J. Biomedical Science and Engineering, 2009, 2, 304-311
10.4236/jbise.2009.25045 Published Online September 2009 (http://www.SciRP.org/journal/jbise/
JBiSE
).
Published Online September 2009 in SciRes. http://www.scirp.org/journal/jbise
Fine-scale evolutionary genetic insights into Anopheles
gambiae X-chromosome
Hemlata Srivastava1, Jyotsana Dixit1, Aditya P. Dash2, Aparup Das
Evolutionary Genomics and Bioinformatics Laboratory, National Institute of Malaria Research, Sector 8, Dwarka, New Delhi-110
077, India. 2Present address: World Health Organization, Southeast Asian Regional Office, New Delhi, India. 1Equal contributions.
Received 28 April 2009; revised 10 May 2009; accepted 15 May 2009.
ABSTRACT
Understanding the genetic architecture of indi-
vidual taxa of medical importance is the first
step for designing disease preventive strategies.
To understand the genetic details and evolu-
tionary perspective of the model malaria vector,
Anopheles gambiae and to use the information
in other species of local importance, we
scanned the published X-chromosome se-
quence for detail characterization and obtain
evolutionary status of different genes. The te-
locentric X-chromosome contains 106 genes of
known functions and 982 novel genes. Majori-
ties of both the known and novel genes are with
introns. The known genes are strictly biased
towards less number of introns; about half of
the total known genes have only one or two in-
trons. The extreme sized (either long or short)
genes were found to be most prevalent (58%
short and 23% large). Statistically significant
positive correlations between gene length and
intron length as well as with intron number and
intron length were obtained signifying the role
of introns in contributing to the overall size of
the known genes of X-chromosome in An. gam-
biae. We compared each individual gene of An.
gambiae with 33 other taxa having whole ge-
nome sequence information. In general, the
mosquito Aedes aegypti was found to be ge-
netically closest and the yeast Saccharomyces
cerevisiae as most distant taxa to An. gambiae.
Further, only about a quarter of the known
genes of X-chromosome were unique to An.
gambiae and majorities have orthologs in dif-
ferent taxa. A phylogenetic tree was constructed
based on a single gene found to be highly
orthologous across all the 34 taxa. Evolutionary
relationships among 13 different taxa were in-
ferred which corroborate the previous and pre-
sent findings on genetic relationships across
various taxa.
Keywords: Anopheles gambiae; Comparative Ge-
nomics; Evolution; Malaria; Orthologous Genes;
X-chromosome
1. INTRODUCTION
Determination of genetic architecture of different taxa in
a vector borne disease model, helps not only in under-
standing the genetic pattern of host-parasite-vector in-
teraction but also adds in devising methods for control
measures. Further, characterization of different genes in
the entire chromosome leads to identification of novel
genes of essential functions and evolutionary process
that governs these genes in populations. These determi-
nations of genetic architecture should start with deep
understanding and evolutionary inference of each indi-
vidual gene of known function at the chromosomal level.
The detail knowledge on the relative size of the genes
[1], differential compositions of coding and non-coding
elements in each gene [2] and contribution of non-coding
DNA to the average length of the gene [3] could easily
be evaluated with such kind of studies. This is further
important when scanning is performed on chromo-
some-to-chromosome basis, so that differential genetic
composition in each chromosome of a species can be
compared [4]. Further, comparing genes among different
taxa exploits both similarities and differences of differ-
ent organisms to infer how Darwinian natural selection
might have acted upon on these elements in the course of
evolution. Considering the genomes as “bags of genes”
and measuring the fraction of orthologs shared between
genomes could provide vital information on the evolu-
tionary history of the genes [5]. Also, if any particular
gene is found to be conserved in many organisms, re-
construction of phylogeny among these organisms is
possible. However, such kinds of studies are possible
only when adequate genome information is at hand.
