The Role Of MicroRNA In Development And Susceptibility To Plasmodium Parasite In Chinese Main Malaria Vector

Anopheles dirus and Anopheles sinensis are the important malaria vectors in China.An.sinensis is still considered as the primary vector of P.vivax malaria due to its wide distribution and high density.An.dirus is an important vector in mountainous areas,and historically an important vector of falciparum malaria.microRNAs(miRNA)with its size about 22 nt,is a regulatory non-coding small RNA molecules.It is not only exists in animals and plants,but also in viruses,bacteria,fungi.It can target 3 ’end of mRNA and result in inhibition mRNA degradation or translation,regulating the expression of genes from the transcription level.It is reported to play critical role in embryonic development,cell proliferation,apoptosis,cell differentiation,metabolism,immunity and many other aspects.The role of miRNAs in the post-regulation of gene expression has also been recognized to contribute to physiological and pathological pathways that affect development,metabolism,host-pathogen interactions,and insecticide resistance.Objective:To compare the ability of the two main methods for identifying miRNA,and to provide methodological basis for subsequent research;to determine the miRNA expression profiles of An.sinensis at each stage of development;to understand the role of miRNA in mosquito vector growth and development,to identificate important development-related miRNAs;to reveale the changes of transcriptome and miRNA in An.sinensis and An.dirus before and after infection with plasmodium,to identificate susceptibility-related miRNAs which would provide a new perspective and theoretical basis for further study on the molecular mechanism of vector-plasmodium interaction.The study included three parts as follows:Part I:BackgroundmicroRNAs(miRNAs)are small non-coding RNAs widely identified in many mosquitoes.They are reported to play important roles in development,differentiation and innate immunity.However,miRNAs in An.sinensis,one of the Chinese malaria mosquitoes,remain largely unknown.MethodsWe investigated the global miRNA expression profile of An.sinensis using Illumina Hiseq 2000 sequencing.Meanwhile,we applied a bioinformatic approach to identify potential miRNAs in An.sinensis.The identified miRNA profiles were compared and analyzed by two approaches.The selected miRNAs from the sequencing result and the bioinformatic approach were confirmed with qRT-PCR.Moreover,target prediction,GO annotation and pathway analysis were carried out to understand the role of miRNAs in An.sinensis.ResultsWe identified 49 conserved miRNAs and 12 novel miRNAs by next-generation high-throughput sequencing technology.In contrast,43 miRNAs were predicted by the bioinformatic approach,of which two were assigned as novel.Comparative analysis of miRNA profiles by two approaches showed that 21 miRNAs were shared between them.Twelve novel miRNAs did not match any known miRNAs of any organism,indicating that they are possibly species-specific.Forty miRNAs were found in many mosquito species,indicating that these miRNAs are evolutionally conserved and may have critical roles in the process of life.Both the selected known and novel miRNAs(asi-miR-281,asi-miR-184,asi-miR-14,asi-miR-nov5,asi-miR-nov4,asi-miR-9383,and asi-miR-2a)could be detected by quantitative real-time PCR(qRT-PCR)in the sequenced sample,and the expression patterns of these miRNAs measured by qRT-PCR were in concordance with the original miRNA sequencing data.The predicted targets for the known and the novel miRNAs covered many important biological roles and pathways indicating the diversity of miRNA functions.We also found 21 conserved miRNAs and eight counterparts of target immune pathway genes in An.sinensis based on the analysis of An.gambiae.ConclusionsOur results provide the first lead to the elucidation of the miRNA profile in An.sinensis.Unveiling the roles of mosquito miRNAs will undoubtedly lead to a better understanding of mosquito biology and mosquito-pathogen interactions.This work lays the foundation for the further functional study of An.sinensis miRNAs and will facilitate their application in vector control.Part II:BackgroundmicroRNAs(miRNAs)are one kind of small non-coding RNAs widely distributed in insects.Many studies have shown that miRNAs play critical roles in development,differentiation,apoptosis,and innate immunity.However,there are a few reports describing miRNAs in An.sinensis,the most common,and one of the dominant malaria mosquito in China.Here,we investigated the global miRNA expression profile across four different developmental stages including embryo,larval,pupal,and adult stages using Illumina Hiseq2500 sequencing.ResultsIn total,164 miRNAs were obtained out of 107.46 million raw sequencing reads.99 of them identified as known miRNAs,and the remaining 65 miRNAs were considered as novel.By analyzing the read counts of miRNAs in all developmental stages,95 miRNAs showed stage-specific expression(q value<0.01&|log2(fold change)|>1)in consecutive stages,indicating that these miRNAs may be involved in critical physiological activity during development.Sixteen miRNAs were identified to be commonly dysregulated throughout four developmental stages.Many miRNAs showed