Codon Usage Analysis of Aminnopeptidase N Gene in Order Diptera

Renu Jakhar, Sanjay Kumar, Neelam Sehrawat, Deepika, Mohini Asija, S.K Gakhar

Multi-Disciplinary Research Unit, PGIMS, Rohtak, Haryana, IndiaIndia
Department of Pathology, Nodal Officer Multi-disciplinary Research Unit, PGIMS, Rohtak, Haryana, IndiaIndia
Department of Genetics, Maharishi Dayanand University, Rohtak, Haryana, IndiaIndia
Department of Medicine, PGIMS, Rohtak, Haryana, IndiaIndia
Department of Health Research, Ministry of Health and Family Welfare, New Delhi, IndiaIndia
Center for Biotechnology, Maharishi Dayanand University, Rohtak, Haryana, India Corresponding Author Renu Jakhar

DOI: https://doi.org/10.17140/phoj.2026.v11.i01.pp203-223.2580 · Published 2026-04-18 · Received 2025-12-14 · Revised 2026-01-19 · Accepted 2026-02-16 · Licence: https://creativecommons.org/licenses/by/4.0 · © 2026 The Author(s)

Also available as PDF (figures and tables appear in the PDF).

Keywords: Anopheles gambiae; Aminopeptidase N; Paralogous gene; Genome, Multigene; Evolution; Aedes ageypti; APN genes; Malaria; Dengue

Introduction

Mosquito-borne diseases continues to remain a life

threatening infectious disease throughout the tropical

region of the world. The global health and socioeco-

nomic burden imposed by the mosquitoes is well estab-

lished, with an estimated 282 million malaria cases and

14 million deaths reported worldwide in 2024 (WHO

Report, 2025). It is a leading infectious disease of hu-

mans worldwide. It kills around 610 000 deaths over

the world every year that account for a large number

of death cases worldwide. In 2024, more than 14.6 mil-

lion dengue cases were reported to WHO, with over

9,000 dengue-related deaths globally. The midgut is

the site where the parasite first interacts with mosquito

tissue and further developmental process takes place.

The mosquito’s midgut represents one of the most chal-

lenging environments for survival of parasites and thus

midgut is a striking site for novel target malaria con-

trol strategies. The work reported suggests that ge-

netic modification of mosquito vectorial capacity is

feasible and represents a major step toward the goal

of containing the spread of malaria. Strong and ubiqui-

tous promoters of midgut could be used to drive the

expression of effector genes that hinders transmission

by either killing or interfering with parasite develop-

ment. The alanyl Aminopeptidase N is the leading

malarial TBV immunogen [1]. A midgut-specific protein,

Aminopeptidase N 1 (APN1) is glycosylphospotidyl in-

ositol anchored protein reported to play an important

role in ookinete invasion of Plasmodium in the An. gam-

biae. The Anopheline Alanyl APN is originally isolated

from the apical brush border microvilli fraction of An.

gambiae midguts [2]. It is a ubiquitous enzyme which is

found in a wide range of organisms from insects to

mammals. APN belongs to a group of membrane-

bound zinc enzymes [3] Dipteran Aminopeptidase N

genes were observed to be highly biased genes among

all the orders of class Insecta as revealed through nu-

cleotide composition. In this study, an attempt has

been made to overview genome-wide identification

of APN genes in Diptera. The study covered compara-

tive analysis of APN nucleotide sequences, nucleotide composition, codon usage bias pattern, amino acid

composition analysis among insects to better under-

stand the evolution of APN and its biological signifi-

cance. A comparative sequence analysis was carried

out to gain better understanding of the evolution of

APN gene. The analysis of complete gene and CDS se-

quences of Aminopeptidase N was performed. The ORF

of APNs belonging to Diptera order of class Insecta have

been retrieved from various databases such as NCBI,

Uniprot KB, vectorbase and flybase database etc and

have been analysed by applying various bioinformatics tools. All the sequences show significant similarity

with each other as revealed after performing BLAST. Al-

though mosquitoes APN genes have diverged consider-

ably from one another, the complete sequence can be

aligned along their entire length. The nucleotide com-

position analysis was performed using Genomics GC%

Content Calculate. The Amino acids composition of all

known Aminopeptidase N proteins revealed mosquito-

specific amino acid usage. The extent of codon bias in

mosquito Aminopeptidase N genes based on the Rela-

tive Synonymous Codon Usage values was also deter-

mined. Gene structure study shows high sequence vari-

ations among them. In addition, nucleotide sequence

alignment, intron analysis and nucleotide composition

was carried out. The genus wise multiple sequence

alignment of APN gene was performed using CLUSTAL

W. Sequence comparisons of the genes showed that,

except in conserved regions, there are many nonsyn-

onymous substitutions, suggesting that the APN genes

are highly evolving genes. Phylogenetic analyses were

also carried out to establish the phylogenetic relation-

ships among different orthologs present in other or-

ganisms. Protein-protein interactions show that APN

is highly connected protein, supporting their role as

hub with glutathione metabolism, Glutathione acts as

insecticide resistance, so disrupting of APN protein or

any other protein, this protein network may block the

whole metabolic pathway of Glutathione. Models of

proteins were also studied to further expand the knowl-

edge of functioning of the gene.

Materials And Methods

Retrieval and analysis of Mosquitoes APN1 ortholo-

gous gene sequences: The APN1 gene sequences of

15 different mosquitoes and 15 non mosquitoes have

been retrieved from Vectorbase, Flybase, UniProt KB

and NCBI databases for comparison. By using these

databases, the functional annotation of genes, num-

ber of nucleotides, gene products, gene orientation

and position, chromosome mapping and amino acids

sequences were analysed in all orthologous.

Sequence analysis: BLAST searches were carried out

to find sequences with similarity in the databases. Se-

quences for alignment and phylogenetic analysis were

retrieved from the Genbank. Multiple sequence align-

ments were performed using the Bioedit tool. MEGA 7

software was used to analyse and establish phyloge-

netic relationships among the mosquitoes APN1 genes

[4].

Conserved domains identification for APN proteins:

The conserved domains were identified in all ortholo-

gous APN sequences by various domain search tools like NCBI CDD (Conserved domain database) [5], Pfam

[6] and InterProScan [7] software, which in turn by

searching significantly close orthologous family mem-

bers. The predicted cleavage sites signal peptides were

analysed by using Signal IP algorithm. Glycosylation

and phosphorylation sites were predicted by using the

Prosite tool [8]. Possible transmembrane domains in

APN proteins were also found by using the TMHMM [9].

The Molecular Weight (MW) and Isoelectric point (pI)

were calculated by Compute pI/MW [10]. Multiple se-

quence alignments were performed with the BIOEDIT

program with all paralogous sequences.

Nucleotide Composition Analysis: General nu-

cleotide composition (A, T, C and G %) and nucleotide

composition at first, second and third position of each

codon were analysed for dipteran insect Aminopepti-

dase N CDS using MEGA6 and Genomics % GC content

calculator. The AT3 and GC3 indices refer to the third

position of synonymous codon and it helps to depict

the biasness of the gene [11].

Synonymous Codon Usage: Relative synonymous

codon usage (RSCU) is calculated as the ratio of the

observed frequency of a codon to the frequency ex-

pected if all synonymous codons of a particular amino

acid are used equally [12]. RSCU values were analysed

using MEGA and CAIcal software The effective number

of codons used by a gene (ENC) is generally used to

measure the bias of synonymous codons [13]. Codon

Adaptive Index (CAI) is a measure of relative adaptive-

ness of codon usage of a gene towards the codon usage

of highly expressed genes. CAIcal software was used to

calculate ENC and CAI values [14].

Multiple sequence alignment and Phylogenetic

analysis: Order wise multiple sequence alignment was

performed with MEGA 6 and BIOEDIT programmes in all

insect APN. This helps to depict conserved characteris-

tic features among these insect species. Phylogenetic

tree were generated by MEGA6 using Bootstrap con-

sensus Neighbour-joining Method [15-16]. These trees

were constructed using APN protein sequences to infer

the evolutionary distances among insects.

Results

Nucleotide Sequence Analysis: To understand the

evolutionary changes of APN gene among Diptera

of insects, the complete coding sequences of fifteen

mosquitoes and non-mosquitoes from Diptera orders

have been obtained from the NCBI/ Genbank and

UniProtKB databases. Further, In silico comparative

analysis was performed. All orthologous sequences

were compared and analysed for conserved sites using

software called MEGA6.06 [14]. The multiple sequence alignments were carried out to detect the conserved

sequences [17]. Dipteran APN from different classes

shows divergence from one another, moreover, APN

genes in mosquitoes reveal strong similarities to each

other (Table 1 and 2). However, the nucleotide coding

sequence was found to have different numbers and po-

sitions of exons (Table 1 and 2). Most of the mosquitoes

APN gene possess almost similar ORF size, within a

range of 3-4 kb except Aedes aegypti. AUG and TAA

(mostly) are used as a start and stop codon, respec-

tively. Nucleotide sequence alignments of dipteran

APNs have shown the presence of limited conservation

at few short coding regions.

Nucleotide Composition of APN: Nucleotide Com-

position analysis of mosquitoes APN coding regions

shows that the Anopheles APN genes were found to

have higher GC content as compared to Aedes and

Culex APN genes. The analysis shows GC content varies

significantly among different species. Among Anophe-

les, An. gambiae has the highest GC content of (54.4%),

while among Aedes and Culex, Culex has more GC

(50.4%) content. The Aedes surprisingly has lower GC

content, lower than 50% and measured with highest

AT content of 51.5% among all mosquitoes. The data

is shown Table 3 for Anopheles, Aedes and Culex re-

spectively. The % GC and AT content of An. gambiae

APN gene was found to be 56.1 and 43.9 respectively,

while the values for GC1s, GC2s and GC3s are 55%, 43%

and 70% respectively. The frequency of codons ending

with G/C is higher than A/T in all mosquitoes. Table

3 shows the distribution of overall GC content of APN

gene in various mosquitoes. The nucleotide composi-

tion analysis of non-mosquitoes was also performed

and found that the APN gene belonging to Drosophila

were found to be GC rich except some other species

(Table 4). Dipterans APN gene was found to be GC rich

and as compared to 1st and 2nd codon position high

GC content was observed at 3rd codon position. This

clearly indicates that the Diptera APN gene is a biased

gene (Table 3 4). The overall nucleotide composition

and composition at the third codon position in APN

gene suggests that compositional constraint might be

influencing the codon usage pattern of these species.

Amino Acids Composition: As APN proteins are re-

ported to play an important role in digestion of blood.

The deduced amino acids composition of insect APN

proteins was determined. The amino acids compo-

sition of mosquito APN is shown in Table 5. Three

amino acids residues; Alanine, leucine and threonine

were found to be very rich in this protein, showing that

these residues may be in high biological demand dur-

ing digestion of blood. There was no other significant

difference found in the amino acids composition of

Acce Len Stop Ex

S Species ssion gth cod on AA Wt PI _M1 _C rich

No no. (bp) on No.

Anoph- AG eles AP 1 3588 ATG TAA 3063 5 1020 113 5.0 1-19 YAH gamb- 004 iae 809

Anoph- AA eles RA 2 3586 ATG TAA 3063 5 1020 113 5.1 1-19 YAH arabi- 016 ensis 470

AM Anoph- EM 3 eles 3599 ATG TAA 3063 5 1020 113 5.1 1-19 YAH merus

Anoph- AFAF 4 eles 015 3704 ATG TAA 3078 5 1025 114 5.0 1-25 YAH farauti 666

Anoph- AQ eles UA 5 3586 ATG TAA 3063 5 1020 113 5.0 1-19 YAH quadria- 016 nulatus 895

Anoph- ASIC 6 eles 009 3688 ATG TAA 3093 5 1030 114 4.9 1-20 LAH sinensis 153 Anoph- AATE eles 7 011 3872 ATG TAA 3072 5 1025 113 4.9 1-20 YAH atroparvus

AD Anoph- AC 8 eles 3612 ATG TAG 3060 5 1019 113 5.1 1-23 YAH darling

Anoph- AM eles AM 9 3805 ATG TGA 2775 5 924 104 5.0 - YAH macu- 007 latus 648

Anoph- AA eles LB 10 3591 ATG TAA 3048 5 1015 113 5.1 1-23 YAH albi- 015 manus 678

Anoph MK eles 11 033 3517 ATG TAA 3084 5 1027 114 5.0 1-25 YAH culicifacies AA Aedes- LF 12 albop 3322 ATG TGA 3003 5 1000 112 4.9 1-23 YAH ictus

AA Aedes- EL 13 2957 ATG TAA 2697 4 898 112 4.8 1-22 YAH ageypti 012

Culex CPIJ quanq- 14 001 3305 ATG TAA 3036 5 1011 113 4.9 1-20 YAH ue-asciatus

Pepti ERA Thre Sta Sig Zn Mol. dase P1 onine rt ORF nal bind

cod (bp) pep ing (kDa) dom dom regi on tide site ain ain on

H366E 292- 597- 887- 519 905 933

H366E 292- 597- 947- 519 905 993

H366E 292- 597- 947- 519 905 993

H366E 292- 597- 949 - 519 904 998

H366E 292- 597- 947 - 519 905 993

H365E 291- 596- 907- 518 902 1006

H361E 287- 592- 942- 514 892 997

H360E 286- 591- 939- 513 894 1003

H356E 282- 587- 509 892

H359E 285- 590- 889- 512 893 999

H372E 280- 603- 956- 525 912 1001

H359E 285- 590- 946- 512 903 974

H359E 285- 590- 512 898

H299E 285- 590- 866 - 512 912 925

Sr. Or Fam ess Species n ORF on ro AA cod cod da no der ily ion

Drosophila FBtr anan 012 2886 5 assae 2736 Drosophila FBgn moja 014 2859 4 vensis 7011

Drosophila 31 virilis 43

Drosophila 31 erecta 80

Drosophila FBgn melan 005 2859 4 ogaster 1233 Drosophila FBgn persi 016 2877 4 milis 1378 Drosophila FBgn grims 012 2880 5 hawi 6717 Drosophila pseuo 30 obsc 89 ura Drosophila FBgn yak 022 2883 5 uba 8109 Drosophila FBgn Dros 35 seche 018 2900 5 ophil 75 llia 1222 idae Drosophila FBgn simul 019 2886 5 ans 2352

Drosophila 30 willistoni 65 Dip 1. MDOA tera Musca dome 89 stica 55

Musc Stomoxys SCAU oidea calcit 003 2871 5 rans 701 Psy Lutzomyia LLOJ chodi long 009 2808 6 dae ipalpis 515

mosquito APN. Furthermore, other insects belonging

to Diptera also have more of the alanine and leucine

amino acids (Table 5). In some non-mosquitoes like

Drosophila grimshawi a significant decline in the num-

Si Pe Acc gn Mat ER Le Ex Int Start Stop pti Mol. al ure AP

Wt. Pe pep 1_ gth s ns on on se_ No. pti tide C M1 de

32 21- 298 – 589 – 4 961 108 1-20 ATG TAG 74 961 510 890

30 22- 291 – 585 – 3 952 107 1-20 ATG TAA 37 952 505 902

FBgn 23- 288 – 582 – 021 2850 4 3 949 109 1-22 ATG TAG 949 485 902 0169 FBgn 25- 294 – 587 – 010 2862 4 3 953 106 1-24 ATG TAG 953 508 888 9703

31 26- 293 – 586 – 3 952 106 1-25 ATG TAA 85 952 507 904

30 26- 294 – 585 – 3 958 107 1-25 ATG TAG 92 958 505 888

33 21- 297 – 588 – 4 959 109 1-20 ATG TAA 48 959 508 897

FBgn 23- 294 – 585 – 007 2877 4 3 958 107 1-22 ATG TAG 958 505 887 6210

35 23- 299 – 591 – 4 960 108 1-22 ATG TAA 04 960 510 888

23- 303 – 595 – 4 966 108 1-22 ATG TAA 966 515 893

35 23- 298 – 590 – 4 961 108 1-22 ATG TAA 66 961 510 888

FBtr 22- 293 – 584 – 024 2871 4 3 956 108 1-21 ATG TAA 956 505 887 3538

106 – 409 – 004 2346 5 4 781 89 - - GGC TAA 330 705

61 25- 283- 586- 4 956 108 1-25 ATG TAG 81 956 507 904

47 22- 270 – 578 – 5 934 105 1-21 ATG TGA 00 934 497 879

ber of alanine residues was noticed with an increase in

arginine and serine residues. The Leucine residues are

present in highest frequency in the amino acid compo-

sition of protein of each species (accounting for 9% of Sno. Species A+T % G+C% AT GC 1. Aedes ageypti 47.0 2. Culex quanquefasciatus 47.4 3. Anopheles darlingi 44.3 4. Anopheles culicifacies 45.0 5. Anopheles arabiensis 44.5 6. Anopheles gambiae 44.5 7. Anopheles merus 44.5 8. Aedes albopictus 46.9 9. Anopheles albimanus 42.2 10. Anopheles atroparvus 43.6 11. Anopheles farauti 41.8 12. Anopheles maculatus 44.4 13. Anopheles quadriannulatus 44.6 14. Anopheles sinensis 43.6 15. Anopheles stephensai 43.1

Table 4. Nucleotide composition of APN gene of non-mosquitoes (Diptera) Sno. Species A+T % G+C% AT GC 1. Drosophila melanogaster 46.6 2. Drosophila mojavensis 44.3 3. Drosophila virilis 45.9 4. Drosophila erecta 45.6

5. Drosophila ananassae 49.7 6. Drosophila persimilis 48.1 7. Drosophila grimshawi 61.3 8. Drosophila pseudoobscura 48.4 9. Drosophila yakuba 51.8 10. Drosophila sechellia 45.3 11. Drosophila simulans 50.3 12. Drosophila willistoni 51.3 13. Musca domestica 50.6 14. Stomoxys calcitrans 54.4 15. Lutzomyia longipalpis 51.0

total amino acids) whereas cysteine residues are least

abundant ( 1%) in the protein of insects (Table 6).

