In vitro analysis of venom from the wasp Nasonia vitripennis: Susceptibility of different cell lines and venom-induced changes in plasma membrane permeability

Abstract

The lethal effects of crude venom prepared from the ectoparasitic wasp Nasonia vitripennis were examined with cultured cells from six insect and two vertebrate species. Venom caused cells from Sarcophaga peregrina (NIH SaPe4), Drosophila melanogaster (CRL 1963), Trichoplusia ni (TN-368 and BTI-TN-5B1-4), Spodoptera frugiperda (SF-21AE), and Lymantria dispar (IPL-Ldfbc1) to round up, swell, and eventually die. Despite similar sensitivities and overlapping LC50 values [0.0004-0.0015 venom reservoir equivalents (VRE)/microl], profound differences were noted at the onset of cytotoxicity among the six insect cell lines: over 80% of the NIH SaPe4 and SF21AE cells were nonviable within 1 h after addition of an LC99 dose of venom, whereas the other cells required a 5-10-fold longer incubation period to produce mortality approaching 100%. In contrast, cells from the grass frog, Rana pipiens (ICR-2A), and goldfish, Carassius auratus (CAR), showed little sensitivity to the venom: six venom reservoir equivalents were needed to induce 50% mortality in ICR-2A cells [50% lethal concentration (LC50) = 0.067 VRE/microl), and 9 VRE did not yield sufficient mortality in CAR cells for us to calculate an LC50. All susceptible cells showed similar responses when incubated with wasp venom: retraction of cytoplasmic extensions (when present), blebbing of the plasma membrane, swelling of the plasma and nuclear membranes, condensation of nuclear material, and eventual cell death attributed to lysis. The rate of swelling and lysis in NIH SaPe4 and BTI-TN-5B1-4 cells exposed to venom appeared to be dependent on the diffusion potential of extracellular solutes (Na+ = choline > sucrose > or = raffinose > K+), which is consistent with a colloid-osmotic lysis mechanism of cell death. When T. ni cells were cotreated with venom and the K+ channel blocker 4-aminopyridine, cell swelling and lysis increased with increasing drug concentration. In contrast, cells from S. peregrina were protected from the effects of the venom when treated in a similar manner. Addition of certain divalent cations (Zn+2 and Ca+2) to the extracellular media 1 h postvenom incubation rescued both BTI-TN-5B1-4 and NIH SaPe4 cells, suggesting that protection was gained from closure of open pores rather than prevention of pore formation. Venom from N. vitripennis displayed no hemolytic activity toward sheep erythrocytes, supporting the view that venom intoxication is not by a nondiscriminate mechanism. A possible mode of action of the venom is discussed.

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

“…As expected (Rivers et al, 1999b), when an LC 99 dose (0.01 VRE/ l) of crude venom was incubated with conXuent monolayers of BTI-TN-5B1-4 cells, the cells were very susceptible to the venom as evidenced by retraction of cytoplasmic extensions and cellular swelling that became apparent within 30 min after the introduction of venom. Ultimately, a complete loss of cell viability occurred by 24 h following venom treatment (Figs.…”

“…BTI-TN-5B1-4 cells (embryos from Trichoplusia ni) (Davies et al, 1993) (also called High Five™) were purchased from Invitrogen (San Diego, CA) and grown in TC-100 insect cell culture media (Sigma Chemical Co. St. Louis, MO) containing 10% fetal bovine serum (FBS) at 27°C as previously described (Rivers et al, 1999b).…”

“…Venom inhibits host cellular immune responses (Rivers et al, 2002a), depresses respiratory metabolism (Rivers and Denlinger, 1994b), triggers increases in lipid levels within hemolymph and fat body (Rivers and Denlinger, 1995;Rivers et al, 1998), and ultimately induces death. At the cellular level, venom induces bleb formation, cellular swelling, and condensation of nuclear material, events which lead to death attributed to cell lysis (Rivers et al, 1999b). This form of oncotic cell death (Ma et al, 2001;Chakrabarti et al, 2003) appears to rely on venom stimulation of G-protein dependent signal transduction pathways that promote mobilization of intracellular calcium from mitochondria and endoplasmic reticulum, elevations in cAMP, and cell death via an oncotic lytic mechanism (Knowles and Ellar, 1987;Rivers et al, 2002bRivers et al, , 2005.…”

Characterization and biochemical analyses of venom from the ectoparasitic waspNasonia vitripennis (Walker) (hymenoptera: Pteromalidae)

“…Similarly, incubation of BTI-TN-5B1-4 cells with fluids from venom glands and reservoirs resulted in cell death (Table 1). Prior to death, the plasma and nuclear membranes swelled, which was followed by cell lysis, a series of events previously shown for crude wasp venom (Rivers et al, 1999b). By contrast, saline, calyx fluid, and fluids from alkaline glands displayed no biological activity in vivo or in vitro (Table 1).…”

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)

“…Venom from N. vitripennis has been the focus of several recent studies (Rivers et al, 1999a(Rivers et al, ,b, 2002 that have examined the intoxication pathways associated with cell death using an in vitro approach (Rivers et al, 1999a). In cultured cells, crude venom from this ectoparasitic wasp appears to evoke cell death by a colloid osmotic lytic mecha- Fig.…”