The impacts of different doses of the plant growth regulator gibberellic acid (GA(3)) in diet on the number of total and differential hemocytes, frequency of apoptotic, and necrotic hemocytes, mitotic indices, encapsulation, and melanization responses were investigated using the greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) larvae. Total hemocyte counts increased in G. mellonella larvae at all treatment doses whereas GA(3) application had no effect on the number of different hemocyte types. The occurrence of apoptosis, necrosis and mitotic indices in GA(3) treated and untreated last instars were detected by acridine orange or ethidium bromide double staining by fluorescence microscopy. While the ratio of necrotic hemocytes increased at all GA(3) treatments, that of late apoptotic cells was only higher at doses >200 ppm when compared with untreated larvae. The percentage of mitotic index also increased at 5,000 ppm. Positively charged DEAE Sephadex A-25 beads were used for analysis of the levels of encapsulation and melanization in GA(3) treated G. mellonella larvae. At four and 24 h posttreatments with Sephadex A-25 bead injection, insects were dissected under a stereomicroscope. Encapsulation rates of larval hemocytes were dependent on the extent of encapsulation and time but not treatment groups. While the extent of melanization of hemocytes showed differences related to time, in general, a decrease was observed at all doses of GA(3) treated larvae at 24 h. We suggest that GA(3) treatment negatively affects hemocyte physiology and cell immune responses inducing cells to die by necrosis and apoptosis in G. mellonella larvae.

Venom from the pupal endoparasitoid Pimpla turionellae L. (Hymenoptera: Ichneumonidae) contains a mixture of biologically active components, which display potent paralytic, cytotoxic, and cytolytic effects toward hosts. Here, we further investigate whether parasitism or envenomation by P. turionellae alters hemocyte numbers of its host Galleria mellonella L. (Lepidoptera: Pyralidae). Total hemocyte counts declined sharply in pupae and larvae of G. mellonella exposed to P. turionellae. These same cellular responses occurred when wasp venom was artificially injected into hosts, suggesting that venom alone induces cytotoxicity in hemocytes. Analysis of the differential hemocyte counts in untreated pupae and larvae revealed that more than half of the circulating hemocytes were granular cells followed by plasmatocytes. Parasitism reduced the number of granular cells while increasing the number of plasmatocytes. This trend was most evident at 4 h postparasitism, and a similar trend was observed with the artificial injection of high (but not low) doses of venom. When isolated larval hemocytes were exposed to a LC99 dose of venom, a differential response was observed for granular cells versus plasmatocytes. Both types of cells displayed some formation of vacuoles within the cytoplasm within 15 min posttreatment. However, the degree of vacuole formation was much more extensive in granular cells at later time points than for plasmatocytes, and granular cells seemed much more susceptible to venom as evidenced by cell death.

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.

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Age‐related progeny production and sex ratio of Apanteles galleriae Wilkinson, a koinobiont, solitary, larval endoparasitoid of two lepidopteran species, Galleria mellonella (L.) and Achoria grisella Fabr., were studied at 25± 1°C, 60 ± 5% relative humidity, and a photoperiod of 12 : 12 h (L : D). The effect of host type on fecundity and sex ratio of the hyperparasitoid Dibrachys boarmiae (Walker), an idiobiont, gregarious pre‐pupae or pupae ectoparasitoid on A. galleriae was also investigated. Total progeny produced by A. galleriae showed very little host‐dependent variation. The mean total number of offspring produced by a female was 232.6 and 239.7 on G. mellonella and A. grisella, respectively. Progeny production and proportion of females per host by A. galleriae decreased directly with parental age. Fecundity of D. boarmiae on A. galleriae pupae reared on G. mellonella and A. grisella was 3.3 and 2.9 respectively. Host type had no significant influence on progeny production and sex ratio of D. boarmiae.