Fortunately, many organisms have been fully sequenced
in recent years providing opportunities to fine-scale un-
derstanding of the genetic architecture of species of
medical and agricultural importance and comparison
across species and taxa [6].

H. Srivastava et al. / J. Biomedical Science and Engineering 2 (2009) 304-311 305
SciRes Copyright © 2009 JBiSE
To this respect, malaria is a devastating disease with
global cases of about 300 to 500 million infections per
year and deaths of about one and half millions [7].
Global efforts to eradicate malaria failed immaturely and
scientist and policy makers are now focusing on the con-
trol of this disease. However, emergence and spread of
drug-resistant parasites and insecticide-resistant vectors
have seriously hampered the efforts and put new chal-
lenges to tackle with the situation [8]. This situation in-
vites a close and deep genetic understanding of both
parasites and vectors and devise new methods for ma-
laria control.
The whole genome sequence information of the mos-
quito Anopheles gambiae [9], the principal vector of
malaria in Africa is available in the public domain.
However, the vector species are not all the same across
different malaria endemic zones in the globe as different
other species of the genus Anopheles are of local impor-
tance. Since controlling the malaria vector is one of the
finest strategies to control malaria, understanding the
genetic composition of different endemic Anopheles
species in different localities is the need of the hour for
development of effective malaria control strategies.
Keeping in view that the whole genome sequence infor-
mation is only available for one of the malaria vector An.
gambiae, utilization of such information might lead to
understanding the genetics of vector potentiality, insecti-
cide resistance, etc. and extend the information to other
species of local and focal importance. In addition, due to
lack of genome information in other Anopheles species,
the information from An. gambiae could be utilized in
designing genetic markers for evolutionary studies in
genes and populations of vectors in the malaria endemic
zones of the globe.
We herewith utilize the whole genome sequence in-
formation of An. gambiae to characterize the whole
X-chromosome for different gene compositions and
fine-scale study of each individual genes of known func-
tion. We performed homology searches of each of the
known genes of An. gambiae X-chromosome in 33 taxa
with published whole genome sequences and also con-
structed phylogenetic tree. The results not only provide
detail understanding on differential compositions of ge-
netic elements in An. gambiae X-chromosome, but also
would help in developing genetic markers to study ge-
netic diversity and population histories of other species
of Anopheles of local importance.
2. MATERIAL AND METHODS
The An. gambiae genome comprises of 3 pairs of
chromosomes, 2 pairs of autosomes and a pair of sex
chromosome. Whole genome sequence information is
available at the public domain for the pest strain of An.
gambiae [9]. We used the Ensemble web database
(www.ensembl.org) from release 45-June 2007, to re-
trieve genetic information on the telocentric X- chromo-
some. We started our scanning for different genes from
one end of the X-chromosome and proceeded till we
reached the end. We looked for genes that have known
functions (known genes) and also genes that are com-
pletely new (novel genes) following the classifications
provided at the Ensemble database. Due to functional
importance, we deeply characterized only the known
genes leaving apart the novel genes. For the convenience
of further analysis, we classified the known genes based
on length of nucleotide bases as, Class 1 ( 0 to 1 kb);
Class 2 (1-2 kb); Class 3 (2-3 kb); Class 4 (3-4 kb);
Class 5 (4-5 kb) and Class 6 (above 5 kb). The composi-
tion of different genes in the An. gambiae X- chromo-
some was determined as per information in the Ensem-
ble web database. Further, information on 33 other taxa
with whole genome sequence information was also
available at the Ensemble web database. We utilized this
information to infer X-chromosome genes of An. gam-
biae having orthologs (orthologs are genes derived from
single ancestral gene in last common ancestor of com-
pared species) and paralogs (paralogous genes develop
by gene duplication in the similar lineage) across 33
different taxa. The genes of An. gambiae with no
ortholog or paralog were considered to be unique genes
to this species. Three criteria were followed to define the
candidate orthologous genes suggested by [8] in the En-
semble web database. First, the sequences of genes
should have highest level of pair-wise identity when
compared with genes in the other genome. Second,
pair-wise identity should be significant (E, the expected
fraction of false positives should be smaller than 0.01)
and third, the similarity extends to at least 60% of one
of the gene. We followed similar procedures to classify
the orthologous genes in An. gambiae X-chromosome.
