BACKGROUND
gene rearrangement is one of the necessary ingredients of genome evolution.
several well studied mechanisms such as chromosomal inversions, translocations, duplications and transpositions are known to have important roles in genomic rearrangement events  <cit> .
reshuffling of genomic dna by gross chromosomal rearrangements generally involves a number of genes that undergo positional relocation in the genome.
in addition to such large scale genomic rearrangements, genomic rearrangements at small scale levels facilitate relocation of genes which are otherwise positionally overlapping in a genome  <cit> .
it has been suggested that transposition mechanisms may contribute to such gene arrangements  <cit> , but the functional and evolutionary significance of such events is largely unknown.
positional overlapping between genes is a common structural feature of prokaryote and mitochondria genomes  <cit> .
however, overlapping genes have also been identified from whole genome sequences of several eukaryotes such as fruit fly, zebrafish, human, chimpanzee, orangutan, marmoset, rhesus, cow, dog, mouse, rat and chicken  <cit> .
studies show that overlapping genes in eukaryotes are extensively rearranged even between closely related species  <cit> .
bhutkar et al.
<dig>  <cit>  compared overlapping genes of drosophila melanogaster and anopheles gambiae with apis mellifera  and suggested that relocalization of overlapping genes may have played a significant role in genome evolution of these insects.
although several other insect genome sequences are now available, overlapping genes of most of these insects have not been studied.
the present study is an effort to investigate overlapping genes of aedes aegypti, the primary global vector of dengue virus, in a comparative manner with those of a. gambiae, a major vector of malaria in subsaharan africa.
understanding genome structure of these mosquitoes has become one of the major interests among insect vector biologists.
at present, the draft genome sequences for three mosquito species have been completed  <cit> .
these projects  have provided new insights on structure, function and evolution of mosquito genes, thus furthering our ability to study mosquito-parasite or mosquito-virus interactions at the molecular level  <cit> .
we identified positional overlapping of genes at the whole genome level in a. aegypti and studied structural differences and evolutionary features by comparisons with orthologous genes of a. gambiae and other selected arthropod genomes.
the primary aim was to test several common hypotheses relating to rearrangement of overlapping genes and determine factors that may have a role in relocalization of overlapping genes in insects.
the results of our investigation show that positional overlapping among genes is a species specific evolutionary process as evident from non-dependency of gene overlapping with species phylogeny, and also show that specific factors, such as introns and repeat sequences, are significantly associated with retention/rearrangement of overlapping genes in mosquitoes.
based on these results, our study suggests that overlapping genes may have played an important role in genome evolution among insects.
methods
official gene sets and extraction of overlapping gene pairs
the overlapping gene pairs of a. aegypti and a. gambiae were identified in a genome-wide manner based on the coordinates of gene boundaries of official gene sets annotated from the genome assemblies.
the other mosquito genome sequence for culex quinquefasciatus was not used for this purpose because of differences in gene annotation of this species compared to a. aegypti or a. gambiae.
that is, while nearly equally percentages  of the official gene sets of a. aegypti as well as a. gambiae have been annotated for gene boundaries that incorporated the 5’ and 3’ untranslated regions, only 15% of the c. quinquefasciatus genes have been annotated in this manner.
thus, incorporating c. quinquefasciatus could have produced biased results in the genome-wide comparison of overlapping gene pairs between a. aegypti and a. gambiae.
however, we have used orthologs of a. aegypti overlapping gene pairs in c. quinquefasciatus and other selected insect species such as drosophila melanogaster, apis mellifera, pediculus humanus, bombyx mori and acyrthosiphon pisum to determine if they are also localized in overlapping positions in the respective genomes.
for genome-wide comparison of overlapping genes, the predicted gene sets of a. aegypti  and a. gambiae  along with coordinates of genes in the reference genome were downloaded from vectorbase .
the one-to-one orthologous genes  were compared to determine if they were also present in overlapping gene pairs across multiple genomes.
to determine the relative position of the orthologous genes, the official gene lists along with their start and end positions in the genome sequences of the other six insects  were downloaded from either vectorbase  or the silkdb database  or the ‘ensembl metazoa 10’ data sets at http://www.biomart.org.
intron analysis
to determine if introns have an association with overlapping between genes, orthologous genes were categorized as intronless and intron-containing genes for overlapping and non-overlapping pairs in the a. aegypti and a. gambiae genomes.
the exon structures predicted for a. aegypti and a. gambiae genes  were used to classify genes into single exon genes  and multi exon genes .
the number of introns in each gene was determined from the number of exons annotated in the genes.
the 2x <dig> contingency analysis of counts of the intronless and intron-containing genes of both categories  was performed using yates chi square tests to determine significance of association between introns and gene overlapping.
transcriptional analysis of overlapping genes
the expressed sequence tags  of a. aegypti and a. gambiae mosquitoes used in this study were largely generated in conjunction with the individual genome sequencing projects .
these ests were used to assist in the annotation of the official gene sets of the two mosquitoes.
we used these ests to investigate expression patterns associated with the overlapping gene pairs.
to further confirm correspondence of ests with overlapping gene pairs, we performed reciprocal blast analyses described as follows.
the est sequences were used to generate a local blast database and then searched by blastn with the sequences of overlapping genes.
the est ‘hits’ that had an e-value =  <dig> were used again as queries in another blastn search against all predicted gene sequences.
if the reciprocal hits matched the same gene that was used as a query in the first blast, it was considered that the est corresponded to that gene.
apart from analyzing the est data, we also analyzed previously performed microarray expression data of a. aegypti <cit>  to determine expression patterns of the overlapping gene pairs.
the a. gambiae microarray expression data was obtained from baker et al.
