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Transcriptome sequencing as a platform to elucidate molecular components of the diapause response in the Asian tiger mosquito, Aedes albopictus.

ABSTRACT: Diapause has long been recognized as a crucial ecological adaptation to spatio-temporal environmental variation. More recently, rapid evolution of the diapause response has been implicated in response to contemporary global warming and during the range expansion of invasive species. Although the molecular regulation of diapause remains largely unresolved, rapidly emerging next-generation sequencing (NGS) technologies provide exciting opportunities to address this longstanding question. Herein, a new assembly from life-history stages relevant to diapause in the Asian tiger mosquito, Aedes albopictus (Skuse) is presented, along with unique methods for the analysis of NGS data and transcriptome assembly. A digital normalization procedure that significantly reduces computational resources required for transcriptome assembly is evaluated. Additionally, a method for protein reference-based and genomic reference-based merged assembly of 454 and Illumina reads is described. Finally, a gene ontology analysis is presented, which creates a platform to identify physiological processes associated with diapause. Taken together, these methods provide valuable tools for analyzing the transcriptional underpinnings of many complex phenotypes, including diapause, and provide a basis for determining the molecular regulation of diapause in Ae. albopictus.

SUBMITTER: Poelchau MF

PROVIDER: S-EPMC3700550 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Transcriptome sequencing as a platform to elucidate molecular components of the diapause response in the Asian tiger mosquito, <i>Aedes albopictus.</i>

Poelchau Monica F MF Reynolds Julie A JA Denlinger David L DL Elsik Christine G CG Armbruster Peter A PA

Physiological Entomology. 20130601 2

Diapause has long been recognized as a crucial ecological adaptation to spatio-temporal environmental variation. More recently, rapid evolution of the diapause response has been implicated in response to contemporary global warming and during the range expansion of invasive species. Although the molecular regulation of diapause remains largely unresolved, rapidly emerging next-generation sequencing (NGS) technologies provide exciting opportunities to address this longstanding question. Herein, a ...[more]

PMID: 23833391

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Project description:BackgroundMany temperate insects survive the harsh conditions of winter by undergoing photoperiodic diapause, a pre-programmed developmental arrest initiated by short day lengths. Despite the well-established ecological significance of photoperiodic diapause, the molecular basis of this crucial adaptation remains largely unresolved. The Asian tiger mosquito, Aedes albopictus (Skuse), represents an outstanding emerging model to investigate the molecular basis of photoperiodic diapause in a well-defined ecological and evolutionary context. Ae. albopictus is a medically significant vector and is currently considered the most invasive mosquito in the world. Traits related to diapause appear to be important factors contributing to the rapid spread of this mosquito. To generate novel sequence information for this species, as well as to discover transcripts involved in diapause preparation, we sequenced the transcriptome of Ae. albopictus oocytes destined to become diapausing or non-diapausing pharate larvae.Results454 GS-FLX transcriptome sequencing yielded >1.1 million quality-filtered reads, which we assembled into 69,474 contigs (N50 = 1,009 bp). Our contig filtering approach, where we took advantage of strong sequence similarity to the fully sequenced genome of Aedes aegypti, as well as other reference organisms, resulted in 11,561 high-quality, conservative ESTs. Differential expression estimates based on normalized read counts revealed 57 genes with higher expression, and 257 with lower expression under diapause-inducing conditions. Analysis of expression by qPCR for 47 of these genes indicated a high correlation of expression levels between 454 sequence data and qPCR, but congruence of statistically significant differential expression was low. Seven genes identified as differentially expressed based on qPCR have putative functions that are consistent with the insect diapause syndrome; three genes have unknown function and represent novel candidates for the transcriptional basis of diapause.ConclusionsOur transcriptome database provides a rich resource for the comparative genomics and functional genetics of Ae. albopictus, an invasive and medically important mosquito. Additionally, the identification of differentially expressed transcripts related to diapause enriches the limited knowledge base for the molecular basis of insect diapause, in particular for the preparatory stage. Finally, our analysis illustrates a useful approach that draws from a closely related reference genome to generate high-confidence ESTs in a non-model organism.

Project description:BackgroundTemporal and stage specific expression of microRNAs (miRNAs) in embryos, larvae, pupae and adults of Aedes albopictus showed differential expression levels across the four developmental stages, indicating their potential regulatory roles in mosquito development. The functional characterization of these miRNAs was not known. Accordingly our study evaluated the functional characterization of three miRNAs, which are temporally up-regulated in the various developmental stages of Ae. albopictus mosquitoes.MethodsmiRNA mimics, inhibitors and negative controls were designed and their knock-in and knock-down efficiency were analyzed by qRT-PCR after transfecting the mosquito cell lines C6/36, and also by injecting in their specific developmental stages. The functional role of each individual miRNA was analyzed with various parameters of development such as, hatching rate and hatching time in embryos, eclosion rate in larvae, longevity and fecundity in the adult mosquitoes.ResultsThe knock-in with the specifically designed miRNA mimics showed increased levels of expression of miRNA compared with their normal controls. We confirmed these findings using qRT-PCR, both by in vitro expression in C6/36 mosquito cell lines after transfection as well as in in vivo expression in developmental stages of mosquitoes by microinjection. The knock-down of expression with the corresponding inhibitors showed a considerable decrease in the expression levels of these miRNAs and obvious functional effects in Ae. albopictus development, detected by a decrease in the hatching rate of embryos and eclosion rate in larvae and a marked reduction in longevity and fecundity in adults.ConclusionThis study carried out by knock-in and knock-down of specifically and temporally expressed miRNAs in Ae. albopictus by microinjection is a novel study to delineate the importance of the miRNA expression in regulating mosquito development. The knock-down and loss of function of endogenously expressed miRNAs by the miRNA inhibitors in specific developmental stages had considerable effects on development, but enhancement of their gain of function was not observed on knock-in of these specific miRNAs. Hence, our study indicates that an optimal level of endogenous expression of miRNA is indispensable for the normal development and maintenance of the vectorial population density and pathogen transmissibility of this mosquito vector.

Dataset Information

Transcriptome sequencing as a platform to elucidate molecular components of the diapause response in the Asian tiger mosquito, Aedes albopictus.

Publications

Transcriptome sequencing as a platform to elucidate molecular components of the diapause response in the Asian tiger mosquito, <i>Aedes albopictus.</i>

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