Localization of intracellular calcium release in cells injured by venom from the ectoparasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) and dependence of calcium mobilization on G-protein activation

Venom from the ectoparasitic wasp Nasonia vitripennis induces cellular injury that appears to involve the release of intracellular calcium stores via the activation of phospholipase C, and culminates in oncotic death. A linkage between release of intracellular Ca2+ and oncosis has not been clearly established and was the focus of this study. When BTI-TN-5B1-4 cells were treated with suramin, an uncoupler of G-proteins, venom-induced swelling and oncotic death were inhibited in a dose-dependent manner for at least 24 h. Suramin also blocked increases in free cytosolic [Ca2+], arguing that venom induces calcium mobilization through G-protein signaling pathways. Endoplasmic reticulum (ER) was predicted to be the source of intracellular calcium release, but labeling with the fluorescent probe ER-tracker revealed no indication of organelle swelling or loss of membrane integrity as would be expected if the Ca(2+)-ATPase pump was disabled by crude venom. Incubation of cell monolayers with calmodulin or nitrendipine, modulators of ER calcium release channels, neither attenuated nor augmented the effects of wasp venom. These results suggest that wasp venom stimulates calcium release from ER compartments distinct from RyRs, L-type Ca2+ channels, and the Ca(2+)-ATPase pump, or calcium is released from some other intracellular store. A reduction of mitochondrial membrane potential delta psi(m) appeared to precede a rise in cytosolic free Ca2+ as evidenced by fluorescent microscopy using the calcium-sensitive probe fluo-4 AM. This argues that the initial insult to the cell resulting from venom elicits a rapid loss of (delta psi(m)), followed by unregulated calcium efflux from mitochondria into the cytosol. Mobilization of calcium in this fashion could stimulate cAMP formation, and subsequently promote calcium release from NAADP-sensitive stores.     

In vivo and in vitro activity of venom from the endoparasitic wasp Pimpla turionellae (L.) (Hymenoptera: Ichneumonidae)

The biological activity of venom from Pimpla turionellae L. (Hymenoptera: Ichneumonidae) was examined in vivo toward larvae and pupae of Galleriae mellonella L. (Lepidoptera: Pyralidae), and in vitro toward bacterial and fungal cultures, as well as cultured insect cells. Pupae of G. mellonella were far more susceptible to the venom than larvae. At low doses of venom [0.1 venom reservoir equivalents (VRE)], pupal abdominal mobility was inhibited within 30 min, and by 24 h, all pupae injected with venom concentrations >0.5 VRE were completely paralyzed. These same doses of venom resulted in an inhibition of adult emergence. Host larvae were far less sensitive to wasp venom as evidenced by all venom injected larvae remaining responsive to mechanical stimulation by 1 h post injection, even at concentrations equivalent to 1 venom reservoir. Eventually (>2 h at 25 degrees C), venom-injected larvae became immobile, then flaccid, and all died within 24 h post-injection. At lower concentrations of wasp venom, the onset of paralysis was delayed by comparison to that evoked by 1 VRE, and few host larvae were able to pupate. Development of host larvae to adult emergence was also reduced in a dose-dependent manner, with eclosion completely prevented at high concentrations (>0.5 VRE) of venom. Venom doses <0.5 VRE did not appear to induce paralysis or alter larval development. When venom was incubated with bacterial or fungal cultures, no antimicrobial activity was detected. However, wasp venom was found to be cytotoxic and cytolytic to cultured cells derived from the cabbage looper Trichoplusia ni Hubner (Lepidoptera: Noctuidae) and the yellow fever mosquito, Aedes aegypti (L.) (Diptera: Culcidae). Though both cell types displayed similar susceptibility in terms of LC50s, the lepidopteran cells responded much more rapidly with regard to the onset of morphological changes and the timing of cell death. A possible mode of action for the venom is discussed.     

