Cloning And Functional Characterization Of Aquaporin Genes In Triboliem Castaneum Larvae

Aquaporins(AQPs)is an inner membrane protein located on the cell membrane.It can promote water and/or other small solutes in and out of cells in many important physiological processes.In the present study,we obtained 9 full length cDNAs encoding putative aquaporin genes using bioinformatics and gene cloning method,The critical roles of TcAQP genes in Tribolium castaneum larvae were elucidated by RNA interference.The main results were shown as fellows:1.Cloning of aquaporin(AQP)genesBy mining of T.castaneum genome and transcriptome data,nine aquaporin-like sequences were identified.According to a phylogenetic comparison with other insect aquaporins,T.castaneum aquaporin(TcAqp)sequences were designated TcBib,TcDrip,TcPrip,TcAqp12L,TcEglp1,TcEglp2,TcEglp3,TcEglp4 and TcEglp5.We compared the aquaporin genes and their corresponding cDNA sequences,and found that alternative splicing events occurred in three of the nine aquaporin genes.Among them Drip,Eglp2 and Eglp5 contained three(A,B and C),two(A and B)and four(A,B,C and D)splicing isoforms,respectively.2.Function analysis of AQP genesThe transcripts of all nine aquaporin genes were easily detectable in foregut,midgut,hindgut-Malpighian tubule complex,fat body and the carcass(except gut and fat body).TcDrip,TcPrip,TcEglpl,TcEglp3 and TcEglp5 were highly transcribed in the hindgut-Malpighian tubule complex;TcEglp2 was abundantly expressed in the fat body;TcEglp4 was greatly expressed in both the fat body and hindgut-Malpighian tubule complex;whereas the highest level of TcAqp12L was detected in the carcass.In contrast,TcBib was evenly expressed in all tested tissues.To dissect the physiological roles of the aquaporin genes,we injected a double-stranded RNA(dsRNA)derived from each of the nine aquaporin genes into fiflhinstar larvae.The mRNA levels of the target aquaporin genes were significantly reduced 3 days after injection,in contrast to the transcript levels of nontarget aquaporin genes.This indicated that injection of dsRNA successfully knocked down its target mRNA.Interestingly,knockdown of TcBib,TcDrip,TcPrip or TcApq12L respectively increased the expression level(s)of TcPrip,TcEglp2/TcEglp3,TcEglp1/TcEglp2 or TcDrip/TcEglp2.Similarly,silencing of TcEglp1,TcEglp2,TcEglp4 or TcEglp5 respectively triggered the expression of TcEglp2,TcEglp4,TcEglp1 or TcEglp2/TcEglp4.However,RNAi of TcEglp3 did not upregulate any other aquaporin genes.Whereas control larvae had a smooth and bright cuticle,from 3 to 9 days after dsEglp3,dsEglp4,dsDrip or dsPrip injection,some of the injected larvae exhibited grey cuticles.Furthermore,the hindguts were observed to protrude from the anus in approximately 40%of the TcEglp3 RNAi larvae,with their fresh wet faeces attached to the hindgut,in contrast to control beetles.Another 20%of the TcEglp3 RNAi specimens did not defaecate normally;wet brown faeces were adhered to the anal area.Similar defective phenotypes were seen in approximately 20%of TcEglp4 or TcDrip RNAi specimens.Six days after dsRNA injection,the surviving larvae were weighed.The larval fresh weights were significantly lighter in the TcEglp3,TcEglp4 and TcPrip RNAi beetles.Moreover,approximately 60,20 and 20%of TcEglp3,TcEglp4 and TcDrip RNAi larvae,respectively,failed to pupate and finally died.Nine days after dsRNA injection,the surviving pupae were weighed.The pupal fresh weights were significantly lighter in the TcEglp3,TcEglp4 and TcPrip RNAi beetles.3.Sugar metabolism in the entomoglyceroporin 3(TcEglp3)knockdown larvaeThe contents of trehalose,glucose and glycogen in the whole bodies of the TcEglp3 RNAi larvae were tested 5 days after dsRNA injection.The concentration of trehalose was increased,whereas the amounts of glucose and glycogen were reduced in the TcEglp3-depleted larvae,compared with those in controls.Moreover,four glycolysis-involved genes encoding hexokinase 2(TcHex2),phosphofructokinase(TcPFK),glyoxalase 1(TcGLO1)and lactate dehydrogenase(TcLDH)were highly transcribed in the TcEglp3 knockdown larvae.Similarly,four trehalose metabolism genes,encoding a trehalose biosynthesis enzyme trehalose-6-phosphate synthase(TcTPS)and three trehalases(TcTRE1,TcTRE2 and TcTRE3),were highly expressed in the TcEglp3-silenced larvae.