<dig> study  <cit> .
the expression data of these studies  <cit>  are publicly available with gene expression omnibus  accession # gse <dig> and gse <dig> at http://www.ncbi.nlm.nih.gov/geo/.
the spearman’s rank correlation test was conducted to ascertain whether the overlapping gene pairs had significantly correlated expression levels throughout the genome.
identification of microsatellites in overlapping genes
in order to determine if there is a significant association of microsatellites with retention or rearrangement of overlapping gene structures between a. aegypti and a. gambiae, we identified microsatellite sequences within the gene pairs in both genomes.
sciroko, a simple sequence repeat  identification program  <cit> , was used to detect both perfect and imperfect mono-, di-, tri-, tetra- and hexa-nucleotide repeats using the default parameters .
the repeats with more than  <dig> consecutive mismatch sites were excluded.
the genes where one or more sites were ambiguous nucleotides  were not used to report microsatellites.
the length of orthologous genes may vary  that may contribute to varying amounts of microsatellite sequences in the orthologous gene copies.
so, instead of comparing the absolute amounts of microsatellite sequences, their relative amounts were compared.
the relative amounts were obtained from the total amount of microsatellites of genes normalized with the alignment length  of the orthologous genes between a. aegypti and a. gambiae.
statistical and computational analyses
all statistical analyses were performed using the r statistical program.
the p-value <  <dig>  was considered statistical significance in all tests unless stated otherwise.
cluster analyses of gene pairs based on overlapping or non-overlapping structures across genomes were based on average correlation of city-block distance estimated using the cluster <dig> program  <cit> .
the phylogenetic analyses were performed by neighbor-joining method using mega <dig>  <cit> .
the evolutionary distances were in the units of the number of base substitutions per site; and they were calculated using the maximum composite likelihood method  <cit> .
the mantel procedure  <cit>  was used to perform linear regression between matrices where the dependent matrix  was permutated  <dig> times to test significance of the observed correlation with the independent matrix  in the genomes used for comparison.
the multi mantel procedure was performed using an algorithm developed by dr. liam j. revell .
maximum likelihood methods described elsewhere  <cit>  were used to estimate the log likelihoods of models assuming either dependency or non-dependency of gene phylogeny with the discrete variation of gene traits .
the likelihood ratio tests were conducted to infer statistical significance of these two models.
a binary logit model was developed to test marginal effects of the rates of synonymous  and non-synonymous  mutations in the orthologous gene pairs between a. aegypti and other select insect genomes .
while each of the gene pairs  were localized in an overlapping manner in the a. aegypti genome, the orthologous genes showed variation in relative localization  in other species.
the dn and ds values of orthologous genes were obtained from metazoan genes database at http://www.biomart.org.
a generalized linear model , fitting the dependent variable  and independent variables , was used in r to estimate the logit coefficients.
RESULTS
identification of overlapping genes
a total of  <dig> and  <dig> overlapping gene pairs were identified in the assembled genomes of a. aegypti and a. gambiae, respectively .
they represent 8-10% of the annotated genes of the two mosquitoes.
the frequencies of overlapping genes of a. aegypti and a. gambiae mosquitoes are within the range of overlapping gene frequencies reported in other eukaryotes  <cit> .
more than two genes  were also found in overlapping locations in both genomes, with the majority of these overlapping gene clusters containing no more than three genes .
these overlapping clustered genes constituted only a minor portion  of the total number of overlapping genes in either of the two genomes.
because of low frequency and also for simplicity of analysis, we have not included the gene clusters in our investigation.
all the analyses performed in this study were based on overlapping gene pairs.
orthology of overlapping genes between a. aegypti and a. gambiae
in a. aegypti as well as in a. gambiae, the overlapping gene pairs are localized either in nested form  or in partially overlapping form  as shown in figure  <dig>  irrespective of whether gene pairs are in e/h or in p/o form, they are predominantly localized in opposite orientation to each other.
three possible patterns of evolution emerged from comparing the orthologs of overlapping genes between the two species: 1) the gene pairs are orthologous between the two species , 2) the gene pair is specific to the species , and 3) one of the genes is specific to the species and the other is common between the two species  .
the number of young-young gene pairs  and the old-old gene pairs  vary between the two species.
the 2x <dig> contingency tests based on the count statistics of old-old and young-young gene pairs between a. aegypti and a. gambiae  shows that there is a significant bias in the distribution of these genes between the two species.
it clearly shows that nearly the same number  of orthologous gene pairs  are present in overlapping manner in both the species, whereas a significantly larger number of a. aegypti specific genes  are in overlapping position compared to a. gambiae .
this shows that the young-young gene pairs show significant variation in overlapping patterns between these mosquitoes.
when the a. aegypti and a. gambiae overlapping genes were compared with d. melanogaster orthologs, consistent patterns were observed .
the data in figure  <dig> shows that the old-old gene pairs  are comparable in numbers between a. aegypti and a. gambiae whereas the young-young gene pairs  vary significantly between the two species.
consistent with the results shown in figure  <dig>  these results also suggest that young genes are major contributors to the positional overlapping of genes in these species.
rearrangement of overlapping genes
one of the common hypotheses about positional overlapping of genes is that selection acts against the retention of gene overlap between genomes  <cit> .
if the above hypothesis is correct, we expect that overlapping genes should be extensively rearranged between a. aegypti and a. gambiae.
to test that expectation, the orthologous  copies of overlapping gene pairs were compared between the two species .
it was found that only  <dig> of the total  <dig> orthologous gene pairs are localized in overlapping manner in both the genomes.
the other  <dig> gene pairs are localized in overlapping manner in one genome but in non-overlapping manner in the other  suggesting that only a fraction of overlapping genes are retained across genomes.
to determine if retention or rearrangement of overlapping localization of genes between a. aegypti and a. gambiae may be associated with loss or gain of terminal exons of genes, we investigated several gene pairs that contain multiple exons in the orthologous gene pairs  and found no discrepancy in annotation of first and last exon of any gene pair between the two species.
the overlapping gene pairs where one gene is embedded in another are represented as e/h pairs.
the gene pairs which are localized in partially overlapping manner with each other are represented as p/o gene pairs.
additionally, we performed specific case studies of retention or rearrangement between the two species.
a short-chain dehydrogenase gene  acts as the host of another protein coding gene aael <dig>  in a. aegypti.
it was found that the corresponding orthologs in a. gambiae genome are also localized in e/h form .
in contrast to this, the gene  of a. aegypti, that codes for a carboxylesterase, is localized in the genome in non-overlapping manner with one of its paralogs  whereas the ortholog in a. gambiae  is localized in p/o manner with the paralog agap <dig> .
these genes associated with retention or rearrangement of positional localization in both the genomes are known to have significant changes in expression during blood feeding, growth and development of these mosquitoes  <cit> .
in another case, we identified multiple genes that are embedded within intron sequences of a single host gene in a. aegypti.
