QTL Determining Diel Flight Activity in Male Culex pipiens Mosquitoes.

Members of the Culex pipiens complex differ in physiological traits that facilitate their survival in diverse environments. Assortative mating within the complex occurs in some regions where autogenous (the ability to lay a batch of eggs without a blood meal) and anautogenous populations are sympatric, and differences in mating behaviors may be involved. For example, anautogenous populations mate in flight/swarms, while autogenous populations often mate at rest. Here, we characterized flight activity of males and found that anautogenous strain males were crepuscular, while autogenous strain males were crepuscular and nocturnal, with earlier activity onset times. We conducted quantitative trait locus (QTL) mapping to explore the genetic basis of circadian chronotype (crepuscular vs. crepuscular and nocturnal) and time of activity onset. One major-effect QTL was identified for chronotype, while 3 QTLs were identified for activity onset. The highest logarithm of the odds (LOD) score for the chronotype QTL coincides with a chromosome 3 marker that contains a 15-nucleotide indel within the coding region of the canonical clock gene, cryptochrome 2. Sequencing of this locus in 7 different strains showed that the C-terminus of CRY2 in the autogenous forms contain deletions not found in the anautogenous forms. Consequently, we monitored activity in constant darkness and found males from the anautogenous strain exhibited free running periods of ~24 h while those from the autogenous strain were ~22 h. This study provides novel insights into the genetic basis of flight behaviors that likely reflect adaptation to their distinct ecological niches.

Yeast interfering RNA larvicides targeting neural genes induce high rates of Anopheles larval mortality.

BACKGROUND: Although larviciding can reduce the number of outdoor biting malaria vector mosquitoes, which may help to prevent residual malaria transmission, the current larvicide repertoire is faced with great challenges to sustainability. The identification of new effective, economical, and biorational larvicides could facilitate maintenance and expansion of the practice of larviciding in integrated malaria vector mosquito control programmes. Interfering RNA molecules represent a novel class of larvicides with untapped potential for sustainable mosquito control. This investigation tested the hypothesis that short interfering RNA molecules can be used as mosquito larvicides. RESULTS: A small interfering RNA (siRNA) screen for larval lethal genes identified siRNAs corresponding to the Anopheles gambiae suppressor of actin (Sac1), leukocyte receptor complex member (lrc), and offtrack (otk) genes. Saccharomyces cerevisiae (baker's yeast) was engineered to produce short hairpin RNAs (shRNAs) for silencing of these genes. Feeding larvae with the engineered yeasts resulted in silenced target gene expression, a severe loss of neural synapses in the larval brain, and high levels of larval mortality. The larvicidal activities of yeast interfering RNA larvicides were retained following heat inactivation and drying of the yeast into user-friendly tablet formulations that induced up to 100% larval mortality in laboratory trials. CONCLUSIONS: Ready-to-use dried inactivated yeast interfering RNA larvicide tablets may someday be an effective and inexpensive addition to malaria mosquito control programmes and a valuable, biorational tool for addressing residual malaria transmission.

Identification of a major Quantitative Trait Locus determining resistance to the organophosphate temephos in the dengue vector mosquito Aedes aegypti.

Organophosphate insecticides (OP) have extensively been used to control mosquitoes, such as the vector Aedes aegypti. Unfortunately, OP resistance has hampered control programs worldwide. We used Quantitative Trait Locus (QTL) mapping to evaluate temephos resistance in two F1 intercross populations derived from crosses between a resistant Ae. aegypti strain (RecR) and two susceptible strains (MoyoD and Red). A single major effect QTL was identified on chromosome 2 of both segregating populations, named rtt1 (resistance to temephos 1). Bioinformatics analyses identified a cluster of carboxylesterase genes (CCE) within the rtt1 interval. qRT-PCR demonstrated that different CCEs were up-regulated in F2 resistant individuals from both crosses. However, none exceeded the 2-fold expression. Primary mechanisms for temephos resistance may vary between Ae. aegypti populations, yet also appear to support previous findings suggesting that multiple linked esterase genes may contribute to temephos resistance in the RecR strain as well as other populations.

