The Early Zygotic Genes And Dosage Compensation In Anopheles Stephensi

Background:The genus Anopheles includes dozens of mosquito species that are important vectors of malaria,one of humankind's deadliest and costliest diseases.Anopheles Stephensi,the Asian malaria mosquito,is a major urban malaria vector in the Middle East and on the Indian subcontinent.Mosquito control contributed significantly to the recent decrease in malaria incidence and mortality.However,insecticide-resistance is widely reported in mosquito populations in malaria-endemic areas of Africa and India leading to considerable interest in developing novel genetic control strategies and techniques that specifically target malaria vectors,including An.stephensi.We are interested in the early embryonic stage when the maternal-to-zygotic transition,as known as embryonic genome activation,occurs in An.stephensi.The maternal-to-zygotic transition includes the syncytial blastoderm and early cellular blastoderm stages,during which the developing embryo is more accessible to genetic,because cell division hasn't begun yet.In other words,the stage from the syncytial blastoderm to early cellular blastoderm is not only important to embryonic development,but also effective to apply novel genetic techniques and strategies.The maternal-to-zygotic transition stage is not only of fundamental importance in embryonic development,but also represents a stage where genetic manipulation could lead to novel mosquito control strategies.In metazoan species,prior to the MZT,the newly formed zygote is transcriptionally inactive and its biological activities are controlled by maternally-deposited RNAs and proteins.During the MZT,transcription of the first set of genes occurs in the nascent zygote while maternally-deposited RNAs and proteins are degraded.The study on the pure early zygotic genes in mosquitos could provide a novel insight into the prevention and control of mosquito-borne infectious diseases.Dosage compensation exists in An.stephensi,who has the X/Y sex determination system.Dosage compensation refers to a mechanism that gene expression on the Xchromosome has been up-regulated in order to keep dosage balance in males,although there is only one copy of the X chromosome but two copies of autosomes.Dosage compensation requires three critical components:sex specificity,the X chromosome specificity,and chromosome-wide up-regulation of transcription.The female-specific sex-lethal(sxl)gene,a primary signal and master switch of sex determination,regulates dosage compensation in Drosophila melanogaster.Loss of function sxl mutants cause female embryonic lethality in D.melanogaster,most likely due to mis-regulation of dosage compensation,thereby up-regulating or over-expressing the X chromosome genes in females.In other model species Bombyx mori and Caenorhabdilis elegans,the master switches of sex-determination Fem/Masc and xo-lethal l,also regulate dosage compensation,respectively,and loss of function of these genes results in sex-specific lethality.The Guyl gene also has been found female-specific lethality.All results mentioned above suggest that mis-regulation of dosage compensation could result in sex-specific lethality,the stability and penetrance of the female-specific lethality conferred by the Guyl transgene suggests that dosage compensation may be explored to develop novel strategies to control mosquito-borne diseases.Objectives:Our goal is to identify pure early zygotic genes and search their upstream motifs in An.stephensi by analyzing RNA sequencing data from four early embryonic time points,provide a novel insight into the prevention and control of mosquito-borne infectious diseases.Our other goal is to establish directly link an embryonic signal expressed from a mosquito Y chromosome to the regulation of dosage compensation and support a mechanism of hyperexpression of X chromosome genes.Moreover,the stability and penetrance of the female-specific lethality conferred by the Guyl transgene suggests that dosage compensation may be explored to develop novel strategies to control mosquito-borne diseases.Methods:We obtained RNA sequencing data in biological triplicates from early An.stephensi embryos at time points 0-1 hr,2-4 hr,4-8 hr and 8-12 hr after egg laying.The RNA sequencing datasets were aligned to An.stephensi genome separately,and differential expressed genes were obtained.We identified two sets of pure early zygotic genes under stringent and relaxed conditions separately,and searched their upstream motifs.To predict their functions,we enriched their gene ontology terms and InterPro annotation names against all genes in An.stephensi.We also compared the gene length and the number of introns between the set of pure early zygotic genes and other An.stephensi genes.The homologs of these pure early zygotic genes in other 12 species have been retrieved from the OrthoDB database,and then the phylogeny of homologous genes was analyzed.We performed a cross to Guyl transgenic males and wild type females to obtain Guyl transgenic females.In Experiment A,we collected transgenic female,wild type female,transgenic male and wild type male progenies,and each genotype group includes 3 biological replicates.In Experiment B,we collected transgenic female and wild type female progenies,and each genotype group includes 4 biological replicates.RNA sequencing datasets from samples above were aligned to An.stephensi genome separately.We used FPKMs as expression level of genes.Wilcoxon rank sum tests were performed to verify whether the median gene expression level of X chromosome genes is different from that of autosomes.In the meantime,we identified differential expressed genes comparing transgenic group to wild type group,and performed Chi square tests to verify whether the ratio of up-regulated to down-regulated X chromosome genes is significantly greater than that of autosomal genes.Furthermore,we looked into the function and distribution of those common up-regulated X chromosome genes in two experiments.Results:Using RNA sequencing data,we identified two sets of pure early zygotic genes under stringent and relaxed conditions,respectively.A GT-rich motif was found in the upstream sequences of the pure early zygotic genes.We show that these pure early zygotic genes are enriched in functional groups related to DNA-binding transcription regulators,cell cycle modulators,proteases,transport,and cellular metabolism.On average these pure early zygotic genes are shorter and have less introns than other An.stephensi genes,which could be activated and transcribed fast during embryonic development stage.There is no or very limited overlap between An.stephensi pure early zygotic genes and D.melanogaster or Aedes aegypti pure early zygotic genes.We also looked into the phylogeny of these pure early zygotic genes and their homologs in other species.Genotyping of 1st instar larvae from transgenic Guyl-expresssing lines indicates late transgenic female hatching and lethality.Only a small percentage of transgenic females are observed as L1 larvae but die shortly after hatching.The median level gene expression of X-linked genes is significantly higher than autosomal genes in Guyl transgenic females while there is no significant difference in median gene expression level between X chromosome and autosomal genes in wild type females.Furthermore,Guyl significantly up-regulates at least 40%of the 996 genes across the X chromosome in transgenic females.382 genes were found in common between the two data sets and they encompass a broad functional range according to gene ontology analysis.These 382 genes represent approximately 40%of the genes on the X chromosome and they are broadly distributed across the X chromosome.Conclusions:The functions of these pure early zygotic genes are related to DNA-binding transcription regulators,cell cycle modulators,proteases,transport,and cellular metabolism.The pure early zygotic genes appear to rapidly turn over within the Dipteran order and even within the Culicidae family.The GT-rich motif could potentially regulate An.stephensi early zygotic genes.We directly link an embryonic signal expressed from a mosquito Y chromosome to the regulation of dosage compensation and support a mechanism that mis-regulation of dosage compensation results in the stable and penetrant female specific lethality,which suggests that dosage compensation may be explored to develop novel strategies to control mosquito-borne diseases.