The Relationship Between The Stunted Gene And Lipid Metabolism And The Analysis Of The Downstream Lipase Gene Function

Previous studies have shown that Stunted is an endogenous ligand of Methuselah(Mth)in Drosophila melanogaster,which broadly regulates the development,longevity,reproduction and stress resistance.Our previous studies displayed thatKnock-down of Tcstunted by RNAi resulted in developmental arrest and defects in ecdysis,pupation and eclosion in T.castaneum.Interestingly,the weight and body length of larvae in the treatment samples are significantly lower thanthe controls.Studies have shown that overexpression of insects triglyceride lipase homologues brummer can lead to a decrease in TAG content in Drosophila melanogaste fat body and produces lean flies,which is similar to the size reduction caused by knockdown of Tcstunted in T.castaneum.These findings indicated Tcstunted may involve in T.castaneum lipid metabolism.Thus,we further explore stunted functions in lipid metabolism and the function of downstream lipase gene of stunted in development,and received the following results.Firstly,we found that knockdown of Tcstunted increased the expression levels of the two lipase genes Tcbrummer and Tclipase3 at 1.9-fold and 2.5-fold,respectively.The content of lipid in ds-stunted samples was significantly changed compared with the controls,among it,the lipid content in the fat body decreased 38.9%,while the lipid content in the hemolymph increased 22.1%.These results suggested that Tcstunted may be involved in fat mobilization.To further investigate its function in lipid content,double-gene RNAi experiments were carried on,which knockdown the Tcstunted&Tcbrummer and Tcstunted&Tclipase3.Our results showed that the lipid content in the fat body and hemolymph in ds-stunted&brummer and ds-stunted&lipase3 group was restored relative to the ds-stunted group,but there was no significant difference in lipid content compare to the control group.These results revealed that Tcstunted affects the fat mobilization by regulating the expression of Tcbrummer and Tclipase3.Interestingly,knockdown of Tcstunted declined TcDgatl expression,a key gene in TAG synthesis pathway.Therefore,we demonstrated that Tcstunted involved in lipid metabolism as well as regulated key genes expression on the TAG synthesis and mobilization pathway in T.castaneum at the transcription level,and further affecting the lipid content,leading to castaneum larval development stagnation ultimately.In addition,we further investigated these genes function during T.castaneum development.The expression patterns analysis showed that the three lipase genes were expressed in different developmental stages and tissues but displayed specific characters:TcDgat1 was highly expressed in early adult and fat body and gut;Tclipase3 was mainly expressed in early adult and gut;Tcbrummer was highly expressed in early pupae and late adult as well as in fat body and gut.RNAi treatments in larval showed that ds-Tclipase3 make larvae could enter the pupal stage,but 96.75%of the pupae could not start eclosion,and finally died in the pupal-imago transition stage in T.castaneum;ds-Tcbrummer resulted in 23.9%larvae not molting,morphologically defects in larvae,growth stagnates in larval stages,eventually dead,besides,there were 54.5%of the larvae can successfully enter the pupal stage but cannot start molting,growth stagnates in the pupa-adult stage,eventually dead;only 15.8%of the larvae can successfully enter the adult stage.RNAi treatments on early adult showed that 100%of the female was not spawning,and the male reproductive capacity was reduced by about 26.8%in ds-TcDgat1 samples.The RNAi of Tclipase3 and Tcbrummer resulted in a decrease of 23%and 58%in egg laying rate,respectively;RNAi of Tcbrummer resulting in 100%eggs could not hatch and it was female mediated,and the vitellogenin(Tcvg)expression level was significantly inhibited.After knockdwon of TcDgat1,Tclipase3 and Tcbrummer gene,TcDgat1 RNAi significantly shorten lifespan,all died in 11.79 ± 0.32 days on starvation condition in T.castaneum.The mean survival lifespan of Tclipase3 and Tcbrummer was higher than the control group,but the survival curve of Tclipase3 was consistent with the control group in response to starving stimulation,and all of them died within 24 days,while knocking down the Tcbrummer gene,all adult died on the 30th day,6 days more than the control group in T.castaneum.These results indicate that TcDgat1,Tclipase3 and Tcbrummer play an important role in the growth,longevity and reproduction in T.castaneum,in addition to lipid metabolism.