Julie A. Reynolds

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

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.

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

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.

Metabolic Restructuring during Energy-Limited States: Insights from Artemia franciscana Embryos and Other Animals

Many life history stages of animals that experience environmental insults enter developmental arrested states that are characterized by reduced cellular proliferation, with or without a concurrent reduction in overall metabolism. In the case of the most profound metabolic arrest reported in invertebrates, i.e., anaerobic quiescence in Artemia franciscana embryos, acidification of the intracellular milieu is a major factor governing catabolic and anabolic downregulation. Release of ions from intracellular compartments is the source for approximately 50% of the proton equivalents needed for the 1.5 unit acidification that is observed. Recovery from the metabolic arrest requires re-sequestration of the protons with a vacuolar-type ATPase (V-ATPase). The remarkable facet of this mechanism is the ability of embryonic cells to survive the dissipation of intracellular ion gradients. Across many diapause-like states, the metabolic reduction and subsequent matching of energy demand is accomplished by shifting energy metabolism from oxidative phosphorylation to aerobic glycolysis. Molecular pathways that are activated to induce these resilient hypometabolic states include stimulation of the AMP-activated protein kinase (AMPK) and insulin signaling via suite of daf (dauer formation) genes for diapause-like states in nematodes and insects. Contributing factors for other metabolically depressed states involve hypoxia-inducible factor-1 and downregulation of the pyruvate dehydrogenase complex. Metabolic similarities between natural states of stasis and some cancer phenotypes are noteworthy. Reduction of flux through oxidative phosphorylation helps prevent cell death in certain cancer types, similar to the way it increases viability of dauer stages in Caenorhabditis elegans. Mechanisms that underlie natural stasis are being used to pre-condition mammalian cells prior to cell biostabilization and storage.

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

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.

Embryonic diapause highlighted by differential expression of mRNAs for ecdysteroidogenesis, transcription and lipid sparing in the cricket Allonemobius socius.

SUMMARY Embryos of the ground cricket, Allonemobius socius, enter diapause 4–5 days post-oviposition and overwinter in this dormant state that is characterized by developmental arrest. Suppressive subtractive hybridization and quantitative real-time PCR reveal eight candidate genes in pre-diapause embryos that show promise as regulators of diapause entry, when compared with embryos not destined for diapause. Identifications are based both on the magnitude/consistency of differential mRNA abundances and the predicted functions of their products when placed in context of the physiological and biochemical events of diapause characterized in our companion paper. The proteins CYP450, AKR and RACK1 (associated with ecdysteroid synthesis and signaling) are consistently upregulated in pre-diapause, followed by major downregulation later in diapause. The pattern suggests that elevated ecdysone may facilitate onset of diapause in A. socius. Upregulation seen for the transcription factors Reptin and TFDp2 may serve to depress transcription and cell cycle progression. Cathpesin B-like protease, ACLY and MSP are three downregulated genes associated with yolk mobilization and/or metabolism that we predict may promote lipid sparing. Finally, embryos that have been in diapause for 10 days show a substantially different pattern of mRNA expression compared with either pre-diapause or embryos not destined for diapause, with the majority of mRNAs examined being downregulated. These transcript levels in later diapause suggest that a number of upregulated genes in pre-diapause are transiently expressed and are less essential as diapause progresses.

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

SUMMARY 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.

Decoupling development and energy flow during embryonic diapause in the cricket, Allonemobius socius

SUMMARY Respiration rate increases 6.3-fold during 15 days of post-oviposition development in embryos of the Southern ground cricket, Allonemobius socius. This ontogenetic increase in metabolism of non-diapause insects is blocked during diapause, such that metabolic rate is only 36% of the rate measured for 15 days developing embryos. Surprisingly, however, there is not an acute metabolic depression during diapause entry at the point when developmental ceases (4–5 days post-oviposition), as measured by blockage of morphological change and DNA proliferation. The results indicate a decoupling of developmental arrest from metabolism. Both non-diapause and diapause embryos have unusually high [AMP]:[ATP] ratios and low [ATP]:[ADP] ratios during early embryogenesis, which suggests embryos may have experienced hypoxia as a result of an insect chorion that limits water loss but may restrict oxygen diffusion. The similar adenylate profiles for these two developmental states indicate the atypical energy status is not a specific feature of diapause. In addition embryos at day 3 have high levels of lactate that decrease as development proceeds up to day 7. Calorimetric-respirometric (CR) ratios of –353 (day 3) to –333 (day 7) kJ mol–1 O2 are consistent with embryos that are aerobically recovering from hypoxia, but are inconsistent with an ongoing anaerobic contribution to metabolism. Superfusing 3-day embryos with O2 enriched air (40% O2) forces these metabolic indicators toward a more aerobic poise, but only partially. Taken together these biochemical data indicate the metabolic poise of A. socius is only partly explained by hypoxia in early development, and that the atypical set points are also intrinsic features of this ontogenetic period in the life cycle.

Transcriptome sequencing as a platform to elucidate molecular components of the diapause response in the Asian tiger 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 for addressing this longstanding question. In the present study, a new assembly from life‐history stages relevant to diapause in the Asian tiger mosquito Aedes albopictus (Skuse) is reported, along with unique methods for the analysis of NGS data and transcriptome assembly. A digital normalization procedure that significantly reduces the 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 for identifying the 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.

Changes in histone acetylation as potential mediators of pupal diapause in the flesh fly, Sarcophaga bullata.

The growing appreciation that epigenetic processes are integral to the responses of many organisms to changes in the environment suggests a possible role for epigenetics in coordination of insect diapause. The results we present suggest that histone modification may be one type of epigenetic process that contributes to regulation of pupal diapause in the flesh fly, Sarcophaga bullata. Reduction in total histone H3 acetylation in diapausing pupae, shifts in mRNA expression profiles of genes encoding histone acetyltransferase (HAT) and histone deacetylase (HDAC) in pre-diapause, diapause and post-diapause flies compared to their nondiapause counterparts, and alterations in HDAC enzyme activity during and post-diapause lend support to the hypothesis that this specific type of histone modification is involved in regulating diapause programming, maintenance, and termination. Transcription of genes encoding HDAC1, HDAC3, HDAC6, and Sirtuin2 were all upregulated in photosensitive first instar larvae programmed to enter pupal diapause, suggesting that histone deacetylation may be linked to the early decision to enter diapause. A 50% reduction in transcription of hdac3 and a corresponding 30% reduction in HDAC activity during diapause suggest that removal of acetyl groups from histones primarily occurs prior to diapause entry and that further histone deacetylation is not necessary to maintain diapause. Transcription of the HDAC genes was quickly elevated when diapause was terminated, followed by an increase in enzyme activity after a short delay. A maternal effect operating in these flies prevents pupal diapause in progeny whose mothers experienced pupal diapause, even if the progeny are reared in strong diapause-inducing short-day conditions. Such nondiapausing pupae had HDAC transcription profiles nearly identical to the profiles seen in nondiapausing pupae generated under a long-day photoperiod. Together, these results provide consistent evidence for histone acetylation and deacetylation as regulators of this insects developmental trajectory.

An experimental and bioinformatics protocol for RNA-Seq analyses of photoperiodic diapause in the Asian tiger mosquito, Aedes albopictus.

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.