the gene aael <dig> that putatively codes for the protein b-cell lymphoma/leukaemia  harbors  <dig> paralogous genes within one intron along with several other paralogous genes that are localized in non-overlapping manner to aael <dig> .
importantly, these genes have been reported to be expressed in a. aegypti <cit> .
moreover, the phylogenetic tree  shows that the embedded genes tend to cluster together and are phylogenetically distinct from the genes located outside the host genes.
this suggests a contrasting rate of evolution of embedded versus non-embedded paralogous copies of the gene aael <dig> in a. aegypti.
gene overlapping is phylogeny independent
having observed that orthologs of overlapping genes are extensively rearranged between species, we hypothesized that phylogenetic relationship has no correlation with the localization pattern  of orthologous genes across species.
if this is true, one would expect that gene overlapping occurs as a trait which should be independent of species phylogenies.
to test this hypothesis, we performed ‘discrete analysis’ of gene overlapping across phylogeny using maximum likelihood method  <cit>  to determine if presence or absence of gene overlapping is correlated with phylogeny among species.
seven insect species  were compared for orthology analysis with a. aegypti overlapping genes: a. gambiae , c. quinquefasciatus , d. melanogaster , a. mellifera , p. humanus , b. mori  and a. pisum .
the results of this analysis showed that the likelihood estimates of the null model  consistently lack statistical significance when tested against the alternate model  .
it clearly shows that there is no apparent relationship of positional overlapping with the phylogeny of the species.
to illustrate this, a representation of phylogeny and gene overlapping pattern is shown in figure  <dig> for orthologs of a. aegypti gene pair aael009614-aael <dig>  among seven other insect species.
it shows that retention or rearrangement of orthologous genes lacks correspondence with the phylogenetic relationships between species.
the likelihoods of test and null models  and the p-values of log likelihood ratio tests are shown.
by comparisons with predicted orthologous genes among sequenced arthropod genomes , we identified a total of  <dig> overlapping gene pairs of a. aegypti where at least one gene of each pair was also present among the other seven insect species.
but only  <dig> of these gene pairs in a. aegypti had both the genes present as orthologs in all the other seven species .
to further confirm that overlapping or non-overlapping localization of genes has no correspondence with presence or absence of orthologs across genomes, we performed hierarchal cluster analysis among the above  <dig> orthologous gene pairs across the eight species .
the potential for correlation between gene orthology and gene positional overlapping was assessed for statistical significance by mantel test .
the correlation was evaluated between binary data of orthologous genes in matrix forms  with the presence or absence of orthology of the gene pairs.
the results showed non-significant correlation between the two  suggesting that gene orthology has no relationship with overlapping localization of genes across species.
role of selection on rearrangement of overlapping genes
another hypothesis about overlapping genes is that mutations occurring within the shared region of overlapping gene pairs would mostly be negatively selected because such mutations may affect adaptation  <cit>  and function of both genes  <cit> .
if this is true, we expect to see a higher frequency of synonymous  changes than non-synonymous  changes between orthologous genes when they are present in overlapping manner.
to test this expectation, the numbers of per site synonymous and non-synonymous changes in  <dig> orthologous gene pairs  were determined among a. aegypti, c. quinquefasciatus, a. gambiae, d. melanogaster and p. humanus.
the ds and dn values are the rate of synonymous and non-synonymous changes, respectively, between a. aegypti gene and the corresponding ortholog of other species mentioned above.
as shown in additional file  <dig>  each of these gene pairs is localized in overlapping manner in a. aegypti.
but, the orthologous genes in the other species are found either in overlapping or non-overlapping manner.
a binary logit model was developed to fit the data of ds and dn values with the occurrence or non-occurrence of positional overlapping between genes among the species.
the dependent variable assumed a value  <dig> when the genes were found in overlapping position but  <dig> when the gene pairs were in non-overlapping position in the genome.
we performed generalized linear model fitting of the data that is represented by y = β0 + xβ + e, where y = dependent variable , x = ds and dn values of both genes, β = coefficient of independent variable, β <dig> represents the value of y when the predictor is equal to zero, and e  is assumed to be independent of x and has a standard logistic distribution with mean zero.
the ds and dn values of both genes  of a. aegypti gene pairs, calculated by aligning the codon sequence to the orthologs of other insect species , were used as the independent variables.
the results of the regression analysis are shown in table  <dig>  it shows estimates of coefficients of each of the four independent  in fitting the model to explain the occurrence of the dependent  variable .
the estimated regression coefficient shows variation  of the outcome with unit change in the predictor variable .
the data in table  <dig> shows that the coefficients of regression are positive for synonymous changes but negative for non-synonymous changes indicating differential effects of synonymous and non-synonymous mutations on overlapping localization between genes.
however, the effects of ds or dn are statistically non-significant in each case  indicating that, in these insect species , the association of synonymous or non-synonymous mutations with overlapping localization of orthologous genes may be a random event.
however, the lack of significance may also be due to differences in the reading frames of orthologous genes.
such differences are known to be associated with bias in codon phases of overlapping prokaryotic genes  <cit> .
however, we have not determined from this study if there is a bias in codon phase distribution of overlapping genes that may influence the rate of synonymous and non-synonymous changes between orthologs.
the regression was performed in relation to presence or absence of positional overlapping of the orthologous gene pairs across species .
association of microsatellites with gene overlapping
it is well recognized that transposition events contribute to positional rearrangement of genes in eukaryotes  <cit> .
and as transposons are known to be intimately associated with simple sequence repeat elements   <cit> , we hypothesized that microsatellites may have a role in positional overlapping of genes.
thus, one of our aims was to determine if there was a significant association between microsatellite contents with rearrangement of overlapping gene pairs between a. aegypti and a. gambiae.
the amounts  of microsatellite sequences were normalized based on length of shared sequences between e/h gene pairs and their rearranged orthologous pairs in a. aegypti and a. gambiae.
the results of the 2x <dig> contingency tests of these data show that positional rearrangement of e/h gene pairs is significantly associated with the amount of microsatellite sequences within the orthologous genes in the two mosquitoes .
one scenario is that the repeat sequences, represented as common motifs between the two genes , are involved in gene rearrangements possibly by facilitating cross over events associated with exchange of the flanking regions between microsatellites  <cit>  that lead to positional rearrangements of genes.