Out of the many orthologs found, one particular gene was
found to be present in all the 34 different taxa (included
An. gambiae) presently studied. We constructed an
un-rooted neighbor-joining (NJ) tree to infer the evolu-
tionary status of different taxa at this conserved gene
(AGAP001043). However, due to high sequence dissimi-
larity, only 13 taxa could be utilized for a meaningful
phylogenetic tree construction. Length of each branch
and bootstrapped values for each internal node were also
estimated using VEGA ZZ software downloaded from
internet (http://www.ddl.unimi.it/vega/index.htm). For
all statistical analyses, the free version of ‘analyze-it’ a
Microsoft Excel add-in was used.
3. RESULTS
Scanning of the whole X chromosome of An. gambiae
revealed the presence of 1088 genes, out of which 982
were novel and 106 were genes of known functions. Due
to functional relevance, the known genes were further
analyzed. These genes were classified based on size (see
materials and methods). Out of the 106 known genes,

306 H. Srivastava et al. / J. Biomedical Science and Engineering 2 (2009) 304-311
SciRes Copyright © 2009
most (62 genes, 58%) were small and thus fall under
Classes 1 and 2. Thus, majority of the known genes in
An. gambiae X-chromosome are small in size (Figure 1).
Only 18% of the total known genes falls under Classes 3,
4, and 5, whereas 23% comes under Class 6 (more than
5 kb). Thus, the distribution of known genes in An. gam-
biae seems to be quite uneven, as small and large sized
genes constitute 81% of the total known genes in the An.
gambiae X-chromosome (Figure 1).
lated Pearson’s correlation coefficient (r) which was
found to be positive and highly statistically significant
(r=0.99, P<0.0001). Moreover, in order to test the hy-
pothesis if the accumulation of introns has considerably
contributed in increasing the length of the gene in gen-
eral, we calculated r value between intron length and
gene length which was found to be positive and highly
statistically significant (r=0.49, P<0.0001) as well. Thus,
it is clear that introns play a major role in the overall
length of genes in the X-chromosome of An. gambiae.
Since genes in the eukaryote genome are often found
to bear introns (non-coding part of the gene, flanked on
each side by the coding parts) and considering An. gam-
biae as a higher eukaryote, we determined the distribu-
tion of exons (coding part of the gene) and introns of
each known gene (Figure 2). The distribution of genes
with different number of introns is shown in Figure 2. It
is interesting to note that almost two-third of the known
genes (79 genes, 74.52%) have either no or very less
number (maximum of three) of introns. Genes having
more than three introns contribute to only 17% of the
total known genes of X-chromosome of An. gambiae.
Further, we looked for size of each intron and exon in
each gene and calculated the average intron and exon
length and their ratio (Figure 3). The average ratio of
exon to intron was higher in genes with less number of
introns (Figure 3), as compared to genes with more
number of introns. Thus, it seems that the average length
of introns in a gene increases with the increase in num-
ber of introns. In order to test this hypothesis, we calcu-
As many as 86 known genes out of 106 total known
genes of An. gambiae X-chromosome were found to
have homologs (both orthologs and paralogs) across 33
different taxa. Out of these 86 genes, 41 have only
orthologs, 3 have only paralogs and 42 have both
orthologs and paralogs. No homologs could be detected
in the rest 20 genes, thus are considered unique to An.
gambiae (Figure 4). Thus, in total, 83 (41+42) have
orthologs and 45 (3+42) have paralogs in the X-chro-
mosome known genes of An. gambiae. However, the
distribution of orthologs varies across 33 different taxa;
Aedes aegypti seems to bear most of the An. gambiae
homologs (78 out of 83 orthologs) and the yeast S. cere-
visiae bears the least (6 out of 83 orthologs) (Figure 5).
