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.
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