Tmc Gene Modulates Egg Laying Behavior Of Brown Planthopper Nilaparvata Lugens And Fruit Fly Drosophila Melanogaster

Transmembrane channel like(TMC)proteins are a novel family of channel-like proteins conserved from Drosophila melanogaster to human.Eight TMC proteins are expressed in vertebrates.Mutations in TMC1 and TMC2 cause deafness and vestibular defects in human and mouse.In invertebrates,TMC protein is critical for detecting food texture and regulating larval locomotion in Drosophila.Our previous studies have showed that sweet neurons inhibit texture discrimination by signaling TMC expressing mechanosensitive neurons in Drosophila.In a recent study,TMC acts in neuron and muscle to promote egg laying and modulates membrane excitability through a background leak conductance.However,whether the tmc gene modulates egg-laying behavior of brown planthopper and fruit fly is still unknown.In this study,we used genetic,behavioral and immunohistochemistry method to illustrate the modulation of tmc gene on egg-laying behavior of brown planthopper and fruit fly.The main results were showed below.1.Cloning and sequence analysis of the TMC gene family of Nilaparvata lugensTMC proteins of individual species can be grouped into three subfamilies A(tmc1,tmc2,and tmc3),B(tmc5 and tmc6),and C(tmc4,tmc7 and tmc8).Different insect species have various tmc transcripts.The Drosophila genome encodes a single tmc homolog.We identified three tmc genes using RT-PCR method from genome and transcriptome database of Nilaparvata lugens.We named them as Nltmc3,Nltmc5 and Nltmc7,according to similar homolog genes of mammals.Sequence analysis indicated that three tmc gene of N.lugens belongs to three subfamilies,respectively.All of them are strongly predicted to encode proteins with 6 to 10 transmembrane domains and a conserved 120-amino-acid sequence that we termed the TMC domain.Among of them Nltmc3 gene was most similar with Drosophila and C.elegans tmc gene.2.Expression profiles of tmc genes in the brown planthopper,Nilaparvata lugensThe expression profiles of tmc genes have been documented in different developmental stages and multiple tissue types of females in the brown planthopper using qRT-PCR method.The results showed that Nltmc3 was relatively higher in old instar nymph stage than in other stages.Interestingly,Nltmc3 was highly expressed in the female reproductive regions particularly in the oviduct that known to be involved in egg-laying behavior.Nltmc5 was relatively higher in macropterous adult female and male than in other developmental stages.Meanwhile,this gene also showed highly expressed in the female reproductive regions particularly in the ovary Nltmc7 was relatively higher in nymphs,brachypterous adult female and male than in other developmental stages.However,this gene showed highly expressed in the male reproductive regions and female copulatory pouch.3.Silencing of Nltmc3 gene leads to defects of egg-laying behavior in the brown planthopperWe performed RNAi injection experiment to knock down three tmc genes of brown planthopper,respectively.Silencing these three tmc genes did not negatively affect the survival of brown planthopper.tmc has been reported involved in the egglaying behavior of C.elegans.We demonstrated that interference of Nltmc3 gene had a strong impact on the egg-laying behavior of female brown planthopper.We observed that knockdown of Nltmc3 impaired ovary development.The amount of eggs in the ovary of dsNltmc3 inj ection is also lower than dsgfp injection.We detected the N1TMC3 immunoreactivity in oviduct of brown planthopper using the developed antibody of N1TMC3.4.tmc modulates egg-laying behavior of DrosophilaWe also investigated the role of tmc in the regulation of egg-laying behavior of model insect Drosophila melanogaster.At first,we knock-in the LexA gene at the start coden of Drosophila tmc gene using CRISPR/Cas9 technology.We got the tmcLexA mutant line.Using this and other mutant lines,we observed the fecundity of Drosophila after TMC gene mutation was significantly lower than that of the control group.We reintroduced the tmc gene back to the tmc1 mutants(by using a tmc-GAL4 line).We found that this manipulation reverted tmc1 mutants to behave more like wildtype animals which indicated that tmc gene plays important role in the egg-laying behavior of Drosophila.Using GAL4/UAS system,we silenced the tmc-GAL4 labeled cells that impairs egg-laying of Drosophila.However,Jilencing of labeled cells have no such effects.Importantly,silencing these tmc-GAL4-expressing neurons in the presence of elav-GAL80,a transgene that blocked GAL4-dependent expression in CNS neurons labeled by tmc-GAL4,still caused animals to show an egg-laying defects.However,activation of tmc-GAL4 labeled cells using theromgenetic method reduced female’s egg-laying rate and made the fly’s abdomen floating.We observed two TMCexpressing cells located on the oviduct.It is still need to be investigated whether them involved in the egg-laying behavior of Drosophila.