Synonymous Codon Usage: Codon bias is the non-

uniform use of synonymous codons which encode

the same amino acid. Some codons are more fre-

quently used than others in several organisms, par-

ticularly in highly expressed genes. The spectacular

diversity of insects makes them a suitable candidate

for analysing the codon usage bias. To understand the

pattern of non-random usage of synonymous codons in

the insects, RSCU of individual codons were compared

between mosquitoes and non-mosquitoes groups of

species. RSCU> 1 represents codons that are used

more frequently than expected whereas <1 represents

codons which are used less frequently. The overall

RSCU values for the 59 codons in Anopheles indicated

that C and G occurred more frequently at the third

codon position (as shown in Table 7). These are TTC,

CTG, ATC, GTG, TCC, TCG, CCG, ACC, ACG, GCC, GCG,

TAC, CAG, AAC, AAG, GAG, TGC, CGC and GGC. Among 61.4 46.2 53.2 38.5 53.6 51.5 48.5 59.2 41.6 52.3 40.9 58.1 49.6 50.4 56.1 30.4 56.0 43.9 69.9 43.4 56.6 55.4 29.6 55.1 44.9 70.1 43.4 56.7 55.9 30.9 55.4 43.8 68.8 44.0 56.0 56.9 30.7 55.4 43.6 69.5 43.9 56.1 56.0 32.3 55.6 43.6 68.1 44.3 55.7 58.3 40.8 53.3 41.1 59.6 48.6 51.3 56.6 22.0 57.8 43.5 77.9 40.4 59.7 55.6 20.4 56.8 44.4 80.0 39.5 60.4 56.7 18.3 58.0 43.7 81.5 38.9 61.1 56.7 33.0 56.0 43.0 67.0 44.6 55.3 55.8 31.4 55.6 43.9 68.1 44.2 55.8 56.0 25.5 56.0 44.3 74.6 41.6 58.3 55.2 23.0 56.4 45.3 77.5 40.3 59.8

61.5 30.9 53.4 38.4 68.8 46.5 53.6 60.6 34.9 55.9 38.1 65.1 46.5 53.4 60.7 38.9 53.8 39.2 61.5 48.5 51.5 60.8 25.0 53.9 39.8 74.8 44.0 56.0 58.8 30.8 48.0 40.2 68.9 53.2 53.2 59.3 24.8 52.2 51.7 75.3 56.1 56.1 47.9 50.1 39.0 40.7 49.5 46.7 46.7 59.7 25.0 52.0 42.2 75.2 55.9 55.9 57.9 29.0 55.9 39.0 71.0 54.0 54.0 60.6 34.9 48.9 42.7 65.5 53.4 53.4 56.9 32.9 49.0 - 67.1 47.1 52.5 59.8 54.8 45.7 39.8 45.1 55.4 44.7 62.7 45.6 50.4 37.2 54.6 54.1 45.8 60.3 40.3 49.4 38.8 59.4 50.5 49.6 60.8 53.4 49.4 39.6 46.7 54.7 45.2

these frequent codons, the two most frequently used

codon having highest values are CTG/Leu and CCG/Pro

and amino acids such as asparagine, lysine, glutamine,

phenylalanine, and tyrosine are encoded by one of the

preferential synonymous codon. Ae. aegypti and Cx.

quinquefasciatus APN genes also exhibit high synony-

mous codon usage bias (Table 7). The number of fre-

quently used codons and rarely used codons varies

among species, for example there were three amino

acids in Ae. aegypti were GAG is preferred over GAA

for glutamic acid and CCG is preferred over CCA for

proline. The APN gene of mosquitoes showed a high

synonymous codon usage bias in comparison to other

insects, preferentially using one or two synonymous

codons (Table 7 8). Three codons (CGC/Arg, ACC/Thr

and UCC/Ser) are used frequently in Diptera. Further-

more some codons are found that are used frequently

in each species of Diptera order of insects (Table 7 8).

Specific codon such as CUA/Leu and GGG/Gly are found

that are avoided in each species of each order. Within

a s p u e y s l y s l h l i A C A G P G H S no Species

1. Aedes aegypti 8.35 0.55 5.34 6.3 3.45 4.9 1.78 5.79 4.12 9.24 2.00 6.12 4.23 3.78 4.45 5.67 8.12 7.57 2.33 5.79 2. Culex quinquefasciatus 9.59 0.59 5.24 6.42 3.65 4.94 2.07 6.13 3.36 9.69 2.07 6.13 3.65 3.06 4.45 6.23 9.59 6.33 1.87 4.84 3. Anopheles darlingi 9.61 0.78 5.78 5.69 3.33 5.20 2.45 4.80 1.96 10.6 2.25 4.71 4.31 3.63 5.49 5.78 10.9 5.88 1.76 4.80 4. Anopheles culicifacies 10.6 1.07 4.96 5.74 3.99 4.96 1.46 4.47 2.23 9.44 2.72 5.06 4.18 3.21 5.55 6.62 9.93 7.10 1.94 4.67 5. Anopheles arabiensis 10.2 0.68 5.09 5.49 3.72 5.98 1.66 4.50 2.64 9.80 2.64 5.39 3.82 3.62 5.09 6.17 10.0 6.66 1.76 4.90 6. Anopheles gambiae 10.2 0.68 5.09 5.58 3.72 5.98 1.76 4.50 2.64 10.0 2.64 5.29 3.72 3.62 4.90 6.17 10.0 6.56 1.76 4.80 7. Anopheles merus 9.90 0.68 5.29 5.58 3.72 5.98 1.66 4.60 2.74 9.60 2.64 5.19 3.82 3.62 5.19 6.37 9.90 6.86 1.76 4.80 8. Aedes albopictus 9 1 5.3 6 3.9 5 2 9. Anopheles albimanus 10.5 0.59 5.51 5.91 3.25 5.32 2.26 4.63 2.56 10.1 2.16 4.23 4.03 3.94 5.32 5.91 9.95 6.79 1.77 5.12 10. Anopheles atroparvus 10.8 0.78 5.36 5.46 3.60 6.24 1.75 5.26 2.04 9.65 2.34 4.97 3.90 3.31 5.36 5.56 10.0 7.12 1.75 4.58 11. Anopheles farauti 10.8 0.58 5.65 5.36 4.09 4.87 1.95 4.39 1.85 9.75 2.24 4.68 4.09 3.41 5.75 5.56 10.2 8.19 1.75 4.68 12. Anopheles maculatus 10.2 0.64 4.76 7.90 4.11 5.30 1.94 5.08 2.16 8.87 2.16 4.97 4.11 3.46 6.70 5.95 8.00 6.16 1.83 5.51 13. Anopheles quadriannulatus 10 0.68 5.29 5.58 3.72 5.88 1.66 4.41 2.64 9.70 2.64 5.19 3.72 3.52 5.19 6.47 10.1 6.86 1.76 4.80 14. Anopheles sinensis 9.90 0.58 4.75 6.40 3.39 5.82 1.45 4.27 2.33 10.1 2.03 5.24 4.07 3.00 5.43 5.53 11.1 7.76 1.84 4.75 15. Anopheles stephensi 10.6 0.68 5.20 5.29 3.82 5.59 1.57 4.71 1.96 10.1 1.96 4.90 3.72 3.33 5.59 6.77 10.5 6.96 1.76 4.80

Table 6. Amino acids composition

a s p u e y s l y s l h l i S no Species A C A G P G H

1. Drosophila melanogaster 8.19 0.73 5.46 6.09 4.62 5.14 2.10 4.09 3.67 10.8 1.36 6.51 3.67 4.62 3.46 7.14 8.29 7.24 1.68 5.04 2. Drosophila mojavensis 9.13 0.73 5.77 5.98 4.30 4.72 2.20 4.30 3.67 10.9 1.68 6.09 3.88 4.93 4.20 6.93 7.56 6.19 1.78 4.93 3. Drosophila virilis 9.16 0.73 5.58 6.63 4.53 4.53 2.31 4.53 3.79 10.7 1.47 6.53 3.58 4.32 3.89 6.63 8.74 5.90 1.79 4.53 4. Drosophila erecta 8.49 0.62 5.24 5.35 4.93 5.56 2.09 3.67 3.67 10.2 1.36 6.71 3.67 4.93 3.56 7.66 7.97 7.66 1.67 4.82 5. Drosophila ananassae 7.59 0.72 5.61 5.20 4.26 4.47 2.08 5.09 2.70 10.8 1.14 6.76 3.64 4.16 4.05 9.78 8.42 5.82 2.28 5.30 6. Drosophila persimilis 8.49 0.73 5.24 6.19 5.03 4.72 1.99 4.82 3.25 11.2 1.36 7.24 3.77 5.03 3.46 6.92 7.13 6.71 1.78 4.82 7. Drosophila grimshawi 5.64 3.87 0.73 1.35 2.92 3.45 3.55 7.21 3.13 10.0 4.81 3.97 7.84 3.76 10.5 10.0 8.15 4.07 2.61 2.19 8. Drosophila pseudoobscura 8.35 0.62 6.15 4.69 4.48 4.90 1.77 4.59 3.02 11.4 1.04 6.88 3.13 5.11 3.86 9.39 8.03 5.01 2.40 5.01 9. Drosophila yakuba 7.60 0.63 5.63 4.68 4.27 4.68 1.88 5.31 3.02 10.7 1.04 6.56 3.13 3.43 4.38 10.1 9.47 5.52 2.18 5.72 10. Drosophila sechellia 9.13 0.73 5.77 5.98 4.30 4.72 2.20 4.30 3.67 10.9 1.68 6.09 3.88 4.93 4.20 6.93 7.56 6.19 1.78 4.93 11. Drosophila simulans 8.22 0.62 5.93 4.37 4.37 4.47 1.76 5.41 3.01 10.5 1.04 6.55 3.32 3.53 4.37 9.78 9.46 5.09 2.39 5.72 12. Drosophila willistoni 8.05 0.83 5.54 6.17 4.70 4.39 2.19 5.33 3.45 9.20 1.15 6.06 3.24 4.81 3.66 8.47 8.99 5.96 2.19 5.54 13. Musca domestica 7.55 0.76 5.63 5.12 3.84 4.73 2.43 5.63 5.12 8.57 2.30 6.91 3.58 4.99 3.58 8.19 6.65 5.76 2.17 6.40 14. Stomoxys calcitrans 8.47 0.62 5.64 5.64 4.18 5.33 2.82 5.43 3.87 8.68 2.40 7.32 4.28 4.18 4.39 6.38 7.21 5.75 2.09 5.23 15. Lutzomyia longipalpis 7.81 0.85 6.42 4.92 4.81 5.56 1.71 6.95 2.03 9.20 2.78 6.10 3.85 3.64 4.49 6.20 8.77 5.78 2.03 5.99

the Insect, the preference shifts accomplished by high

bias are seen in mosquitoes of Diptera and show pref-

erence shifts in three amino acids phenylalanine, histi-

dine and threonine.

Effect of base composition on codon bias: Because

of differences in mutational bias, the percentage of GC

content varies greatly among different species, even

in the species from the same order. An earlier study

shows that genes in the Hymenoptera genome are gen-

erally localized in GC poor regions whereas among

dipteral genomes such bias is not evident [18]. To de-

termine the association of GC biasness with codon

bias among different species, we move to the non-

directional codon bias measures effective number of

codon (ENC), which depends on nucleotide composi-

tion of genes [13]. An ENC value varies from 20 to 61,

when ENC=20 means that only one codon is used for

each amino acid (extreme codon bias) and ENC =61

means all codons are equally likely to code the amino

acid (no biased usage of codon). The dipterans and

particularly the mosquitoes were found to have the

highest codon usage bias with an average ENC value of

45 and also with the highest average GC content at third

codon position; 77.9 (Table 9). The greater the extent

of codon preference in a gene, the lesser is the corre-

sponding ENC value. The ENC values range from 40 to

56 in mosquitoes. Lower ENC value means more bias

(Table 9 10). The average for the non-mosquito species

s u t n o n g r r l p r e y e e s r l r e h a r y Il L L M A P G A S T V T T

6 3 9 2 5.7 4 4 4 7 9 7 2 5

of APN in non-mosquito Diptera

s u t n o n g r r l p r e y e e s r l r e h a r y Il L L M A P G A S T V T T

was 47.4. It was found that there was a positive corre-

lation between GC content and the degree of synony-

mous codon usage bias measured by ENC. The highest

synonymous codon usage bias was found in An. albi-

manus APN with an ENC of 40.4 and GC3s is 78% (9-10).

CAI is a directional measure of codon usage bias similar

to relative codon bias score unlike ENC. Thus compar-

ison between CAI and ENC provides a good quantita-

tive assessment between nucleotide composition and

codon bias selection. This gene shows increase in CAI

value with decrease in ENC value in Diptera. CAI is used

to calculate the level of gene expression. The average

CAI value ranges from 0.5 to 0.7, a level close to 1.0,

which might give a high expression level of genes ac-

cording to the index definition. Drosophila willistoni

is found to have lower codon biases with highest ENC

value.

s u t ia c s s a ie s f i d i e a n a i t u g f i c n i e p q i i b A y n l c b s i r i m o n g l a e u a u r a

in o

a q d c a g d . . . . . .

m o e u n n n n A C A C A A A A TTT 0.77 0.54 0.29 0.2 0.47 0.37 Phe (F) TTC 1.23 1.46 1.71 1.8 1.53 1.63 TTA 0.14 0.12 0.11 0.31 0.18 0.29 TTG 1.16 1.35 1.21 0.74 0.84 0.82 CTT 1.23 1.04 0.5 0.49 0.42 0.47 Leu (L) CTC 0.8 0.86 0.94 0.74 1.02 0.99 CTA 0.29 0.37 0.61 0.49 0.66 0.52 CTG 2.39 2.27 2.64 3.22 2.88 2.91 ATT 1.5 1.16 0.49 0.59 0.85 0.85 Ile (I) ATC 1.21 1.74 2.27 1.96 1.89 1.89 ATA 0.29 0.1 0.24 0.46 0.26 0.26 Met (M) AUG 1 1 1 1 1 1 GTT 1.35 1.5 0.53 0.22 0.59 0.66 GTC 0.88 0.75 1.4 1.1 0.88 0.9 Val (V) GTA 0.47 0.63 0.33 1.1 0.47 0.48 GTG 1.29 1.13 1.73 1.59 2.06 1.97 TCT 0.47 1.24 0.71 0.44 0.48 0.38 TCC 1.41 1.24 0.51 1.24 1.33 1.43 TCA 0.35 0.38 0.31 0.44 0.29 0.19 Ser (S) TCG 1.53 0.86 2.24 1.94 2 2 AGT 1.88 0.86 1.02 0.79 1.05 1.05 AGC 0.35 1.43 1.22 1.15 0.86 0.95 CCT 0.95 0.32 0.36 0.19 0.41 0.42 CCC 0.95 0.76 0.55 1.02 0.72 0.74 Pro (P) CCA 1.37 1.3 0.64 0.28 1.03 0.95 CCG 0.74 1.62 2.45 2.51 1.85 1.89 ACT 0.93 0.66 0.5 0.47 0.55 0.66 ACC 1.53 1.73 1.39 1.22 1.53 1.4 Thr (T) ACA 0.6 0.41 0.57 0.39 0.27 0.27 ACG 0.93 1.2 1.54 1.92 1.65 1.67 GCT 1.07 1.03 0.69 0.59 0.76 0.72 GCC 1.12 1.48 1.51 1.36 1.18 1.22 Ala (A) GCA 1.07 0.78 0.82 0.77 0.95 0.88 GCG 0.75 0.7 0.98 1.28 1.1 1.18 TAT 0.58 0.73 0.37 0.58 0.44 0.45 Tyr (Y) TAC 1.42 1.27 1.63 1.42 1.56 1.55 TAA(*) 3 3 0 3 3 3 STOP TAG(*) 0 0 3 0 0 0 CAT 1.25 0.86 0.64 1.2 1.18 1 His (H) CAC 0.75 1.14 1.36 0.8 0.82 1 CAA 0.94 0.65 0.59 0.42 0.27 0.27 Gln (Q) CAG 1.06 1.35 1.41 1.58 1.73 1.73 AAT 0.91 0.55 0.67 0.5 0.65 0.63 Asn (N) AAC 1.09 1.45 1.33 1.5 1.35 1.37 AAA 0.97 0.94 0.3 0.52 0.3 0.3 Lys (K) AAG 1.03 1.06 1.7 1.48 1.7 1.7 GAT 1.21 0.98 1.15 1.06 1.04 1 Asp (D) GAC 0.79 1.02 0.85 0.94 0.96 1 GAA 1.19 1.48 0.55 0.85 0.64 0.7 Glu (E) GAG 0.81 0.52 1.45 1.15 1.36 1.3 TGT 0.8 1 0.5 1.27 0.86 0.86 Cys (C) TGC 1.2 1 1.5 0.73 1.14 1.14 s u t a l s s s u s u n i u u v u s i c s e t t n s c n r i a a i n i a a t l i s r s e u p p u u n h r m o a c d o i a e p e b b r r a n e l l t a u i t m a a a f m q s s . . . . . . . . .

