- Research article
- Open Access
Stage-specific transcription during development of Aedes aegypti
© Harker et al.; licensee BioMed Central Ltd. 2013
Received: 17 October 2012
Accepted: 10 July 2013
Published: 22 July 2013
Aedes aegypti is the most important global vector of dengue virus infection in humans. Availability of the draft genome sequence of this mosquito provides unique opportunities to study different aspects of its biology, including identification of genes and pathways relevant to the developmental processes associated with transition across individual life stages. However, detailed knowledge of gene expression patterns pertaining to developmental stages of A. aegypti is largely lacking.
We performed custom cDNA microarray analyses to examine the expression patterns among six developmental stages: early larvae, late larvae, early pupae, late pupae, and adult male and female mosquitoes. Results revealed 1,551 differentially expressed transcripts (DETs) showing significant differences in levels of expression between these life stages. The data suggests that most of the differential expression occurs in a stage specific manner in A. aegypti. Based on hierarchical clustering of expression levels, correlated expression patterns of DETs were also observed among developmental stages. Weighted gene correlation network analysis revealed modular patterns of expression among the DETs. We observed that hydrolase activity, membrane, integral to membrane, DNA binding, translation, ribosome, nucleoside-triphosphatase activity, structural constituent of ribosome, ribonucleoprotein complex and receptor activity were among the top ten ranked GO (Gene Ontology) terms associated with DETs. Significant associations of DETs were also observed with specific KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway modules. Finally, comparisons with the previously reported developmental transcriptome of the malaria vector, Anopheles gambiae, indicated that gene expression patterns during developmental processes reflect both species-specific as well as common components of the two mosquito species.
Our study shows that genes involved in the developmental life cycle of A. aegypti are expressed in a highly stage-specific manner. This suggests that transcriptional events associated with transition through larval, pupal and adult stages are largely discrete.
Mosquito (Culicidae) development, as characteristic of all holometabolous insects, proceeds through embryonic, larval, pupal, and adult stages that reflect considerable morphological and physiological differences. These stages also exhibit distinct niche partitioning as larvae and pupae are aquatic while adults are free-flying and terrestrial. In addition, following an estimated ~192-230 million years of divergence among the major mosquito lineages , it is anticipated that individual species might have evolved in molecular pathways of developmental processes as seen throughout the evolution of insect metamorphosis . Larvae of all mosquito species progress through four instars that include periods of continuous growth interrupted by shedding of the old cuticle or ecdysis [3, 4]. The molting process begins with physical separation of the epidermis from the old endocuticle, a process known as apolysis. In response to hormonal changes by increasing their rate of protein synthesis during this period, the epidermal cells secrete a lipoprotein that forms the cuticulin layer to insulate and protect them from the molting fluid's digestive action. The cuticulin layer becomes part of the new exoskeleton's epicuticle. When the new exoskeleton is ready, the old exoskeleton splits open. Ecdysis (shedding old exoskeleton) continues to fully expand the new exoskeletons. After ecdysis, sclerites harden and darken within the exocuticle, the process known as sclerotization, which gives the exoskeleton its final texture and appearance. With the completion of the four instars of larval molting and sclerotization, metamorphosis, the transformation from larvae to pupae to adult stages, begins. It includes complex processes that involve larval and pupal tissue histolysis and remodeling leading to adult tissue formation. The cascades of transcriptional events associated with insect ecdysis and metamorphosis are controlled by coordinated ecdysteroid and juvenile hormone (JH) activities [5–7].
The mosquito, Aedes aegypti, is the principal global vector for dengue viruses. Dengue fever (DF) is caused by infection with dengue virus throughout the subtropics and tropics, with >2.5 billion people at risk. An annual incidence of ~50 million cases and ~500,000 cases of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) results in ~24,000 deaths per year [8–11]. No effective vaccines are currently available and no drug treatments exist. Thus mosquito control remains the most effective strategy for controlling dengue and other mosquito-borne diseases, in spite of resistance to insecticides in specific populations . A. aegypti maintains a strong association with humans, breeding in virtually any container that holds water long enough for larval/pupal development , and because of a strong dietary preference for human blood  it is capable of completing the entire life cycle within human dwellings.