stage-specific expression,such as asi-miR-2943 was exclusively expressed in the egg stage,and asi-miR-1891 could not be detected in larval stage.The expression of six selected differentially expressed miRNAs identified by qRT-PCR were consistent with our sequencing results.Furthermore,5296 and 1902 target genes were identified for the dysregulated 68 known and 27 novel miRNAs respectively by combining miRanda and RNAhybrid prediction.GO annotation and KEGG pathway analysis for the predicted genes of dysregulated miRNAs revealed that they might be involved in a broad range of biological processes related with the development,such as membrane,organic substance transport and several key pathways including protein processing in endoplasmic reticulum,propanoate metabolism and folate biosynthesis.Thirty-two key miRNAs were identified by microRNA-gene network analysis.ConclusionThe present study represents the first global characterization of An.sinensis miRNAs in its four developmental stages.The presence and differential expression of An.sinensis miRNAs imply that such miRNAs may play critical roles in An.sinensis life cycle.A better understanding of the functions of these miRNAs will have great implication for the effective control of vector population and therefore interrupting malaria transmission.Part Ⅲ:BackgroundIn this study,we performed conjoint analysis of small RNA-seq and mRNA-seq for two important malaria mosquitoes in response to plasmodium infection.We examined gene expression at 1 day post-infection,a remarkable time point of infection establisment in which a female mosquito become infectious to provide clues to the susceptibility of different vectors.ResultsResults showed changes in the abundance of a large number of transcripts and miRNAs following infection,with 671 transcripts and 20 miRNAs commonly changed in An.dirus and 29 transcripts and 9 miRNAs commonly changed in An.sinensis.In An.,Gene ontology analysis revealed that most of the altered genes are involved in single-organism process,MCM complex and vitamin binding.KEGG analysis revealed that most of the altered genes are involved in Cell cycle,Purine metabolism and Insect hormone biosynthesis.In An.sinensis,Gene ontology analysis revealed that most of the altered genes are involved in cell-matrix adhesion,cell-substrate adhesion and biological adhesion.KEGG analysis revealed that most of the altered genes are involved in ABC transporters,Protein digestion and absorption and Calcium signaling pathway.The comprehensive transcriptomic analysis of two vectors upon parasite infection not only improved the genome annotation but also provide a detailed catalogue of gene expression.The outcomes provide a basic understanding of vector responses to plasmodium parasite and help to determine factors involved in susceptibility of different mosquito response against the parasite.ConclusionThis result forms a strong foundation for the study of genes involving the mosquito susceptibility to parasite and their regulation.Furthermore,understanding the molecular genetic basis of mosquito susceptibility has important implications for vector control.Although miRNAs are reported to play a role in development in C.elegans,fruit flies and other insects,and significant stage-specific expression was observed for a number of miRNAs in various mosquito species.However,the role of miRNAs in metamorphosis and development is far less understood and remains an enigma.For some specific or novel miRNAs,their functional roles still need to be confirmed by biological experiments.Furthermore,only very limited specific target genes have been identified for small part of miRNAs.It is important to note that the result in this work is a key step towards improving our understanding of the complexity and regulation mode of miRNAs in An.sinensis embryogenesis and metamorphosis.Further analysis of stage-specific miRNA expression and functions would be helpful to decipher the complex genetic network that controls mosquito at a crucial stage.An.sinensis is the most common and wide-distributed malaria vector exhibiting a variety of ecological behaviors and feeding habits throughout the China.It is also of great medical and veterinary importance.Thus,identification of key miRNAs required for gene regulation throughout life cycle will not only help to a better understanding of its developmental biology but also may help to conceive novel approaches to control this vector.However,the function and molecular mechanism of these miRNAs are still need to be further investigated.The comprehensive transcriptomic analysis of two vectors upon parasite infection and post blood-feeding not only provided a detailed catalogue of gene expression but also improved the genome annotation.The outcomes provide a basic understanding of vector responses to plasmodium parasite and help to determine factors involved in susceptibility of different mosquito response against the parasite.The results presented may form a strong foundation for the study of genes involving the mosquito susceptibility to parasite and their regulation.Furthermore,understanding the molecular genetic basis of mosquito susceptibility may advance our understanding of parasitic habits within mosquito vectors and has important implications for vector control.