Characterization of Venom and Oviduct Components of Parasitoid Wasp Asobara japonica

Abstract

During natural parasitization, Asobara japonica wasps introduce lateral oviduct (LO) components into their Drosophila hosts soon after the venom injection to neutralize its strong toxicity; otherwise, the host will die. Although the orchestrated relationship between the venom and LO components necessary for successful parasitism has attracted the attention of many researchers in this field, the molecular natures of both factors remain ambiguous. We here showed that precipitation of the venom components by ultracentrifugation yielded a toxic fraction that was inactivated by ultraviolet light irradiation, boiling, and sonication, suggesting that it is a virus-like entity. Morphological observation of the precipitate after ultracentrifugation showed small spherical heterogeneous virus-like particles 20-40 nm in diameter. The venom's detrimental effect on D. melanogaster larvae was not directly neutralized by the LO components but blocked by a hemolymphal neutralizing factor activated by the LO factor. Furthermore, we found that A. japonica venom and LO components acted similarly on the larvae of the common cutworm Spodoptera litura: the venom injection caused mortality but coinjection of the LO factor protected S. litura larvae from the venom's toxicity. In contrast, D. ficusphila and D. bipectinata, which are closely related to D. melanogaster but non-habitual host species of A. japonica, were not negatively affected by A. japonica venom due to an intrinsic neutralizing activity in their hemolymph, indicating that these species must have acquired a neutralizer of A. japonica venom during evolution. These results give new insights into the characteristics of both the venom and LO components: A. japonica females have utilized the virus-like toxic venom factor to exploit a wider range of host species after the evolutionary process enabled them to use the LO factor for activation of the host hemolymph neutralizer precursor, although the non-habitual host Drosophila species possess an active intrinsic neutralizer in their hemolymph.

Fig 1. Survival rates of Drosophila melanogaster larvae one day after injection of indicated samples.

(A) PBS, venom (V), venom plus lateral oviduct (V+LO), venom irradiated with UV for 10 min (UV-V10), venom irradiated with UV for 3 min (UV-V3), venom plus 10 μg/ml psoralen irradiated with UV for 3 min (pUV-V3), venom sonicated for 5 min (sonicV), venom boiled for 5 min (boilV), trypsinized venom (trpsV), DNase-treated venom (DNaseV), Benzonase-treated venom (BenzV), or RNase-treated venom (RNaseV) was injected. Each value represents the mean ± S.D. for six independent experiments performed separately. Significant differences are indicated by Tukey’s HSD (**P<0.01). (B) PBS, venom (V), pellet (pelV) or supernatant (supV) after centrifugation of venom at 120,000 g or 450,000 g was injected. Other explanations are as in (A).

Fig 2. Survival rates of Drosophila melanogaster larvae one day after injection of PBS, venom (V), venom plus non-immunized IgG (V+ctlIgG), or venom plus anti-venom pellet (after centrifugation) IgG (V+VpelIgG).

Venom components were treated with IgG for 3 h before injection into test D. melanogaster larvae. Each value represents the mean ± S.D. for five independent experiments performed separately. Significant differences are indicated by Tukey’s HSD (**P<0.01).

Fig 3. Transmission electron micrographs of ultracentrifuged precipitate of venom.

A. japonica venom was centrifuged at 450,000 g for 1 h, and the pellet fraction was observed by transmission electron microscopy after negative-staining with aqueous phosphotungstic acid. Scale bar indicates 100 nm.

Fig 4. Survival rates of Drosophila melanogaster larvae one day after injection of indicated samples.

(A) PBS, venom (V), or venom plus lateral oviduct (V+LO) was injected at indicated intervals before or after injection of venom. Survival rates of test larvae injected the LO factor 2 h after the venom injection: 12.6 ± 9.5% at 48 h, 8.5 ± 5.4% at 72 h, 0% at 96 h. Each value represents the mean ± S.D. for seven independent experiments performed separately. Significant differences are indicated by Tukey’s HSD (*P<0.05, **P<0.01). (B) PBS, venom (V), venom pretreated with lateral oviduct (LO-V), venom plus lateral oviduct (V+LO), or hemolymph of D. melanogaster larvae that had been pretreated with PBS (PBS-Hl) or lateral oviduct (LO-Hl) was injected. Each value represents the mean ± S.D. for six independent experiments performed separately. Significant differences are indicated by Tukey’s HSD (**P<0.01).

Fig 5. Effects of A. japonica venom and LO components on survival rates and growth of Spodoptera litura larvae.

(A) Life span curves of S. litura larvae after injection of PBS, venom (V), or venom plus lateral oviduct (V+LO). The amount injected was increased with the increasing ratio of S. litura larva weight to Drosophila larva weight. Third instar larvae of S. litura (body weight: 1.50 ± 0.10 mg) were used for this experiment. Each value represents the mean ± S.D. for ten independent experiments performed separately. Significant differences compared to the value at 0 day are indicated by Tukey’s HSD (**P<0.01). (B) Body weights of surviving S. litura larvae one day after injection of PBS, venom (V), or venom plus lateral oviduct (V+LO). Other explanations are as in (A).

Fig 6. Survival rates of Drosophila melanogaster (D.m.), Drosophila bipectinata (D.b.), and Drosophila ficusphila (D.f.) larvae one day after injection of A. japonica venom.

Each value represents the mean ± S.D. for eight independent experiments performed separately. Significant differences are indicated by Tukey’s HSD (**P<0.01).

Fig 7. Survival rates (A) and hemolymph protease activities (B) of Drosophila melanogaster one day after injection of indicated samples. PBS, venom (V), or venom plus larval hemolymph of D. melanogaster (V+D.m.Hl), D. bipectinata (V+D.b.Hl), or D. ficusphila (V+D.f.Hl).

Survival rates of test larvae injected the venom factor together with D. ficusphila hemolymph: 49.3 ± 10.6% at 48 h, 48.6 ± 11.1% at 72 h, 46.9 ± 11.5% at 96 h. Each value represents the mean ± S.D. for eight independent experiments performed separately. Significant differences are indicated by Tukey’s HSD (*P<0.05, **P<0.01).