Fine-scale evolutionary genetic insights into Anopheles gambiae X-chromosome

doi:10.4236/jbise.2009.25045

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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.

Email: [email protected]

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

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

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

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

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

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

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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