Effects Of Mulberry Leaf Metabolites On The Silkworm And Their Interaction With Silkworm Gustatory Receptors

The Chinese sericulture industry seeks to transition to a reduced-labor,artificial feed sericulture process.An important component of this transformation will be to modify the feeding habits of silkworm larvae.Silkworm larvae must adapt to artificial food to achieve artificial food consumption at all stages,reduce labor costs and obtain economic benefits.However,a key element of silkworm larval food acceptance is the gustatory receptor and its main mechanism is unclear.Analysis of the interaction between the gustatory receptors of silkworm and the main metabolites of mulberry leaves may reveal the molecular mechanism of silkworm feeding habits.It can also provide a basis for understanding the dependence of the silkworm on mulberry leaves,and help to improve silkworm larval development on artificial feeding.In this study,four types of mulberry leaf metabolites were extracted and their effects on the growth and development of silkworm larvae were investigated.Five silkworm gustatory receptor genes that were highly expressed in the mouth parts of larvae were screened.The function of these five gustatory receptors was studied by using Xenopus oocytes combined with a two-electrode voltage clamp system,and the interaction between silkworm gustatory receptors and mulberry leaf metabolites was explored.The main results are as follows:1.Effects of mulberry leaf metabolites on silkworm larvae growth and development.Flavonoids,alkaloids,triterpenes,and polysaccharide metabolites were extracted from mulberry leaves,and feeding experiments were carried out on third-instar larvae to observe their effects on growth and development.The four metabolites of mulberry leaves influenced silkworm growth and development.Mulberry leaf flavonoids reduced the growth rate and food utilization rate of fifth instar larvae.Alkaloids significantly improved food intake,growth rate,food utilization rate,and food conversion rate of larvae.Triterpenoids improved the growth rate,food utilization rate,and food conversion rate of the third instar larvae but reduced the growth rate and food utilization rate of fifth instar larvae.Polysaccharides significantly increased the food intake of the third instar larvae,improved the food intake,growth rate,and food conversion rate at the fifth instar larvae.The four metabolites all reduced food digestibility in fifth instar larvae.Silkworm larvae had feeding preferences for the different metabolites of mulberry leaves.We believe that it is important to understand how silkworm gustatory receptors identify the metabolites of mulberry leaves.2.Screening of silkworm gustatory receptor genes and analysis of the transmembrane domain.Five candidate gustatory receptor genes that were expressed relatively highly in the mouth parts of silkworm larvae were screened.These were Bm Gr22,Bm Gr43,Bm Gr53,Bm Gr61,and Bm Gr63.Domain analysis on the website of transmembrane domain prediction(https://dtu.biolib.com/Deep TMHMM)showed that Bm Gr22,Bm Gr53 and Bm Gr63 have seven transmembrane domains,while Bm Gr43 and Bm Gr61 have six transmembrane domains,all of which conform to the structural characteristics of the gustatory receptor family.The full-length sequences of these five candidate silkworm gustatory receptor genes were cloned and ligated into cloning vectors for functional verification.3.The functions of gustatory receptors in silkworm larvae.The full-length sequences of five candidate silkworm receptor genes were introduced into the Xenopus oocyte expression vector p T7 Ts and the corresponding c RNAs were transcribed,in vitro,to express the five silkworm gustatory receptor genes in Xenopus oocytes.The oocytes were infiltrated with mulberry leaf extract and the current changes were recorded using a two-electrode voltage clamp.Three silkworm gustatory receptors,Bm Gr43,Bm Gr53,and Bm Gr63,responded strongly to the metabolites of mulberry leaves,while the response intensity of Bm Gr22 was relatively weaker.These results indicate that there is an interaction between the gustatory receptors of the silkworm and the metabolites of the mulberry leaves during feeding and that Bm Gr43,Bm Gr53,and Bm Gr63 play important roles in this process.This result will allow advanced studies on the function of silkworm gustatory receptors.The silkworm gustatory perception mechanism will help clarify the mechanism of silkworm food acceptance and consumption and help to develop an artificial food for silkworm breeding.