n e n n n n n n n A A A A A A A A A 0.42 0.56 0.24 0.22 0.29 0.26 0.47 0.34 0.21 1.58 1.44 1.76 1.78 1.71 1.74 1.53 1.66 1.79 0.18 0.07 0.06 0.06 0.06 0.29 0.18 0.06 0.06 0.73 1.4 0.93 0.91 0.78 0.88 0.67 1.2 0.58 0.43 0.63 0.06 0.48 0.06 0.8 0.42 0.23 0.47 0.98 1.12 1.34 0.97 1.38 0.88 0.97 1.03 1.05 0.67 0.42 0.52 0.12 0.3 0.8 0.61 0.4 0.41 3 2.37 3.09 3.45 3.42 2.34 3.15 3.09 3.44 0.96 1.36 0.26 0.72 0.8 0.64 0.87 0.41 0.69 1.79 1.42 2.74 2.11 2.13 1.98 1.93 2.39 2.19 0.26 0.22 0 0.17 0.07 0.38 0.2 0.2 0.13 1 1 1 1 1 1 1 1 1 0.74 1.08 0.64 0.55 0.43 0.56 0.57 0.8 0.34 0.8 1.03 0.87 1.1 0.95 1.12 0.86 1.1 0.85 0.46 0.59 0.41 0.38 0.38 0.91 0.51 0.6 0.51 2 1.3 2.09 1.97 2.24 1.4 2.06 1.5 2.31 0.46 0.79 0.1 0.11 0.11 0.22 0.36 0.21 0.35 1.57 1.5 1 1.79 1.89 1.31 1.36 1.47 1.48 0.09 0.71 0.2 0.11 0 0.44 0.18 0.21 0.17 1.94 0.79 2.3 2.53 1.89 1.75 2.09 1.89 1.74 1.02 1.06 0.7 0.32 0.42 1.53 1 0.84 0.96 0.92 1.15 1.7 1.16 1.68 0.76 1 1.37 1.3 0.41 0.76 0.29 0.4 0.1 0.53 0.42 0.29 0.32 0.82 1.05 0.49 1 1.14 0.84 0.74 0.76 0.95 1.03 1.05 0.59 0.3 0.19 0.74 0.95 0.57 0.42 1.74 1.14 2.63 2.3 2.57 1.89 1.89 2.38 2.32 0.59 0.75 0.4 0.54 0.27 0.43 0.65 0.52 0.41 1.35 1.58 1.31 1.79 1.75 1.51 1.38 1.57 1.38 0.32 0.4 0.36 0.23 0.27 0.22 0.35 0.28 0.07 1.74 1.27 1.94 1.44 1.71 1.84 1.62 1.63 2.13 0.79 0.72 0.49 0.29 0.29 0.59 0.75 0.55 0.29 1.11 1.57 1.64 1.77 1.44 1.52 1.18 1.88 1.76 0.99 1.11 0.34 0.36 0.4 0.72 0.98 0.59 0.62 1.11 0.6 1.53 1.59 1.87 1.18 1.1 0.98 1.32 0.45 0.72 0.35 0.3 0.25 0.59 0.49 0.37 0.24 1.55 1.28 1.65 1.7 1.75 1.41 1.51 1.63 1.76 3 0 3 3 3 0 3 3 3 0 0 0 0 0 0 0 0 0 1.18 0.7 0.7 0.56 0.5 1 1.18 0.4 1.25 0.82 1.3 1.3 1.44 1.5 1 0.82 1.6 0.75 0.32 1.1 0.35 0.24 0.23 0.31 0.28 0.32 0.18 1.68 0.9 1.65 1.76 1.77 1.69 1.72 1.68 1.82 0.68 0.67 0.33 0.24 0.17 0.74 0.64 0.41 0.32 1.32 1.33 1.67 1.76 1.83 1.26 1.36 1.59 1.68 0.43 0.63 0.31 0.19 0.32 0.6 0.44 0.25 0.4 1.57 1.38 1.69 1.81 1.68 1.4 1.56 1.75 1.6 1 1.06 1.14 0.44 0.93 0.91 1 0.73 0.72 1 0.94 0.86 1.56 1.07 1.09 1 1.27 1.28 0.67 1.11 0.53 0.68 0.73 0.82 0.77 0.73 0.74 1.33 0.89 1.47 1.32 1.27 1.18 1.23 1.27 1.26 0.86 0.8 0.33 0 0.67 0.67 0.86 0.33 0.86 1.14 1.2 1.67 2 1.33 1.33 1.14 1.67 1.14 Amino Acids Sequence Alignment: Multiple sequence

alignment of APN protein sequences were performed

separately to know the conservation patterns within an

order. The complete sequences of amino acids of APN

belonging to Diptera order have been analysed. Se-

quence analysis of APN indicates all are evolved from

common ancestors. The amino acids sequence align-

ment of is shown in Fig.1 2. The areas of highly con-

served regions for amino acid were clearly observed.

From the MSA it is clear that residues GAMEN, HEXXH

and NEGFA are highly conserved, showing that these

conserved sites are present in almost all the aligned se-

quences. All these three motifs are found conserved in

Anopheles at similar positions. These repeats were also

found in other mosquito species and showed homol-

ogy to other insects. Glycosylation sites are also found

conserved in all mosquitoes. Four cysteine residues

are also found conserved in all insects. GPI prediction

program exhibited the presence of GPI anchor signal se-

quence at the C-terminus. Such a sequence was found

to be identical with An. gambiae and An. stephensi.

Furthermore analysis of the C-terminal part of all in-

sect APNs revealed a high degree of conservation of

tyrosine residues.

Phylogenetic Analysis: Coding regions of AcAPN1

were examined for Phylogenetic analysis to study the

evolutionary patterns among mosquitoes. This analy-sis

includes 15 selected mosquitoes for the APN1 gene. The

evolutionary pattern on phylogenetic tree showed that

Anopheles, Aedes and Culex APN1 gene may have

originated from common ancestors and revealed close

similarity to each other. The relationship among all

three genus of mosquito is more strikingly represented

in two clades: Anopheles and Aedes/ Culex, this show

Aedes and Culex shows close similarity than Anophe-

les. The evolutionary relationship of APN1 protein

derived from different Anopheles was also evaluated,

among this Anopheles group An. culicifacies found

to be placed nearer to An. stephensi and An. gam-

biae, to these which is in agreement with the pep-

tide sequences of these genera as all contains GAMEN

catalytic domain (Fig. 3). The tree topology shows

that the Anopheles APN1 shares orthologs from other

mosquitoes such as Aedes and Culex. Phylogenetic tree

based on APN protein of fifteen species of Diptera order of

Non mosquitoes were analysed separately to know the

evolutionary relationship of organisms within an

order. Phylogenetic tree also supported the grouping

of species into two major clades. Phylogenetic analy-

sis of APN indicates that all are evolved from common

ancestors (Fig. 3 4).

Discussion

Conserved DNA sequences of APN gene have been re-

trieved from 30 mosquitoes and non-mosquito species

from NCBI, GenBank etc and has been analysed with

various bioinformatic tools. The APN gene sequences

consist of a start site, ATG followed by TAA (in most

species) as termination codon site. The nucleotide

sequences of all APN insects were found to have dif-

ferent numbers and positions of exons. The ORF re-

gion of APN gene almost falls in between 2-3 kb. The

present investigation highlights comparative studies

on nucleotide composition and codon bias between

30 species belonging to order Diptera of class Insecta.

As synonymous is not uniform during the translational

process, the identification of the codon usage pattern

is important to understand the translational selection

of codons in protein coding genes in various insects’

species. The overall RSCU values in mosquitoes indi-

cated that C/G occurred more frequently at the 3rd

codon position whereas in Hymenoptera, Lepidoptera

and Hemiptera insect orders most frequent codon ends

with A/T at the 3rd position (Sharma et al., 2014). In

Anopheles and Culex species APN gene was found to

be CG rich, whereas in Aedes AT richness was observed.

Also for comparison, coding sequences of APN gene

were taken from various insects to perform nucleotide

composition analysis. All insect species were found to

be AT rich except Diptera. The analysis shows that at

1st and 2nd codon positions, the A content was found

higher than the T content except for the 3rd codon po-

sition. Dipterans APN gene was found to be GC rich and

as compared to 1st and 2nd codon position high GC

content was observed at 3rd codon position. These ob-

servations clearly indicate that the Diptera APN gene is

a biased gene. The pattern of GC contents of the honey-

bee genome and its relationship with codon usage was

studied [19]. This showed that genes located in GC poor

regions showed much larger deviation in both codon

usage bias and amino acid bias than genes located in

GC rich regions. The codon context pattern in the honey

bee genome indicates that T and G combination occurs

more frequently for adjacent 1st and 3rd positions of

codon pairs than A and C combinations [20]. It thus sug-

gested that such a pattern of codon context bias might

have evolved in order to minimize the effect of nu-

cleotide mutations coding regions. Except methionine

and tryptophan, the APN1 amino acid composition re-

vealed mosquito-specific amino acid usage. This study

shows the amino acid alanine is the amino acid that

accounts for the greatest usage and cysteine accounts

for the least usage in the genes of Diptera. The amino

acid composition analysis of other orders of APN re-

veals the presence of thymine residues in high content s u t a i c s s a i f i c s e d i e a n i t g a u f e i c p q n i i b A s y n l i b g i r l m o n a e u a u r a n o a q d c a g

i d

. . . . . .

m o e u n n n n A C A C A A A A

STOP TGA(*) 0 0 0 0 0 0 Trp (W) TGG 1 1 1 1 1 1 CGT 1.2 2.13 1.5 2 1.62 1.56 CGC 1.35 1.33 2.68 2 2.42 2.4 CGA 1.8 1.07 0.64 0.21 0.81 0.96 Arg (R) CGG 0.6 0.4 0.75 1.26 0.69 0.72 AGA 1.05 0.53 0.21 0.21 0.35 0.24 AGG 0 0.53 0.21 0.32 0.12 0.12 GGT 1 1.28 1.96 1.57 1.38 1.31 GGC 1.18 0.64 0.98 1.18 1.44 1.57 Gly (G) GGA 1.73 2.08 0.75 0.63 0.85 0.79 GGG 0.09 0 0.3 0.63 0.33 0.33

[21]. The present study compares the codon usage pat-

terns between different types of species from Diptera

order of insects. As synonymous codon usage is not uni-

form during the translation process, the identification

of the codon usage pattern is important to understand

the translational selection of codons in protein coding

genes among related species. A significant high synony-

mous codon usage bias was observed in mosquito APN

gene belonging to the order Diptera, by predominantly

using one or two optimal synonymous codons over

others. Similar results observed in non-mosquitoes

species of Diptera order. In this study, the overall RSCU

values for the 59 codons in mosquitoes indicated as

C and G (C is more) occurs more frequently at the 3rd

codon position. These results indicate that codon us-

age pattern in the mosquitoes is mostly contributed by

compositional constraints. This main factor affects the

synonymous codon usage bias reported as in the case

of D. melanogaster [22-23]. In Drosophila generally G

and especially C are favoured at synonymous sites in

biased genes. In Anopheles, the codon (CTG/Leu and

CCG/Pro) are used more frequently. Amino acids like

phenylalanine, tyrosine, glutamine, asparagine, lysine

and cysteine are almost solely encoded by one syn-

onymous codon. The codon bias of Aedes ageypti and

Anopheles gambiae mosquito’s genome was analysed

[24]. Genome wide analysis of relative synonymous

codon usage value of gene indicated that only a select

set of codon was optimized in these mosquitoes. Op-

timized and rare codons were also identified in both

species. Results support that high codon bias occurs

in highly expressed genes. The comparison shows that

although the pattern of codon usage of the Culicinae s u t a l s s s s u ie s u i u u u n s t v t n n r i s n c a a a i e s i a t l i s p p u r h i c u m u n r o i o a c d p a e e b b r r a n e l l t a u i t m a a a f m q s s . . . . . . . . .

n e n n n n n n n A A A A A A A A A

0 3 0 0 0 3 0 0 0 1 1 1 1 1 1 1 1 1 1.58 1.4 0.78 0.87 0.51 0.87 1.7 1.71 0.63 2.49 1.12 2.56 2.84 2.24 1.94 2.38 2.14 2.74 0.91 1.53 0.22 0.33 0.41 0.77 0.91 0.54 0.84 0.57 0.7 2.11 1.31 2.34 0.87 0.57 0.96 1.16 0.23 0.42 0.22 0.44 0.2 1.26 0.23 0.32 0.21 0.23 0.84 0.11 0.22 0.31 0.29 0.23 0.32 0.42 1.25 1.2 1.93 1.63 1.28 1.39 1.4 1.47 1.33 1.57 0.8 1.26 1.5 1.6 1.63 1.33 1.13 1.61 0.92 1.68 0.44 0.63 0.48 0.82 0.87 0.87 0.7 0.26 0.32 0.37 0.25 0.64 0.16 0.4 0.53 0.35

mosquitoes (Ae. ageypti and Cu. quinquefasciatus) is

similar to the Anopheline mosquito, there was varia-

tion in the usage of individual codons [25]. Because half

of the codons are more biased in An. gambiae than in

Ae. aegypti and Cu. quinquefasciatus, while the other

half of codon tends to be more biased in Culicinae than

An. gambiae. Detailed analysis of codon bias among

twelve species of Drosophila sequenced genome was

done[26], It was found that one species, D. willistoni

has unusually low codon usage bias pattern in the us-

age of synonymous codons. While G or C ending codons

were preferentially used in other species, D. willistoni

genes showed a shift toward A or T ending codon prefer-

ence and high ENC value. Genome sequences of 22 in-

sect species of order Diptera and Hymenoptera where

was known [27]. The study revealed that in Diptera

more frequent codon ends with G or C mostly at 3rd

codon position whereas in Hymenoptera insect order,

most frequent codon contain A or T at its 3rd position.

Also in Lepidoptera, analysis of codon usage bias of

Bombyx mori revealed that most of the preferentially

codons end with A or T at the 3rd codon position [28].

Codon usage on two Hemiptera insect species namely,

Bemisia tabaci and Homalodisca coagulata was stud-

ied [21]. They found that most frequent codons end

with A or C at 3rd codon position. The above said stud-

ies suggest that the third position of codon seems to af-

fect codon bias. The results of our analysis are in agree-

ment with these results in similar manner in species of

Diptera orders. This finding may be the result of compo-

sitional constraint that occurs in codon usage patterns

in these insect species. The extent of codon bias in

mosquitoes APN genes based on the RSCU value was

Table 10. Relative Synonymous Codon Usage (RSCU) of APN gene in non-mosquitoes (Diptera) is s e s a s d a i

i

n g l c s e o i s A a v n s a m

s

a a i t i o n n j l l c s a i e r n o n o e r r e i i d a m m v e p

m o

. . . . . .