High-throughput cis-regulatory element discovery in the vector mosquito Aedes aegypti.

BACKGROUND: Despite substantial progress in mosquito genomic and genetic research, few cis-regulatory elements (CREs), DNA sequences that control gene expression, have been identified in mosquitoes or other non-model insects. Formaldehyde-assisted isolation of regulatory elements paired with DNA sequencing, FAIRE-seq, is emerging as a powerful new high-throughput tool for global CRE discovery. FAIRE results in the preferential recovery of open chromatin DNA fragments that are not bound by nucleosomes, an evolutionarily conserved indicator of regulatory activity, which are then sequenced. Despite the power of the approach, FAIRE-seq has not yet been applied to the study of non-model insects. In this investigation, we utilized FAIRE-seq to profile open chromatin and identify likely regulatory elements throughout the genome of the human disease vector mosquito Aedes aegypti. We then assessed genetic variation in the regulatory elements of dengue virus susceptible (Moyo-S) and refractory (Moyo-R) mosquito strains. RESULTS: Analysis of sequence data obtained through next generation sequencing of FAIRE DNA isolated from A. aegypti embryos revealed >121,000 FAIRE peaks (FPs), many of which clustered in the 1 kb 5' upstream flanking regions of genes known to be expressed at this stage. As expected, known transcription factor consensus binding sites were enriched in the FPs, and of these FoxA1, Hunchback, Gfi, Klf4, MYB/ph3 and Sox9 are most predominant. All of the elements tested in vivo were confirmed to drive gene expression in transgenic Drosophila reporter assays. Of the >13,000 single nucleotide polymorphisms (SNPs) recently identified in dengue virus-susceptible and refractory mosquito strains, 3365 were found to map to FPs. CONCLUSION: FAIRE-seq analysis of open chromatin in A. aegypti permitted genome-wide discovery of CREs. The results of this investigation indicate that FAIRE-seq is a powerful tool for identification of regulatory DNA in the genomes of non-model organisms, including human disease vector mosquitoes.

Multiple QTL Determine Dorsal Abdominal Scale Patterns in the Mosquito Aedes aegypti.

The mosquito, Aedes aegypti (L.) originated in Sub-Saharan Africa as a dark form sylvan species (A. aegypti formosus). Evolution of A. aegypti aegypti type form as a human commensal facilitated its colonization of most semitropical and tropical areas. We investigated the genetic basis for abdominal white scale presence that represents the diagnostic for sylvan A. aegypti formosus (scales absent), from type form (scales present) and A. aegypti queenslandensis form (dense scaling). We performed quantitative trait locus (QTL) mapping using 3 criteria for scale patterns among 192 F1 intercross progeny from matings between a queenslandensis type and an aegypti type form. Results identified 3 QTL determining scale patterns and indicated that classification criteria impact robustness of QTL LOD support. Dark- and light-colored forms exist in sympatry, but vary in multiple phenotypic characteristics, including preferences for vertebrate host, oviposition container, house-entering behavior, and dengue vector competence. Markers associated with 2 QTL regions reflected major reductions in recombination frequencies compared with the standard type form linkage map, suggestive of inversion polymorphisms associated with observed linkage disequilibrium between type-specific characteristics. Understanding the genic basis for differences in A. aegypti forms could inform efforts to develop new mosquito and arboviral disease control strategies.

Whole transcriptome responses among females of the filariasis and arbovirus vector mosquito Culex pipiens implicate TGF-ß signaling and chromatin modification as key drivers of diapause induction.

