Molecular cloning and expression of a hexamerin cDNA from the honey bee, Apis mellifera.

A cDNA encoding a hexamerin subunit of the Africanized honey bee (Apis mellifera) was isolated and completely sequenced. In the deduced translation product we identified the N-terminal sequence typical of the honey bee HEX 70b hexamerin. The genomic sequence consists of seven exons flanked by GT/AT exon/intron splicing sites, which encode a 683 amino acid polypeptide with an estimated molecular mass of 79.5 kDa, and pI value of 6.72. Semi-quantitative RT-PCR revealed high levels of Hex 70b message in larval stages, followed by an abrupt decrease during prepupal-pupal transition. This coincides with decaying titers of juvenile hormone (JH) and ecdysteroids that is the signal for the metamorphic molt. To verify whether the high Hex 70b expression is dependent on high hormone levels, we treated 5th instar larvae with JH or 20-hydroxyecdysone (20E). In treated larvae, Hex 70b expression was maintained at high levels for a prolonged period of time than in the respective controls, thus indicating a positive hormone regulation at the transcriptional level. Experiments designed to verify the influence of the diet on Hex 70b expression showed similar transcript amounts in adult workers fed on a protein-enriched diet or fed exclusively on sugar. However, sugar-fed workers responded to the lack of dietary proteins by diminishing significantly the amount of HEX 70b subunits in hemolymph. Apparently, they use HEX 70b to compensate the lack of dietary proteins.

Characterization and expression of the Hex 110 gene encoding a glutamine-rich hexamerin in the honey bee, Apis mellifera.

An N-terminal amino acid sequence of a previously reported honey bee hexamerin, HEX 110 [Danty et al., Insect Biochem Mol Biol 28:387-397 (1998)], was used as reference to identify the predicted genomic sequence in a public GenBank database. In silico analysis revealed an ORF of 3,033 nucleotides that encompasses eight exons. The conceptual translation product is a glutamine-rich polypeptide with a predicted molecular mass of 112.2 kDa and pI of 6.43, which contains the conserved M and C hemocyanin domains. Semiquantitative and quantitative RT-PCR with specific primers allowed for an analysis of mRNA levels during worker bee development and under different physiological conditions. Concomitantly, the abundance of the respective polypeptide in the hemolymph was examined by SDS-PAGE. Hex 110 transcripts were found in high levels during the larval stages, then decreased gradually during the pupal stage, and increased again in adults. HEX 110 subunits were highly abundant in larval hemolymph, decreased at the spinning-stage, and remained at low levels in pupae and adults. In 5th instar larvae, neither starvation nor supplementation of larval food with royal jelly changed the Hex 110 transcript levels or the amounts of HEX 110 subunit in hemolymph. In adult workers, high levels of Hex 110 mRNA, but not of the respective subunit, were related to ovary activation, and also to the consumption of a pollen-rich diet.