Investigation On The Interaction Between A Silkworm Gustatory Receptor BmGr63 And Flavonoid Glycosides In Mulberry

The long-term co-evolution has occurred between phytophagous insects and their host plants,and the relationship is close and complex.When insects are in search of food,they are faced with the highly complex mixtures that are present in plants.In these plant-derived mixtures,there are nutrients such as carbohydrates or amino acids,which are required for the growth and development of insects.The secondary metabolites used by plants for chemical defenses are also mixed in.Therefore,the ability to recognize compounds and make evaluation is essential for phytophagous insects.The insects are equipped with sensitive chemosensory systems(including olfactory and gustatory systems,etc.)to perceive a series of information produced by plants and the surrounding environment.And the insect gustatory systems are mainly used to identify nonvolatile compounds.When insects attempt to bite the leaves,the gustatory sensilla mainly located on mouthparts come into direct contact with plant sap released by chewing.The chemicals enter the gustatory sensilla through the single hole and contact the dendrites of the gustatory receptor neurons(GRNs)located in it.The GRN dendrites extend to the sensory opening,and the gustatory receptors(Gr)located on are then stimulated by the chemicals to produce electrical signals.The electrical signals are transmitted to the central nervous system through axons in the form of pulses.Thus,the corresponding behavior of insects will be regulated.On the basis of Gr,the feeding stimulants and feeding deterrents are perceived through gustatory sensilla.If this plant is identified as a host plant,the continuous feeding will be conducted.And from a broader perspective,throughout the insecta development,there is a general tendency for phytophagous insects to evolve toward specialists.The research argues that such a shift would be more sufficient for insects to select host plants,and it can also alleviate the pressure caused by natural enemies.The evolution of the insect nervous system and chemosensory mechanisms plays an indispensable role in the process of feeding transformation.The elucidation about the host plant recognition of phytophagous insects from the perspective of phytochemistry and molecular biology can help to deeply understand insect feeding,which in turn can provide biological evidence for beneficial insect use and pest control.The silkworm,as a representative species of Lepidoptera,has developed a strong feeding preference for mulberry leaves after a long period of domestication.And the silkworm is typically oligophagous.The silkworm-mulberry feeding mechanism has been studied as a representative in the interaction between a phytophagous insect and its host plant.To fully reveal this feeding mechanism,it requires both chemical and molecular investigations: on the one hand,it is necessary to identify and classify the chemical compounds in mulberry leaves.Then the effects of different chemicals on silkworms feeding should be explored,which provides the chemical basis for studying the silkworm-mulberry feeding mechanism.On the other hand,it is important to understand the silkworm chemosensory system from the molecular level,and identify chemosensory receptors including Grs.Further identification of the interaction between silkworm Grs and corresponding chemical ligands in mulberry leaves should be conducted.The related studies not only help to elucidate the silkworm-mulberry feeding mechanism from the molecular level,but also provide reference for investigation about the relationship establishment and interaction between phytophagous insects and their host plants.Following experiments would be conducted in the paper:(1)The coding sequences of Bm Grs expressed in silkworm larval mouthparts were cloned.And then both prediction tools HMMTOP and TOPCONS were used to analyze the transmembrane domains and N-terminus direction of cloned Bm Grs.The phylogenetic tree using Gr protein sequences from Lepidoptera was constructed to evaluate their evolutionary relationships.q RT-PCR was conducted to determine relative expression levels of the Bm Grs in 4th and 5th larval mouthparts.The highly expressed Bm Gr was selected for further function investigation.For this selected Bm Gr,the cellular localization experiments and transmembrane structures were performed to verify whether it matches the structural characteristics of Grs.q RT-PCR was used to depict its tissue expression profiles.(2)The highly expressed Bm Gr was ectopically expressed in oocytes.Combining with two-electrode clamp voltage experiments,the currents induced by mulberry extracts and 20 metabolites were recorded.The ligands were initially screened.