role of introns in positional overlapping of genes
in a. aegypti and a. gambiae, most of the embedded genes  are localized within introns of host genes.
thus, intron loss/gain could contribute to gene relocalization.
in that case, we expect that intron loss/gain between one-to-one orthologous genes should be significantly associated with rearrangement of overlapping gene pairs between the two mosquito species.
to determine if introns have an association with retention/rearrangement of overlapping genes between the two species, the number of introns among orthologous gene pairs listed in table  <dig> were quantified.
based on count statistics of introns between one-to-one orthologous gene pairs between a. aegypti and a. gambiae, we found that that loss/gain of introns is significantly  associated with retention or rearrangement of overlapping gene pairs between the two mosquitoes .
the rearranged genes show significant loss of introns compared to the orthologous gene pairs located in overlapping positions and vice versa suggesting that introns may have a role in gene overlapping and rearrangement.
although intron-mediated gene recombination  <cit>  and references therein or other mechanisms such as intron-transposition  <cit>  may be likely mechanisms for these processes, further investigations are needed to determine the exact role of introns in positional overlapping of genes.
expression of overlapping genes
overlapping expression of more than one gene is well known in eukaryotes  <cit> ,  <cit> .
we analyzed the expressed sequence tags  datasets of a. aegypti and a. gambiae to determine if overlapping gene pairs may have overlapping transcripts.
using reciprocal blastn searches, we identified several ests of a. aegypti that represented the likely transcription product of overlapping gene pairs .
although many of these gene pairs are oriented in opposite direction to each other, ests were also observed for gene pairs oriented in same direction.
whether these gene pairs are co-transcribed or co-regulated by common upstream/downstream sequences  <cit>  are not known from this study.
however, identification of ests of overlapping gene sequences clearly shows that these sequences are expressed.
moreover, we show that the annotation of overlapping genes is unaffected whether good evidence of expression  is available or not.
the dataset of overlapping genes was also analyzed based on availability or non-availability of est evidence.
we found no significant difference in the number of genes that localized in positionally overlapping manner between the two groups .
in a. gambiae, the est dataset didn’t reveal such transcripts except for a single gene pair.
although transcripts of overlapping genes were not available in the est collections of a. gambiae, we found evidence of expression of these genes  from published microarray data  <cit> .
generally, overlapping transcripts are processed by post-transcriptional events to produce individual transcripts of the genes  <cit> .
to assess the expression level of individual gene transcripts of overlapping gene pairs, we examined the microarray expression data of a. aegypti <cit> .
because overlapping genes are predominantly localized in opposite orientation to each other in the genome , we compared expression level of gene pairs  which are either oppositely oriented or oriented in same direction to each other.
it was found that the expression levels of overlapping genes in opposite orientation lack significant correlation, whereas the overlapping genes which are oriented in the same direction to each other show statistically significant correlation  .
most of these genes code for known proteins and have been annotated with start and stop codons suggesting that these genes are not annotation artifacts, although a few genes were annotated as hypothetical proteins.
nevertheless, these results suggested that when the two genes are localized in overlapping manner and also oriented in the same direction, their expression may be co-regulated leading to similar transcription levels.
on the other hand, when the two genes are localized in overlapping manner, but oriented in the opposite direction, their transcripts may have differential regulation.
in fact, it is well documented that overlapping genes when transcribed in the opposite directions, give rise to sense-antisense transcript pairs which are differentially regulated to play a role in a variety of processes, including mrna splicing and stability, rna editing, genomic imprinting and control of translation .
CONCLUSIONS
the results from this study provide insight into the common prevailing theories of origin and evolution of positionally overlapping genes.
these are particularly important for better understanding of distribution and structure of overlapping genes in the genomes of a. aegypti and a. gambiae.
the genome sequences of both a. gambiae and a. aegypti contain gaps that could affect our estimates of overlapping genes in the genome assemblies, but we find this unlikely based on our observation that the overlapping genes are distributed throughout the genome in each species without any bias to specific chromosomal region of a. gambiae or specific supercontigs of a. aegypti .
furthermore, our estimated frequencies of overlapping genes in mosquitoes are within the range of overlapping gene frequencies reported in other eukaryotes  <cit> .
thus, it is unlikely that there may be large numbers of genes missing because of gaps in sequencing that are positionally overlapping.
nevertheless, the dynamic patterns of positional rearrangement of overlapping genes suggest that these genes may have important roles in genome evolution of vector mosquitoes.
importantly, the information from this investigation may help us in further studies pertaining to evolution and functional characterization of antisense transcripts among overlapping genes in mosquitoes.
availability of supporting data
the manuscript is accompanied with the following listed additional files in the form of supporting data for this study.
competing interests
the authors declare that they have no competing interests.
authors' contributions
conceived and designed the experiments: skb.
analyzed the data: skb.
contributed reagents/materials/analysis tools: skb, dws.
wrote the paper: skb, dws.
both the authors read and approved the final manuscript.
authors' information
skb is a research assistant professor in the department of biological sciences and the eck institute for global health at the university of notre dame, indiana.
he has a broad interest in insect genomics and evolution with emphasis on disease transmitting vector species.
dws is a professor of biological sciences and the director of eck institute for global health at the university of notre dame, indiana.
his work focuses on genetic and genomic analysis of mosquito vector competence to various pathogens as well as on development and application of molecular tools to investigate population biology of mosquitoes.
supplementary material
additional file 1
list of e/h and p/o gene pairs of a. aegypti and a. gambiae.
click here for file
additional file 2
overlapping and non-overlapping patterns of orthologous genes between a. aegypti and a. gambiae.
click here for file
additional file 3
list of gene pairs showing overlapping or non-overlapping localization in a. aegypti and a. gambiae genomes but have no changes in exon structure.
click here for file
additional file 4
list of one-to-one orthologous genes of a. aegypti overlapping genes in other insect species and comparison of overlapping patterns across genomes.
click here for file
additional file 5
comparison of cluster patterns of retention or rearrangement of gene overlapping  with that of presence or absence of orthology of the corresponding gene pairs  among different insects.
red color indicates presence and black color indicates absence of overlapping/ orthology between genes.
click here for file
additional file 6
list of common microsatellite motifs associated with the overlapping gene pairs that are rearranged between the two mosquitoes.
click here for file
additional file 7
list of gene pairs and overlapping ests in a. aegypti.
click here for file
additional file 8
number of gene pairs identified in positionally overlapping patterns with or without evidence of expressed sequence tags.
click here for file
additional file 9
expression level of gene pairs that are localized in positionally overlapping manner in a. gambiae genome.
click here for file
additional file 10
expression level of a. aegypti overlapping genes.
click here for file
acknowledgements
this work was supported, in part, by grants ai <dig> and ai <dig> from the national institute of allergy and infectious diseases , national institutes of health .