Culex pipiens mosquitoes are important disease vectors inhabiting temperate zones, worldwide. The seasonal reduction in temperature and photoperiod accompanying late summer and early fall prompts female mosquitoes to enter diapause, a stage of developmental arrest and physiological conditioning that enhances survival during the winter months. To investigate the molecular mechanisms underlying diapause induction, we used custom whole transcriptome microarrays to identify differences in gene expression following exposure to nondiapause (long days, 25 °C) and diapause-inducing (short days, 18 °C) environmental conditions. Using a two-way ANOVA, we identified 1130 genes that were differentially expressed. We used the expression of these genes across three time points to construct a gene co-expression network comprising five modules. Genes in modules 1, 2, and 3 were largely up-regulated, while genes in modules 4 and 5 were down-regulated when compared to nondiapause conditions. Pathway enrichment analysis of the network modules revealed some potential regulatory mechanisms driving diapause induction. Module 1 was enriched for genes in the TGF-ß and Wnt signaling pathways; module 2 was enriched for genes involved in insect hormone biosynthesis, specifically, ecdysone synthesis; module 3 was enriched for genes involved in chromatin modification; and module 5 was enriched for genes in the circadian rhythm pathway. Our results suggest that TGF-ß signaling and chromatin modification are key drivers for the integration of environmental signals into the diapause induction phase in C. pipiens mosquitoes.

P53-mediated rapid induction of apoptosis conveys resistance to viral infection in Drosophila melanogaster.

Arthropod-borne pathogens account for millions of deaths each year. Understanding the genetic mechanisms controlling vector susceptibility to pathogens has profound implications for developing novel strategies for controlling insect-transmitted infectious diseases. The fact that many viruses carry genes that have anti-apoptotic activity has long led to the hypothesis that induction of apoptosis could be a fundamental innate immune response. However, the cellular mechanisms mediating the induction of apoptosis following viral infection remained enigmatic, which has prevented experimental verification of the functional significance of apoptosis in limiting viral infection in insects. In addition, studies with cultured insect cells have shown that there is sometimes a lack of apoptosis, or the pro-apoptotic response happens relatively late, thus casting doubt on the functional significance of apoptosis as an innate immunity. Using in vivo mosquito models and the native route of infection, we found that there is a rapid induction of reaper-like pro-apoptotic genes within a few hours following exposure to DNA or RNA viruses. Recapitulating a similar response in Drosophila, we found that this rapid induction of apoptosis requires the function of P53 and is mediated by a stress-responsive regulatory region upstream of reaper. More importantly, we showed that the rapid induction of apoptosis is responsible for preventing the expression of viral genes and blocking the infection. Genetic changes influencing this rapid induction of reaper-like pro-apoptotic genes led to significant differences in susceptibility to viral infection.

Genomic composition and evolution of Aedes aegypti chromosomes revealed by the analysis of physically mapped supercontigs.

BACKGROUND: An initial comparative genomic study of the malaria vector Anopheles gambiae and the yellow fever mosquito Aedes aegypti revealed striking differences in the genome assembly size and in the abundance of transposable elements between the two species. However, the chromosome arms homology between An. gambiae and Ae. aegypti, as well as the distribution of genes and repetitive elements in chromosomes of Ae. aegypti, remained largely unexplored because of the lack of a detailed physical genome map for the yellow fever mosquito. RESULTS: Using a molecular landmark-guided fluorescent in situ hybridization approach, we mapped 624 Mb of the Ae. aegypti genome to mitotic chromosomes. We used this map to analyze the distribution of genes, tandem repeats and transposable elements along the chromosomes and to explore the patterns of chromosome homology and rearrangements between Ae. aegypti and An. gambiae. The study demonstrated that the q arm of the sex-determining chromosome 1 had the lowest gene content and the highest density of minisatellites. A comparative genomic analysis with An. gambiae determined that the previously proposed whole-arm synteny is not fully preserved; a number of pericentric inversions have occurred between the two species. The sex-determining chromosome 1 had a higher rate of genome rearrangements than observed in autosomes 2 and 3 of Ae. aegypti. CONCLUSIONS: The study developed a physical map of 45% of the Ae. aegypti genome and provided new insights into genomic composition and evolution of Ae. aegypti chromosomes. Our data suggest that minisatellites rather than transposable elements played a major role in rapid evolution of chromosome 1 in the Aedes lineage. The research tools and information generated by this study contribute to a more complete understanding of the genome organization and evolution in mosquitoes.