Pathological and ultrastructural changes in cultured cells induced by venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

The ectoparasitic wasp Nasonia vitripennis produces a proteinaceous venom that induces death in fly hosts by non-paralytic mechanisms. Previous in vitro assays have suggested that the primary cause of cell and tissue death is oncosis, a non-programmed cell death (PCD) pathway characterized by cellular swelling and lysis. However, ultrastructural analyses of BTI-TN-5B1 cells exposed to LC₉₉ doses of wasp venom revealed cellular changes more consistent with apoptosis and/or non-apoptotic PCD than oncosis or necrosis: By 3 h after incubation with venom, susceptible cells displayed indentations in the nuclear membranes, large nucleoli, and extensive vacuolization throughout the cytoplasm. In the vast majority of venom treated cells, annexin V bound to the plasma membrane surface within 15 min after treatment, a characteristic consistent with translocation of phosphatidylserine to the cell surface during the early stages of apoptosis. Likewise, mitochondrial transmembrane potential was depressed in cells within 15 min in venom-treated cells, an event that occurred in the absence of mitochondrial swelling or rupturing of cristae. Active caspase 3 was detected by fluorescent labeling in nearly all venom treated cells 3 h after exposure to venom, and in turn, the potent caspase 3 inhibitor Z-VAD-FMK attenuated the morphological changes elicited by wasp venom and afforded protection to BTI-TN-5B1-4 cells.     

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.     

Characterization of phenoloxidase activity in venom from the ectoparasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

Crude venom isolated from the ectoparasitic wasp Nasonia vitripennis was found to possess phenoloxidase (PO) activity. Enzyme activity was detected by using a modified dot blot analysis approach in which venom samples were applied to nylon membranes and incubated with either L-DOPA or dopamine. Dot formation was most intense with dopamine as the substrate and no activators appeared to be necessary to evoke a melanization reaction. No melanization occurred when venom was incubated in Schneider's insect medium containing 10% fetal bovine serum or when using tyrosine as a substrate, but melanization did occur when larval or pupal plasma from the fly host, Sarcophaga bullata, was exposed to tyrosine. Only fly larval plasma induced an enzyme reaction with the Schneider's insect medium. The PO inhibitor phenylthiourea (PTU) and serine protease inhibitor phenylmethylsulfonylfluoride (PMSF) abolished PO activity in venom and host plasma samples, but glutathione (reduced) only inhibited venom PO. Elicitors of PO activity (sodium dodecyl sulfate and trypsin) had no or a modest effect (increase) on the ability of venom, or larval and pupal plasma to trigger melanization reactions. SDS-PAGE separation of crude venom followed by in-gel staining using L-DOPA as a substrate revealed two venom proteins with PO activity with estimated molecular weights of 68 and 160 kDa. In vitro assays using BTI-TN-5B1-4 cells were performed to determine the importance of venom PO in triggering cellular changes and evoking cell death. When cell monolayers were pre-treated with 10 mM PTU or PMSF prior to venom exposure, the cells were protected from the effects of venom intoxication as evidenced by no observable cellular morphological changes and over 90% cell viability by 24 h after venom treatment. Simultaneous addition of inhibitors with venom or lower concentrations of PMSF were less effective in affording protection. These observations collectively argue that wasp venom PO is unique from that of the fly hosts, and that the venom enzyme is critical in the intoxication pathway leading to cell death.     

两种金小蜂毒液对菜粉蝶蛹血细胞延展、存活及包囊作用的影响

活体微注射测定结果表明,将0.5毒囊当量（venom reservoir equivalent, VRE）的蝶蛹金小蜂毒液注射于其寄主菜粉蝶蛹体内,注射后4～24 h寄主浆血细胞和颗粒血细胞的延展、存活和对Sephadex A50微珠的包囊能力显著下降;以0.002～0.02 VRE/μL的该蜂毒液处理其离体寄主血细胞均能产生同样的生理效应。该毒液抑制90%菜粉蝶蛹浆血细胞和颗粒血细胞延展的浓度各为0.00076 VRE/μL和0.00804 VRE/μL。可见,蝶蛹金小蜂毒液能显著抑制其寄主血细胞的延展和包囊作用,并导致血细胞的死亡。然而,同样条件下丽蝇蛹集金小蜂毒液对其非自然寄主菜粉蝶蛹的血细胞延展、存活和包囊作用则无任何效应。可见,寄生蜂毒液的生理作用具有明显的寄主特异性。     

Effects of host (Boettcherisca peregrina) copper exposure on development, reproduction and vitellogenesis of the ectoparasitic wasp, Nasonia vitripennis

Effects of copper (Cu) accumulation by the flesh fly Boettcherisca peregrina (R.-D.) (Diptera: Sarcophagidae) on the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) were investigated experimentally by exposing host larvae to contaminated diets with final Cu concentrations of 400 μg/g and 800 μg/g diet fresh weight (DFW), respectively. Results showed that Cu can be transferred along food chains to secondary consumers (parasitoids) in small amounts, resulting in negative effects on parasitoid growth and development (body weight and developmental duration) as well as fecundity (number of offspring per female). Copper exposure also inhibited vitellogenesis of parasitoids from Cu-contaminated host pupae. It is suggested that the decreased fecundity and inhibition of vitellogenesis of N. vitripennis resulted from poor host nutritional state rather than from direct effects of Cu stress.     