To date, gene expression studies pertaining to A. aegypti development are limited [15–21]. In the malaria vector mosquito Anopheles gambiae, microarray studies have been performed to study life cycle developmental transcriptome [22, 23]. These independent studies identified a total of 1,571  and 560 A. gambiae transcripts, respectively, that showed differential regulation specific to development. Comparative global expression analyses with Drosophila melanogaster revealed a strong positive correlation of development-related expression between orthologous genes . However, a genome-scale transcriptional analysis of A. aegypti life cycle development is lacking.
A draft whole genome sequence is available for A. aegypti. As part of the genome sequencing effort a large collection of expressed sequenced tags (ESTs) derived from a broad range of tissues and strains was generated. Here we employed a custom cDNA-based microarray platform that represents 9,504 unique EST contig assemblies. We compared transcriptional profiles across the A. aegypti life cycle including early and late larvae, early and late pupae, mixed adults, and adult males and females. Where possible, we also compared our results for A. aegypti with those previously reported for similar stages in A. gambiae.
This study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol was approved by the University of Notre Dame Institutional Animal Care and Use Committee (Study #11-036).
Aedes aegypti Liverpool IB-12 strain was reared at 26°C with 84% relative humidity and in a 16-h light/8-h dark cycle with 1-h crepuscular periods. Larvae were reared on a bovine liver powder (MP Biomedical) suspension as the food source and adults were provided a 5% sugar solution ad libitum. The larval density was 500 per 1,500 cm3 in all the rearing to prevent crowding effects. The detailed protocol on rearing and maintenance of A. aegypti is provided elsewhere .
Microarray content and design
Custom microarrays were generated from 9,504 unique cDNA amplicons as previously described . The cDNAs were obtained from a number of A. aegypti strains and included tissue-specific and pathogen response-specific origins generated as part of the genome sequence annotation effort . Consensus EST assemblies and associated cDNA clones were downloaded at the A. aegypti Gene Index .
Microarray hybridization and analysis
Five developmental comparisons were investigated: 1) early larvae-late larvae, 2) late larvae-early pupae, 3) early pupae-late pupae, 4) late pupae-adult mixture, and 5) adult male-adult female. Hybridization experiments were carried out following the two step protocol as recommended by the manufacturer (Genisphere). All of the hybridization comparisons included one dye-swap in order to eliminate dye fluorescence bias. The entire experiment was performed with a total of three biological replicates. After hybridization and washing, the microarray slides were scanned at two wavelengths, 532 and 635 nm, using the GenePix Pro 4200A scanner (Molecular Devices Corp).
Spot intensity data was quantified using the segmentation and data analysis software GenePix Pro 6.0 (Molecular Devices Corp). The average signal intensities were normalized with an intensity dependent (Lowess) normalization using GeneSpring GX 7.3 software (Agilent). Statistical analysis of the data was conducted using Significance Analysis of Microarrays (SAM) . All the raw as well as processed expression data of the microarray experiments have been deposited in ArrayExpress under the accession number E-TABM-385.
Annotation information and gene ontology (GO) data for the transcripts were obtained at VectorBase . Fisher’s exact test was used to determine significant associations of GO terms with the differentially expressed genes. The numbers of significant and non-differentially expressed transcripts (DETs) associated with each GO term were compared with the respective counts of genes with all other GO terms for the entire gene set. Similar comparisons were also made for GO terms associated with DETs for each developmental stage. Association of the DETs with A. aegypti pathways was determined at KEGG . All statistical tests were conducted using the statistical analysis package R . The modular expression patterns were predicted by weighted gene correlation network analysis of DETs using default parameters with the WGCNA program . The expression fold-changes of transcripts among the five pairs of developmental stages were clustered using hierarchical clustering method (average linkage) implemented in Cluster 3.0 software . The rank order correlation of fold-changes was used to determine clusters among genes (columns) and stages (rows). The clusters were viewed by the TreeView program (http://www.eisenlab.org/eisen/).
