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. 2013 Sep 4;3(9):1493-509.
doi: 10.1534/g3.113.006742.

The developmental transcriptome of the mosquito Aedes aegypti, an invasive species and major arbovirus vector

Omar S Akbari  1 , Igor Antoshechkin, Henry Amrhein, Brian Williams, Race Diloreto, Jeremy Sandler, Bruce A Hay
Affiliations

The developmental transcriptome of the mosquito Aedes aegypti, an invasive species and major arbovirus vector

Omar S Akbari et al. G3 (Bethesda). .

Abstract

Mosquitoes are vectors of a number of important human and animal diseases. The development of novel vector control strategies requires a thorough understanding of mosquito biology. To facilitate this, we used RNA-seq to identify novel genes and provide the first high-resolution view of the transcriptome throughout development and in response to blood feeding in a mosquito vector of human disease, Aedes aegypti, the primary vector for Dengue and yellow fever. We characterized mRNA expression at 34 distinct time points throughout Aedes development, including adult somatic and germline tissues, by using polyA+ RNA-seq. We identify a total of 14,238 novel new transcribed regions corresponding to 12,597 new loci, as well as many novel transcript isoforms of previously annotated genes. Altogether these results increase the annotated fraction of the transcribed genome into long polyA+ RNAs by more than twofold. We also identified a number of patterns of shared gene expression, as well as genes and/or exons expressed sex-specifically or sex-differentially. Expression profiles of small RNAs in ovaries, early embryos, testes, and adult male and female somatic tissues also were determined, resulting in the identification of 38 new Aedes-specific miRNAs, and ~291,000 small RNA new transcribed regions, many of which are likely to be endogenous small-interfering RNAs and Piwi-interacting RNAs. Genes of potential interest for transgene-based vector control strategies also are highlighted. Our data have been incorporated into a user-friendly genome browser located at www.Aedes.caltech.edu, with relevant links to Vectorbase (www.vectorbase.org).

Keywords: Aedes aegypti; Medea; chikungunya; dengue fever; gene drive; malaria; population replacement; transcriptomes; yellow fever.