These are at the highest and lowest ends of the homol-
ogy prediction of 83 X-chromosome genes of An. gam-
biae.
Since majority of the known genes of X-chromosome
of An. gambiae are either short or large in size (Figure
1), we were interested to know the distribution pattern of
different types of genes based on homology predictions
(orthologous, paralogous and unique) in different classes
based on size (Figure 1). The details of such distribution
are shown in Figure 6, which seems to be random.
Whereas the orthologous genes are slightly abundant in
Classes 2 and 6, the paralogous genes show a clear pat-
tern of decreasing abundance from Class 1 to Class 6. In
contrast unique genes are found to be much prevalent in
Class 2 and least in Class 6 (Figure 6). We further
looked at the distribution of these three types of genes
(orthologous, paralogous and unique) based on the
number of introns they posses (Figure 7) and found that
Figure 1. Classification of known genes of An. gambiae X-
chromosome based on size (nucleotide base pair).
Figure 3. Average exon to intron ratio of An. gambiae X-
chromosome known genes.
Figure 2. Distribution of An. gambiae X-chromosome
known genes according to the number of introns.
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H. Srivastava et al. / J. Biomedical Science and Engineering 2 (2009) 304-311 307
SciRes Copyright © 2009 JBiSE
Figure 4. Distribution of different gene types (based on homology prediction) in
X-chromosome of An. gambiae.
Figure 5. Distribution of different taxa showing number of shared genes with An. gambiae.
Figure 6. Distribution of orthologous, paralogous and unique
genes of An. gambiae X-chromosome across different classes
as in Figure 1.
Figure 7. Distribution of orthologous, paralogous and unique
genes of An. gambiae X-chromosome based on intron number.
most of the orthologous genes are biased towards more
number of introns (>2) and majority of unique genes are
without introns. There seem to be no biasness on the
distribution of paralogous genes based on the number of
introns, although a comparatively less number of para-
logous genes were found to be without introns. In con-
trast, most of the unique genes are found to be without
intron and the number decreases with increase in the
number of introns. However, no significant correlation
was found to exist between these two variables (r=-0.04,
P=0.88).
In the process of homology prediction, we found a
single gene (AGAP001043) of An. gambiae having
orthologs in all other 33 taxa. This situation provided us
an opportunity to look for evolutionary history, based on
this conserved gene across all the 34 taxa. For this we
constructed un-rooted neighbor joining (NJ) tree with only
the coding sequences (exons) considering all 34 taxa, but
due to lots of non-homologous sequences, no proper
alignment among sequences could be obtained. Thus we
went on deleting taxa with more number of
non-homologous stretches and ended-up with only 13 taxa
where meaningful alignment and construction of a phy-
logenetic tree was possible (Figure 8). Length of each
branch leading to taxa was calculated as also the strength
of each internal node through estimation of bootstrapped
trees (Figure 8). Although in most cases, known closely
related species came together in a single clade, (e.g. Aedes
aegypti, An. gambiae and Drosophila melanogaster), the
separation of human and chimpanzee branch is somehow
an interesting observation (Figure 8). The strength of

308 H. Srivastava et al. / J. Biomedical Science and Engineering 2 (2009) 304-311
SciRes Copyright © 2009 JBiSE
Figure 8. Phylogenetic tree with bootstrap values (in bold font) and branch length (in normal font)
in13 different taxa.
each internal node was found to be absolute in all the
cases, signifying the phylogenetic tree is robust enough
to be considered significant.
4. DISCUSSION
Although about two third part of the world is colonized
by different species of mosquitoes and various species of
the genus Anopheles is of great abundance, whole ge-
nome sequence information from only a single species,
An. gambiae is so far available at the public domain.