A C D D D D D D TTT 0.59 0.54 0.59 1.02 0.64 0.54 Phe (F) TTC 1.41 1.46 1.41 0.98 1.36 1.46 TTA 0.12 0.12 0.12 0.24 0.06 0 TTG 1.46 0.98 1.46 1.29 1.29 0.56 CTT 0.7 0.4 0.7 0.12 0.43 0.5 Leu (L) CTC 0.29 0.69 0.29 0.82 0.49 1.01 CTA 0.64 0.75 0.64 0.71 0.12 0.17 CTG 2.8 3.06 2.8 2.82 3.61 3.76 ATT 1.69 1.17 1.69 1.4 1.29 0.91 Ile (I) ATC 1.23 1.54 1.23 1.26 1.63 2.02 ATA 0.08 0.29 0.08 0.35 0.09 0.07 Met (M) AUG 1 1 1 1 1 1 GTT 0.58 0.61 0.58 0.86 0.66 0.44 GTC 1.04 1.02 1.04 1.29 1.15 1.81 Val (V) GTA 0.29 0.34 0.29 0.21 0.05 0.38 GTG 2.09 2.03 2.09 1.64 2.14 1.38 TCT 0.26 0.82 0.26 0.76 0.08 0.36 TCC 2.12 1.18 2.12 1.43 2.3 2 TCA 0.18 1 0.18 0.29 0.25 0.09 Ser (S) TCG 0.79 0.55 0.79 1.05 0.58 1.36 AGT 0.35 0.55 0.35 0.38 0.41 0 AGC 2.29 1.91 2.29 2.1 2.38 2.18 CCT 0.46 0.65 0.46 1.29 0.34 0.44 CCC 2.29 1.73 2.29 1.53 2.4 2.56 Pro (P) CCA 0.69 0.65 0.69 0.71 0.57 0.56 CCG 0.57 0.97 0.57 0.47 0.69 0.44 ACT 0.76 1.22 0.76 0.48 0.47 0.76 ACC 2.13 1.44 2.13 1.64 2.37 2 Thr (T) ACA 0.51 0.61 0.51 0.92 0.37 0.47 ACG 0.61 0.72 0.61 0.96 0.79 0.76 GCT 0.82 0.87 0.82 0.84 0.69 0.81 GCC 2.56 1.98 2.56 1.93 2.57 2.57 Ala (A) GCA 0.31 0.51 0.31 0.46 0.2 0.44 GCG 0.31 0.64 0.31 0.55 0.4 0.3 TAT 0.83 1.11 0.83 1.44 0.74 0.43 Tyr (Y) TAC 1.17 0.89 1.17 0.56 1.26 1.57 TAA(*) 1 0 1 0 0 0 STOP TAG(*) 0 0 0 0 0 3 CAT 0.8 0.76 0.8 1.45 0.9 0.53 His (H) CAC 1.2 1.24 1.2 0.55 1.1 1.47 CAA 0.41 0.34 0.41 0.54 0.34 0.25 Gln (Q) CAG 1.59 1.66 1.59 1.46 1.66 1.75 AAT 0.68 0.97 0.68 1.13 0.72 0.49 Asn (N) AAC 1.32 1.03 1.32 0.87 1.28 1.51 AAA 0.4 0.74 0.4 0.39 0.29 0.19 Lys (K) AAG 1.6 1.26 1.6 1.61 1.71 1.81 GAT 1.04 1.13 1.04 1.32 0.8 1 Asp (D) GAC 0.96 0.87 0.96 0.68 1.2 1 GAA 0.52 0.46 0.52 0.48 0.27 0.34 Glu (E) GAG 1.48 1.54 1.48 1.52 1.73 1.66 TGT 0.57 0.29 0.57 0.86 0.67 0.57 Cys (C) TGC 1.43 1.71 1.43 1.14 1.33 1.43

determined by the effective number of codons (ENC).

is s p n l a a a r a r p r c t i e u i i g t c t c n i s s l b i e a o w s c l o ia n a l n o m s a h o a l a o i s d b l t y y e is d x u u h u l m im e k c l a o s m i c o r a e i s m z g p y s s w t u o . . . . . . t u D D D D D D M S L 1.14 0.47 0.54 0.54 0.71 0.84 0.53 0.75 0.67 0.86 1.53 1.46 1.46 1.29 1.16 1.47 1.25 1.33 1 0.07 0.06 0.18 0.12 0.41 0 0.14 0.42 1.69 0.65 0.93 0.98 0.89 1.77 4.03 2.89 1.19 0.56 0.38 0.47 0.4 0.65 0.75 0.45 0.58 1.33 0.75 0.98 0.93 0.69 0.65 0.41 0.72 0.72 2.16 0.94 0.44 0.35 0.75 0.59 1.43 0.18 0.43 0.35 1.06 3.49 3.26 3.06 3.09 1.23 0.63 1.23 0.56 0.78 0.82 0.94 1.17 0.75 1.29 1.5 1.38 1.43 1.26 1.91 1.76 1.54 2.08 1.06 1.43 1.21 1.34 0.96 0.27 0.29 0.29 0.17 0.65 0.07 0.4 0.23 1 1 1 1 1 1 1 1 1 0.82 0.25 0.53 0.61 0.82 1.33 1.6 1.02 1.78 0.82 1.58 1.28 1.02 1.06 0.84 1.33 1.09 0.52 0.92 0.25 0.15 0.34 0.33 0.7 0.36 0.51 0.3 1.44 1.92 2.04 2.03 1.8 1.12 0.71 1.38 1.41 1.13 0.4 0.12 0.82 0.32 0.67 0.47 0.69 0.72 0.81 1.67 1.92 1.18 1.98 1.19 1.88 1.57 0.83 1.13 0.27 0.31 1 0.19 1.33 0.47 0.69 1.24 0.75 1.33 1.42 0.55 1.15 0.89 0.75 1.28 0.41 1.13 0.73 0.56 0.55 0.77 0.96 1.03 1.08 1.86 1.06 1.6 1.67 1.91 1.6 0.96 1.41 0.69 0.93 1.28 0.4 0.4 0.65 0.75 0.9 0.71 0.78 0.78 0.59 2.4 1.73 1.73 1.88 1.29 2.14 2.63 1.56 1.55 0.67 0.65 0.65 0.75 1.42 0.71 0.49 1.56 0.59 1.2 1.2 0.97 0.63 0.39 0.43 0.11 0.11 0.87 0.52 0.75 1.22 0.84 1.16 0.85 0.64 0.59 0.41 1.82 1.85 1.44 1.63 0.88 2.31 2.26 0.73 1.54 0.57 0.4 0.61 0.44 1.16 0.46 0.64 1.9 1.18 1.09 1.01 0.72 1.1 0.79 0.38 0.46 0.78 0.25 0.99 0.87 0.87 0.96 1.25 1.22 0.64 1.21 0.52 2.85 2.19 1.98 2.13 1.56 2.31 2.91 1.15 1.7 0.3 0.44 0.51 0.56 1.04 0.34 0.3 1.42 0.96 0.6 0.38 0.64 0.35 0.16 0.14 0.15 0.22 1.05 0.75 0.62 1.11 0.76 1.43 1.4 1 0.82 0.95 1.25 1.38 0.89 1.24 0.57 0.6 1 1.18 0.96 0 0 0 0 0 3 0 0 0.75 0 0 0 0 0 0 3 0 0.82 0.71 1 0.76 0.59 1.43 0.95 0.81 1.38 1.18 1.29 1 1.24 1.41 0.57 1.05 1.19 0.63 1.06 0.45 0.42 0.34 0.47 0.83 1.23 1.1 1 0.94 1.55 1.58 1.66 1.53 1.17 0.77 0.9 1 1.16 0.7 0.63 0.97 0.76 1.31 1.07 1.2 1.26 0.84 1.3 1.37 1.03 1.24 0.69 0.93 0.8 0.74 1.2 0.28 0.28 0.74 0.34 1.03 1.1 0.92 0.84 0.8 1.72 1.72 1.26 1.66 0.97 0.9 1.08 1.16 1.14 0.98 1.41 1.13 1.3 1.32 1.27 1.07 1.53 0.86 1.02 0.59 0.87 0.7 0.68 0.73 0.93 0.47 1.38 0.36 0.67 0.46 0.57 1.25 1.7 1.07 1.26 0.62 1.64 1.33 1.54 1.43 0.75 0.3 0.93 0.74 0.59 0.67 0.29 0.67 0.75 1.33 0.67 0.33 0.86 1.41 2 1.33 1.71 1.33 1.25 0.67 1.33 1

The APN gene of mosquitoes has a codon bias ranging s t i s e s a s a i d n g l i s i c s e o v a A a n s t m s a a i i n j l l c s o n a i r o o e r e

in

n i r e

d

a m m v e p m o . . . . . .

A C

D D D D D D

STOP TGA(*) 0 0 0 0 0 0 Trp (W) TGG 1 1 1 1 1 1 CGT 1.64 1.65 1.64 1.62 1.24 1.27 CGC 2.91 3.45 2.91 2.43 3 4 CGA 0.36 0.15 0.36 0.97 0.53 0 Arg (R) CGG 0.18 0.6 0.18 0.81 0.18 0.36 AGA 0 0 0 0 0 0.18 AGG 0.91 0.15 0.91 0.16 1.06 0.18 GGT 1.39 1.24 1.39 1.86 0.83 0.8 GGC 0.98 2.04 0.98 1.67 1.58 2.31 Gly (G) GGA 1.47 0.53 1.47 0.19 1.51 0.71 GGG 0.16 0.18 0.16 0.28 0.08 0.18

Table 12. Effective number of synonymous codons and GC content of APN gene in mosquitoes (Diptera) from ENC from 40.4 (An. albimanus) to 56.2 (An. gam- biae), the larger the extent of codon preference in a gene shows synonymous codon usage, the smaller the corresponding ENC value. It was found that there was a positive correlation between GC content and degree of

synonymous codon usage bias measured by ENC.

The highest synonymous codon usage bias of An. albi-

manus APN with an ENC of 40.4 indicates the biased

GC3s (77.9). This gene shows increase in CAI value with

decrease in ENC value in Diptera. The average CAI value

was found as 0.7, a level close to 1.0, which might give

a high expression level of genes according to the index

definition. Many highly expressed genes show high syn-

onymous codon usage bias for fast and accurate trans-

lation. In conclusion, codon bias is most prominent in

highly expressed genes for translational competency

and accuracy [29-30]. The results revealed the simi-

lar pattern was observed from other highly expressed

genes [31]. So, higher codon biasness is found in APN1

gene among various mosquitoes and it is clear that

APN1 is one of the highly expressed gene plays impors i s p n l a a a r r a r p e c t i u i i g c t c i s s l n i e o w b a s a l o i n c a n m o a l o s a h l a t o i s d b e l s y y i d u u h u l x m k l a o m i e c m i o r s a e i c s m z g p y s s w o t . . . . . . u t u D D D D D D M St Lu

0 1.29 0 0 0 0 0 0 3 1 1 1 1 1 1 1 1 1 0.48 0.81 1.14 1.65 1.57 1.89 3 2.86 1.29 0.89 2.11 2.71 3.45 1.86 1.89 1.93 2.43 1.86 0.83 1.14 0.14 0.15 0.57 0.51 0 0.14 0.71 0.83 0.16 0.71 0.6 0.71 0.34 0.21 0.14 0.14 1.25 0.49 0.43 0 0.57 0.86 0.64 0.14 1 1.72 1.3 0.86 0.15 0.71 0.51 0.21 0.29 1 0.97 1.02 1.16 1.24 1.02 1.71 2.05 1.73 1.08 1.21 1.79 1.96 2.04 1.77 0.86 1.73 1.65 0.77 1.09 0.68 0.89 0.53 1.21 1.43 0.22 0.47 1.69 0.73 0.51 0 0.18 0 0 0 0.16 0.46

Species CAI ENC GC3s GC % Culex quinquefasciatus 0.749 52.1 58.1 50.4 Aedes ageypti 0.771 54.5 53.6 48.5 Anopheles darling 0.783 45.4 69.9 56.6 Anopheles gambiae 0.527 56.2 69.4 47.8 Anopheles arabiensis 0.734 47.9 68.8 56 Anopheles merus 0.661 48.2 68.1 55.8 Anopheles culicifacies 0.735 46.2 70 56.6 Anopheles albimanus 0.737 40.4 77.9 59.7 Aedes albopictus 0.705 55.4 59.5 51.4

tant in blood digestion and parasite invasion processes.

APN amino acid sequence analysis shows the presence

of high amino acid conservation among specific areas in

all insects, all aligned confidently along their entire

lengths. These include M1 Peptidase and ERAP1-like

C- terminal domains. The zinc ions play an important

role in the catalysis mechanism of metalloproteases

like aminopeptidase. Multiple alignment results of dif-

ferent APN sequences showed the highest similarity

between the Dipteran APN sequences [32]. In this study

multiple alignments showed three major conserved

sites in almost all the aligned sequences Where HEXXH

motif is also conserved, which is responsible for pep-

tidase activity [33-34]. The zinc metalloproteases like

Aminopeptidase N among various phyla were shown

to bear a signature sequence motif HEXXH where they

bind the zinc atom in tetrahedral fashion. The highly

conserved HEYAH amino acid residues are found in all

insects [35]. Here the histidine residue acts as ligands

of zinc ion where GLU plays a catalytic role. The GAMEN

domain signature was also found in all insects and showed homology to each other. Many workers have

determined hydrolysis and catalytic activities of APNs.

Above said site GAMEN found as Bastatin binding site in

APN of E. coli [36]. Third motif WWDNL found as a pep-

tide binding site in human APN and it was found that

this motif is also important in tumor angiogenesis and

binding with peptides [37]. Also, these conserved sites

are reported for Cry toxin protein binding region on

APN [38]. The entire above said three motifs are found

to be conserved in APN of M. sexta and H. armigera the

similar sites were predicted by Conseq and Consurf

servers [39]. APN protein of all the insects encoding

amino acids sequence within 900-1000 amino acids

and weights of APN protein was found in between 90 to

100 kDa. Another important finding is that APN follow

secretary pathway, as amino terminal is encoded by

signal sequences. Moreover, in the present investiga-

tion, GPI prediction program exhibited the presence

of GPI anchor signal sequence at the C-terminus. Such

sequence was found to be more conserved in dipterans

and lepidopterans. Besides, the amino acid sequence

also contained four Cysteine residues. Cys residues are

important in the structural integrity of proteins by form-

ing disulphide bridges, which in turn helps to keep the

molecules intact and to maintain the conformation of

the active sites. These residues help in enhancing the

stability of the proteins [40]. These residues are highly

conserved among APN molecules of higher mammals

(rat, rabbit, pig and human), was detected [41].The

post translational modification i.e. phosphorylation

was found at serine, threonine and tyrosine residues

conferring protection from proteolytic degradation.

Protein-protein interaction results show that APN pro-

tein connected to glutathione synthase may involve

in metabolic pathways for metabolism of Glutathione.

Glutathione makes mosquito insecticide resistance, so Species CAI ENC GC3s GC % Drosophila mojavensis 0.714 45.8 65.5 53.5 Drosophila ananassae 0.801 40.4 74.8 55.9 Drosophila virilis 0.789 48.0 61.5 51.5 Drosophila erecta 0.790 40.8 74.8 56.0 Drosophila sechellia 0.796 42.1 70.8 54.2 Drosophila melanogaster 0.782 43.7 68.8 53.5 Drosophila persimilis 0.768 41.5 75.3 56.1 Drosophila grimshawi 0.661 55.2 52.2 47.4 Drosophila pseudoobscura 0.718 42.0 75.2 55.9 Drosophila yakuba 0.739 45.1 70.9 54.0 Drosophila simulans 0.748 48.0 67.1 52.9 Drosophila willistoni 0.808 55.2 45.1 44.7 Musca domestica 0.815 42.9 54.6 45.8 Lutzomyia longipalpis 0.777 52.7 46.7 49.5 Stomoxys calcitrans 0.748 45.7 59.4 45.3

disrupting of APN protein or any other protein, this

protein network may block the whole metabolic path-

way of Glutathione [42]. Amino acid sequence analysis

of the Dipterian APN gene shows that they all can be

aligned confidently along their entire lengths. Spe-

cific areas of relatively high amino acid conservation

were observed. The ORF’s range among all APN was

found from 2 to 3.5 kb, encoding a 900-1000 amino

acids sequence. All APN protein molecular weights

were found to be above 90 kDa. Three important do-

mains namely Aminopeptidase N like, M1 Peptidase

and ERAP1-like C- terminal domain were identified in

all APNs. All APNs are found to have four conserved

Cysteine residues. Signal peptide sequence of 15-30

amino acids was present at N terminal end and GPI

anchored region (hydrophobic amino acid residues)

at C terminal end of APN protein. The APN analysis

revealed the three conserved regions, gluzicin APN mo-

tif GAMEN, second one is Zinc- binding/ gluzicin motif

HEXXH, which is a part of a typical catalytic site for the

majority zinc dependent metallopeptidase and is re-

quired for enzymatic function, the third one is zinc bind-

ing ligand which is conserved in the sequence motif

NEXFA. Highly conserved region is present in domain I,

about 64 amino acid residues believed to be important

for Cry 1 toxin binding. The Phylogram was constructed

using the mosquitoes APN sequences and similar se-

quences of non-mosquitoes origin retrieved from the

NCBI database. Undoubtedly, the ancestry species of

the gene should be more closely related to sequences

from a similar genus. In the present investigations the

An. gambiae and An. stephensi seems to be the closest

relatives of the An. culicifacies [43]. Out of the non-

mosquitoes species, with no surprise are closely re-

lated to each other as they are the latest to diversify on

the path of the evolutionary journey. Phylogenetic tree ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. .................................. | Anopheles_gambiae -------------------------------MVRLLLLVSLGLCCLLASADRPPYKSSGIYEEEPQLPGASDLSGGVASDEIGVAPAQAVDERYRLPTTSIPIHYDLHLRTEIHRNERTFTGTV Anopheles_arabiensis ----------------------------MVRSLLLVSLGLCCLLASADRPPYKSSGIYEEEPQLPGASDLSGGVASDEIGVAPAQAVDERYRLPTTSIPIHYDLHLRTEIHRNERTFTGTV Anopheles_merus ----------------------------------MVRSLLLVSLGLCCLLASADRPPYKSSGIYEEEPQLPGASDLSGGVASDEIGVVPAQPVDERYRLPKTSIPIHYDLHLRTEIHRNERTFTGTV Anopheles_quadriannulatus-------------------MVRSLLLVSLGLCCLLASADRPPYKSSGIYEEEPQLPGASDLSGGVASDEIGVAPAQAVDERYRLPTTSIPIHYDLHLRTEIHRNERTFTGTV Anopheles_maculatus -------------MYLFEDNRFARAERPPFKSSSGLYEDEPQLAASEQEGDGT ------ FVGIVPAQPVDEAYRLPKTSIPIHYDLHLTTEIHRNERQFSGTV Anopheles_stephensai Anopheles_culicifacies MVRFWILVSLGLCCLVAPNDRFVRAEVRPPFKTGGIYEDEPQLLLDESERSDASVEQGAVVPAQAVDERYRLPKTSVPIHYDLHLRTEIHRNERTFTGSV Anopheles_farauti -MVRSLILATLGLWCLLGPANVFTRAERPPFKASGLYDQEPELAVTDP------------ DTVAVAVPVQAVDERYRLPKTSVPLHYDLHLRTEIHRNERTFQGSV Anopheles_atroparvus ------MVRALIVVSIGVCCLLLAGAERPPFKSRGIHGED------- ELAEAGAASEVTGVAPSQAVDETYRLPKTSVPVHYDLHLRTEIHINERTFDGSV Anopheles_sinensis ------MVRALSVVSIGLCWLLLAGAERPPFRNVGINGDDE--LLGRPVEAEAASEVTGVAPVQAVDETYRLPKTSVPLHYDLHLRTEIHRNERTFTGSV Anopheles_darlingi ---MVRPLVIVSIGLWCLLGAGELLAERPPFKTDAIHAEEEE ------------- PVPSAEQAIVPAQDVDESYRLPQTSIPIHYAVHLTTNIHRNERTFTGTV Aedes_albopictus ---MVRPLVIVSIGLWCLLGAGELLAERPPYKTDTVHAEEE --------------------- PVVSAEQAFVPAQDVDESYRLPKASVPIHYDVHLTTNIHRNDRTFTGTV Aedes_ageypti ------------------------------------------- MIVLKWSVVLWGLTLATYG-TVAERPLSDVSDNVDPPQIAPAFAVE-ESEIIPLQEVDESYRLPKTSYPTHYELRLRTEVHTGNRQFDGTV Anopheles_albimanus --------------------------------- MIALKWSVVFLGLALTAFGTAAAERPLSGEFNVDDPPQIAPAFAAE-ESEIVPLQAVDETYRLPKTSYPTHYDIKLHTEVHTGERQFQGTV Culex_quanquefasciatus -------------------------------MIAPKSILLCVLAVAGAALAADRPLSKSADVDPPLLIAPEIEAFGDDTAVPFQEIDEAYRLPTTITPTRYDVLLRTEVHTGDRAFEGTV Clustal Consensus