Disruption of pupariation and eclosion behavior in the flesh fly, Sarcophaga bullata Parker (Diptera: Sarcophagidae), by venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

The action of venom from the ectoparasitic wasp, Nasonia vitripennis, was monitored by examining alterations in patterned muscular movements characteristic of pupariation and eclosion behavior in the flesh fly, Sarcophaga bullata. Venom injected into larvae prior to pupariation caused a dose-dependent delay in pupariation. Eventually, such larvae did pupariate, but puparia were abnormally formed. Barographic records revealed that all elements of pupariation behavior were present in venom-injected larvae, but pupariation behavior was not well synchronized with tanning, thus implying that the venom caused disruption in the temporal organization of central motor programs. When larvae were ligated and injected with venom posterior to the ligature, no response was evident in the posterior region, suggesting that the venom does not directly stimulate muscles or neuromuscular junctions. Injection of exogenous ecdysteroid into venom-injected larvae restored some elements of pupariation behavior, consistent with ecdysone's role in stimulating the release of anterior retraction factor and puparium tanning factor, two factors that are released from the CNS to regulate pupariation. When the venom was injected into newly emerged imagoes, the duration of extrication behavior was shortened, whereas all phases of post-eclosion behavior were lengthened. These observations imply that the venom affects CNS centers that regulate the muscular systems engaged in extrication and post-eclosion behavior.     

Absence of chemical alarm in a primitively eusocial wasp (Belonogaster petiolata,Hymenoptera: Vespidae)

Summary The hypothesis thatBelonogaster petiolata (fam. Vespidae) is able to communicate alarm chemically, using odours released with the venom, was tested in bioassays involving presentation of artificial targets to a wasp colony, simultaneously with crushed venom apparatuses. The odour of venom did not lower the threshold of attack and visual stimuli alone (particularly a black, moving object) were sufficient to release attack. Venom odour on a previously stung target probably does not play a role in focusing further attacks on such a target. The results therefore support the null hypothesis that a venom-based alarm pheromone is absent in this species.     

The mode of action of venom from the endoparasitic wasp Pimpla hypochondriaca (Hymenoptera: Ichneumonidae) involves Ca+2‐dependent cell death pathways

The endoparasitoid Pimpla hypochondriaca injects venom during oviposition to condition its lepidopteran hosts. Venom is a complex mixture of proteins and polypeptides, many of which have been identified as enzymes, including phenoloxidase, endopeptidase, aminopeptidase, hydrolase, and angiotensin‐converting enzyme. Constituents of the venom have been shown to possess cytolytic and paralytic activity, but the modes of action of factor(s) responsible for exerting such effects have not been deciphered. In this study, we examined the mode of action of isolated venom using cultured cells (BTI‐TN‐5B1‐4). A series of blockage and inhibition assays were performed using a potent inhibitor (phenylthiourea, PTU) of venom phenoloxidase, and anti‐calreticulin antibodies. Monolayers exposed to venom alone were highly susceptible with more than 84.6±2.3% dead within 15 min. Susceptible cells displayed a retraction of cytoplasmic extensions, rounding, and swelling prior to lysis in more than half (55.7±1.7%) of the dying cells. Within 15 min of exposure to venom, cells displayed qualitative increases in [Ca⁺²]_i as evidenced by staining with the calcium‐sensitive probe fluo‐4 AM, and mitochondrial membrane potential (ΔΨ_m) was undetectable by 5 min post‐treatment with venom. These venom‐mediated changes occurred regardless of whether an external source of calcium was present, or whether venom was pre‐treated with PTU. In contrast, venom toxicity was attenuated by treatment with anti‐calreticulin antibodies. Not only did fewer cells die when exposed to antibody‐treated venom but also cell swelling diminished and no increases in intracellular calcium were detected. A possible mode of action for the venom is discussed. © 2009 Wiley Periodicals, Inc.     