Role of semaphorin-1a in the developing visual system of the disease vector mosquito Aedes aegypti.

BACKGROUND: Despite the devastating impact of mosquito-borne illnesses on human health, very little is known about mosquito developmental biology, including development of the mosquito visual system. Mosquitoes possess functional adult compound eyes as larvae, a trait that makes them an interesting model in which to study comparative developmental genetics. Here, we functionally characterize visual system development in the dengue and yellow fever vector mosquito Aedes aegypti, in which we use chitosan/siRNA nanoparticles to target the axon guidance gene semaphorin-1a (sema1a). RESULTS: Immunohistochemical analyses revealed the progression of visual sensory neuron targeting that results in generation of the retinotopic map in the mosquito optic lobe. Loss of sema1a function led to optic lobe phenotypes, including defective targeting of visual sensory neurons and failed formation of the retinotopic map. These sema1a knockdown phenotypes correlated with behavioral defects in larval photoavoidance. CONCLUSIONS: The results of this investigation indicate that Sema1a is required for optic lobe development in A. aegypti and highlight the behavioral importance of a functioning visual system in preadult mosquitoes.

Influence of mosquito genotype on transcriptional response to dengue virus infection.

The mosquito Aedes aegypti is the principal vector that transmits dengue virus (DENV) to humans. The primary factors that trigger a susceptible or refractory interaction of A. aegypti with DENV are not well understood. In this study, our aim is to characterize the influence of vector genotype on differential gene expression of susceptible vs. refractory A. aegypti strains to DENV infection. To accomplish that, we identified differential expression of a set of complementary DNAs (cDNAs; n = 9,504) of the D2S3 (susceptible) and Moyo-D (refractory) strains of A. aegypti to DENV serotype 2 (JAM1409) and compared these results to the differential expression of cDNAs in a different susceptible vector genotype (Moyo-S) relative to the same refractory genotype (Moyo-D) identified from our previous study. We observed that, although the number of differentially expressed transcripts (DETs) was similar in both the studies, about ~95% of the DETs were distinct between Moyo-D/D2S3 vs. Moyo-D/Moyo-S. This suggested that A. aegypti response, to infection of a given genotype of dengue, is largely dependent upon the vector genotype. However, we observed a set of common DETs among the vector strains that were associated with predicted functions such as endocytosis, regulation of autophagy, peroxisome, and lipid metabolism that may be relatively universal in conferring mosquito response to DENV infection.

Bicluster pattern of codon context usages between flavivirus and vector mosquito Aedes aegypti: relevance to infection and transcriptional response of mosquito genes.

The mosquito Aedes aegypti is the primary vector of dengue virus (DENV) infection in most of the subtropical and tropical countries. Besides DENV, yellow fever virus (YFV) is also transmitted by A. aegypti. Susceptibility of A. aegypti to West Nile virus (WNV) has also been confirmed. Although studies have indicated correlation of codon bias between flaviviridae and their animal/insect hosts, it is not clear if codon sequences have any relation to susceptibility of A. aegypti to DENV, YFV and WNV. In the current study, usages of codon context sequences (codon pairs for neighboring amino acids) of the vector (A. aegypti) genome as well as the flaviviral genomes are investigated. We used bioinformatics methods to quantify codon context bias in a genome-wide manner of A. aegypti as well as DENV, WNV and YFV sequences. Mutual information statistics was applied to perform bicluster analysis of codon context bias between vector and flaviviral sequences. Functional relevance of the bicluster pattern was inferred from published microarray data. Our study shows that codon context bias of DENV, WNV and YFV sequences varies in a bicluster manner with that of specific sets of genes of A. aegypti. Many of these mosquito genes are known to be differentially expressed in response to flaviviral infection suggesting that codon context sequences of A. aegypti and the flaviviruses may play a role in the susceptible interaction between flaviviruses and this mosquito. The bias in usages of codon context sequences likely has a functional association with susceptibility of A. aegypti to flaviviral infection. The results from this study will allow us to conduct hypothesis-driven tests to examine the role of codon context bias in evolution of vector-virus interactions at the molecular level.

