from male parasitoids have similar effects but the location and function of such a factor(s) in males are unknown.     

Effect of the venom of the endoparasitoid,Apanteles kariyai Watanabe,on the cellular defence reaction of the host,Pseudaletia separata Walker

Although a venom apparatus is present in all female braconid wasps, the function of the venom injected into the host at the time of oviposition by the endophagous members of this group is unknown. Eggs laid by females of Apanteles kariyai, from which the venom apparatus had been removed, were encapsulated, which suggests that the fluid is necessary to enable the parasitoid eggs to escape the cellular defence reaction of the host. Studies with anti-venom serum demonstrated that the venom is attached to the surface of the egg. However, injection of DEAE-Sephadex A-50 particles (Sephadex particles) revealed that the venom alone is insufficient to inhibit the encapsulation reaction. Calyx and venom fluids together seem to be essential for evasion of the host defence reaction by the parasitoid eggs. Eighty-five % Sephadex particles, injected together with calyx and venom fluids, fail to become fully encapsulated, whereas 46% of particles injected with only the calyx fluid avoided encapsulation. Furthermore, when eggs from the lateral oviduct were injected into unparasitized larvae, together with the calyx and venom fluids, a few eggs developed successfully although they had undergone no mechanical distortion.     

Proteins from Mucuna pruriens and enzymes from Echis carinatus venom: characterization and cross-reactions

Mucuna pruriens seeds have been widely used against snakebite in traditional medicine. The antivenin property of a water extract of seeds was assessed in vivo in mice. The serum of mice treated with extract was tested for its immunological properties. Two proteins of Echis carinatus venom with apparent molecular masses of 25 and 16 kDa were detected by Western blot analysis carried out using IgG of mice immunized with extract or its partially purified protein fractions. By enzymatic in-gel digestion and electrospray ionization-mass spectrometry/mass spectrometry analysis of immunoreactive venom proteins, phospholipase A(2,) the most toxic enzyme of snake venom, was identified. These results demonstrate that the observed antivenin activity has an immune mechanism. Antibodies of mice treated with non-lethal doses of venom reacted against some proteins of M. pruriens extract. Proteins of E. carinatus venom and M. pruriens extract have at least one epitope in common as confirmed by immunodiffusion assay.     

Functional morphology of the sting apparatus of the spider wasp Cryptocheilus versicolor (Scopoli, 1763) (Hymenoptera: Pompilidae)

The females of the spider wasps (Hymenoptera: Pompilidae) hunt spiders to provision their larvae. The genital structures of pompilid females are modified in a sting that is used for paralyzing the prey (spiders) and defense. The skeleto‐muscular structure of the sting apparatus of a typical representative of the family (Cryptocheilus versicolor) is examined. The shape of sclerites, their relative positions and articulations are described. Some morphological adaptations are described for the first time. The wide anal arc of the tergum IX provides a stiff support for the muscles that move the valvulae. The resilin structures in the areas of articulation support the work of muscles and in some cases replace them. The 1st valvulae form a venom duct along their entire length, which provides the delivery of the venom to a specific point. An unpaired flap in the venom duct provides a dose of venom in the sting. This mechanism probably enhances the speed and accuracy of the wasp's sting movements. Functions of muscles and interactions of the structures of the sting apparatus of C. versicolor are discussed.     

Recovery of proteins on a milligram scale from polyacrylamide electrophoresis gels,exemplified by purification of a retinol-binding protein

An apparatus suitable for the recovery of proteins from polyacrylamide gels on a milligram scale by displacement electrophoresis (isotachophoresis) is described along with a buffer system that is suitable for this purpose with most proteins. The technique is illustrated by the recovery of a protein from a 15% polyacrylamide gel. The recovery was almost quantitative and the eluted protein showed little contamination upon quantitative amino acid analysis and automatic Edman degradation.