 gene rearrangement is one of the necessary ingredients of genome evolution. several well studied mechanisms such as chromosomal inversions, translocations, duplications and transpositions are known to have important roles in genomic rearrangement events. reshuffling of genomic dna by gross chromosomal rearrangements generally involves a number of genes that undergo positional relocation in the genome. in addition to such large scale genomic rearrangements, genomic rearrangements at small scale levels facilitate relocation of genes which are otherwise positionally overlapping in a genome. it has been suggested that transposition mechanisms may contribute to such gene arrangements, but the functional and evolutionary significance of such events is largely unknown. positional overlapping between genes is a common structural feature of prokaryote and mitochondria genomes. however, overlapping genes have also been identified from whole genome sequences of several eukaryotes such as fruit fly, zebrafish, human, chimpanzee, orangutan, marmoset, rhesus, cow, dog, mouse, rat and chicken. studies show that overlapping genes in eukaryotes are extensively rearranged even between closely related species. bhutkar et al. compared overlapping genes of drosophila melanogaster and anopheles gambiae with apis mellifera and suggested that relocalization of overlapping genes may have played a significant role in genome evolution of these insects. although several other insect genome sequences are now available, overlapping genes of most of these insects have not been studied. the present study is an effort to investigate overlapping genes of aedes aegypti, the primary global vector of dengue virus, in a comparative manner with those of a. gambiae, a major vector of malaria in subsaharan africa. understanding genome structure of these mosquitoes has become one of the major interests among insect vector biologists. at present, the draft genome sequences for three mosquito species have been completed. these projects have provided new insights on structure, function and evolution of mosquito genes, thus furthering our ability to study mosquito-parasite or mosquito-virus interactions at the molecular level. we identified positional overlapping of genes at the whole genome level in a. aegypti and studied structural differences and evolutionary features by comparisons with orthologous genes of a. gambiae and other selected arthropod genomes. the primary aim was to test several common hypotheses relating to rearrangement of overlapping genes and determine factors that may have a role in relocalization of overlapping genes in insects. the results of our investigation show that positional overlapping among genes is a species specific evolutionary process as evident from non-dependency of gene overlapping with species phylogeny, and also show that specific factors, such as introns and repeat sequences, are significantly associated with retention/rearrangement of overlapping genes in mosquitoes. based on these results, our study suggests that overlapping genes may have played an important role in genome evolution among insects. methods official gene sets and extraction of overlapping gene pairs the overlapping gene pairs of a. aegypti and a. gambiae were identified in a genome-wide manner based on the coordinates of gene boundaries of official gene sets annotated from the genome assemblies. the other mosquito genome sequence for culex quinquefasciatus was not used for this purpose because of differences in gene annotation of this species compared to a. aegypti or a. gambiae. that is, while nearly equally percentages of the official gene sets of a. aegypti as well as a. gambiae have been annotated for gene boundaries that incorporated the 5’ and 3’ untranslated regions, only 15% of the c. quinquefasciatus genes have been annotated in this manner. thus, incorporating c. quinquefasciatus could have produced biased results in the genome-wide comparison of overlapping gene pairs between a. aegypti and a. gambiae. however, we have used orthologs of a. aegypti overlapping gene pairs in c. quinquefasciatus and other selected insect species such as drosophila melanogaster, apis mellifera, pediculus humanus, bombyx mori and acyrthosiphon pisum to determine if they are also localized in overlapping positions in the respective genomes. for genome-wide comparison of overlapping genes, the predicted gene sets of a. aegypti and a. gambiae along with coordinates of genes in the reference genome were downloaded from vectorbase. the one-to-one orthologous genes were compared to determine if they were also present in overlapping gene pairs across multiple genomes. to determine the relative position of the orthologous genes, the official gene lists along with their start and end positions in the genome sequences of the other six insects were downloaded from either vectorbase or the silkdb database or the ‘ensembl metazoa 10’ data sets at http://www.biomart.org. intron analysis to determine if introns have an association with overlapping between genes, orthologous genes were categorized as intronless and intron-containing genes for overlapping and non-overlapping pairs in the a. aegypti and a. gambiae genomes. the exon structures predicted for a. aegypti and a. gambiae genes were used to classify genes into single exon genes and multi exon genes. the number of introns in each gene was determined from the number of exons annotated in the genes. the 2x contingency analysis of counts of the intronless and intron-containing genes of both categories was performed using yates chi square tests to determine significance of association between introns and gene overlapping. transcriptional analysis of overlapping genes the expressed sequence tags of a. aegypti and a. gambiae mosquitoes used in this study were largely generated in conjunction with the individual genome sequencing projects. these ests were used to assist in the annotation of the official gene sets of the two mosquitoes. we used these ests to investigate expression patterns associated with the overlapping gene pairs. to further confirm correspondence of ests with overlapping gene pairs, we performed reciprocal blast analyses described as follows. the est sequences were used to generate a local blast database and then searched by blastn with the sequences of overlapping genes. the est ‘hits’ that had an e-value =  were used again as queries in another blastn search against all predicted gene sequences. if the reciprocal hits matched the same gene that was used as a query in the first blast, it was considered that the est corresponded to that gene. apart from analyzing the est data, we also analyzed previously performed microarray expression data of a. aegypti to determine expression patterns of the overlapping gene pairs. the a. gambiae microarray expression data was obtained from baker et al. study. the expression data of these studies are publicly available with gene expression omnibus accession # gse and gse at http://www.ncbi.nlm.nih.gov/geo/. the spearman’s rank correlation test was conducted to ascertain whether the overlapping gene pairs had significantly correlated expression levels throughout the genome. identification of microsatellites in overlapping genes in order to determine if there is a significant association of microsatellites with retention or rearrangement of overlapping gene structures between a. aegypti and a. gambiae, we identified microsatellite sequences within the gene pairs in both genomes. sciroko, a simple sequence repeat identification program, was used to detect both perfect and imperfect mono-, di-, tri-, tetra- and hexa-nucleotide repeats using the default parameters. the repeats with more than consecutive mismatch sites were excluded. the genes where one or more sites were ambiguous nucleotides were not used to report microsatellites. the length of orthologous genes may vary that may contribute to varying amounts of microsatellite sequences in the orthologous gene copies. so, instead of comparing the absolute amounts of microsatellite sequences, their relative amounts were