Quantitative real-time PCR analysis
Expression levels of a randomly selected set of genes were measured using quantitative real-time PCR (qRT-PCR) analysis using SYBR Green dye technology (Applied Biosystems). Primer Express Software version 3.0 (Applied Biosystems) was used to design primers (Additional file 1). All amplifications and fluorescence quantification were performed using an ABI 7500 Fast System Sequence Detector System (Applied Biosystems) and the Sequence Detector Software version 1.3 (Applied Biosystems). The reactions were performed in a total volume of 25 μl containing 12.5 μl of SYBR Green PCR Master Mix, 10 ng of cDNA (the same samples used in microarrays), 300 nmol of each primer, and nuclease free water. Reactions were performed with the following conditions: 50°C for 2 min, 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 15 s, annealing and extension at 60°C for 1 min. Three biological replicates were performed for each assay. Melting curves of the data points were used to determine the specificity of the PCR reaction. Data was used from assays only when PCR efficiency was greater than 95%. Expression values were obtained by using the delta-delta cycle threshold (∆∆CT) method  using the ribosomal protein S17 (RpS17) gene as the reference control .
The developmental transcriptome data of A. gambiae from a previous study  were used for comparisons with our current microarray data for A. aegypti. The A. gambiae microarray expression data were downloaded at VectorBase  and were compared with DETs at five life stages (Lb, Le, P, M, F) of A. gambiae. The different life stages of A. gambiae were represented as La, Lb, Lc, Ld and Le stages for larvae whereas P, M and F represented the pupae, male- and female- adults stages in that study . For comparison with A. aegypti, the specific stages were chosen that generally approximated to early larvae, late larvae, early pupae and adult male and female stages of A. aegypti. Our objective of this analysis was to detect if larvae, pupae and adult stages of both the mosquitoes have signature gene expression patterns, as it is difficult to ascertain that the chosen developmental times corresponded to the same exact life stages of both the species. A total of three comparisons (Lb-Le, Le-P, and M-F) between the A. gambiae data and our data for A. aegypti were performed. The fold change of gene expression levels were compared between the two data sets. The orthologous genes between the two species were obtained from Biomart data included in VectorBase . Only genes that were 1-to-1 orthologs were considered (n = 8,325), and from these the list of genes expressed in both organisms in the similar developmental stages were identified.
Identification of differentially expressed transcripts related to development
The DETs were determined at five developmental stages of A. aegypti: early larvae – late larvae (EL-LL), late larvae – early pupae (LL-EP), early pupae – late pupae (EP-LP), late pupae – adult male and female mix (LP-AdultMix) and adult male – adult (AM-AF). The significance levels of differential expression for each comparison were assessed by SAM analysis where the significance threshold (δ) ranged within 0.34 to 0.51, while the false discovery rate ranged from 4.9 to 5.5%. The minimum significant fold change was 1.9 for the five comparisons. To validate the microarray data, expression patterns for nine randomly selected genes were determined using qRT-PCR. The results revealed highly similar trends between qRT-PCR and microarray data for the expression levels of the genes (Additional file 2).