Inheritance of Resistance to Deltamethrin in Aedes aegypti (Diptera: Culicidae) From Cuba.

The development of pyrethroid resistance in Aedes aegypti (L) (Diptera: Culicidae) is a serious concern because major A. aegypti control programs are predominantly based on pyrethroid use during epidemic disease outbreaks. Research about the genetic basis for pyrethroid resistance and how it is transmitted among mosquito populations is needed. The objective of this study was to determine how deltamethrin resistance is inherited in the Cuban A. aegypti-resistant reference strain. Here, a field population of A. aegypti from Santiago de Cuba (SAN-F14), subjected to 14 generations of selection for high deltamethrin resistance level (91.25×), was used to prepare reciprocal F1 and backcross progeny with the insecticide-susceptible Rockefeller strain. Bioassays with larvae were performed according to World Health Organization guidelines. The activities of metabolic enzymes were assayed through synergist and biochemical tests. The null hypothesis of the parallelism test between the two probit regression lines of the reciprocal F1 (susceptible females × resistant males and vice versa) was not rejected at the 5% significance level (P = 0.42), indicating autosomal inheritance. The LC50 response of both F1 progenies to deltamethrin was elevated but less than the highly resistant SAN-F14 strain. DLC values for the F1 progenies were 0.91 and 0.87, respectively, suggesting that deltamethrin resistance in the SAN-F14 strain is inherited as an autosomal incompletely dominant trait, involving at least two factors, which implies a faster development of deltamethrin resistance in larvae and lost product effectiveness. Metabolic enzymes including esterases and cytochrome P-450 monooxygenases but not glutathione-S-transferases were involved in deltamethrin resistance in larvae.

Insecticide Resistance and Metabolic Mechanisms Involved in Larval and Adult Stages of Aedes aegypti Insecticide-Resistant Reference Strains from Cuba.

Studies were conducted to compare levels of insecticide resistance and to determine the metabolic resistance mechanisms in larval and adult stages of Aedes aegypti from Cuba. Three insecticide-resistant reference strains of Ae. aegypti from Cuba were examined. These strains were derived from a Santiago de Cuba strain isolated in 1997; it was previously subjected to a strong selection for resistance to temephos (SAN-F6), deltamethrin (SAN-F12), and propoxur (SAN-F13) and routinely maintained in the laboratory under selection pressure up to the present time, when the study was carried out. In addition, an insecticide-susceptible strain was used for comparison. The insecticide resistance in larvae and adults was determined using standard World Health Organization methodologies. Insecticide resistance mechanisms were determined by biochemical assays. The esterases (α EST and ß EST) and mixed function oxidase (MFO) activities were significantly higher in adults than in the larvae of the three resistant strains studied. The association of resistance level with the biochemical mechanism for each insecticide was established for each stage. The observed differences between larval and adult stages of Ae. aegypti in their levels of insecticide resistance and the biochemical mechanisms involved should be included as part of monitoring and surveillance activities in Ae. aegypti vector control programs.

Requirement for commissureless2 function during dipteran insect nerve cord development.

BACKGROUND: In Drosophila melanogaster, commissureless (comm) function is required for proper nerve cord development. Although comm orthologs have not been identified outside of Drosophila species, some insects possess orthologs of Drosophila comm2, which may also regulate embryonic nerve cord development. Here, this hypothesis is explored through characterization of comm2 genes in two disease vector mosquitoes. RESULTS: Culex quinquefasciatus (West Nile and lymphatic filiariasis vector) has three comm2 genes that are expressed in the developing nerve cord. Aedes aegypti (dengue and yellow fever vector) has a single comm2 gene that is expressed in commissural neurons projecting axons toward the midline. Loss of comm2 function in both A. aegypti and D. melanogaster was found to result in loss of commissure defects that phenocopy the frazzled (fra) loss of function phenotypes observed in both species. Loss of fra function in either insect was found to result in decreased comm2 transcript levels during nerve cord development. CONCLUSIONS: The results of this investigation suggest that Fra down-regulates repulsion in precrossing commissural axons by regulating comm2 levels in both A. aegypti and D. melanogaster, both of which require Comm2 function for proper nerve cord development.