(3)The RNAi was adopted and used in feeding assays,the interaction between highly expressed Bm Grs and ligands was demonstrated in vivo.The main results obtained in this research are as follows:1.The c DNA of 5th larval mouthparts by reverse transcription was used as templates to get accurate coding sequences of Bm Grs.And the full-length coding sequences of 36 Bm Grs were cloned.The cloned protein sequences were submitted to two prediction websites(HMMTOP and TOPCONS)for structural analysis.It was shown that the transmembrane domains of these Bm Grs were ranged from five to nine.The intracellular N-terminus or extracellular N-terminus was observed.The phylogenetic tree of Gr from five Lepidoptera(including Bombyx mori,Manduca sexta,Heliconius melpomene,Plutella xylostella,and Pieris rapae)revealed that the Gr proteins were divided into four clades as the CO2 clade,fructose/ inositol clade,sugar clade,and bitter clade(function remained unknown).Bm Gr1–3 were clustered in the CO2 clade,Bm Gr4–8 belonged to sugar clade and Bm Gr9–10 were in the fructose/ inositol clade.The remaining Bm Grs were divided into bitter clades with uncharacterized functions.In the constructed evolutionary tree,bitter receptors constitute the largest clade,and most Grs diverged later than the divergence of Lepidoptera species.q RT-PCR was performed to investigate relative expression levels of Bm Grs in 4th and 5th silkworm larval mouthparts.The results indicated that Bm Gr63 was highly expressed in both larval mouthparts.Then the Bm Gr63 was selected as the candidate receptor for further functional characterization.According to the prediction results of HMMTOP,there was 7 transmembrane domains in Bm Gr63 and the N-terminus was intracellular.The recombinant plasmids of Bm Gr63-m Scarlet were transfected into HEK293 T cells,and it was localized on membranes.These results were consistent with Grs functioning as membrane proteins.The c DNA of 5th larval tissues by reverse transcription was used as templates to depict the tissue distribution.The results indicated that the highest expression levels of Bm Gr63 were observed in the labrum.Bm Gr63 also expressed at relatively higher levels in mandible,labium,maxilla,and thoracic leg and was repressed in midgut.2.The identification of ligands is an important part of the functional validation of Grs.Bm Gr63 was taken for this study,and the oocyte expression system and two-electrode voltage clamp were used.Firstly,the mulberry extracts were used as stimulants to record the induced current of oocytes expressing Bm Gr63.It showed that Bm Gr63 could interact with different concentrations of mulberry extracts in a dose-dependent manner.Further,the voltage clamp recording was used to clarify which classes of mulberry metabolites could interact as ligands with Bm Gr63.Twenty tested metabolites,included mulberry sugars,flavonoids,alkaloids,amino acids,phenylpropanoids,and plant hormones.The inward currents of oocytes expressing Bm Gr63 were recorded by different metabolites.The results indicated that Bm Gr63 could interact with two flavonoid glycosides,and a strong inward current induced by isoquercetin was observed.The structural analog astragalin induced a weaker response while the response to rutin was very weak.After then,the extracts and the standard solution were analyzed using an UPLC-MS/MS system to identify their secondary structures.Isoquercetin was detected in mulberry extracts using the standard as a reference,and the concentration of isoquercetin in the mulberry extracts used in this experiment was determined as 1.437×10-3 M.According to the metabolite database of mulberry leaves,the content of isoquercetin was determined in 91 mulberry resources and differences in isoquercetin content were shown among these mulberry resources.The resources with higher isoquercetin content were mainly cultivated resources.In contrast,wild resources accounted for the majority of resources with lower isoquercetin content.3.To study the stimulating or deterrent effects of isoquercetin on silkworm feeding,diet assays were conducted.The food intake increased as 10-4 M,5×10-4 M,and 10-3 M isoquercetin was supplemented in the artificial diets(the p values were 0.573,0.060,and 0.023).It was demonstrated that mulberry isoquerccetin was silkworm feeding stimulants.In addition,it was necessary to confirm whether Bm Gr63 was involved in the recognition to isoquercetin in vivo.In Bm Gr63 RNAi lines,the no way choice and dual choice feeding assays were conducted.It was shown that,in Bm Gr63 knock-down lines,the feeding preference to isoquercetin in silkworms was weakened.We then investigated whether Bm Gr63 affected food intake.The preliminary feeding assays were conducted.Knock-down of Bm Gr63 significantly decreased the food consumed by silkworm larvae,which indicated the function of Bm Gr63 in identification to host plants.The above results indicated that the Bm Gr63 with high expression levels in silkworm 4th and 5th larval mouthparts could interact with mulberry flavonoid glycosides.The induced response by isoquercetin was relatively strong.Combined with experiments in vivo,mulberry isoquercetin function as feeding stimulant.And the Bm Gr63 was involved in the recognition to isoquercetin in vivo.In the Bm Gr63 knock-down lines,the food consumed by silkworm larvae was significantly down-regulated.It was indicated that the Gr from bitter clade could be activated by plant feeding stimulants,and the biological function of bitter Gr in identification to host plants.This report should help to explain the silkworm-mulberry feeding mechanism from molecular levels and facilitate understanding of the specific evolutionary relationships between phytophagous insects and their host plants.