Numbers of significant DETs identified from the microarray analysis
Different patterns of differentially expression of transcripts where significant changes in expression level are evident in multiple developmental stages of A. aegypti
No. of transcripts
Developmental stages investigated
EL-LL + LL-EP
EL-LL + EP-LP
EL-LL + LP-AdultMix
LL-EP + LP-AdultMix
EP-LP + LL-EP
EP-LP + LP-AdultMix
AM-AF + EL-LL
AM-AF + LL-EP
AM-AF + EP-LP
AM-AF + LP-AdultMix
EL-LL + LL-EP + LP-AdultMix
EL-LL + EP-LP + LL-EP
EL-LL + EP-LP + LP-AdultMix
EP-LP + LL-EP + LP-AdultMix
AM-AF + EL-LL + LL-EP
AM-AF + EL-LL + EP-LP
AM-AF + EL-LL + LP-AdultMix
AM-AF + LL-EP + LP-AdultMix
AM-AF + EP-LP + LL-EP
AM-AF + EP-LP + LP-AdultMix
EL-LL + EP-LP + LL-EP + LP-AdultMix
AM-AF + EL-LL + LL-EP + LP-AdultMix
AM-AF + EL-LL + EP-LP + LL-EP
AM-AF + EL-LL + EP-LP + LP-AdultMix
AM-AF + EP-LP + LL-EP + LP-AdultMix
Correlated expression patterns of DETs
Gene annotation and functional assignments
List of gene ontology (GO) terms significantly associated with the differentially expressed transcripts of A. aegypti during development
Integral to membrane
Structural constituent of ribosome
Regulation of transcription, DNA-dependent
Structural molecule activity
Phosphoprotein phosphatase activity
Cellular amino acid biosynthetic process
Cellular iron ion homeostasis
Ferric iron binding
Iron ion transport
Structural constituent of cuticle
G-protein coupled receptor protein signaling pathway
Intracellular membrane-bounded organelle
Protein catabolic process
Clathrin coat of coated pit
COPI vesicle coat
‘de novo’ IMP biosynthetic process
Galactose metabolic process
G-protein coupled receptor activity
Methionine biosynthetic process
Mitochondrial intermembrane space protein transporter complex
Non-membrane spanning protein tyrosine phosphatase activity
Protein import into mitochondrial inner membrane
Pyrroline-5-carboxylate reductase activity
Signal transducer activity
Comparison with developmental transcriptome of Anopheles gambiae
Although very little is known on molecular aspects of developmental processes of any mosquito, A. aegypti is emerging as a model organism for developmental biology studies . The genome sequence of A. aegypti, along with that for two other mosquito species, C. quinquefasciatus (vector of lymphatic filariasis and West Nile virus)  and A. gambiae (major malaria vector) , have greatly enhanced our understanding of several aspects of mosquito biology . A comparative genomic analysis of developmental genes in these mosquitoes with Drosophila melanogaster indicated that while orthologs for most D. melanogaster developmental genes are present in mosquitoes, some key genes in D. melanogaster are not represented. The present investigation was initiated to profile transcriptional changes across the different stages of A. aegypti development. Our results represent the first efforts toward uncovering and understanding temporal patterns of gene expression underpinning the processes of larval morphogenesis, pupation and transition to adult stages of A. aegypti.
We observed that the majority of DETs (1,173 of 1,551; 75.6%) showed significant differential expression for only a single developmental stage comparison. The differentially expressed genes within life stages were characterized by specific metabolic processes. The earlier stages of the life cycle (larvae and pupae) were significantly associated with KEGG pathway genes related to development, transcription, amino acid metabolism and carbohydrate metabolism, whereas genes related to lipid- and energy-metabolism were significantly associated with the later developmental stages such as pupae-adult transition and between males and females within the adult stage (Additional file 6).
The developmental processes involve several interesting pathways as revealed by analysis of KEGG pathway genes of the differentially expressed transcripts (Figure 5). The genes that were differentially expressed between developmental stages largely represented pathways involved in processing of genetic information such as translation and folding, sorting and degradation of RNA and proteins, and specific metabolisms such as carbohydrate metabolism and energy metabolism as well as several signal transduction processes. The developmental processes of genetic information in A. aegypti involved differentially expressed genes related to ribosome, ribosome biogenesis, RNA transport and surveillance of mRNA; these collectively represent different events of protein translation. The post-translational events required for protein folding, sorting and degradation (such as protein processing in the endoplasmic reticulum, proteasome and ubiquitin mediated proteolysis) represented another major component of A. aegypti development. Among the carbohydrate and energy metabolism pathways, sugar metabolism, glycolysis, propanoate metabolism and oxidative phosphorylation related genes were among the top-three ranking KEGG pathways represented by the differentially expressed genes. It is long established that energy metabolism is intricately associated with developmental stages of insects . In addition, lipid metabolism (primarily glycerolipid, sphingolipid and fatty acid metabolism) genes were represented by the differentially expressed transcripts between different stages of development. This is consistent with earlier studies that suggest significance of sugar and lipid metabolism in developmental processes of insects [4, 22, 41–43]. Furthermore, several signal transducing genes were also differentially expressed representing pathways such as Notch signaling, Hedgehog signaling, WNT signaling and others (Additional file 6).