GIS-Enhanced Survey of Potential Aedes aegypti and Aedes albopictus Artificial Oviposition Containers Distributed across Communities in Trinidad, West Indies.

Dengue and other arboviruses remain a global threat, and enhanced efforts to control the mosquitoes that transmit them are urgently needed. A survey of potential manmade Aedes aegypti (L.) and Aedes albopictus (Skuse) oviposition containers was performed in four communities near the end of the typical dry season in 2018 in Trinidad, West Indies. The purpose was to conduct individual premise surveys and use GIS mapping to visualize premises within communities that had Aedes-positive containers, as this information could be used for the prioritization of mosquito control efforts in potential high risk areas as the wet season progressed. Accessible premises were surveyed following standard inspection protocols used by the Insect Vector Control Division (IVCD), Ministry of Health (MOH). The results indicated that two of the four locations would be at high risk for arbovirus transmission going into the wet season. The GIS mapping of premises with Aedes-positive containers facilitated the identification of potential hot spots for arbovirus transmission risk within communities that should be prioritized for enhanced monitoring and vector control efforts, emphasizing the need to increase community participation in standard surveys by IVCD.

DNA Barcoding Analysis of Trinidad Haemagogus Mosquitoes Reveals Evidence for Putative New Species.

Background: Haemagogus janthinomys is a primary sylvan vector of yellow fever virus and the emerging Mayaro virus. However, despite its medical importance, there is a dearth of data on the molecular taxonomy of this mosquito species. Methods: In this study, DNA barcoding analysis was performed on 64 adult female mosquitoes from Trinidad morphologically identified as Hg. janthinomys. The mitochondrial cytochrome c oxidase I (COI) gene and ribosomal DNA internal transcribed spacer 2 (ITS2) region of the mosquitoes were PCR amplified and sequenced, and molecular phylogenies inferred. Results: The BLASTN analysis showed that only 20% (n = 13/66) of COI sequences had high similarity (>99% identity) to Hg. janthinomys and the remaining sequences had low similarity (<90% identity) to reference GenBank sequences. Phylogenetic analysis of COI sequences revealed the presence of four strongly supported groups, with one distinct clade that did not align with any reference sequences. Corresponding ITS2 sequences for samples in this distinct COI group clustered into three clades. Conclusions: These molecular findings suggest the existence of a putative new Haemagogus mosquito species and underscore the need for further, more in-depth investigations into the taxonomy and classification of the Haemagogus genus.

Overlapping genes of Aedes aegypti: evolutionary implications from comparison with orthologs of Anopheles gambiae and other insects.

BACKGROUND: Although gene overlapping is a common feature of prokaryote and mitochondria genomes, such genes have also been identified in many eukaryotes. The overlapping genes in eukaryotes are extensively rearranged even between closely related species. In this study, we investigated retention and rearrangement of positionally overlapping genes between the mosquitoes Aedes aegypti (dengue virus vector) and Anopheles gambiae (malaria vector). The overlapping gene pairs of A. aegypti were further compared with orthologs of other selected insects to conduct several hypothesis driven investigations relating to the evolution and rearrangement of overlapping genes. RESULTS: The results show that as much as ~10% of the predicted genes of A. aegypti and A. gambiae are localized in positional overlapping manner. Furthermore, the study shows that differential abundance of introns and simple sequence repeats have significant association with positional rearrangement of overlapping genes between the two species. Gene expression analysis further suggests that antisense transcripts generated from the oppositely oriented overlapping genes are differentially regulated and may have important regulatory functions in these mosquitoes. Our data further shows that synonymous and non-synonymous mutations have differential but non-significant effect on overlapping localization of orthologous genes in other insect genomes. CONCLUSION: Gene overlapping in insects may be a species-specific evolutionary process as evident from non-dependency of gene overlapping with species phylogeny. Based on the results, our study suggests that overlapping genes may have played an important role in genome evolution of insects.