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. .................................. | Anopheles_gambiae GIQLQVVQATDKLVMHNRGLVMSSAKVSSLPNGVTGAPTLIGDVQYSTDTTFEHITFTSPTILQPGT-YLLEVAFQGRLATNDDGFYVSSYVADNGERRY Anopheles_arabiensis GIQLQVVQATDKLVMHNRGLVMSSAKVSSLPNGVTGAPTLIGDVQYSTDTTFEHITFTSPTILQPGT-YLLEVAFQGRLATNDDGFYVSSYVADNGERRY Anopheles_merus GIQLQVVQATDKLVMHNRGLVMSSAKVSSLPNGVTGAPTLIGDVQYSTDTTFEHITFTSPTILQPGT-YLLEVAFQGRLATNDDGFYVSSYVADNGERRY Anopheles_quadriannulatus GIQLQVVQATDKLVMHNRGLVMSSAKVSSLPNGVTGAPTLIGDVQYSTDTTFEHITFTSPTILQPGT-YLLEVAFQGRLATNDDGFYVSSYVADNGERRY Anopheles_maculatus AIQLQVLQATDQLVLHNRRLEIASAKISSLPNGVNGAPTLIGDATYSTNTTIEHITFTCPGILQPGF-YWLEVTFEGILATNDDGFYVSSYVADSGERRY Anopheles_stephensai VISSARISSLPNERQFSGSVAIQLQVLETTDNGVTGAPVLIGDATYSTNTTIEHITFTSPNILQPGF-FWLEVSFVGQLATNDDGFYVSSYVADSGERRY Anopheles_culicifacies AIQLQVLEATSTLVLHNRALSIASAKLSSLPNGVTGTPTPIGDATFSTDTTFEHITFTSPMLLQPGY-YLLEVSFEGRLATNDDGFYVSSYVADSGERRY Anopheles_farauti GIQLQVLEVTDRLVLHNRNLVITSAKLSALPDGVTGAPQLIGDVTYATDATFEHITFTSPSVLQPGF-YLLEVVYEGRLATNDDGFYVSSYVNDRDERVY Anopheles_atroparvus GITLQVLEATNQLVLHNRALSISTAKISSLPNGVDGAPTLIGDVTNSYDTRVEHLTFTSPVVLQPGY-YLLEVTFQGRLTTNDDGFYVSSYVNDLGERRY Anopheles_sinensis AIKLQVLETTDKLVLHNRGLSILTAKVSSLPNGVEGAPTLIGDATFSLDTRVEHLTFTSPTILQTGF-YLLEVTFEGRLTTNDDGFYVSSYVTDAGERRY Anopheles_darlingi AIKLQVVEPTDNLVLHQLGLQLSTAKISSLPAGIDGPPTLIGDVTFATDTRVAHLTLTSPSVLLAGY-YLLEIDYSGRLSTDDDGFYVSSYVADNGERRY Aedes_albopictus AIKLQVIEPTDKLVLHQRGLQLSAARISSLLAGIDGPATPIGDVTFATETQRAHLTLTAAMVLQPGY-YQVEIDYSGRLSTDDDGFYVSSYVADNGERRY Aedes_ageypti AIHLNVVEATNAIVVHYRSLTIQNAKLAFIPT-PEADPQQLNDPTWTYDAKVEQLSFNSETLLNPGS-YILTVEYNGRLSNSEDGFYISSYVNKDGVTKY Anopheles_albimanus AIDLNVVEATNSIVVHHRSLTIQSAKLVLVPT-AGE-PQELNDPTWTYDTRVEQLSFNSVNLLSPGS-YILTVTYAGRLSNNQDGFYMSSYVDSQNRTVY Culex_quanquefasciatus DIYFTVLEATNEIVIHQRDLAIQSYVLYSVG Clustal Consensus * :. . : : :

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. .................................. | Anopheles_gambiae LATTQFESTSARMAFPCYDEPGLKATFTVSITHSLSYKAISNMPQKTTTDIETDMRTTFFEKTPAMSTYLLAFVVSDFQLRLSGAQRVYVRPNAFNEATF Anopheles_arabiensis LATTQFESTSARMAFPCYDEPGLKATFTVSITHSLNYKAISNMPQKTTTDIETDMRTTYFEKTPAMSTYLLAFVVSDFQFRVSGPQRVYVRPNAFNEAAF Anopheles_merus LATTQFESTSARMAFPCYDEPGLKATFTVSITHSRSYKAISNMPQKTTTDIETDMRTTDFEKTPAMSTYLLAFVVSDFLFRVSGTQRVYVRPNAFNEAAF Anopheles_quadriannulatus LATTQFESTSARMAFPCYDEPGLKATFTVSITHSRSYKAISNMPQKTTTDIETDMRTTDFEKTPAMSTYLLAFVVSDFLFRVSGTQRVYVRPNAFNEATF Anopheles_maculatus LATTQFESTSARMAFPCYDEPALKATFTVSITHSLTYTAISNMPENRRSDADG-MRTTVFEKTPIMSSYLLAFVVSDFQYRIATPQRVYVRPNAFQQASF Anopheles_stephensai LATTQFESTSARMAFPCYDEPALKATFTVSLTHSITYSAISNMRENRRSDADG-MRTTVFEIDGTQRVYVRPNAFQQATFAKTPIMSSYLLAFVVSDFLY Anopheles_culicifacies LATTQFESTSARMAFPCYDEPALKATFTVSITHSATYSAISNMPQQETFNEDA-MRTTVFQRTPLMSTYLLAFVVSDFQYRINGPQRVYVRPNAIQEATF Anopheles_farauti LATTQFESTSARMAFPCYDEPALKATFTVSITHINAYSAISNMPVASTVDLADSMRTTAFQRTPLMSTYLLAFVVSDFQFRISGTQRVFVRPNAFNEASF Anopheles_atroparvus LATTQFESTSARMAFPCYDEPALKATFTVSITHHSSYFAISNMPVTGIIIVDGDMRTTRFNQSPAMSTYLLAFVVSDFLFRTSGTQRVYVRPNAFNEAGF Anopheles_sinensis LATTQFESTSARMAFPCYDEPALKATFTVSITHSRSYFAISNMPVNTLTDVEPDMRTTLFNPTPAMSSYLLAFVVSDFLFRQSGTQRVYARPNAFDEIPF Anopheles_darlingi LATTQFESTSARRAFPCYDEPALKATFTITITHGSLYGAISNMPEDTR-TVNGEMATTKFETTPLMSTYLIAFVVSDFLRRGDNQHRVFARPNAFQELPF Aedes_albopictus LATTQFESTSARRAFPCYDEPALKATFTITITHGALYGAISNTEQVSR-IVSGESATTKFKTTPLMSSYLIAFVVSDFLNRGSDKHRVFARPNAFQELPF Aedes_ageypti LATTQFESTSARMAFPCYDEPGLKATFALWITHDVLYTANSNMPYTSTIDG--DIRVTQFEVTPKMSTYLLAFVVSDFQRLGTLEHSVYARPNAIDEVVF Anopheles_albimanus LATTQFESTSARMAFPCYDEPALKATFALQITHSRFYTANSNMEYDFDIDG--DYRISKFKVTPIMSTYLLAFVVSDFAYKGPPTHRVHARVNAINEVDF Culex_quanquefasciatus LATTQFESTSAREAFPCFDEPNLKARFTIKILHHGSYSARSNMGVAKEEDIGNGYKITTFGITPLMSTYLLAFVVSDFESIGTDTQRVYVRSNALDEATF Clustal Consensus ************ ****:*** *** *:: : * * * ** : * *: . ...: . ...: :

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. .................................. | Anopheles_gambiae ALEAGVKILKVLDDHLGIPYDTYMPKLDQIAIPDFAAGAMENWGLVTYR-EQALLFNPAVSTYRGKTNVATTIAHEYAHQWFGNLVSPEWWEYIWLNEGF Anopheles_arabiensis ALEAGVKILKVLDDHLGIPYDTYMPKLDQIAIPDFAAGAMENWGLVTYR-EQALLFNPAVSTYRGKTNVATTIAHEYAHQWFGNLVSPEWWEYIWLNEGF Anopheles_merus ALEAGVKILKVLDDHLGIPYDTYMPKLDQIAIPDFAAGAMENWGLVTYR-EQALLFNPTVSTYRGKTNVATTIAHEYAHQWFGNLVSPEWWEYIWLNEGF Anopheles_quadriannulatus ALEAGVKILKVLDDHLGIPYDTYMPKLDQIAIPDFAAGAMENWGLVTYR-EQALLFNPAVSTYRGKTNVATTIAHEYAHQWFGNLVSPEWWEYIWLNEGF Anopheles_maculatus ALEAGVKILEVLDEHLGIPYSTYMPQLYQIAVPDFAAGAMENWGLVTYR-EQALLFDPAVSTYRTKTNIATTIAHEYAHQWFGDLVTPEWWEYIWLNEGF Anopheles_stephensai RLEAGVKILKVLDEHLGIPYSTYMPTLNQIAVPDFAAGAMENWGLVTYKREQLLLFDPAVSTYRTKTNIATTIAHEYAHQWFGDLVTPEWWEYIWLNEGF Anopheles_culicifacies ALEAGVKILKALDDHLDIPYSAYMPKLFQIAVPDFAAGAMENWGLVTYR-EQLLLFNSAVSTYRTKTNIVTTVAHEYAHQWFGDLVTPEWWEYIWLNEGF Anopheles_farauti ALEAGVKILAALDEHLGIAYGAHMPKLDQIAVPDFAAGAMENWGLVTYR-EELLLFKPAANTYRTKTNIATTVAHEYAHQWFGDLVTPEWWEYIWLNEGF Anopheles_atroparvus ALEAGVKILKVLDDYLGIDYAAQMPKLDQIAVPDFAAGAMENWGLVTYR-EQLLLFNPDVSTYRTKTNIATTIAHEYAHQWFGDLVTPEWWEYIWLNEGF Anopheles_sinensis ALEAGVKILKALDDYLDIPYSSVMPKLDQIAVPDFAAGAMENWGLVTYR-EQALLFNPTVSTYRTKVNVATTIAHELAHQWFGDLVSPEWWEYIWLNEGF Anopheles_darlingi ALEASRLILQVLDDHLGVPYTNYMEKIDQVALPDFAAGAMENWGLVTYR-EQLLLFNPEVNSYRTKTNIATTISHEYAHQWFGDLVTPQWWEYIWLNEGF Aedes_albopictus ALEASKLILPVLDEHLYVPYMSYMEKIDQVALPDFAAGAMENWGLVTYR-EQLLLFNPEVNTYRTKTNIATTIAHEYAHQWFGDLVTPQWWEYIWLNEGF Aedes_ageypti AVEAGQKILEKLDAHLGIGYYDHMPQMKQFAIPDFAAGAMENWGLVTYR-EQYLLFNPELSTYRTKTNIATVIAHEYAHQWFGNLVSPEWWEYIWLNEGF Anopheles_albimanus AVEAGQIILEKLDTHLGIGYYDHMPTMKQFAIPDFAAGAMENWGLVTYR-EQYLLFNQELSTYRTKTNIATVIAHEYAHQWFGNLVSPEWWEYIWLNEGF Culex_quanquefasciatus AQEAGEKILAALDSHLKMKYSEQMPEMKQFAIPDFAAGAMENWGLVTYR-EQYLLFNPALSTYRTKTNIATIIAHEYAHQWFGNLVSPEWWEYIWLNEGF Clustal Consensus **. ** ** :* : * * : *.*:****************: *: ***. .:** *.*:.* ::** ******:**:*:***********

based on APN protein of non-mosquitoes were anal-

ysed separately to know the evolutionary relationship LPKTSVPIHYDLHLTTEIHRQLVLHTRGL

** : * :* : * *::* . : 110 120 130 140 150 160 170 180 190 200

ATDVELAAPAMTYNALYNKVTFTSGTLLTAGENYKLVIRFNGKLANDEDGFYISSYVDDNGVTKY : : :: ::::.. :* .* : : : : * *:..:****:**** . . * 210 220 230 240 250 260 270 280 290 300

310 320 330 340 350 360 370 380 390 400

of organisms. Phylogenetic analysis of APN indicates

that all are evolved from common ancestors. Phyloge- ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. .................................. | Anopheles_gambiae ATLYEFYALDMAYPGQEYWELFNQQVIQYAMGQDGQASTRPMNWNAATPGEISALFDRVAYDKSGSVLNMMRHVLGDDNWKAGLKAYLTDRALQGAVDEQ Anopheles_arabiensis ATLYEFYALDMAYPGQEYWELFNQQVIQYAMGQDGQASTRPMNWNAATPGEISALFDRVAYDKSGSVLNMMRHVLGDDNWKAGLKAYLTDRALQGAVDEQ Anopheles_merus ATLYEFYALDMAYPGQEYWELFNQQVIQYAMGQDGQASTRPMNWNAATPGEISALFDRVAYDKSGSVLNMMRHVLGDDNWKAGLKAYLSDRALQGAVDEQ Anopheles_quadriannulatus ATLYEFYALDMAYPGQEYWELFNQQVIQYAMGQDGQASTRPMNWNAATPGEISALFDRVAYDKSGSVLNMMRHVLGDDNWKAGLKAYLTDRALQGAVDEQ Anopheles_maculatus ATLYEFYAAHLAYPEAEYWELFNPQVIQAAMVPDGLATTRPMNWNAATPGEISALFDRVAYPKSGSVLNMMRHVLGEENWKAGLNAYLTDRALSVAVDEQ Anopheles_stephensai ATLYEFYAAHLAYPEAEYWELFNTQVIQAAMVPDGLASTRPMNWNAATPGEISALFDRVAYG SVLNMMRHVLGEENWKAGLNTYLNDRALSVAVDEQ Anopheles_culicifacies RTLYEFYAAHLAYPEMEYWELFNTQVIQAAMVPDALATTRPMNWNAATPGEISALFDRVAYPKSGSVLNMMRHVLGEENWRSGLKVYLEDRALATAVDEQ Anopheles_farauti ATLYEFYATHLAYPEQEYWELFNLQVIHAAMVPDALVSTRPMNWNAATPGEISALFDRVAYPKSGSVLNMMRHVLGDDNWRAGLRTYLTDRTYKSANDEQ Anopheles_atroparvus ATLYEYYATHLAYPEHEYWELFNTQVIQAAMVPDGLASTRPMNWNAATPGEISALFDRVAYPKSGSVLNMMRHVLGDDNWRAGLKIYLEARSLKEANDEQ Anopheles_sinensis ATLYEYYATDLAYPGMEYWELFNTQVIQAAMVPDGQASTRPMNWNAATPGEIAALFDRVAYPKSGSVLNMMRHVLGDDNWRAGLKIYLEDRALKEANDEQ Anopheles_darlingi ATLYEYLATDLAYPGMEYWELFNTQVIQAAMVPDGLASTRPMNYNAATPGEISALFDRVAYPKSGSVLNMMRHVLGDDNWRAGLRTYLLDRALNKATDED Aedes_albopictus ATLYEYLATDMAYPGMEYWELFNTQVIQAAMVPDGLASTRPMNYNAATPAEISALFDRVAYPKSGSVLNMMRHVLGDDNWREGLRIYLLDRALKGATDED Aedes_ageypti ATLYEYYATHLAYPEVGYWELFNTQVIQAAMVPDGLETTRPMTWNADTPRSIASLFDRVAYPKSGSVLNMMRNVLGEINWTAGLKAYLTARQFDGANADH Anopheles_albimanus ATLYEYYATHMAYPEVGYWELFNTQVIQAAMVPDGLDSTRPMTWNADTPASIGSLFDRVAYPKSGSVLNMMRNVLGEDNWTAGLKAYLTARQFDTANSDH Culex_quanquefasciatus ATLYEYLATHEAYPNMEYWELFNLQVIQAAMVPDGLESTRPMTWNADSPQSIARLFDRVAYPKSGSVLNMMRNIVGDENWATGLRLYLQARQYKGANVEH Clustal Consensus ****: * . *** ****** ***: **

