Publication details

Nitric oxide production by hemocytes of larva and pharate prepupa of Galleria mellonella in response to bacterial lipopolysacharide: cytoprotective or cytotoxic?


KRISHNAN Natraj HYRŠL Pavel ŠIMEK Vladimír

Year of publication 2006
Type Article in Periodical
Magazine / Source Comparative Biochemistry and Physiology, Part C
MU Faculty or unit

Faculty of Science

Description Nitric oxide production by the hemocytes of the last instar larvae and sessile pharate prepupa of Galleria mellonella (Lepidoptera: Pyralidae) was demonstrated and compared in response to preparations of bacterial lipopolysaccharide (LPS) from Escherichia coli using the Griess reaction. Augmented, dose dependent nitric oxide production was observed in the pharate prepupal hemocytes compared to larval hemocytes. This was reversed partially in a dose dependent manner with the use of the specific inhibitor S-methyl thiourea (SMT) of inducible nitric oxide synthase (iNOS). A decrease in NO production was also observed when non-selective inhibitors such as NG Nitro-L-arginine (L-NAME) and N-ů-Nitro L-arginine (L-NNA) were used, albeit, the inhibition was not to the extent of SMT. Use of the entomopathogenic bacterium Photorhabdus asymbiotica also resulted in enhanced NO production by hemocytes of both stages. Use of SMT, the specific inhibitor of iNOS, alone or in combination with P. asymbiotica resulted in significantly decreased levels of NO production. However, it was observed that phenoloxidase activity (a cascade for innate immune responses) in both cases was independent of NO production stimulation. Use of NO donors such as S-nitroso-N-acetyl-penicillamine (SNAP) and Diethylenetriamine NO adduct (DETA/NO) at various concentrations (100 to 500ľM) resulted in the lysis of hemocytes dose dependently. The nitrite production in these cases was however similar to LPS stimulation (10 ľg/ml) and 1.5 3 fold lower than those observed upon P. asymbiotica (2.5 x 107 cfu/ml) stimulation. Survival analysis (Kaplan-Meier) was done following injection of P. asymbiotica alone or in combination with SMT which revealed that only 12.5% of co-injected larvae of G. mellonella survived in comparison to 28.6% survivors in P. asymbiotica alone injected groups till the end of the study. In contrast, in pharate prepupa the co-injected lots survived longer (median survival 28 hours) than the P. asymbiotica alone injected individuals (median survival 24 hours), however, both co-injected and P. asymbiotica injected groups showed 100% mortality at the end of the study. Based on the above we propose that NO production is part of a defense arsenal in the hemocytes of this insect and does signal innate immune responses in both stages, but, probably the extended NO production /overproduction by pharate prepupal hemocytes could result in cytotoxic rather than cytoprotective effects compared to larval hemocytes.

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