Expression and DNA methylation of phospholipase A2 in Thai native honeybees (Hymenoptera: Apidae)

Abstract

Phospholipase A₂ (PLA2; EC 3.1.1.4) is a lipolytic enzyme and is one of the important components of the honeybee venom. Total RNA was extracted from four native honeybees in Thailand: Apis andreniformis, A. florea, A. cerana indica and A. dorsata. In each species, pupae’s whole body, house bees’ and foragers’ abdomen were used for RNA source. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed PLA2 transcript expression levels were highest in house bees in all four tested species and were also highly expressed in foragers of A. florea and A. dorsata. However, there was no detectable PLA2 transcript expression in pupae. Furthermore, the crude protein extracts were isolated from the whole bodies of black-eyed pupae and abdomen of house bees. The PLA2 activity and specific activity levels in crude protein extracts mirrored the transcript pattern, being very low in the black-eyed pupae (0.01 to 0.05 μmol min^–1 mL^–1 and 0 Units (U)/mg) and higher in the house bees (1.46 to 5.64 μmol min^–1 mL^–1 and 1.78 to 2.26 U/mg in A. andreniformis, A. cerana indica and A. dorsata but only 0.29 μmol min^–1 mL^–1 and 0.72 U/mg in A. florea). DNA methylation in PLA2, investigated by bisulfite treatment and pyrosequencing, revealed a higher percentage of DNA methylation in A. florea and A. dorsata pupae (27.5 and 12%) than in house bees (9.4 and 7.0%). On the contrary, this was not the case in A. andreniformis or A. cerana indica, where no marked differences in the net DNA methylation level were found between developmental stadia. However, the percentage of DNA methylation was studied in about 120-bp fragment corresponding to the gene body. Thus, if DNA methylation is involved in the control of PLA2 expression in all four honeybee species, it may involve methylation of other sites outside of the investigated fragment and/or more specific localized CG islands.