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. .................................. | Anopheles_gambiae LYAGLQSAIEGKGVLPNGVTVAQIMRTWTNEAGYPVLNVRRSYDTGDVIISQERFYNDRKVPNTNIWMIPYNYVHQAKADFNEFDDFQWLATKAARIETT Anopheles_arabiensis LYAGLQSAIEGKGVLPNGVTVAQIMRTWTNEAGYPVLNVRRSYDTGDVIISQERFYNDRKVPNTNIWMIPYNYVHQAKADFNEFDDFQWLATKAARIETT Anopheles_merus LYAGLQSAIEGKGVLPNGVTVGQIMRTWTNEAGYPVLNVRRSYDTGDVIISQERFYNDRKVPNTNIWMIPYNYVHQAKADFNEFDDFQWLATKAARIETT Anopheles_quadriannulatus LYAGLQSAIEGKGVLPNGVTVSQIMRTWTNEAGYPVLNVRRSYDTGDVIISQERFYNDRKVPNTNIWMIPYNYVHQAKADFNEFDDFQWLATKAARIETT Anopheles_maculatus LYEGLQSAIEGKGVLPNGVTVAQIMRTWTNEAGYPVLTVRRSYDTGDVIISQERFYSDRKVPNTNIWMIPYNYVQQARADFNEFDDFEWLATKAARIETT Anopheles_stephensai LYTALQSAIEGKDVLPNGVTVAQIMRTWANEAGYPVLSVRRSYDTGDVIISQERFYSDRKVPNTNVWMIPYNYVQQARADFNEFDDFQWLATKAARIATT Anopheles_culicifacies LYTSLQSVIEGKNVLPGDVTVAQIMRTWANEAGYPVLNVRRSYDTGDVIISQERFYSDRKVPNTNVWMIPYNFVQQARADFNEFDDFQWLATKAARISTT Anopheles_farauti LYQGLQSAIEGKGVLPGGVTVAQIMRTWTTEAGYPVLNVRRSYDTGDVIISQERFYSDRKIPNANVWMIPYNFVQRARADFNEFDDFQWLATKAARIETG Anopheles_atroparvus LYAGLQAAIEGKGVLPAGVTVAQIMRTWTTEAGYPVLNVRRSYDTGDVIISQERFYSDRKVPNSNIWMIPYNMVQQSRADFNEFDDFAWLATKAARLQTS Anopheles_sinensis LYRGLQSAIEGKNVLPAGVTVAQIMRTWTTEAGYPVLNVRRSYDTGDVIISQERFYSDRKVPNENVWMIPYNMVNQARADFNEFDDFEWLASKAARIQTT Anopheles_darlingi LYRGLQSAIEGKGVLPDGVTVGQIMRTWTNEAGYPVLNVRRSYDTGEIIISQERFYSDRKIPNTNIWMIPYNYVHQAVADFNEFDDFKWLATKADRFTTT Aedes_albopictus LYNALQQAIEGKGVLPAGVTVGQIMRTWTNEAGYPVLNVRRSYDTGEIIISQERFYSDRKIPNANIWMIPYNYVHQAVADFNEFDDFKWLATKADRFMTT Aedes_ageypti LYVGLQSAIQGNNVLPEGVTVKDIMDTWANEKGYPVLSVRRTYETGDIIISQERFISDRKVPNTNVWMIPYNYVHQSKADFDDLSTFSWLSTKAARINTE Anopheles_albimanus LYTGLQSAIQGKNVLPDGVTVKDIMESWATEKGYPVLSVRRTYETGSVIISQERFISDRKVPNTNIWMIPYNFVRQSRADFDDLSTFSWLSTKAARISTD Culex_quanquefasciatus LHEGLQAAIEGKNILPDGMTIKQVMDSWTTEKGYPVLNVRRSYDTGDVIISQERFISDRKVPNTNVWMLPYNYVNQSVADFHDTNTYSWLTTKAARISTD Clustal Consensus *: .** .*:*:.:** .:*: ::* :*:.* *****.***:*:**.:******* .***:** *:**:*** *.:: ***.: . : **::** *: *

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. .................................. | Anopheles_gambiae VPANEWIVFNKQQVGYYRVNYDEHNWELITNALHENWASIHRLNRAQLIDDAYWLARSGRLDLRVALRFMTYLRNEREYAPWTAANVALTYFNNRLRGTA Anopheles_arabiensis VPANEWIVFNKQQVGYYRVNYDERNWELITNALHENWASIHRLNRAQLIDDAYWLARSGRLDLRVALRFMTYLRNEREYAPWTAANVALTYFNNRLRGTA Anopheles_merus VPANEWIVFNKQQVGYYRVNYDERNWELITNALHENWASIHRLNRAQLIDDAYWLARSGRLDLRVALRFMTYLRNEREYAPWTAANVALTYFNNRLRGTA Anopheles_quadriannulatus VPANEWIVFNKQQVGYYRVNYDERNWELITNALHENWASIHRLNRAQLIDDAYWLARSGRLDLRVALRFMTYLRNEREYAPWTAANVALTYFNNRLRGTA Anopheles_maculatus VPATEWIIFNKQQVGYYRVNYDDHNWELITNALIDNFAAVHRLNRAQLIDDAYWLARSGRLDLRVALRLMTYLRGEREYAPWTAANVALSYFNSRLRGTE Anopheles_stephensai VPATEWLIFNKQQVGYYRVNYDDRNWELITNALIDNLAAVHRLNRAQLIDDAYWLARSGRLDLRVALRLMTYLRGEREYAPWAAANTALSYFNSRLRGTE Anopheles_culicifacies VPANEWIIFNKQQVGYCCVPYAVRNWKLITNAWWENCAGCIRLNRALLIDDANWLARSGRLDMRVALNLMTYLRGEREYAPWTAASVVLSYFNSRLRGTA Anopheles_farauti VPADEWIIFNKQHVGYYRVNYDDHNWELITQALFDNYASIHRLNRAQLIDDAYWLARSGRLDMRVTLRLMTYLRNEREYAPWTAANVVLTYFNSRLRGTA Anopheles_atroparvus VPATEWIVCNKQQVGYYRVNYDDRNWELITDALIANWASIHRLNRAQLIDDAYWFARSGRLDMRITLRLMTYLRNEEEYAPWTAANVALTYFNNRLRGTA Anopheles_sinensis VPENEWIIFNKQQVGYYRVNYDERNWELITNALIANWASVHRLNRAQLIDDAYWLARSGRLDMRITLRLLTYLRHEEEYAPWTAANVVLSYFNSRLRGTA Anopheles_darlingi VPAHEWIIFNKQEVGYYRVNYDDHNWELITNALHECFGCIHRLNRAQLIDDAYWLARSGRLDLRIALRLMTYLHNEREYSPWAAADVALSYFNSRLRGTE Aedes_albopictus VPAHEWIIFNKQEVGYYRVNYDDHNWELITNALHASYGSVHRLNRAQLIDDAYWLARSGRLDLRIALRLMAYLQHEREYSPWASADVALSYFNSRLRGTE Aedes_ageypti VPANEWIIFNKQQVGYYRVNYDANNWELITNALINNLSSIDRLNRAQLIDDAYWLARSGRLDIEVLMKLLTYLKNETEYAPWTAANNVLSYFNGKLRGTP Anopheles_albimanus VSANEWIIFNKQQVGYYRVNYDDVNWELITNALINNVDSIHRLNRAQLIDDAYWLARSGRLDLEILMKLLTYLPNETEYAPWTAANNVLSYFNGKLRGTP Culex_quanquefasciatus VPDHLWIIFNIQQVGFYRVNYDERNWELITNSLITNWASVNRLNRAQLIDDAFWLARSERLDLEIFMKLLTYLKDEREYAPWTAASNALSYFNGKLRGTN Clustal Consensus *. *:: * *.**: * * **:***::

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. .................................. | Anopheles_gambiae EYHNFLIFVDALIEDIYSLLTIDAVSPDDTLLHKYLVQTISTWACSMGYTDCLMKTAALLKAEASGTGP-AVHPDIASVTYCYGMRSALESEFQYLYRKM Anopheles_arabiensis EYHNFLIFVDALIEDIYSLLTIDAVSPDDTLLHKYLVQTISTWACSMGYTDCLMKTAALLKAEASGTGP-AVHPDIASVTYCYGMRSALESEFQYLYRKM Anopheles_merus EYHNFLIFVDALIEDIYSLLTIDAVSPDDTLLHKYLVQTISTWACSMGYTDCLMKTAALLKAEASGTGP-AVHPDIASVTYCYGMRSAAESEFQYLYRKM Anopheles_quadriannulatus EYHNFLVFVDALIEDIYSLLTIDAVSPDDTLLHKYLVQTISTWACSMGYTDCLMKTAALLKAEASGTGP-AVHPDIASVTYCYGMRSALESEFQYLYRKM Anopheles_maculatus NYHDFLVFASALIENIYELLTIDVVSPNDTLLHKYLVQTISTWACSLGYKDCLERTGALLRAEAAGTGP-AVHPDIATVTYCYGMHTTGETEYQYLYRKM Anopheles_stephensai NYHDFILFVDALIEELYGLLTIDVVSPNDSLLHKYLVQTITSWACSLGYKDCLERTGALLRAGASGTGP-AVHPDIATVTYCYGMRTTGAAEYQYLYRKM Anopheles_culicifacies AYHDFLLFVDALIENIYGMLTIDVVSPNDTLLHKYLVQTISTWACTIGYKDCAEKTAALLKAEVASAGQTPVHPDIASVTYCYGMRSANDAEYQYFYRKM Anopheles_farauti AYHDFLVFVDALIENIYGLLAIDAVAEEDTLLHKYLVQTISTWACASGYTDCLTKTRALLTAEATSTTGFTVHPDIASVTYCYGMRSVGETEFQYLYRKM Anopheles_atroparvus QYHDFLQFVDALIENIYGKLTIDAVADDETLLHKYLKQTISTWACMIGYTDCLTRTAALLSREALGTGP-AVHPDIATVTYCYGMRNIGEPEFQYIYRKM Anopheles_sinensis QYHDFLQFVDALIENVYSKLKIDEVQENETLLHKYLKQTISTWACTIGYTDCLTRTGALLRGEALNTGP-AVPADIATVTYCYGMRNADEAVYQYIYGKL Anopheles_darlingi AYEHFTTFVHSLTEPLYSLLNIDAVDAQDTLLHKYLVQTISSWACRLDHPDCLARTKTLLTSEAS--GRAMVHADLSTVTYCYGMRTAGEQEYQYVYRKM Aedes_albopictus AYEHFSTFVQSLVVPLYSLLNIEAVDPQDSLLHKYLVQTISSWACRLDHPDCLARTKALLATEAS--GGAKVHPDLATVTYCYGMRTAGEQEYQYVYRKM Aedes_ageypti AYKDFTTMVDHLIKKVYKTLDVTAVSDTEPLLHKYLKQTISTWACLIGNEDCLKRTKEALQKEVTEGIP--VHPDVATVVYCNGLRTADVAEYQYLYKRI Anopheles_albimanus DYENFTTLVNHLIRKVYATLEVDSVSESEPLLHKYLKQTISTWACMVGNEDCLKRTKAALQKEVTEGVK--VHPDIATVVYCQGLRTADAAEYQHLYKRI Culex_quanquefasciatus EYHDFLIMVHRLIKYVYSTLSVATVSETESMLHKYLKQTITNWACIVGDLECLRLTKEALENEAHNGVM--VHPDVSSVVYCHGLRDADENVFVYLYNRI Clustal Consensus *..* :. * :* * : * :.:***** ***:.*** . :* * * . * .*:::*.** *:: : :.* :: *. :****.:** :* .*. ******* *******:::*: ** **. ** * * :.

510 520 530 540 550 560 570 580 590 600

610 620 630 640 650 660 670 680 690 700

***** ***** *:*** ***:.: :.:::** * **:**::*. .*:***.:****

710 720 730 740 750 760 770 780 790 800 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. .................................. | Anopheles_gambiae MNSKNLAERTMLIDSLGCSNNKEFLKAFLTTALG-SGTGVEINYRADERRRVVQAIYSGGRTGVDALIEFLMDPALVNEFVSTLSTSTLNSALSAIASRT Anopheles_arabiensis MNSKNLAERTMLIDSLGCSNNKEFLKAFLTTALG-SGTGVEINYRADERRRVVQAIYSGGRTGVDALIEFLMDPALVNEFVSTLSTSTLNSALSAIASRT Anopheles_merus MNSKNLAERTMLIDSLGCSNNKEFLKAFLTTALG-SGTGVEINYRADERRRVVQAIYSGGRTGVDALIEFLMDPALVNEFVSTLSTSTLNSALSAIASRT Anopheles_quadriannulatus MNSKNLAERTMLIDSLGCSNNKEFLKAFLTTALG-SGTGVEINYRADERRRVVQAIYSGGRTGVDALIEFLMDPALVNEFVSTLSTSTLNSALSAIASRT Anopheles_maculatus MDSKNLAERTMLIDALGCSNKTEFLTSFLSTALG-SGTGTEINYRADERRRVVQAVYSGGRVGVDALIEFLMDPNLANEFVSALSASTLNSALSAIASRT Anopheles_stephensai MDSKNLAERTALIDALGCSTNKEFLTSFLSTALG-TGTGVEINYRADERRRVVQAVYSGSRVGVDALIEYLMDPAVVNEFVSILSTSTLNSALSAIASRT Anopheles_culicifacies MDSKNLAERTMLIDSLGCTTNKEYLMAFLTTALG-SGTGVEINYSADERRRMKQDVYSGSRAGVDALIDFLMDPTLVNDFFSILSTSTLNSALSAIASRT Anopheles_farauti MDSRNIAERAMLIDSLGCSHNKEYLKAFLTTALG-SGTGVEINYRADERRRVVQAVYSGSRIGVDALIEFLMDPQLVNEFVSMLSTSTLNSALSAIASRT Anopheles_atroparvus MDSKNLAERTALIDSLGCSANKEFLHSFILTALG-NGN--EINYRADERRRVVQAVYSGGRAGVDALIEFLMDGMLVNEFVSTLGASTLNSALSAIASRT Anopheles_sinensis MASKNLAERTALIDSMGCSYNKEHLHAFLLTALG-NGN--EINYSTEERRRVVQAVYSGSRVGVDALIEFLMEGTYINDFVSTLGTSTLNSALSAIASRS Anopheles_darlingi MDSKNLVERTMLIDALGCSWNKEYLHSFIMTALG-NTE INYRADERRRVIQAVYSGSRAGVDTLIEFLSNAQLTSEFVSTLGQSTLSSTLSAIASRT Aedes_albopictus MNSKNLAERTMLIDALGCSWNKEYLKALIMTAIG-NSE INYRDDERRRVVQAVYSGSRAGVDTLIEFFSDAQLTREFVSGLGQSVLNSALSAIASRT Aedes_ageypti YPTQNWAFRSMIISALGCSENKQFLKDFLQTAIGGSGSGVEINYKTAERTQIVQAVYSGGRAGVDALIEFLRDPNMLREFVAILDVNTLNSALSNIASRT Anopheles_albimanus YSTQNWAFRSMIITALGCSENKEFLKAFLQTAIASNGAGVEINYKSAERTQIVQAVYSGGRAGVDALIEFLRDPDMVREFISRLGINTLNSAISNIASRT Culex_quanquefasciatus YKTQNWAFRMQIIDALGCSRNQKFLVDFLHTALG SGSEINYKASERTRIIQSVYSGSRVGVDALIEFLSNTNNVNDFALRLGINTLNSAIANIASRT Clustal Consensus ::* . * :* ::**: : :.* :: **:. *** ** :: * :***.* ***:**::: : :* *. ..*.*::: ****:

