ABSTRACT
De novo biosynthesis of male sex pheromone from two bumblebee species (Bombus terrestris and Bombus lucorum) was studied by using in vitro incubations of labial glands (LGs) with radioactive [1,2-(14)C]acetate and deuterated [D(3)]acetate. The labeled substrate was incorporated into several types of compounds, such as terpenic alcohols, fatty acids, esters, and hydrocarbons. A similar incubation of [1,2-(14)C]acetate with fat bodies (FB) led to the formation of fatty acids, triacylglycerols (TAG), and hydrocarbons. To support the results from in vitro incubations, PCR analysis of fatty acid synthase (FAS) transcripts in LG and FB was performed. Relative quantification of FAS transcription levels revealed that the abundance of mRNA from the FAS gene is a function of the age of B. terrestris males. A comparison of the relative FAS mRNA gene transcription level in FB and LGs of B. terrestris and B. lucorum males proved that high biosynthetic activity takes place in the LGs of both species. Together, these results indicate that pheromone components are synthesized de novo in the LG.
Subject(s)
Bees/metabolism , Sex Attractants/biosynthesis , Acetic Acid/chemistry , Acetic Acid/metabolism , Animals , Carbon Radioisotopes/chemistry , Chromatography, Thin Layer , Deuterium/chemistry , Fat Body/enzymology , Fat Body/metabolism , Fatty Acid Synthases/genetics , Fatty Acid Synthases/metabolism , Fatty Acids/biosynthesis , Fatty Acids/chemistry , Male , Mass Spectrometry , RNA, Messenger/metabolism , Salivary Glands, Minor/enzymology , Salivary Glands, Minor/metabolism , Sex Attractants/analysisABSTRACT
Quantitative real-time polymerase chain reaction (PCR) is an accurate and sensitive technique for gene expression analysis. However, it requires data normalization using reference genes. Here we assessed the stability of eight reference genes in the labial gland and fat body of the bumblebees Bombus terrestris and Bombus lucorum of different ages. To date, no reference genes have been identified for these species. Our data show that arginine kinase (AK) and phospholipase A2 (PLA2) are the most stable genes in both tissues of B. terrestris. The most stable genes for the labial gland and fat body of B. lucorum were found to be elongation factor 1alpha (EEF1A) and PLA2.