This situation possesses great constraint on researchers
working on species of non-African importance. The pre-
liminary objective of this study is to understand genomic
architecture of An. gambiae through fine-scale dissection
of the X-chromosome and utilize the information in de-
veloping nuclear DNA markers for estimating genetic
diversity to infer both population demography and natu-
ral selection in species of importance in non-African
malaria vectors. To start with, we have studied the X-
chromosome, since it has several advantages over the
autosomes. For example, X-chromosome is known to
bear a rich resource of easily accessible genetic data, and
provides a unique tool for population genetic studies
[10]. Further, majority of population genomic studies
have been undertaken on the model organism, Droso-
phila utilizing X-linked genes [11,13].
We have considered genes of known function for de-
tail characterization and analyses as they are the store
house of important identifiable characters. More than a
half of the known genes in An. gambiae X-chromosome
are of short length and about 40% of the total known
genes are engaged in housekeeping functions. House-
keeping genes are known to be often compact in size,
which is attributed to selection for economy in transcrip-
tion and translation [14,15]. Although a direct correla-
tion between the function and size of the genes was not
found (data not shown), abundance of shorter genes in
An. gambiae might be due to the fact that most of the
genes are engaged in housekeeping functions. However,
long-sized genes (of more than 5kb length) were also
abundant to certain extent (20%) in this chromosome. In
contrast, genes of intermediate length (2kb to 5kb) were
very less in occurrence. As far our knowledge goes, no
study has reported this trend in the distribution of vari-
able gene length across a single chromosome in any or-
ganism.
Majority of genes in An. gambiae X-chromosome
shows a clear tendency of having either no or a very few
(about three) introns (Figure 2). It has been suggested

H. Srivastava et al. / J. Biomedical Science and Engineering 2 (2009) 304-311 309
SciRes Copyright © 2009 JBiSE
Based on evolutionary status, known genes in An.
gambiae fall in all three categories (orthologous,
paralogous and unique). Further, there is a clear distinc-
tion on the size, intron number and non-coding DNA
content in different gene categories. Orthologous genes
are comparatively larger in length and also contain more
number of introns. These observations corroborate the
fact that conserved or orthologous genes show more
number of introns [23], possibly due to their slow evolu-
tion rate. Further, these types of genes tend to gain more
and lose fewer introns [24]. In contrast to the characters
of orthologous genes, no clear pattern was observed for
the paralogous genes. This observation fits to the find-
ings in the malaria parasite, Plasmodium falciparum and
P. yoelli yoelii, where duplicate genes or paralogous
genes in both the species exhibit a dramatic acceleration
of intron gain/loss and protein evolution in comparison
with orthologous genes, suggesting increased directional
and/or relaxed selection in duplicate genes [25]. In con-
trast, the unique genes are mostly intron-less and might
be evolving at a faster rate loosing the non-coding por-
tions; hence homologs of these genes are no more rec-
ognizable [26].
that intronless gene families can evolve rapidly either by
gene duplication or by reverse transcription/integration
[16]. In Oryza and Arabidopsis, intronless genes have no
homology and perform species-specific functions that
are unique in respective species [17]. Thus, the findings
of biasness towards either no or a few introns in majority
of genes of known functions point towards the role of
natural selection in reducing the size or presence of in-
trons, thus reducing the overall transcriptional cost for
effective expression. Findings of genes with very few
numbers of introns in eukaryotic genome in general [18],
further corroborate our contention.
The present study revealed the presence of only four
genes with an appreciably high number of introns (9-11).
Three of these four genes belong to nicotinic acetylcho-
line receptor gene families [19] which are the known
targets of insecticides, neonicotinoids [20], as well as
naturally derived spinisyns [21]. The presence of such
high number of introns in these genes shows that they
require more variability in their protein products and
more number of introns make these genes more versatile
in their expression by increasing the chances of alterna-
tive splicing and less 5′splice site biasness [22]. These
genes are of much importance as far as growing insecti-
cide resistance in almost all species of malaria vectors is
concerned. Understanding of molecular mechanisms of
resistance and molecular evolutionary studies should be
targeted towards these genes for better understanding on
evolution of insecticide resistance in malaria vectors.