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. .................................. | Anopheles_gambiae NNVEEMNKLNALITALGSRVNSQTAANLRTTAQANLDWVNGFEGLMLSNFLAEFAAETLLTTTAAPETTTVTAGPTTTTTTPSSAVTTT----------- Anopheles_arabiensis NNVEEMNKLNALITALGSRVNSQTAANLRTTAQANLDWVNGFEGLMLSNFLAEFAAETLLTTTAAPGTTTVTAGPTTTTTTPSSAVTTT----------- Anopheles_merus NNVEEMNKLNALITALGSRVNSQTAANLRTTAQANLDWVDGFEGLMLSNFLAEFVAETQLTTTAAPETTTVTAGPITTTTTPSSAVTTT----------- Anopheles_quadriannulatus NNVEEMNKLNALITALGSRVNSQTAANLRTTAQANLDWVDGFEGLMLSNFLAEFVAETLSTTTAAPETTTVTAGPTTTTTTPSSAVTTT----------- Anopheles_maculatus NNEAEMN---KVRKHFIVGERERERERERERERERERERESATSSEIN---------------------------------------------------- Anopheles_stephensai NNEAEMN---KLLTALGSRITSQTATNLRNTAQSNLDWVNGFEGLMLTNFLSEFVTDIPNTTTDAPSTTTAATQSTTTGAG-ATTTTTT----------- Anopheles_culicifacies NNEEEMNRLNALLNALGTRVSSQTATNVRNAAQTNVDWANGFEGLMLSNFLAEYVAGIETDTTTAPATTTTASSMSSTTAGPGPVTTTT----------- Anopheles_farauti NNDAELAQLNTLITALGSRVSSQTATNLRNTAQDNLNWVNGFEGLMMSSFLTEFVAEITNPTEPTDTTTVAPVTTTTAAPAPVTTTTVAP --------P Anopheles_atroparvus NNVAELNQLNVLITALGSRVTSQTATNLRNSAQSNIDWTEGFEGLMLTSFLSEFLVELETTTTVAPVTTTVGPATTMAPG-TTAAPPATTTAPGTTAAPP Anopheles_sinensis NNVDELNKLNALITALGDRVTPQTATNLRNSVQTNIAWTEGYEGLMLTSFVTEYLVELETTTTEAPTTTTTAATTTTTAGPGVTTTTGAPGVTTTTGAPG Anopheles_darlingi NNAAEMAQLNALITTLGDRVNPTTATNLRNSAQANLDWVNGFEGLMLTDFLAEYVKDLQTTTTTAQPTTTTVTVTTSSTTQATNPPITTSPAP PMT Aedes_albopictus NNAAELVQLNTLLTTLGDSITPTTATNVRNSAQTNLDWVNGFEGLMLTDFLAEYVKDLQQTTTAEPTTTTVAGPTTTTTQAPG---VTTTESP AVT Aedes_ageypti NTQEELDMVRSKIEVR Anopheles_albimanus NNQEELHMLNDLLTDLGDNITPQTAESARATVQANFAWQQSSEGLLTVNFVEEYVDSIVPEPPTTTEGPTVTPGPTTVSQAP------------------ Culex_quanquefasciatus NNAEELEKLNNLLTSLGSHITPATASAARATVQANLDWQQSHEGLLTKTFIADYVNTVEPETTTQGTETTTLPSPITTTTAAPAPGETTT---------- Clustal Consensus *. *:

....|....|....|....|....|....|....|....|. ..................| Anopheles_gambiae --TGP-TSVTTTVVQTTTEEDDGAATIGLSIAALLVSITVHLLMG Anopheles_arabiensis --TRP-TSVTTTVVQTTTEEDDGAATIGLSIAALLVSITVHLLMG Anopheles_merus --TRP-TSVTTTVVQTTTEEDDGAATIGLSIAALLVSITVHLLMG Anopheles_quadriannulatus --TRP-TSVTTTVVQTTTEEDDGAATIGLSIAALLVSITVHLLMG Anopheles_maculatus Anopheles_stephensai --PAPTTTVTTTTVQTTTDDDGGAATIGLSIAALLVTIAVHLLG- Anopheles_culicifacies --AAPPTTVTTTTVRTTTEEDGGAATIGLSIAALLVSITIHLLG- Anopheles_farauti AVTTTVPVTTTQVQTTTVDDDDGAATISVSIAAVLVAITVQLLG- Anopheles_atroparvus ATTTGAVTTTTIQTTTVDDDDGGAATIGVSIAALLVAITVQLLG- Anopheles_sinensis VTTTASPAPTVTTTTVQTTTEGGAATISVSLAALLVVVTVQLLG- Anopheles_darlingi TTGTAPITTTQAQQTTTEDDDGGAATVTVSLAALLLAIATHLLG- Aedes_albopictus TTTTAASTTTTQTQETTTEDDGGAATITVSLAAVLLAIATHLLG- Aedes_ageypti Anopheles_albimanus ------------------------------ STTTAGSTTTEDDGGAATIAVSITLLVSAVAVALFN- Culex_quanquefasciatus --TPSGGSTTTLAPTTTTEDPDGAATIAVSLTLLISAVIVTLIN- Clustal Consensus \\

Figure 1. Amino acid sequence alignment of APN in mosquitoes (Diptera). Signal peptide was underlined at N terminal region. Yellow represents GAMEN motif, Blue represents HEXXH, Zn binding region, green represents NEGFA conserved regions, red represents conserved Cys residues, royal blue colour represents glycosylation sites and pink represents GPI anchored region at C terminal end.

netic tree also supported the grouping of each order of

non-mosquitoes into two major clades. In Particular,

in Diptera two major clades belonged to Anopheline

and Culicinae. The phylogenetic relationship followed

by aligning the amino acid sequences from various in-

sect species belonging to six distinct orders on one

scale showed that the Lepidoptera and Diptera APN se-

quences situated into a single cluster [44]. The accumu-

lation of similar sequences from other insects species 910 920 930 940 950 960 970 980 990 1000

1010 1020 1030 1040 reveal closer relative of the mosquitoes. Coleoptera

and Hymenoptera were found in another single cluster.

However, Hemiptera are situated in separate clades.

Conclusion

In silico analysis of the APN gene among six orders

of class Insecta was also carried to understand more

about APN genes and their evolution. A comparative ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. .................................. | Drosophila_persimilis --MAMWLLNSSLLLLLLAAVSLQARLILPQ------------------ DDGQLETKEQFFATPRDDIDEGNYRLPNNTAPESYKVELWTNVHSGARDFNGSVQIDLQVL Drosophila¬_pseudoobscura --MAMWLLNSSLLLLLLAAVSLQARLILPQ ------------------ DDGQLETKGQFFATPRDDIDEGNYRLPNNTAPESYKVELWTNVHSGARDFNGSVQIDLQVL Drosophila_sechellia --MARWLLNSFSLILLLAVGSLQARLILPLSPDDGPRDQGKMETKGAALFAAPRVDDNSAGNYRLPNTTEPESYNVELWTNVHNGDTQFTGTVNIDIRVL Drosophila_simulans --MARWLLNSFSLILLLAVGSLQARLILPLSPDD------------- GQLETKGAALFAAPRVDDNSAGNYRLPNTTEPESYNVELWTNVHNGDTQFTGTVNIDIRVL Drosophila_yakuba --MARWLLNSFSLILLLAVGSLQARLILPLSLDD ------------- GQLETKGAALYATPRVDDNSAGNYRLPNTTEPESYNVELWTNVHTGDDQFNGIVNIDIRVL Drosophila_ananassae ----MAMWLLKSCILFLAVASSQARLIPPLSLDIED Drosophila_willistoni --MAKWLLNSCLLLLLGAVSLQAGLIIPHTDVEQIET ------------------KGYQVITPRVDDNSPDNYRLPNNSIPVSYNVELWTNVHEGDTNFNGTVKIDIQIV Drosophila_grimshawi ---MMRWLITSGLLLLLAASLQAGLLPPPIDNESQ---------- LETKGYQQVSLNTPREDDNNDENYRLPNNTVPIAYDVELWTQVDTGIREFKGIVKIDLQVQ Drosophila_mojavensis ----MARVFTPILILLLVLATTHAAVVRQFPPFER-----------LQQQLIENPNLRAAQRADEQTYRLPNNTEPLEYNIDLTTNVHDGTTEFQGTVTITLKVL Drosophila_virilis ---MARVLVTILALLLVVATTAHAAVVRNFPPFEQ------LQQLEN--PNSRLAPRADEDTYRLPNNTEPVEYTVELHTNVHTGDTAFSGTVAISINVL Drosophila_erecta MSRVQVGGMSLFLVLLLAISATQAAVVRQYPVFDN ------LQHLEN-PMQRSVVPFADEDNYRLPNDTIPSHYAVSLSTNVHTGDTVFNGTVAITLSVS Drosophila_melanogaster MSCVQVGGMSVFLVLLLAISATQAAVVRQYPILDH ----------LQHLEN-PMQRLVVPLADEDNYRLPYDTIPSHYAVSLSTNVHTGDTVFNGTVAITLSVL Stomoxys_calcitrans --------------------------------------------------------------------------------------------------------------------------------------NTTKPTHYDIDLSTMVHEGTRAFVGTVKIHVTFE Musca_domestica Clustal Consensus

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. .................................. | Drosophila_persimilis ELSSSITLHYRQTSNFTASIISRD-EVAQAIELDVATDTVREFLTLTPKLATVTFAANTNWTLSIDYNGTLRGDMGGFYISSYTDDDSTVHYLATTQFES Drosophila¬_pseudoobscura ELSSSITLHYRQTSNFTASIISRD-EVAQAIELDVATDTDREFLTLTPKLATVTFAANTNWTLSIDYNGTLRGDMGGFYISSYTDDDSTVHYLATTQFES Drosophila_sechellia SETSNITLHYRQTSNFEATIISRDGATPTAIPLTVTPELQREFLVLTRTTAGEAFGANTNWTITINYTGIHRSDMGGFYISSYTDDDGKQHFLATTQFES Drosophila_simulans SETSNITLHYRQTSNFEATIISRDVATPTAIPLTVTPELQREFLVLTRTTAGEAFGANTNWTITINYTGIHRSDMGGFYISSYTDDDGKQHFLATTQFES Drosophila_yakuba NETSNITLHYRQTSNFEASIISRDVVTPTSIPLTVTTELQREFLVLTRTTAGEAFGANTNWTITIKYNGTHRTDMGGFYLSSYTDDDGKQHFLATTQFES Drosophila_ananassae EPTTEIKLHFLQTSDHVATIISRDQTDPTAIPLTVANETLRQFLILTRPDSTEPFEANTNWTLSITYNGTLRSDMGGFYISSYTGVDNQVHYLATTQFES Drosophila_willistoni EASVNITLHARQTSNFVATIIDRDNATATEINLEVDASDTTREFLVLSAEG-LTFDPNTNWTLTIKYTGLLRLDMGGFYMSSYTEN-NTVHYLATTQFES Drosophila_grimshawi TASNTITLHAKETENYKATLKSKDNAASSSIPLKWN-VGTREFLQLTPVDTTLTFEKDSNWTLTIEYEGSLRTDNGGFYLSTYKDDDDATHYLATTQFES Drosophila_mojavensis ETSSDIVVHARQLAILGATIRKSDAAESTDVALDIVEDKEREFLHLS-ALG-LTFEEDSIWLLTIKYTGNLRLDNGGFYLSRYTDETGATKYLATTQFES Drosophila_virilis ETSTIIVVHARQLENFTATIRKD--GETTDTALEITHDTDREFLKLS-AKD-LTFEEGTSWLLTIKYEGNLRTDNGGFYLSKYTDENGATKYLATTQFES Drosophila_erecta STTSKIVVHARQLENFTASIIQQGVTGAVAQDLVYEYEAEREFLTFS-KTG-LSFPEDTTWILTIKYQGHLRKDNGGFYLSTYTDEQGNTKYLATTQFES Drosophila_melanogaster NTTTKIVVHARQLENFTASIIQQGVTEAVAQELVYEYEAEREFLTFS-KTG-LTFPEDTTWILTINYQGHLRTDNGGFYLSTYTDEEGNTKYLATTQFES Stomoxys_calcitrans EATTSIVLHARQLIITAATIQFG TTAPEDLEPTYEEEREFLTLT-RKTNTPFPKDSQWVVTLDYNGQLRLDNGGFYLSTYNDANGNVRHLATTQFES Musca_domestica -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------GNERYLAITQFES Clustal Consensus

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. .................................. | Drosophila_persimilis VESSLAGYFGVPFALPKLDQAGIPDFSAGAMENWGLATYREQYMWWNKQNSTINQKTNIANIIGHEYAHMWFGDLVSIKWWTYLWLKEGFATLFSYESND Drosophila¬_pseudoobscura VESSLAGYFGVPFALPKLDQAGIPNFSAGAMENWGLATYREQYMWWNKQNSTINQKTNIANIIGHEYAHMWFGDLVSIKWWTYLWLKEGFATLFSYESND Drosophila_sechellia VESSLASYFGVPFALPKLDQAGIPDFSAGAMENWGLATYRERYMWWNKQNSTINLKTNIANIIGHEYAHMWFGDLVSVKWWTYLWLKEGFATLFSYESND Drosophila_simulans VESSLASYFGVPFALPKLDQAGIPDFSAGAMENWGLATYREQYMWWNKQNSTINLKTNIANIIGHEYAHMWFGDLVSVKWWTYLWLKEGFATLFSYESND Drosophila_yakuba VEASLAGYFGVPFALPKLDQAGIPDFSAGAMENWGLATYREQYMWWNEQNSTINLKTNIANIIGHEYAHMWFGDLVSVKWWTYLWLKEGFATLFSYESND Drosophila_ananassae VESSLASYFGVPFPLPKLDQAGIPDFSAGAMENWGLATYREQYMWWTKENSTINLKTNIANIIGHEYAHMWFGDLVSIQWWTYLWLKEGFATLFSYESND Drosophila_willistoni VESSLASYFGVPFALPKLDQAGIPDFSAGAMENWGLATYREQYMWWTKEESTINLKTNIANIIGHEYTHMWFGDLVSVKWWTYLWQKEGFATLFSYESND Drosophila_grimshawi VEASLAKYYDVPFPLPKLDQAGIPQFSAGAMENWGIATYREQYMWWTKEKSTINLKTSIANIIGHEYAHMWFGDLVSLKWWTYLWLKEGFATLFSYESND Drosophila_mojavensis VEKRLSGYFGVPFALPKLDQAAIPDFAAGAMENWGLATYREEYLLYNVENSTTSTQTNIATIEAHEDAHMWFGDLVAIEWWSYLWLKEGFATLFENLAVN Drosophila_virilis VEKRLSGYFGVPFALPKLDQAAIPDFAAGAMENWGLATYREEYLLYNIENSTTNTQTNIATIEAHEDAHMWFGDLVAIEWWSYLWLKEGFATLFENLAVH Drosophila_erecta VEKRLSGYFGVPYTLPKLDQAGIPDFAAGAMENWGLATYREEYLLYNTENSTTSTQTNIATIEAHEDAHMWFGDLVAIEWWSFLWLKEGFATLFENLAVD Drosophila_melanogaster VEKRLSGYFDVPFALPKLDQAGIPDFAAGAMENWGLATYREEYLLYNTENSTTSTQTNIATIEAHEDAHMWFGDLVAIEWWSFLWLKEGFATLFENLAVD Stomoxys_calcitrans ILERLAEYYGVPFALPKIDQAAIPDFAAGAMENWGLATYREEYMLYNKDHSTITTQTNIANIIAHEYAHQWFGNYVAIQWWTYLWLKEGFATLFSYKATD Musca_domestica ILERLAEYYNVDFALPKIDQAAIPDFAAGAMENWGLATYREEYMLYNKDTSAVTTQTNIASIIAHEYCHQWFGNYVAVHWWTYLWLKEGFATLFSYKAVD Clustal Consensus : *: *:.* :.***:***.**:*:********:*****.*: :. : *: . :*.**.* .** * ***: *::.**::** ********. : .

