Function and evolution of a gene family encoding odorant binding-like proteins in a social insect, the honey bee (Apis mellifera)

Abstract

The remarkable olfactory power of insect species is thought to be generated by a combinatorial action of two large protein families, G protein-coupled olfactory receptors (ORs) and odorant binding proteins (OBPs). In olfactory sensilla, OBPs deliver hydrophobic airborne molecules to ORs, but their expression in nonolfactory tissues suggests that they also may function as general carriers in other developmental and physiological processes. Here we used bioinformatic and experimental approaches to characterize the OBP-like gene family in a highly social insect, the Western honey bee. Comparison with other insects shows that the honey bee has the smallest set of these genes, consisting of only 21 OBPs. This number stands in stark contrast to the more than 70 OBPs in Anopheles gambiae and 51 in Drosophila melanogaster. In the honey bee as in the two dipterans, these genes are organized in clusters. We show that the evolution of their structure involved frequent intron losses. We describe a monophyletic subfamily of OBPs where the diversification of some amino acids appears to have been accelerated by positive selection. Expression profiling under a wide range of conditions shows that in the honey bee only nine OBPs are antenna-specific. The remaining genes are expressed either ubiquitously or are tightly regulated in specialized tissues or during development. These findings support the view that OBPs are not restricted to olfaction and are likely to be involved in broader physiological functions.

Figure 1.

The alignment of the predicted polypeptides encoding OBPs in Apis mellifera. Conserved residues are highlighted and the signal peptides are in boxes. The rectangular shapes above the alignment represent the α-helices in AmelOBP1 secondary structure. The splice sites are labeled with separators: Vertical ones indicate splice sites between codons; backward slanted separators point out splice sites within codons after the first base.

Figure 2.

Phylogeny of the OBP protein family in Apis mellifera. An unrooted tree was constructed with aligned protein sequences from the honey bee using neighbor-joining. The OBP protein family is composed of two color-coded subgroups, the C-minus subfamily (dark gray) on chromosome 15 and OBPs clustered on chromosome 9 (light gray). The stars and squares indicate nodes with 95% and 80%–95% bootstrap supports respectively.

Figure 3.

Positive selection on the C-minus OBP family. The top panel shows the number of non-synonymous substitutions divided by the number of synonymous substitutions when this ratio is >1. Amino acids detected to be significantly under positive selection by both BEB and REL (see Methods) are in black. The bottom panel shows the residues (dots) forming either the binding pocket or its lip in four OBPs for which the secondary structures are known. Those residues that correspond to positively selected amino acids of the C-minus OBPs in the top panel are enclosed.

Figure 4.

Global expression patterns of Apis mellifera OBPs. The levels of expression are illustrated by three grades of grayscale relative to the ribosomal protein S8: Light gray indicates low level of expression defined as more than 2× background and less than half of the S8 level; dark gray indicates medium level of expression (between half and twice the level of S8), and black stands for high level of expression defined as more than 2× the level of S8. The expression of S8 was always more than 2× the background value. OBPs are shown on the y-axis and the examined tissues on the x-axis: an_fo, forager antennae; an_qu, queen antennae; an_dr, drone antennae; br_d1, newly emerged bee brain; br_fo, forager brain; cu_d1, newly emerged bee head's cuticle; cu_fo, forager head's cuticle; lg_d1, newly emerged bee legs; tx_d1, newly emerged bee thorax; tg_d6, 6-d-old bee tergites; st_d6, 6-d-old bee sternites; fb_d6, 6-d-old bee fat bodies; ov_qu, queen ovaries; eggs, eggs; lary, young larvae, stage 1 and 2; laro, old larvae, stage 5; pupy, young pupae, white-eyed; pupo, old pupae, dark-eyed.