We found strong positive correlations between gene
length and intron length and intron length and intron
number, which clearly suggests that size of the X-
chromosome known genes in An. gambiae, is somehow
dependent on accumulation of introns. These findings
are in complete agreement with the general pattern ob-
served in eukaryotic genome, where enlargement of ge-
nome size and decrease in genome compactness with
increase in number and size of introns was supposed to
be a general pattern during evolution of eukaryotes [5].
We detected a close genetic affinity at the X-chro-
mosome known genes among three taxa of insects; An.
gambiae, Ae. aegypti and D. melanogaster. This is not
surprising, as three of them belong to the similar order
Diptera. Similar observations have been made in earlier
studies [27] as also among Drosophila species with
comparative genomic approaches [6]. Although S. cere-
visiae was found to be farthest in homology, six genes of
S. cerevisiae show homology to An. gambiae genes. The
function of these genes was found to be tyrosyl-t RNA
synthetase, peroxiredoxin dependent peroxidase and
glutathione reductase, one was putative membrane
bound O-acyltransferase. However, these genes possess
very few introns. Although it is apparent that gene size
has increased through accumulation of introns in higher
taxa, it is surprising that these six genes remained con-
served in the long evolutionary process from yeast to
insects. It is probable that, these genes might be under
some functional constraint and natural selection saved
their compositions across different lineage.
We considered a gene present in all the 34 taxa to in-
fer evolutionary history through phylogenetic tree con-
struction. The pattern in the tree was mostly found to
comply with general patterns of phylogenetic relation-
ships among organisms. The placement of An. gambiae,
D. melanogaster and Ae. aegypti in a single clade further
corroborates our observation of close genetic relatedness
among these three taxa (see above). However, it was
surprising to note that human and chimpanzee are placed
in two separate clades. Although, human and chimpan-
zee are genetically close to each other [28], gross differ-
ences at several genes have also been observed [29].
Included in these genes is a gene responsible for malaria
susceptibility in humans and chimpanzee [30]. These
two taxa differ in their susceptibility to malaria due to
human specific loss of N-glycolylneuraminic acid,
which is present in primates. It might then be true that
genomic difference between two species only can be
traceable at selected genes and the presently studied
gene is one of them. This gene codes for a protein which
is involved in signal transduction process and partici-
pates in transmission of developmental information by
associating with alpha or beta catenin. Whereas basic
function of this gene is conserved, little functional dif-
ferences exist across almost all taxa. Further in depth
study revealed that however, the gene function is con-
served in taxa falling in a particular clade found here,
whereas few differences were observed across clades in
the phylogenetic tree (Figure 8). Thus, functional con-

310 H. Srivastava et al. / J. Biomedical Science and Engineering 2 (2009) 304-311
SciRes Copyright © 2009 JBiSE
servation of this gene is exactly overlaps with the posi-
tion of taxa in the phylogenetic tree.
In conclusion, the present study not only provides
fine-scale views to the genetic architecture of the X-
chromosome of the model malaria vector of African
importance, but also reveals several interesting features
on evolutionary insights into genes and taxa of different
taxonomic status. The information is of great importance,
especially to the population geneticists, to understand
genetic diversity and infer the respective roles of de-
mography and natural selection in evolution of genes in
different Anopheles species populations of local impor-
tance.
5. ACKNOWLEDGEMENTS
Extramural funding from the Indian Council of Medical Research
(ICMR), New Delhi in the form of an Ad-hoc research grant to AD is
thankfully acknowledged. We thank Dr. Neena Valecha for her kind
support to HS during the initial phase of the study. Lily Basu, Deep-
shikha Lal, Garima Goyal and Suchita Singh helped in organizing the
initial work elements.
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