抗苏云金杆菌毒素Cry1Ac粉纹夜蛾细胞系的特性研究

为了从离体细胞水平探讨昆虫对苏云金芽孢杆菌杀虫晶体蛋白的部分抗性机制,本文采用活化的Cry1AC毒素对粉纹夜蛾BTI-TN-581-4细胞连续筛选86代,获得了高水平抗性细胞,研究了其某些特性。它对Cry1C产生了低水平的交互抗性,对低渗溶液的耐受性显著增强,双向电泳图谱表明抗性细胞膜蛋白组分发生了明显的变化。膜蛋白组分的变化可能导致了筛选细胞的耐低渗透压和抗Cry1C。     

Assay and properties of the hemolysis activity of pure venom from the nematocysts of the acontia of the sea anemone Aiptasia pallida

Pure venom from the acontial nematocysts of the sea anemone Aiptasia pallida was isolated and an assay for the hemolysis activity of the venom devised. The assay is rapid, sensitive, and reproducible. Venom concentrations as low as 0.1 μg protein/ ml were accurately assayed. The properties of the hemolysis activity were analyzed using techniques similar to those used to study enzyme-catalyzed reactions. The biochemical events underlying venom-induced lysis required the direct participation of millimolar levels of Ca²⁺. The slight variability of the apparent K_m for Ca²⁺ at different venom concentrations appeared to be due to the release of some material(s) from lysing cells. Both Sr²⁺ and Mg²⁺ weakly substituted for Ca²⁺. Inhibition of lysis by EDTA was reversed by Ca²⁺. Small monovalent cations, such as Na⁺ or K⁺, appeared to be involved in the venom-induced alteration of the red cell membrane so that lysis could occur. The venom's hemolysis activity was stabilized in solution only if the concentration of the venom proteins was high while also in the presence of at least the equivalent of 0.15 m NaCl.     

The venom of Ampulex compressa--effects on behaviour and synaptic transmission of cockroaches

1. The solitary wasp Ampulex compressa stings a cockroach, Periplaneta americana, twice. 2. The first sting into the ventral thorax results in a transient paralysis. During this paralysis the wasp stings the suboesophageal ganglion, which gradually results in a permanent deactivation. 3. The venom gland is a paired and highly branched organ, with a common ductus venatus. The large lumen is lined with a folded cuticula. No venom reservoir is present. 4. Extract of the venom gland induces a slow contraction of the guinea pig ileum. 5. The agonist present in the venom cannot be identified with a known agonist. 6. Venom gland extract blocks synaptic transmission from the cercal nerve to giant neurons in the sixth abdominal ganglion of the cockroach. 7. The block develops gradually, like the gradual appearance of the effects of the sting into the suboesophageal ganglion on the behaviour of the cockroach.     

啮小蜂毒液对寄主亚洲玉米螟蛹发育的影响

假寄生和毒液注射实验表明啮小蜂Tetrastichussp .毒液能永久麻痹亚洲玉米螟蛹Ostriniafurnacalis,较高剂量的毒液对亚洲玉米螟蛹有不同程度的麻痹作用。麻痹的蛹在室温下较长时间内不腐烂和干化。低剂量毒液能延长亚洲玉米螟蛹期 1～ 3d ,导致畸形成虫。啮小蜂初产卵转移寄主实验证实毒液可以帮助啮小蜂卵在亚洲玉米螟蛹体内的发育 ,啮小蜂初产卵在转移寄主体内发育到羽化时间为 2 5d ,比它们在原寄主内的羽化时间增加 8d左右 ,初产蜂卵与 1个毒液储液囊当量的毒液同时注入转移寄主体内 ,初产卵的羽化时间明显缩短 ,死亡率降低     

Comparative recombinant protein production of eight insect cell lines

Summary A recombinantAutographa californica baculovirus expressing secreted alkaline phosphatase (SEAP) gene was used to evaluate the expression of a secreted glycoprotein in eight insect cell lines derived fromSpodoptera frugiperda, Trichoplusia ni, Mamestra brassicae andEstigmene acrea. Because cell density was found to influence protein production, SEAP production was evaluated at optimal cell densities for each cell line on both a per cell and per milliliter basis. On a per cell basis, theT. ni-derived BTI-TN-5B1-4 cells produced a minimum of 20-fold more SEAP than theS. frugiperda-derived Sf9 or Sf21 cell lines and a minimum of 9-fold more than any of the other cell lines growing in serum-containing medium. On a per milliliter basis, BTI-TN-5B1-4 cells produced a minimum of fivefold more SEAP than any of the other cell lines tested. Using cell lines that were adapted to serum-free medium, SEAP yields were the same or better than their counterparts in serum-containing medium. At 3 days postinoculation, extracellular SEAP activity ranged from 59 to 85% of total SEAP activity with cell lines grown in serum-free and serum-containing media.     

