Molecular Mechanisms Of Wiebesia Pumilae Recognizing Specific VOCs Released By Ficus Pumila Var.pumila

Mutualisms between plants and pollinating insects contribute critically to stabilizing ecosystem structure and functioning.Establishment of these mutualisms predominantly relies on the accurate recognition of plants' volatile organic compounds(VOCs)by insect olfactory.Both plants' VOCs and insect olfactory are highly diverse and complex,strongly limiting the exploration of how pollinating insects recognize host plants.As a classic type of obligate pollination mutualisms,Ficus species are only pollinated by their specific pollinating wasps,whose larvae can only develop in the syconia(figs)of the host Ficus species.Host identification of pollinating wasps crucially depends on their recognition of specific VOCs released by host figs.Therefore,chemical communications play a key role in co-adaptation and coevolution in fig–pollinator mutualisms,but the molecular mechanisms underlying such specific recognition are still unclear.Uncovering these mechanisms will expand our understanding of the diversification and maintenance of insects and plants.Here,we focused on Ficus pumila var.pumila and its pollinating wasp Wiebesia pumilae.We used a combination of dynamic headspace(DHS),gas chromatography mass spectrometry(GC-MS),gas chromatography electroantennogram detection(GCEAD)and Y-tube olfactometer to identify the specific VOCs attracting W.pumilae.Then,we identified the repertoires of both odorant-binding proteins(OBPs)and odorant receptors(ORs)based on the high-quality genome of W.pumilae.The key olfactory proteins participating in the recognition of the attractants were identified and validated using a series of methods including transcriptomic and proteomic analyses,motif prediction,prokaryotic expression,and surface plasma resonance(SPR)assays.Finally,the evolution of these key olfactory genes was analyzed via testing selective pressures for orthologous genes.The main results and conclusions are as following:(1)Using GC-MS,we found that female and functionally male figs of F.pumila released 51 and 38 VOCs at the receptive stage.Among them,decanal,nonanal and linalool were proved to be active components by GC-EAD.Y-tube assays showed that decanal was the only attractive VOC.The relative content of decanal was 1.44% in receptive female figs and 1.10% in receptive functionally male figs,and was not detected in figs at pre-receptive stage.These results indicated that F.pumila figs attracted W.pumilae by decanal.(2)Using the Illumina sequencing and Pac Bio sequencing techniques,we assembled the high-quality genome for W.pumilae to pseudo-chromosomes,with the contig N50 of 10.9 Mb.Twelve OBPs of W.pumilae were identified based on BLASTP,TBLASTN,and sequence characteristics of OBPs.Wpum OBP2 was predicted to be the main decanal-binding protein by conserved motif analysis and binding affinity experiments using SPRs.Moreover,high expression of Wpum OBP2 gene and protein was found in transcriptomes and proteomes.Comparing with the orthogroups of OBP genes of W.pumilae,Ceratosolen solmsi,Eupristina verticillata,and Valisia javana,there were no huge differentiations in the OBP repertoires among four fig wasps,and Wpum OBP2 gene was not a species-specific gene.Using the selective pressure analysis,we also found that Wpum OBP2 gene of W.pumilae suffered significantly stronger selective constraints than its orthologs in the other three fig wasps,and thus Wpum OBP2 was highly conserved,preventing functional redundancy.These results indicated that Wpum OBP2 is the main OBP of W.pumilae to recognize decanal.(3)Sixty-seven ORs were identified from the genome of W.pumilae,using BLASTP and TBLASTN and according to domains of ORs.Phylogenetic and conserved motif analyses revealed that Wpum OR65 was associated with specific recognition of decanal.The phylogenetic tree also showed that Wpum OR16 was the odorant receptor co-receptor(ORco)of Wpum OR65.There were only a few OR genes with high expression in OR repertoire of W.pumilae in transcriptomes.Comparing the orthogroups of ORs gene of the four fig wasp species,we found that Wpum OR65 gene was not a species-specific gene.There were no significant differences in the selective constraints between Wpum OR65 gene and its ortholog in E.verticillata(Ever OR1),with no positive selection sites in Wpum OR65.However,clustering results showed that the OR repertoires of four fig wasps were highly differentiated,providing the molecular basis for olfactory preferences.These results demonstrated that Wpum OR65 was the main odorant receptor for the specific recognition of decanal and Wpum OR16 might be the ORco assisting this recognition.In conclusion,our results confirmed that decanal was the attractive chemical compound,with Wpum OBP2,Wpum OR16 and Wpum OR65 being the olfactory proteins in W.pumilae genome which participated in the specific recognition of decanal.Our study provides a paradigm for uncovering the molecular mechanisms of chemical communications in pollination mutualisms and novel insights into plant-insect interactions.