Cytotoxic effects of parasitism and application of venom from the endoparasitoid Pimpla turionellae on hemocytes of the host Galleria mellonella

Abstract

In parasitoid species devoid of polydnaviruses and virus‐like particles, venom appears to play a major role in suppression of host immunity. Venom from the pupal endoparasitoid Pimpla turionellae L. (Hymenoptera: Ichneumonidae) has previously been shown to contain a mixture of biologically active components, which display potent paralytic, cytotoxic, and cytolytic effects toward lepidopteran and dipteran hosts. The current study was undertaken to investigate if parasitism and/or envenomation by P. turionellae affects the frequency of apoptotic and necrotic hemocytes, hemocyte viability and mitotic indices in Galleria mellonella L. (Lepidoptera: Pyralidae) pupae and larvae. Our study indicates that parasitism and experimental envenomation of G. mellonella by P. turionellae resulted in markedly different effects on the ratio of apoptotic hemocytes circulating in hemolymph depending on the host developmental stages. The ratio of early and late apoptotic hemocytes increased in G. mellonella pupae and larvae upon parasitization and at high doses of venom when compared to untreated, null and Phosphate Buffered Saline (PBS) injected controls. In contrast, an increase in necrotic hemocytes was only observed in parasitized pupae at 24 h and no difference was observed in larvae. The lowest hemocyte viability values were observed with pupae as 69.87%, 69.80%, and 72.47% at 4, 8, and 24 h post‐parasitism. The ratio of mitotic hemocytes also decreased in pupae and larvae upon parasitization and at high doses of venom. Staining of hemocytes with annexin V‐FITC revealed green fluorescent ‘halos’ along the plasma membranes of venom treated cells within 15 min following exposure to venom. By 1 h post‐venom – treatment, the majority of hemocytes displayed binding of this probe, indicative of early stage apoptosis. These same hemocytes also displayed a loss of plasma membrane integrity at the same time points as evidenced by accumulation of propidium iodide in nuclei.

Venom-Induced Immunosuppression: An Overview of Hemocyte-Mediated Responses

“…In certain host parasitoid systems, the reduction of hemocytes in circulation was caused by cell death [27,63,[82][83][84][85]. Apoptosis and/or oncosis appear to be necessary means to manipulate the host to ensure successful development of parasitoid larvae [27,63,[82][83][84][85]. Recently, studies have been conducted to examine the ability of P. turionellae venom to induce cell death in the circulating hemocytes of the natural host G. mellonella at the larval and pupal stage [82].…”

“…Apoptosis and/or oncosis appear to be necessary means to manipulate the host to ensure successful development of parasitoid larvae [27,63,[82][83][84][85]. Recently, studies have been conducted to examine the ability of P. turionellae venom to induce cell death in the circulating hemocytes of the natural host G. mellonella at the larval and pupal stage [82]. The occurrence of apoptosis in venom-treated, parasitized, and untreated host larvae and pupae was detected using acridine orange/ethidium bromide double staining method ( Figure 2) [82].…”

“…Recently, studies have been conducted to examine the ability of P. turionellae venom to induce cell death in the circulating hemocytes of the natural host G. mellonella at the larval and pupal stage [82]. The occurrence of apoptosis in venom-treated, parasitized, and untreated host larvae and pupae was detected using acridine orange/ethidium bromide double staining method ( Figure 2) [82]. This method of detecting apoptosis is based on the loss of plasma membrane integrity as cells die [86].…”

“…Cells were identified as viable (green nucleus with red-orange cytoplasm with an intact membrane), early apoptotic (cell membrane still continuous but chromatin condensation and an irregular green nucleus are visible), late apoptotic (ethidium bromide penetrates through altered cell membrane and stains the nuclei orange, while fragmentation or condensation of chromatin is still observed), and necrotic (orange nucleus with intact structure) [86,87]. Also venom-induced apoptosis was detected using an Annexin V-FITC and propidium iodide apoptosis detection kit [82].…”

“…The kit relies on cells undergoing early apoptosis translocating membrane phosphatidylserine (PS) to the cell surface [82].…”