Identification And Phylogenetic Analyses Of Mediterranean Fruit Fly BHLH Genes And Insect ASC Genes

Basic helix-loop-helix(bHLH)genes are a group of proteins that constitute a super family of transcription factors found in eukaryotic organisms ranging from small organisms such as yeast to large ones such as human.They play a key role in the regulation of eukaryotic developmental processes including neurogenesis,sex determination,myogenesis,cell fate determination and sensing environmental signals.The name bHLH is due to the presence of a highly conserved structural motif which is made up of about 60 amino acids divided into a basic region(b)and two helices separated by an intervening loop region of varying length(HLH).Currently,bHLH proteins are divided into six major groups based on their DNAbinding specificities.Besides,they are classified into 45 families according to their roles in regulating various developmental and physiological processes.Ceratitis capitata,commonly known as the Mediterranean fruit fly,is a species of fruit fly that is vastly distributed around the world.They feed on and also reproduce in fruits,thereby causing extensive physical and economical damages to fruits.To curb the damages caused by the medfly,it is important to understand the biology and mechanism the medfly undergoes to cause damage to the fruits.The whole genome sequencing project for C.capitata was completed in September 2016.However,no study has been conducted to establish the complete bHLH gene list in Mediterranean fruit fly.Therefore,we conducted Blast searches and phylogenetic analyses to establish the repertoire of C.capitata bHLH(CcbHLH)genes.Achaete-scute complex(ASC)genes encode bHLH proteins that play an essential role in neurogenesis.Insect ASC genes are currently grouped into two distinct families,ASCa and ASCb.However,a number of identified insect ASC genes are not orthologous to known ASCb family genes from other animals such as nematode and mammal.Besides,many ASC genes have been annotated differently in GenBank(www.ncbi.nlm.nih.gov).Therefore,we conducted in silico cloning of ASC genes from 41 insect species and analyzed their evolutionary relationships with known ASC genes from other animals.Major achievements of our work in the above two aspects are as follows:1.Identification of CcbHLH proteins.59 CcbHLH genes were identified in genome of the Mediterranean fruit fly,which is the same with those of Drosophila melanogaster.Annotations to all 59 proteins encoded by CcbHLH genes are available in GenBank.However,only 21 CcbHLH proteins have been annotated in consistence with our classification.The rest 38 CcbHLH proteins are either annotated differently with our classification or merely named as hypothetical proteins.Therefore,our work provides useful information for updating annotations to 38 CcbHLH proteins.2.Special CcbHLH genes.Among the 59 Mediterranean fruit fly bHLH genes,three CcbHLH genes of the H/E(spl)family do not form orthologous clade with correspondent D.melanogaster genes,suggesting that different mechanisms could have been employed in evolution of these genes.Besides,compared to other insect species,Mediterranean fruit fly and D.melanogaster have much higher number of bHLH genes in H/E(spl)family.These two species have 11 H/E(spl)genes while other insect species have four to eight ones.3.Identification and classification of insect ASC genes.Totally 108 ASC genes were identified in 41 insect species.Phylogenetic analyses demonstrated that 14 insect ASC genes does not fall into the two existing bHLH families named ASCa and ASCb.Moreover,bHLH motif amino acids of these 14 ASC genes are obviously different with those of ASCa and ASCb family genes.Therefore,a new ASC gene family named ASCc was put forward to classify these new ASC genes,and the gene in this family is named Asl(Achaete-scute-like).The naming method can be used to correct inappropriate annotations of insect ASC genes in GenBank and to classify newly identified insect ASC genes accurately.4.Evolution of insect ASC genes.Our survey data showed that ASC gene composition and location are greatly diversified between different orders or even between different families of insects.Further phylogenetic analyses revealed that insect ASC genes have diverged greatly during evolution,and different orders/families of insects have followed different routes of evolution.Firstly,ASC genes in Hymenoptera and Hemiptera as well as Phthiraptera diverged soon after they split from the common ancestor of insects.Secondly,recent gene duplication events occurred independently in Lepidoptera and Diptera.Thirdly,ASC genes in Coleoptera and Blattodea have the same composition and location,suggesting that Coleoptera has closer genetic relationship with Blattodea than with other insect orders.Finally,Asl gene is present in specific orders/families of insects,suggesting that Asl gene loss might have occurred during evolution of insects in certain families of Hemiptera,Diptera and Blattodea.In summary,our present work would facilitate correction of inappropriate annotations to bHLH genes of Mediterranean fruit fly and classification of newly identified insect ASC genes.Furthermore,it brings better understanding on evolutionary relationships between CcbHLH genes with other insect bHLH genes and between insect ASC genes of different lineages.