We also observed differential expression of several proteases at different stages of A. aegypti development (Additional file 3). Roles for proteases in the developmental process are known in Xenopus laevis, specific ciliates and arthropod species [44–46]. During earlier development stages of A. aegypti, several genes encoding different types of proteases were significantly differentially expressed but expression changes of the same genes were not significant at the late developmental stages. Two proteasome related genes (AAEL003871 and AAEL007049) were differentially expressed between early pupal to late pupal stages suggesting their possible role in transition from pupal to adult development. Because many proteases have immune related functions in insects and the fact that immunity varies with age , it is possible that proteases may have a significant role in the aging processes of the mosquito. Consistent with that, we also identified differentially expressed genes such as AAEL006571 and AAEL010083 which are associated with the Toll and IMD signaling pathways as well as several ras and rab GTPases (AAEL006091, AAEL012071, AAEL013620, AAEL013139) at different stages of A. aegypti development.
Genes related to odorant binding (e.g. AAEL003525, AAEL003315 and AAEL006424) were differentially expressed only at the onset of adulthood and are likely associated with development of smell and sense related capabilities necessary for host seeking and other behaviors in newly emerged adults. Adult male and female specific developmental genes were particularly interesting. We found three important gene functions that were significantly associated with differentially expressed genes between males and females. They included genes related to intracellular protein transport (AAEL006091, AAEL003106), vesicle-mediated transport (AAEL003106, AAEL014423) and DNA replication (AAEL012826, AAEL007457, and AAEL010644). Many of these, particularly genes related to actin cytoskeleton organization (AAEL012283), cellular component organization (AAEL012283), receptor activity (AAEL009110) as well as genes related to intracellular protein trafficking (AAEL003106, AAEL006091) play roles in the innate immune response, including response to dengue virus infection , in the adult mosquitoes. Because only adult females transmit disease causing pathogens to vertebrates, such as different flaviviruses to vertebrate hosts, differential expression of such genes between males and females may reflect in part their roles in determining vector competence to different pathogens in adult females. Furthermore, application of insecticides such as pyrethroids and organ-ophosphates is routinely practiced to control of A. aegypti larval and adult, respectively. Research suggests that resistance developed to these compounds can have confounding effects on development of A. aegypti.
A comparative genomic analysis between D. melanogaster and mosquito developmental pathways identified several key genes that reflect conserved developmental processes in mosquitoes as well fruit flies . Two particular genes, 14-3-3zeta (AAEL006885) and modifier of mdg4 (AAEL010576), which are conserved as 1:1 orthologs between D. melanogaster and A. aegypti (also represented as single copy orthologs in the A. gambiae and C. quinquefasciatus genomes) were significantly up-regulated during the transition from early larval to late larval stages of the mosquito. Identification of these genes and pathways related to development implies key roles for these genes in evolution of development within mosquitoes and fruit flies. At the same time, several key genes that are known to play roles in the development of fruit flies were not identified from our study. That can be attributed not only to the fact that we utilized custom cDNA microarrays that do not represent all the annotated genes in A. aegypti, but may also be due in part to extensive divergence of many developmental genes within dipterans [36, 49–51].
In regard to comparison with the A. gambiae developmental transcriptome, our results suggest that conservation of gene expression between the two species decreases as the mosquitoes develop to later developmental stages. The percentage of genes that are conservatively expressed during adult stage is ~5-fold less compared to that we observed at the larval stages between the two mosquitoes. It is likely that many of the genetic components related to developmental processes have undergone evolutionary changes between the two species.
This is the first report on an effort to characterize the developmental transcriptome of A. aegypti. Our results show that genes involved in the developmental programs of this mosquito are highly stage-specific and that the molecular events associated with transitions through the larval, pupal and adult stages are largely discrete. Comparison with the A. gambiae developmental transcriptome suggests that gene expression during developmental processes reflects both common as well as distinct patterns between the two mosquito species.
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