an-experimental-bioinformatics-protocol-for-rna-seq-analyses

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, <em>Aedes albopictus</em>

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

Georgetown University

Many 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.

A de novo transcriptome of the Asian tiger mosquito, Aedes albopictus, to identify candidate transcripts for diapause preparation.

The Asian tiger mosquito, Aedes albopictus, is a medically important invasive species whose geographic distribution has expanded dramatically during the past 20 years, and one of the key elements of its success is its capacity to survive long distance transport as a diapausing pharate first instar larva, encased within the chorion of the egg. We report that pharate larvae entering diapause are larger and contain 30% more lipid than their nondiapausing counterparts. To improve our understanding of the molecular regulation of lipid metabolism during diapause, we assessed the relative mRNA abundance of 21 genes using qRT-PCR. Elevated expression of lipid storage droplet protein 2 during embryonic development likely contributes to the higher amounts of lipid we noted in diapausing individuals. The conservation of lipids during diapause is reflected in downregulation of genes involved in lipid catabolism, including lipase 2, lipase 3, lipase 4, acyl-CoA dehydrogenase 4, and isovaleryl-CoA dehydrogenase. Two genes involved in fatty acid synthesis and modification, Δ(9)-desaturase, and fatty acyl-CoA elongase, were both upregulated in diapausing pharate larvae, suggesting roles for their gene products in generating unsaturated fatty acids to enhance membrane fluidity at low temperatures and generating precursors to the surface hydrocarbons needed to resist desiccation, respectively. Together, the results point to substantial distinctions in lipid metabolism within the embryo as a consequence of the diapause program, and these differences occur both before the actual onset of diapause as well as during the diapause state.

Transcript profiling reveals mechanisms for lipid conservation during diapause in the mosquito, Aedes albopictus.

Landscape genetic studies use spatially explicit population genetic information to determine the physical and environmental causes of population genetic structure on regional scales. Comparative studies that identify common barriers to gene flow across multiple species within a community are important to both understand the evolutionary trajectories of populations and prioritize habitat conservation. Here, we use a comparative landscape genetic approach to ask whether gradients in temperature or precipitation seasonality structure genetic variation across three codistributed tree species in Central America, or whether a simpler (geographic distance) or more complex, species-specific environmental niche model is necessary to individually explain population genetic structure. Using descriptive statistics and causal modelling, we find that different factors best explain genetic distance in each of the three species: environmental niche distance in Bursera simaruba, geographic distance in Ficus insipida and historical barriers to gene flow or cryptic reproductive barriers for Brosimum alicastrum. This study confirms suggestions from previous studies of Central American tree species that imply that population genetic structure of trees in this region is determined by complex interactions of both historical and current barriers to gene flow.

Differential effects of landscape-level environmental features on genetic structure in three codistributed tree species in Central America.

Seasonal environments present fundamental physiological challenges to a wide range of insects. Many temperate insects surmount the exigencies of winter by undergoing photoperiodic diapause, in which photoperiod provides a token cue that initiates an alternative developmental programme leading to dormancy. Pre-diapause is a crucial preparatory phase of this process, preceding developmental arrest. However, the regulatory and physiological mechanisms of diapause preparation are largely unknown. Using high-throughput gene expression profiling in the Asian tiger mosquito, Aedes albopictus, we reveal major shifts in endocrine signalling, cell proliferation, metabolism, energy production and cellular structure across pre-diapause development. While some hallmarks of diapause, such as insulin signalling and stress response, were not important at the transcriptional level, two genes, Pepck and PCNA, appear to show diapause-induced transcriptional changes across insect taxa. These processes demonstrate physiological commonalities between Ae. albopictus pre-diapause and diapause strategies across insects, and support the idea of a genetic 'toolkit' for diapause. Observations of gene expression trends from a comparative developmental perspective suggest that individual physiological processes are delayed against a background of a fixed morphological ontogeny. Our results demonstrate how deep sequencing can provide new insights into elusive molecular bases of complex ecological adaptations.

Deep sequencing reveals complex mechanisms of diapause preparation in the invasive mosquito, Aedes albopictus.

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.

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

Dormancy is a crucial adaptation allowing insects to withstand harsh environmental conditions. The pre-programmed developmental arrest of diapause is a form of dormancy that is distinct from quiescence, in which development arrests in immediate response to hardship. Much progress has been made in understanding the environmental and hormonal controls of diapause. However, studies identifying transcriptional changes unique to diapause, rather than quiescence, are lacking, making it difficult to disentangle the transcriptional profiles of diapause from dormancy in general. The Asian tiger mosquito, Aedes albopictus, presents an ideal model for such a study, as diapausing and quiescent eggs can be staged and collected for global gene expression profiling using a newly developed transcriptome. Here, we use RNA-Seq to contrast gene expression during diapause with quiescence to identify transcriptional changes specific to the diapause response. We identify global trends in gene expression that show gradual convergence of diapause gene expression upon gene expression during quiescence. Functionally, early diapause A. albopictus show strong expression differences of genes involved in metabolism, which diminish over time. Of these, only expression of lipid metabolism genes remained distinct in late diapause. We identify several genes putatively related to hormonal control of development that are persistently differentially expressed throughout diapause, suggesting these might be involved in the maintenance of diapause. Our results identify key biological differences between diapausing and quiescent pharate larvae, and suggest candidate pathways for studying metabolism and the hormonal control of development during diapause in other species.

RNA-Seq reveals early distinctions and late convergence of gene expression between diapause and quiescence in the Asian tiger mosquito, Aedes albopictus.

Article	Year
A de novo transcriptome of the Asian tiger mosquito, Aedes albopictus, to identify candidate transcripts for diapause preparation. BMC genomics\| PubMed ID: 22185595	2011
Transcript profiling reveals mechanisms for lipid conservation during diapause in the mosquito, Aedes albopictus. Journal of insect physiology\| PubMed ID: 22579567	2012
Differential effects of landscape-level environmental features on genetic structure in three codistributed tree species in Central America. Molecular ecology\| PubMed ID: 22988889	2012
Deep sequencing reveals complex mechanisms of diapause preparation in the invasive mosquito, Aedes albopictus. Proceedings. Biological sciences / The Royal Society\| PubMed ID: 23516243	2013
Transcriptome sequencing as a platform to elucidate molecular components of the diapause response in the Asian tiger mosquito, Aedes albopictus. Physiological entomology\| PubMed ID: 23833391	2013
RNA-Seq reveals early distinctions and late convergence of gene expression between diapause and quiescence in the Asian tiger mosquito, Aedes albopictus. The Journal of experimental biology\| PubMed ID: 23913949	2013

Monica F. Poelchau

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