Expression and evolutionary divergence of the non-conventional olfactory receptor in four species of fig wasp associated with one species of fig

Abstract

Background: The interactions of fig wasps and their host figs provide a model for investigating co-evolution. Fig wasps have specialized morphological characters and lifestyles thought to be adaptations to living in the fig's syconium. Although these aspects of natural history are well documented, the genetic mechanism(s) underlying these changes remain(s) unknown. Fig wasp olfaction is the key to host-specificity. The Or83b gene class, an unusual member of olfactory receptor family, plays a critical role in enabling the function of conventional olfactory receptors. Four Or83b orthologous genes from one pollinator (PFW) (Ceratosolen solmsi) and three non-pollinator fig wasps (NPFWs) (Apocrypta bakeri, Philotrypesis pilosa and Philotrypesis sp.) associated with one species of fig (Ficus hispida) can be used to better understand the molecular mechanism underlying the fig wasp's adaptation to its host. We made a comparison of spatial tissue-specific expression patterns and substitution rates of one orthologous gene in these fig wasps and sought evidence for selection pressures.

Results: A newly identified Or83b orthologous gene was named Or2. Expressions of Or2 were restricted to the heads of all wingless male fig wasps, which usually live in the dark cavity of a fig throughout their life cycle. However, expressions were widely detected in the antennae, legs and abdomens of all female fig wasps that fly from one fig to another for oviposition, and secondarily pollination. Weak expression was also observed in the thorax of PFWs. Compared with NPFWs, the Or2 gene in C. solmsi had an elevated rate of substitutions and lower codon usage. Analyses using Tajima's D, Fu and Li's D* and F* tests indicated a non-neutral pattern of nucleotide variation in all fig wasps. Unlike in NPFWs, this non-neutral pattern was also observed for synonymous sites of Or2 within PFWs.

Conclusion: The sex- and species-specific expression patterns of Or2 genes detected beyond the known primary olfactory tissues indicates the location of cryptic olfactory inputs. The specialized ecological niche of these wasps explains the unique habits and adaptive evolution of Or2 genes. The Or2 gene in C. solmsi is evolving very rapidly. Negative deviation from the neutral model of evolution reflects possible selection pressures acting on Or2 sequences of fig wasp, particularly on PFWs who are more host-specific to figs.

Figure 1

Alignment of Or83b orthologous amino acids in insects. Conservation of amino acid sequence is displayed as a sequence logo. The relative frequency with which an amino acid appears at a given position is reflected by the height of its one-letter amino acid code, with the total height at a given position proportional to the level of sequence conservation. Transmembrane domains (TM 1–7) and extracellular (EC) and intracellular domains (IC) are numbered and indicated. Threonine (T) and tyrosine (Y) residues that are sites of potential phosphorylation are enclosed in heavy yellow boxes.

Figure 2

The maximum parsimony tree of Or2 genes from fig wasps. The MP phylogram was constructed based on 1st + 2nd codon positions. The reliability of each tree node was assessed by bootstrap proportions with 1000 replications. Branch lengths are proportional to change.

Figure 3

Olfactory tissue-specific expression of Or2 genes in fig wasps. RT-PCR was performed with cDNAs prepared from different species and tissues of fig wasp. Reaction products were visualized by ethidium bromide staining and UV illumination. Bands were the expected size based on the primer design. A no-template negative control ensured the specificity of the amplification. Actin was amplified from each sample as a control for RNA integrity. CsmOr2, Ceratosolen solmsi Or2; AbOr2, Apocrypta bakeri Or2; PpOr2, Philotrypesis pilosa Or2; PsOr2, Philotrypesis sp. Or2; A, antenna; T, thorax; L, leg; Abd, abdomen; H, head; Neg, negative control.