Stage-specific transcription during development of Aedes aegypti.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Association of microsatellite pairs with segmental duplications in insect genomes.

BACKGROUND: Segmental duplications (SDs), also known as low-copy repeats, are DNA sequences of length greater than 1 kb which are duplicated with a high degree of sequence identity (greater than 90%) causing instability in genomes. SDs are generally found in the genome as mosaic forms of duplicated sequences which are generated by a two-step process: first, multiple duplicated sequences are aggregated at specific genomic regions, and then, these primary duplications undergo multiple secondary duplications. However, the mechanism of how duplicated sequences are aggregated in the first place is not well understood. RESULTS: By analyzing the distribution of microsatellite sequences among twenty insect species in a genome-wide manner it was found that pairs of microsatellites along with the intervening sequences were duplicated multiple times in each genome. They were found as low copy repeats or segmental duplications when the duplicated loci were greater than 1 kb in length and had greater than 90% sequence similarity. By performing a sliding-window genomic analysis for number of paired microsatellites and number of segmental duplications, it was observed that regions rich in repetitive paired microsatellites tend to get richer in segmental duplication suggesting a "rich-gets-richer" mode of aggregation of the duplicated loci in specific regions of the genome. Results further show that the relationship between number of paired microsatellites and segmental duplications among the species is independent of the known phylogeny suggesting that association of microsatellites with segmental duplications may be a species-specific evolutionary process. It was also observed that the repetitive microsatellite pairs are associated with gene duplications but those sequences are rarely retained in the orthologous genes between species. Although some of the duplicated sequences with microsatellites as termini were found within transposable elements (TEs) of Drosophila, most of the duplications are found in the TE-free and gene-free regions of the genome. CONCLUSION: The study clearly suggests that microsatellites are instrumental in extensive sequence duplications that may contribute to species-specific evolution of genome plasticity in insects.

Nucleotide substitutions in dengue virus serotypes from Asian and American countries: insights into intracodon recombination and purifying selection.

BACKGROUND: Dengue virus (DENV) infection represents a significant public health problem in many subtropical and tropical countries. Although genetically closely related, the four serotypes of DENV differ in antigenicity for which cross protection among serotypes is limited. It is also believed that both multi-serotype infection as well as the evolution of viral antigenicity may have confounding effects in increased dengue epidemics. Numerous studies have been performed that investigated genetic diversity of DENV, but the precise mechanism(s) of dengue virus evolution are not well understood. RESULTS: We investigated genome-wide genetic diversity and nucleotide substitution patterns in the four serotypes among samples collected from different countries in Asia and Central and South America and sequenced as part of the Genome Sequencing Center for Infectious Diseases at the Broad Institute. We applied bioinformatics, statistical and coalescent simulation methods to investigate diversity of codon sequences of DENV samples representing the four serotypes. We show that fixation of nucleotide substitutions is more prominent among the inter-continental isolates (Asian and American) of serotypes 1, 2 and 3 compared to serotype 4 isolates (South and Central America) and are distributed in a non-random manner among the genes encoded by the virus. Nearly one third of the negatively selected sites are associated with fixed mutation sites within serotypes. Our results further show that of all the sites showing evidence of recombination, the majority (~84%) correspond to sites under purifying selection in the four serotypes. The analysis further shows that genetic recombination occurs within specific codons, albeit with low frequency (< 5% of all recombination sites) throughout the DENV genome of the four serotypes and reveals significant enrichment (p < 0.05) among sites under purifying selection in the virus. CONCLUSION: The study provides the first evidence for intracodon recombination in DENV and suggests that within codons, genetic recombination has a significant role in maintaining extensive purifying selection of DENV in natural populations. Our study also suggests that fixation of beneficial mutations may lead to virus evolution via translational selection of specific sites in the DENV genome.