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Figures

Figure 1
Figure 1
Global dynamics of gene expression. The number of expressed (FPKM > 1) AAEL genes (blue) and AAEL and AAEL-NIP transcripts (red) and NTR gene (green), and NTR transcripts (purple) were plotted across all 42 developmental time points (A). Correlation matrix of all 42 poly (A+) RNA seq time points throughout development for AAEL genes and NTRs. Each developmental stage is most highly correlated with its adjacent time point across all embryogenesis. A decrease in correlation is observable in the 36−48hr ovary and 60−72hr ovary, 52−56hr to 56−60hr embryo. The scale bar indicates the coefficient of variation value between samples 0−1 (B). The expression heat map indicates the number of AAEL genes and NTRs that are fivefold upregulated between each sample. The number of AAEL genes and NTRs that are 5 fold up-regulated can be determined by matching the criteria with respect to the sequence of the row tissue (left) to the column tissue (top). For example, there are 10,762 (yellow, highest number of expressed genes and this value is 1) genes and NTRs that have 5-fold more transcriptional activity in the 24hr BF ovary tissue (left) compared with the NBF ovary tissue (top). In addition, there are 4302 (0.399 value in chart) genes and NTRs (blue), which have 5-fold more transcriptional activity in the NBF ovary tissue (left) compared with the 24-hr BF ovary tissue (top). These two statements are mutually exclusive and therefore each cell represents a different set of genes (C). Hierarchical clustering heat map of AAEL genes and NTRs, illustrating the various patterns of gene expression across all developmental time points. Scale bar indicates the FPKM z scores (D). For A−D, The major developmental groups are indicated by color bars and are organized left to right, as follows: M (brown, male testes, male carcass), Fc (purple, NBF Female Carcass, and multiple time points PBM: 12hr, 24hr, 36hr, 48hr, 60hr, and 72hr), O (red, NBF ovaries, and multiple ovarian time points PBM: 12hr, 24hr, 36hr, 48hr, 60hr and 72hr), E (green, embryo, 0-2hr, 2-4hr, 4-8hr, 8-12hr, 12-16hr, 16-20hr, 20-24hr, 24-28hr, 28-32hr, 32-36hr, 36-40hr, 40-44hr, 44-48hr, 48-52hr, 52-56hr, 56-60hr, 60-64hr, 64-68hr, 68-72hr and 72-76hr embryos), L (light blue, larvae, 1st, 2nd, 3rd and 4th instar larvae stages), and P (light orange, male and female pupae).
Figure 2
Figure 2
Soft clustering, principal component analysis, and totals. Twenty AAEL gene expression profile clusters were identified through soft clustering. Each gene is assigned a line color corresponding to its membership value, with red (1) indicating high association. The major developmental groups are indicated by symbols on the X axis, and are organized as in Figure 1, B−D (A). Principal component analysis shows relationships between the 20 clusters, with thickness of the blue lines between any two clusters reflecting the fraction of genes that are shared (B, thickness of blue lines). n, the number of genes in each cluster.
Figure 3
Figure 3
Developmental time course of TE expression profiles. Developmental expression profiles of different TE families, indicating RPKM values (y-axis) across all 42 developmental time points (x-axis). The top 10 TE families with the greatest expression levels are indicated from 1 (highest) to 10 (lowest). All families of TEs are indicated in the table in the lower left, with the number of elements in each family indicated in parentheses. Small numbers associated with specific boxes identify highly expressed TE families from upper plot of RPKM values (A). A circular pie chart indicating the percentage of the annotated genome occupied by each TE class (outer circle), and sublcass (inner circle) (B).
Figure 4
Figure 4
Small RNA distribution and clustering. Length distributions for small RNAs that map to the genome are indicated as percentages of the total reads mapping to the genome, for each library. Results from both unique and multimapping are shown. Samples are indicated to the right. Dotted line corresponds to the length distribution for previously annotated Aedes miRNAs included in mirBase (A). Heat map representation of all previously annotated miRNAs in A. aegypti, and 38 newly discovered miRNAs. Scale bar indicates FPKM Z-scores (B). Genome browser snap shot of a 54,727-bp genomic region, dense in 27−32bp mapped fragments, on supercontig 1: 1,174,222-1,228,948. All small RNA libraries are uniquely mapped (C). Color bar graph depicting the log2 RPM (reads per million) of each miRNA expressed in the 9 samples indicated to the right, organized in order from most to least abundant (D). The sample color scale for (A−C) is identical, as depicted in C, and is as follows: male testes and AG (red), male carcass (orange), 72-hr BF-F-carcass (blue), 2−4 hr embryo (green), 0−2 hr embryo (brown), 72-hr BF-ovary (purple), 48-hr Bf-ovary (light green), 24-hr BF-ovary (light red), and NBF ovary (black).
Figure 5
Figure 5
Small RNA mapping results and expression profiles to features. Percentage of reads mapping to the genome or annotated features; results are shown using multi-mapping (unlimited alignments) and unique mapping (single alignment) for the samples indicated on the X axis. The fraction of reads corresponding to novel transcribed features is indicated in red (A). Of the small RNAs that map to known features, the percent of small RNAs mapping to specific features is indicated for both multi and unique mapping (B). RPM values (y-axis) for each small RNA library (x-axis) were quantified against all annotated TE element families in A. aegypti for both multi and unique mapping. Numbers highlight the TEs for which small RNAs are most abundant across all samples (C).
Figure 6
Figure 6
piRNA production by a single locus and RNA expression profiles for genes involved in small RNA production. (A) An example of piRNAs mapping specifically to the 3′UTR of AAEL007686 is shown. (B) The expression dynamics across development of genes important for processing of different small RNAs, including miRNA, siRNAs, and piRNAs are shown as a heat map (log2 FPKM).
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