compared. the relative amounts were obtained from the total amount of microsatellites of genes normalized with the alignment length of the orthologous genes between a. aegypti and a. gambiae. statistical and computational analyses all statistical analyses were performed using the r statistical program. the p-value <  was considered statistical significance in all tests unless stated otherwise. cluster analyses of gene pairs based on overlapping or non-overlapping structures across genomes were based on average correlation of city-block distance estimated using the cluster program. the phylogenetic analyses were performed by neighbor-joining method using mega. the evolutionary distances were in the units of the number of base substitutions per site; and they were calculated using the maximum composite likelihood method. the mantel procedure was used to perform linear regression between matrices where the dependent matrix was permutated times to test significance of the observed correlation with the independent matrix in the genomes used for comparison. the multi mantel procedure was performed using an algorithm developed by dr. liam j. revell. maximum likelihood methods described elsewhere were used to estimate the log likelihoods of models assuming either dependency or non-dependency of gene phylogeny with the discrete variation of gene traits. the likelihood ratio tests were conducted to infer statistical significance of these two models. a binary logit model was developed to test marginal effects of the rates of synonymous and non-synonymous mutations in the orthologous gene pairs between a. aegypti and other select insect genomes. while each of the gene pairs were localized in an overlapping manner in the a. aegypti genome, the orthologous genes showed variation in relative localization in other species. the dn and ds values of orthologous genes were obtained from metazoan genes database at http://www.biomart.org. a generalized linear model, fitting the dependent variable and independent variables, was used in r to estimate the logit coefficients.  identification of overlapping genes a total of and overlapping gene pairs were identified in the assembled genomes of a. aegypti and a. gambiae, respectively. they represent 8-10% of the annotated genes of the two mosquitoes. the frequencies of overlapping genes of a. aegypti and a. gambiae mosquitoes are within the range of overlapping gene frequencies reported in other eukaryotes. more than two genes were also found in overlapping locations in both genomes, with the majority of these overlapping gene clusters containing no more than three genes. these overlapping clustered genes constituted only a minor portion of the total number of overlapping genes in either of the two genomes. because of low frequency and also for simplicity of analysis, we have not included the gene clusters in our investigation. all the analyses performed in this study were based on overlapping gene pairs. orthology of overlapping genes between a. aegypti and a. gambiae in a. aegypti as well as in a. gambiae, the overlapping gene pairs are localized either in nested form or in partially overlapping form as shown in figure  irrespective of whether gene pairs are in e/h or in p/o form, they are predominantly localized in opposite orientation to each other. three possible patterns of evolution emerged from comparing the orthologs of overlapping genes between the two species: 1) the gene pairs are orthologous between the two species, 2) the gene pair is specific to the species, and 3) one of the genes is specific to the species and the other is common between the two species. the number of young-young gene pairs and the old-old gene pairs vary between the two species. the 2x contingency tests based on the count statistics of old-old and young-young gene pairs between a. aegypti and a. gambiae shows that there is a significant bias in the distribution of these genes between the two species. it clearly shows that nearly the same number of orthologous gene pairs are present in overlapping manner in both the species, whereas a significantly larger number of a. aegypti specific genes are in overlapping position compared to a. gambiae. this shows that the young-young gene pairs show significant variation in overlapping patterns between these mosquitoes. when the a. aegypti and a. gambiae overlapping genes were compared with d. melanogaster orthologs, consistent patterns were observed. the data in figure  shows that the old-old gene pairs are comparable in numbers between a. aegypti and a. gambiae whereas the young-young gene pairs vary significantly between the two species. consistent with the results shown in figure  these results also suggest that young genes are major contributors to the positional overlapping of genes in these species. rearrangement of overlapping genes one of the common hypotheses about positional overlapping of genes is that selection acts against the retention of gene overlap between genomes. if the above hypothesis is correct, we expect that overlapping genes should be extensively rearranged between a. aegypti and a. gambiae. to test that expectation, the orthologous copies of overlapping gene pairs were compared between the two species. it was found that only of the total orthologous gene pairs are localized in overlapping manner in both the genomes. the other gene pairs are localized in overlapping manner in one genome but in non-overlapping manner in the other suggesting that only a fraction of overlapping genes are retained across genomes. to determine if retention or rearrangement of overlapping localization of genes between a. aegypti and a. gambiae may be associated with loss or gain of terminal exons of genes, we investigated several gene pairs that contain multiple exons in the orthologous gene pairs and found no discrepancy in annotation of first and last exon of any gene pair between the two species. the overlapping gene pairs where one gene is embedded in another are represented as e/h pairs. the gene pairs which are localized in partially overlapping manner with each other are represented as p/o gene pairs. additionally, we performed specific case studies of retention or rearrangement between the two species. a short-chain dehydrogenase gene acts as the host of another protein coding gene aael in a. aegypti. it was found that the corresponding orthologs in a. gambiae genome are also localized in e/h form. in contrast to this, the gene of a. aegypti, that codes for a carboxylesterase, is localized in the genome in non-overlapping manner with one of its paralogs whereas the ortholog in a. gambiae is localized in p/o manner with the paralog agap. these genes associated with retention or rearrangement of positional localization in both the genomes are known to have significant changes in expression during blood feeding, growth and development of these mosquitoes. in another case, we identified multiple genes that are embedded within intron sequences of a single host gene in a. aegypti. the gene aael that putatively codes for the protein b-cell lymphoma/leukaemia harbors paralogous genes within one intron along with several other paralogous genes that are localized in non-overlapping manner to aael. importantly, these genes have been reported to be expressed in a. aegypti. moreover, the phylogenetic tree shows that the embedded genes tend to cluster together and are phylogenetically distinct from the genes located outside the host genes. this suggests a contrasting rate of evolution of embedded versus non-embedded paralogous copies of the gene aael in a. aegypti. gene overlapping is phylogeny independent having observed that orthologs of overlapping genes are extensively rearranged between species, we hypothesized that phylogenetic relationship has no correlation with the localization pattern of orthologous genes across species. if this is true, one would expect that gene overlapping occurs as a trait which should be independent of species phylogenies. to test this hypothesis, we performed ‘discrete analysis’ of gene overlapping across phylogeny using maximum likelihood