Mode of action of the venom of the ectoparasitic wasp,Euplectrus comstockii, in causing developmental arrest in the European corn borer,Ostrinia nubilalis

Euplectrus comstockii Howard (Hymenoptera: Eulophidae), is an ectoparasitic, gregarious wasp which parasitizes the larval stage of several important lepidopteran pests. Parasitization of both natural and unnatural hosts prevents molting in the parasitized instar. Here we report the effect of wasp venom on the European corn borer (unnatural host), an important pest of corn and other vegetables. Venom collected from venom glands of adultE. comstockii, when injected intoO. nubilalis 5th instars, inhibited the growth rate, development and molting of the injected larvae. The observed effect on molting was dose and age dependent. When 3rd, 4th and 5th instarO. nubilalis were envenomated by adult wasps, the larvae also were developmentally arrested and failed to undergo a molt. However, 3rd and 4th instars underwent apolysis (separation of the epidermis from the old cuticle) and produced new cuticle. Fifth instars did not. A premolt hemolymph ecdysteroid peak was not observed in these experimental 5th instars, but injections of 20-hydroxy-ecdysone induced apolysis and new cuticle formation. Envenomated 4th instars (on becoming pharate 5th instars) exhibited a premolt hemolymph ecdysteroid peak. HPLC/RIA revealed that 20-hydroxyecdysone was present in the hemolymph of these pharate 5th instars. Thus, in the European corn borer, the mode of action of the venom depended upon the instar parasitized. Our results support the presence of a venom component(s) that, in 4th instar hosts, inhibited ecdysis, but did not prevent hemolymph ecdysteroid levels from increasing sufficiently to stimulate apolysis. In 5th instars, the same, or perhaps, a different component(s) ofE. comstockii venom prevented the synthesis/release of ecdysteroid by inhibiting a previously unknown molt-regulating physiological event that occurs between days 3 and 4 of the instar. Deceased     

Towards a comprehensive view of the primary structure of venom proteins from the parasitoid wasp Pimpla hypochondriaca

Venom from the parasitoid wasp Pimpla hypochondriaca has potent in vivo activity against insect haemocytes and disrupts host immune responses. Using hybridisation techniques, and more recently random sequence analysis, we had previously identified cDNAs encoding 10 venom proteins from this wasp and deduced their primary structures. We have now extended the random sequence analysis and discovered a further nine cDNAs encoding proteins with predicted signal sequences. The mature proteins were calculated to have masses of between 4 and 22 kDa. Post-signal sequence residues predicted from the cDNAs matched those derived by Edman degradation from venom proteins separated using gel filtration and reverse phase chromatography, confirming that the cloned cDNAs encode proteins which are secreted into the venom sac. Proteins containing at least six cysteine residues were abundant and seven of these cysteine-rich venom proteins, cvp1-7, were identified. The sequences of some of these proteins were similar, or contained similar cysteine arrangements, to Kunitz type protease inhibitors, pacifastin, the trypsin inhibitor domain protein family, atracotoxin and omega-conotoxin, respectively, which occur in a diverse range of animals including spiders, molluscs, humans and grasshoppers. Two small venom proteins, svp1 and svp2, as well as cvp7 did not have similar sequences to proteins in the GenBank protein database suggesting they may be highly specialised venom components. The random sequencing approach has provided a rapid means of determining the primary structure of the majority of Pimpla hypochondriaca venom proteins.     

Rho 1 participates in parasitoid wasp eggs maturation and host cellular immunity inhibition

Endoparasitoid wasps introduce venom into their host insects during the egg-laying stage. Venom proteins play various roles in the host physiology, development, immunity, and behavior manipulation and regulation. In this study, we identified a venom protein, MmRho1, a small guanine nucleotide-binding protein derived from ovary in the endoparasitoid wasp Microplitis mediator and found that knockdown of its expression by RNA interference caused down-regulation of vitellogenin and juvenile hormone, egg production, and cocoons formation in the female wasps. We demonstrated that MmRho1 entered the cotton bollworm's (host) hemocytes and suppressed cellular immune responses after parasitism using immunofluorescence staining. Furthermore, wasp MmRho1 interacted with the cotton bollworm's actin cytoskeleton rearrangement regulator diaphanous by yeast 2-hybrid and glutathione s-transferase pull-down. In conclusion, this study indicates that MmRho1 plays dual roles in wasp development and the suppression of the host insect cellular immune responses.