method to determine if presence or absence of gene overlapping is correlated with phylogeny among species. seven insect species were compared for orthology analysis with a. aegypti overlapping genes: a. gambiae, c. quinquefasciatus, d. melanogaster, a. mellifera, p. humanus, b. mori and a. pisum. the results of this analysis showed that the likelihood estimates of the null model consistently lack statistical significance when tested against the alternate model. it clearly shows that there is no apparent relationship of positional overlapping with the phylogeny of the species. to illustrate this, a representation of phylogeny and gene overlapping pattern is shown in figure  for orthologs of a. aegypti gene pair aael009614-aael among seven other insect species. it shows that retention or rearrangement of orthologous genes lacks correspondence with the phylogenetic relationships between species. the likelihoods of test and null models and the p-values of log likelihood ratio tests are shown. by comparisons with predicted orthologous genes among sequenced arthropod genomes, we identified a total of overlapping gene pairs of a. aegypti where at least one gene of each pair was also present among the other seven insect species. but only of these gene pairs in a. aegypti had both the genes present as orthologs in all the other seven species. to further confirm that overlapping or non-overlapping localization of genes has no correspondence with presence or absence of orthologs across genomes, we performed hierarchal cluster analysis among the above orthologous gene pairs across the eight species. the potential for correlation between gene orthology and gene positional overlapping was assessed for statistical significance by mantel test. the correlation was evaluated between binary data of orthologous genes in matrix forms with the presence or absence of orthology of the gene pairs. the results showed non-significant correlation between the two suggesting that gene orthology has no relationship with overlapping localization of genes across species. role of selection on rearrangement of overlapping genes another hypothesis about overlapping genes is that mutations occurring within the shared region of overlapping gene pairs would mostly be negatively selected because such mutations may affect adaptation and function of both genes. if this is true, we expect to see a higher frequency of synonymous changes than non-synonymous changes between orthologous genes when they are present in overlapping manner. to test this expectation, the numbers of per site synonymous and non-synonymous changes in orthologous gene pairs were determined among a. aegypti, c. quinquefasciatus, a. gambiae, d. melanogaster and p. humanus. the ds and dn values are the rate of synonymous and non-synonymous changes, respectively, between a. aegypti gene and the corresponding ortholog of other species mentioned above. as shown in additional file each of these gene pairs is localized in overlapping manner in a. aegypti. but, the orthologous genes in the other species are found either in overlapping or non-overlapping manner. a binary logit model was developed to fit the data of ds and dn values with the occurrence or non-occurrence of positional overlapping between genes among the species. the dependent variable assumed a value when the genes were found in overlapping position but when the gene pairs were in non-overlapping position in the genome. we performed generalized linear model fitting of the data that is represented by y = β0 + xβ + e, where y = dependent variable, x = ds and dn values of both genes, β = coefficient of independent variable, β represents the value of y when the predictor is equal to zero, and e is assumed to be independent of x and has a standard logistic distribution with mean zero. the ds and dn values of both genes of a. aegypti gene pairs, calculated by aligning the codon sequence to the orthologs of other insect species, were used as the independent variables. the results of the regression analysis are shown in table  it shows estimates of coefficients of each of the four independent in fitting the model to explain the occurrence of the dependent variable. the estimated regression coefficient shows variation of the outcome with unit change in the predictor variable. the data in table  shows that the coefficients of regression are positive for synonymous changes but negative for non-synonymous changes indicating differential effects of synonymous and non-synonymous mutations on overlapping localization between genes. however, the effects of ds or dn are statistically non-significant in each case indicating that, in these insect species, the association of synonymous or non-synonymous mutations with overlapping localization of orthologous genes may be a random event. however, the lack of significance may also be due to differences in the reading frames of orthologous genes. such differences are known to be associated with bias in codon phases of overlapping prokaryotic genes. however, we have not determined from this study if there is a bias in codon phase distribution of overlapping genes that may influence the rate of synonymous and non-synonymous changes between orthologs. the regression was performed in relation to presence or absence of positional overlapping of the orthologous gene pairs across species. association of microsatellites with gene overlapping it is well recognized that transposition events contribute to positional rearrangement of genes in eukaryotes. and as transposons are known to be intimately associated with simple sequence repeat elements, we hypothesized that microsatellites may have a role in positional overlapping of genes. thus, one of our aims was to determine if there was a significant association between microsatellite contents with rearrangement of overlapping gene pairs between a. aegypti and a. gambiae. the amounts of microsatellite sequences were normalized based on length of shared sequences between e/h gene pairs and their rearranged orthologous pairs in a. aegypti and a. gambiae. the results of the 2x contingency tests of these data show that positional rearrangement of e/h gene pairs is significantly associated with the amount of microsatellite sequences within the orthologous genes in the two mosquitoes. one scenario is that the repeat sequences, represented as common motifs between the two genes, are involved in gene rearrangements possibly by facilitating cross over events associated with exchange of the flanking regions between microsatellites that lead to positional rearrangements of genes. role of introns in positional overlapping of genes in a. aegypti and a. gambiae, most of the embedded genes are localized within introns of host genes. thus, intron loss/gain could contribute to gene relocalization. in that case, we expect that intron loss/gain between one-to-one orthologous genes should be significantly associated with rearrangement of overlapping gene pairs between the two mosquito species. to determine if introns have an association with retention/rearrangement of overlapping genes between the two species, the number of introns among orthologous gene pairs listed in table  were quantified. based on count statistics of introns between one-to-one orthologous gene pairs between a. aegypti and a. gambiae, we found that that loss/gain of introns is significantly associated with retention or rearrangement of overlapping gene pairs between the two mosquitoes. the rearranged genes show significant loss of introns compared to the orthologous gene pairs located in overlapping positions and vice versa suggesting that introns may have a role in gene overlapping and rearrangement. although intron-mediated gene recombination and references therein or other mechanisms such as intron-transposition may be likely mechanisms for these processes, further investigations are needed to determine the exact role of introns in positional overlapping of genes. expression of overlapping genes overlapping expression of more than one gene is well known in eukaryotes,. we analyzed the expressed sequence tags datasets of a. aegypti and a. gambiae to determine if overlapping gene pairs may have overlapping transcripts. using reciprocal blastn searches, we identified several ests of a. aegypti that represented the likely transcription product of overlapping gene pairs. although many of these gene pairs are oriented in opposite direction to each other, ests were also observed for gene pairs oriented in same direction. whether these gene pairs are co-transcribed or co-regulated by common upstream/downstream sequences are not known from this study. however, identification of ests of overlapping gene sequences clearly shows that these sequences are expressed. moreover, we show that the annotation of overlapping genes is unaffected whether good evidence of expression is available or not. the dataset of overlapping genes was also analyzed based on availability or non-availability of est evidence. we found no significant difference in the number of genes that localized in positionally overlapping manner between the two groups. in a. gambiae, the est dataset didn’t reveal such transcripts except for a single gene pair. although transcripts of overlapping genes were not available in the est collections of a. gambiae, we found evidence of expression of these genes from published microarray data. generally, overlapping transcripts are processed by post-transcriptional events to produce individual transcripts of the genes. to assess the expression level of individual gene transcripts of overlapping gene pairs, we examined the microarray expression data of a. aegypti. because overlapping genes are predominantly localized in opposite orientation to each other in the genome, we compared expression level of gene pairs which are either oppositely oriented or oriented in same direction to each other. it was found that the expression levels of overlapping genes in opposite orientation lack significant correlation, whereas the overlapping genes which are oriented in the same direction to each other show statistically significant correlation. most of these genes code for known proteins and have been annotated with start and stop codons suggesting that these genes are not annotation artifacts, although a few genes were annotated as hypothetical proteins. nevertheless, these results suggested that when the two genes are localized in overlapping manner and also oriented in the same direction, their expression may be co-regulated leading to similar transcription levels. on the other hand, when the two genes are localized in overlapping manner, but oriented in the opposite direction, their transcripts may have differential regulation. in fact, it is well documented that overlapping genes when transcribed in the opposite directions, give rise to sense-antisense transcript pairs which are differentially regulated to play a role in a variety of processes, including mrna splicing and stability, rna editing, genomic imprinting and control of translation.  the results from this study provide insight into the common prevailing theories of origin and evolution of positionally overlapping genes. these are particularly important for better understanding of distribution and structure of overlapping genes in the genomes of a. aegypti and a. gambiae. the genome sequences of both a. gambiae and a. aegypti contain gaps that could affect our estimates of overlapping genes in the genome assemblies, but we find this unlikely based on our observation that the overlapping genes are distributed throughout the genome in each species without any bias to specific chromosomal region of a. gambiae or specific supercontigs of a. aegypti. furthermore, our estimated frequencies of overlapping genes in mosquitoes are within the range of overlapping gene frequencies reported in other eukaryotes. thus, it is unlikely that there may be large numbers of genes missing because of gaps in sequencing that are positionally overlapping. nevertheless, the dynamic patterns of positional rearrangement of overlapping genes suggest that these genes may have important roles in genome evolution of vector mosquitoes. importantly, the information from this investigation may help us in further studies pertaining to evolution and functional characterization of antisense transcripts among overlapping genes in mosquitoes. availability of supporting data the manuscript is accompanied with the following listed additional files in the form of supporting data for this study. competing interests the authors declare that they have no competing interests. authors' contributions conceived and designed the experiments: skb. analyzed the data: skb. contributed reagents/materials/analysis tools: skb, dws. wrote the paper: skb, dws. both the authors read and approved the final manuscript. authors' information skb is a research assistant professor in the department of biological sciences and the eck institute for global health at the university of notre dame, indiana. he has a broad interest in insect genomics and evolution with emphasis on disease transmitting vector species. dws is a professor of biological sciences and the director of eck institute for global health at the university of notre dame, indiana. his work focuses on genetic and genomic analysis of mosquito vector competence to various pathogens as well as on development and application of molecular tools to investigate population biology of mosquitoes. supplementary material additional file 1 list of e/h and p/o gene pairs of a. aegypti and a. gambiae. click here for file additional file 2 overlapping and non-overlapping patterns of orthologous genes between a. aegypti and a. gambiae. click here for file additional file 3 list of gene pairs showing overlapping or non-overlapping localization in a. aegypti and a. gambiae genomes but have no changes in exon structure. click here for file additional file 4 list of one-to-one orthologous genes of a. aegypti overlapping genes in other insect species and comparison of overlapping patterns across genomes. click here for file additional file 5 comparison of cluster patterns of retention or rearrangement of gene overlapping with that of presence or absence of orthology of the corresponding gene pairs among different insects. red color indicates presence and black color indicates absence of overlapping/ orthology between genes. click here for file additional file 6 list of common microsatellite motifs associated with the overlapping gene pairs that are rearranged between the two mosquitoes. click here for file additional file 7 list of gene pairs and overlapping ests in a. aegypti. click here for file additional file 8 number of gene pairs identified in positionally overlapping patterns with or without evidence of expressed sequence tags. click here for file additional file 9 expression level of gene pairs that are localized in positionally overlapping manner in a. gambiae genome. click here for file additional file 10 expression level of a. aegypti overlapping genes. click here for file acknowledgements this work was supported, in part, by grants ai and ai from the national institute of allergy and infectious diseases, national institutes of health.
based on the results, our study suggests that overlapping genes may have played an important role in genome evolution of insects. 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. 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. gene rearrangementculicidaegenome evolutionpositionally overlapping genesnegative selection in this study, we investigated retention and rearrangement of positionally overlapping genes between the mosquitoes aedes aegypti and anopheles gambiae. gene overlapping in insects may be a species-specific evolutionary process as evident from non-dependency of gene overlapping with species phylogeny.
 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 and anopheles gambiae. 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.  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.  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. gene rearrangementculicidaegenome evolutionpositionally overlapping genesnegative selection 