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. .................................. | Drosophila_persimilis IAFPEWNTYQIYHVNDYNSALLNDAAGAATPMTHYVQTPSEISSRYNTFSYAKPASVLFMFKNAWTDSVFRLGLNKYLTKNQFNSCDEWDLFESLQEAAN Drosophila¬_pseudoobscura IAFPEWNTYQIYHVNDYNSALLNDAAGSATPMTHYVQTPSEISNRYNTFSYAKPASVLFMFKNAWTDSVFRLGLNKYLTKNQYNSCDEWDLFESLQEAAN Drosophila_sechellia IAFPLWDTYQIFHVSDYNSALINDALASAVPMTHYVQTPTEISNRYNTFSYAKPASVLYMFKNAWTDKVFRTGLNKYLTKNKYTSCDEWDLFASFQESAD Drosophila_simulans IAFPLWDTYQIFHVSDYNSALINDALASAVPMTHYVQTPTEISNRYNTFSYAKPASVLYMFKNAWTDKVFRTGLNKYLTKNKYTSCDEWDLFASFQESAD Drosophila_yakuba IAFPLWDTYQIYHVNDYNSALLNDALASAVPMTHYVQTPSEISNRYNTFSYAKPASVLYMFKNAWSDKVFRTGLNKYLTKNQYTSCDEWDLFASFQESAD Drosophila_ananassae IAFPEWDTYQIYHVNDYNSALLNDASTSAVPMTHYVQTPTEISGRYNTFSYAKPSAVLYMFKNAWTDRVFRAGLNKYLTKNQYTSCDEWDLFAALQESAD Drosophila_willistoni IAFPEWDTYQIFHVADYHSALMNDAGENAVPMTHYVQTPNEISNRYNTYSYAKPASVLYTFKNAWTDKVFRLGVHKYLDQHKFTSCDEWDLFASFQEAAD Drosophila_grimshawi IAFPEWDTYQIFHVSDYNSALINDASISTP-MTHYVQTPREISGRYGTISYAKPASVLYMFKNAWTDDVFRRGLNLYLTQFAYTSADEWDLFSSLQKAAD Drosophila_mojavensis LAYPEWDIFQTFHTGSYQSAMVSDGNPSVRPMTYYVEKPSDISLLYDSIAYAKAGSVLDMWRHALTNTVFQQGLHNYLVEKAYSAANETDLFNAIALAAR Drosophila_virilis LAYPEWDIFQTFHAGSYQSALIADANPNVRPMTTFVEKPSEISQLYDSIAYAKAGSVLDMWRHALTNTVFQQGLHNYLVTKAYSAANETDLFDAISLAAR Drosophila_erecta LAYPEWDIFQTFHAGSYQSALVNDASANARPMSHFVQKPSEIALLYDSVSYAKAGSVLDMWRHALTNTVFQRGLHNYLVDNQFTAANPSKLFDGIAKAAV Drosophila_melanogaster LAYPEWDIFQTFHAGSYQSALVNDASANARPMSHFVQKPSEIALLYDSVSYAKAGSVLDMWRHALTNTVFQRGLHNYLVDNQFTAANPSKLFEAIAKAAV Stomoxys_calcitrans AAYPEWGIWQQFHTDDYQGALNNDAGVSPRPMTHYVQKPQEISRLYDTVSYAKAGSVLNMWNHALTDTVFQRGLHNYLTANNFSAAVEDKLFTAIQIASK Musca_domestica AAYPEWGVWQQFQTDDYQSALTNDASDYPRPMTHYVQKPSEIAMSYGTVTYAKAGSVLNMWNHALTDKVFQRGLHNYLDANQYSSAVEEQLFLAIQTAAK Clustal Consensus *:* *. :* ::. .*:.*: *. *: :*:.* :*: *.: :***..:** :.:* :: **: *:: ** :.:. .** .: ::

sequence analysis was carried out to gain a better un-

derstanding of the evolution of APN gene. The conser-

vation of residues was considerable and widespread

throughout the coding region. However, the high level

of divergence was observed in the size and positions of ---------- VQLETKGTYSLISPRADDNSEGNYRLPNNTAPEAYRVELWTNVHNGTREFNGSVQIDIQVL

110 120 130 140 150 160 170 180 190 200

. :.** ***** 310 320 330 340 350 360 370 380 390 400

410 420 430 440 450 460 470 480 490 500

introns. This could be ascribed to the presence of repet-

itive DNA elements. Great sequence and structural di-

versity suggests that APN genes are highly evolving

with selective functional constraints. APN sequences

were found that contain conserved HEXXH and GAMEN ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. .................................. | Drosophila_persimilis EQGLSLPTTVANIFSSWSQQAGYPLLTVERNYDAGTFTVKQQRYLADKDAANQNTWYVPINFATASSPDYRNTSASHYLLNISEVAITDAQIASDDWLIL Drosophila¬_pseudoobscura EQGLSLPTTVANIFSSWSHQAGYPLLTVERNYDAGTFTVKQQRYLADKDAANQNTWYVPINFATASSPDNRNTSASHYLLNISEVAITDAQIGSDDWLIL Drosophila_sechellia ELGFKLPASVDDIFSSWSHQAGYPLLTVTRNYNAGTITITQKRYVANKTDTNTATWYVPLNFATANKYDYRDTSATHYLLNVTETVISDVEASSDDWILV Drosophila_simulans ELGFKLPASVDDIFSSWSHQAGYPLLTVTRNYNAGTITITQKRYVANNTDTNTATWYVPLNFATANKYDYRDTSATHYLLNVTETVISDVEASSDDWIVV Drosophila_yakuba ELGFKLPTTVDNIFSSWSHQAGYPLLTVTRNYEAGTITITQKRYVANKSDTNTATWYVPLNFATASKYDYRNTTATHYLLNVTETVISDVDASSTEYIIV Drosophila_ananassae EHGLSLPTSVDNIFSSWSQKAGYPLLTVTRNYATGTFTVKQQRYLAVD-DSNQITWYVPINFATASNPDYRNTSASHYLLNTTEVEISDVAIASEDWIIF Drosophila_willistoni ELNFALPASVADIFSSWSHQAGYPLLTVERNYDSGSFTVKQQRYVEDKNATNDKTWYVPINYVTASQSDYRDTSASNYLLNTAELTIN-AQIDSSDFLLL Drosophila_grimshawi ELGHKLPVALADIFSSWSKQAGFPLLTVERNYQDGTFTIKQQRYLNNKETPNEDTWYVPINYATASNPDYRNTVASHYLLKVKESTVSDAKIANDDWLIL Drosophila_mojavensis ELNYEVPALVDDMMSSWTRQGGLPLLTVERNYNDGSFTVKQEQYTNNKDAKGDKTWYVPVNYALQSNPDFRRTEATHYLHKDQELSISDAALKSDDWLIL Drosophila_virilis ELNYAVPALVGDMLSSWTRQGGVPLITVERNYNNGSFTIKQEQYTNDKTFTSEKLWYVPVNYAVASNPDFRNTEATHYLLNETEKSVADAQLTNDDWLIL Drosophila_erecta EEDYAVPATVHNMMGSWTNQGGVPLLTVTRNYETGDFTVKQSVYTNDKDYTNDKLWYVPINYADFSNPDFRNTEATHYLLNQSEITINAGLDPTN-WLVL Drosophila_melanogaster EENYEVKATVPDMMGSWTNQGGVPLLTVTRNYDNGSFTVKQSVYTNDKDYTNDKLWYVPINYAESKNPDFRNTEATHYLLNQSEITIYSDLDQTN-WLIL Stomoxys_calcitrans EENYEIPAKIEDMMGSWTHQGGYPLLTVKRNYDDGSFSVTQQAFHNNETINSDKSWYIPFNYATQSNADFRDTEATNYLLNVKSVSVGAKLGKEE-WLIL Musca_domestica EENYEIPAKIEDMLGSWSRQGGYPLLTVTRNYNNGSFTVTQSSFHNNKNTVTQKSWYIPFNYAHQSNPDFRDTEASHYLLNVPSVRVDAKLDKED-WLIL Clustal Consensus * . : . : :::.**:.:.* **:** *** * :::.*. : .**:*.*:. .. * * * *::** : . : :::.

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. .................................. | Drosophila_persimilis NKQSSGYYRILYDNQNYQLLAQVLADQPYKIHVRNRAQLLYDTYIFLTSGRLNHSTLFDLLSYLKNETEYAPWSTANTILTVFDRYLRGDEQYSQFTSFV Drosophila¬_pseudoobscura NKQSSGYYRILYDNQNYQLLAQVLADQPYKIHVRNRAQLLYDTYIFLTSGRLNHSTLFDLLSYLKNETEYAPWSTANTILTVFDRYLRGDEQYSQFSSFV Drosophila_sechellia NIQNSGYYRTLYDTQNYGLITAALKSQPWRFHPRNRAQLLYDTYIFVSTDRLSHSILLELLGYLTNEQQYAPWSTANTILTVYDRYLRGDDLYYHFQNFV Drosophila_simulans NIQNSGYYRTLYDTQNYGLITAALKSQPWRFHPRNRAQLLYDTYIFVSTDRLSHSILLELLGYLTNEQQYAPWSTANTILTVYDRYLRGDDLYYHFQNFV Drosophila_yakuba NIQNSGYFRTLYDIPNYGLITSALKSQPHRFHPRNRAQLLYDTYIFVTTDRLSHSILLELLGYLVNEQQYAPWSTANTILTVYDRYLRGDDSYYHFQDFV Drosophila_ananassae NLKNAFYYRTLYDLDNYDRIVDGLNNNHHKIHVRNRAQLLYDAYIFVTTGRLSHSVLLRLLTYLKYEEQYAPWSNANTILTVFNRYLRGDESYGNFQTFV Drosophila_willistoni NKQSAYYQRVLYDQQNYDLIAQALLETPYKFHPRNRAQLIAETYRFAATGRLSQSTFLQIVAYLKNEDQYAPWSFANSIFPTYDQYLRGDDQYEHFRLYV Drosophila_grimshawi NKQSTGYYRILYDEENYRRIAQGLIEKPYAFHTRNRAQLMHDAYRFVDTGRIGDGILLQLMVYLRKEDQYAPWSTANSILNVYDKYLRGHEQYVRFSDFV Drosophila_mojavensis NKQSTGYYRINYDTQNWQLISNGLADRPHKIHPRNRAQLIDDAYRFATSGRLSHYVLLQLLTYLPQETQYAPWSTANSVISLFNRYLSGDEAYENFQFYV Drosophila_virilis NKQSTGYYRINYDERNWQLIIEGLANRSHKIHPRNRAQLISDAYRFATSNRLSHALLLQLLTYLPQENQYAPWSAANTAITLFDRYLSSDESYEHFQFYV Drosophila_erecta NKKSTGYYRVLYDAQNYKLITNALTTRPHKIDSRNRAQFINDLYRFVTSGRVPHATLLELLTYLPQEDQYSPWSATNTVITLLNRYLSGDADYGNFQFYV Drosophila_melanogaster NKKSTGYYRVLYDAQNYQLITSALITRPHKIDPRNRAQLINDLYRFATSGRVPHATLLELLTYLPQEDQYSPWSATNTVITLLNRYLSGDADYGNFQFYV Stomoxys_calcitrans NKQSTGYYRINYDEQNWKLIINGLIHHPFKIHPRNRAQLMHDAYRFSASNRLNHAILLEMLTYLPSEVEYAPWSTANGIINIYNRYLGGDNDYKVFQEFV Musca_domestica NKQSTGYYRINYDEENWKLIIDGLIKSPYKIHPRNRAQLMHDAYRFSASNRLAHSVLLDMMTYLPKEDQYAPWSTANGIINVYNRYLSGDAKYYDFQMFV Clustal Consensus * :.: * * ** *: : * :. *****:: : * * :.*: . :: :: ** * :*:*** :* : ::** .. * * :*

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. .................................. | Drosophila_persimilis VQQIDGIFNRLGVNEIPGEHYLNNYLRVVLISLACQVGSESCYEQSALKLKQFLLNGIHIEATIRTQTFCGGLRKTEDDIFNLVLQNLGASSDATDRSLF Drosophila¬_pseudoobscura VQQIDGIFNRLGVNEIPGEHYLNNYLRVVLISLACQVGSESCYEQSALKLKQFLLNGIHIEATIRTQTFCGGLRKTEDDIFNLVLQNLGASSDATDRSLF Drosophila_sechellia RLLIDPIFEQIGVNEIPGEHYLNNYLRIVLVSLACQVGSNDCYNQSAKKLSEYLYNGTAIEATLKTQAYCAGLRSTTNDIYSRVQSDLLSSSDSTDRSLF Drosophila_simulans KLLIDPIFDQIGVNEIPGEHYLNNYLRIVLVSLACQVGSNDCYNQSAKKLSEYLYNGTAIEATLKTQAYCAGLRSTTNDIYSRVQSDLLSSSDSTDRSLF Drosophila_yakuba RGLIEPIFDKIGVNEIPGEHYLNNYLRIVLVSLACQVGSNDCYSQSAKKLSEYLYNGTAIEATLKTQAYCAGLRSTTNDIYSRVQSDLLSSTDSTDRSLF Drosophila_ananassae QELIDPVFEKLGVNEIPGEHYLNNYLRIVLVSLACQVGSVECHTASARKLSEFLFDGIVIEPTLRTQAYCAGLRNPTDVVYERVISDLLATTDATDRSLF Drosophila_willistoni VELVEDIFDKIGVHESTGEHYLNKYLRVVLINLACQVGTPACYEQTHQKLKEFQQQGIAIEPTIRTQVYCGGLRQAEDSTFNQVLSDLFASTVSTDRSLF Drosophila_grimshawi IELVEDIFEKLGVHEIPGEHYLNNYLRVTLVSLACQVGSQMCTQQSHQKLKQFLEDSIPIEPTLRTQAYCAGLRQAEDSTFNLAFDKLLSSNVASDRSLF Drosophila_mojavensis AQLVSEQFDKFGVNDQSGEQHLVKFTRNAVINVACLAGLKSCLDETNAKLQALIEHGTPIEPNLQTPVYCNGLKQADDKTFDFVYDKLMHSNNQAERRLL Drosophila_virilis ATLVSDQFDKFGVNDIDGEQHLVKFTRNVVINLACLAGLKSCLDETNVKLQALVNQGTPIEPNLQTPVYCNGLKQADDKTFNFVYDKLMDSNDQAERRLL Drosophila_erecta GELVSQQFDKFGVNDNKGDHHLVPFTRNVLINLACLAGLENCLTETTAKLTALVEKGTRIEPNLQSQVYCNGLKQANDKTFDFVFNKLLNSNDQAERRLL Drosophila_melanogaster RELVSLQFDKFGVNDNKGDHHLVPFTRNVLINLACLAGLENCLTETTAKLTALVEEGTKIEPNLQSQVYCNGLKQADDKTFDFVFNKLLNSTDQAERRLL Stomoxys_calcitrans AEIVTPIYEKLGINDVPGEHHYQKFTRNVAINMACLAGINSCLKETNDKLKDLVEHDVAIEPNVQSQIYCNGLKQSSNDVFDFVYNKLMDSDDQALRRIL Musca_domestica AEIVTPIYEKLGINDVPGEHHYQKYTRNAVINMACLAGIQSCLTETKNKLKALVQHGTPIEANVQSQIYCNGLKQSGDDVFQYVFNQLMDSTDQAFRRVL Clustal Consensus : ::::*::: *::: : * . :.:** .* * : ** .. **..::: :* **:.. : :. . ..* : : * ::

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. .................................. | Drosophila_persimilis VSSLGCSQVPSQLETFFSSSFNESNGLTDSERTSLLNAAYSRSEVGLTASLDFLESNWRDYAQLN --- GSTKPLDLALRGIATYVVSTKQQTRLNALVED Drosophila¬_pseudoobscura VSSLGCSQVPSQLETLFSSSFNESNGLTDSERTSLLNAAYSRSEVGLTASLDFLESNWRDYAQLN --- GSTKPLDLALRGIATYVVSTKQQTRLNALVED Drosophila_sechellia ISSLGCSGSTSQLLDFLRLSLDTANSLSYSERTSLLNSAYSRSEIGLTATLDFLETNWEAYAKLS -------- DSAKPLDAALRGIATYVVSEKQKTRLNALVDV Drosophila_simulans ISSLGCSGSTSQLLDFLRLSLDAANSLSYSERTSLLNSAYSRSEIGLTASLEFLETNWEAYAKLS -------- DSAKPLDAALRGIATYVVSEKQKTRLNALVDV Drosophila_yakuba ISSLGCSSSTSQLIGFLNLSLNTANSLSSSERFSLLNSAYSRSEIGVTASLEFLEASWLDYAKLS --------- ETSKPLDSALRGIATYVVSEKQKTRLNALVDV Drosophila_ananassae ISSLGCSLQASQLGNFLNLSLDTSNGLSYSQRTSLLNAAYSQSEDGVTASLEFLESNWEAYAQLSS--DSAKPLDLALRGIATYVVSEKQKTRLNALVEE Drosophila_willistoni ISSLGCSQTAEQIERFISSSIDTSNSLTNAERTTLLNSAYSRGEVGITESLKFLESNWEAYANVYP-ESTGKPLDAALRGIATYVVSETQKTRLENLVEE Drosophila_grimshawi ISSLGCSHNAAQLKKLVESSIDNANGLSYSERTSLLPAAYPRSEAGLTASLEFLESNWKAYAELST-NPQQKPLDTVLRGMAQSVVSESQKNRLNALVAT Drosophila_mojavensis ISALGCAQNEKQLEKYVSSSIDTNNQLRTQERYTLLTPTYSRGSVGLNVCIDFLLNNWEKYAQLNAGFGGVNPLYSDILGMSSYVVNDSQRAKLQELIDA Drosophila_virilis ISALGCSQNEDQLMKYVSSSIDQSNKLRTQERYTLLSPTYSRGEVGLLVCIDFLLEHWQEYGNLNPGFGGNNPLAADIESMAAYVVTESQKQKLQLLIDT Drosophila_erecta ISALGCSQNAAQLEKFVSSSIEPQG-LRDQERITVLSPVYSRGEVGLLVAVEFLKENWEQYGQLNSGFGGVNPLYSDIVGISAYTVNDKQKTALQELVDL Drosophila_melanogaster ISALGCSQNTAQLEKFVYSSIEPEG-LRDQERITVLSPVYSRGEVGLLVAIEFLQKNWEKYGQLNSGFGGVNPLYSDIVGISAYTVNDKQKDALQELVDT Stomoxys_calcitrans INSLGCSQNKQQLDKFVSSSIDHDNELRVQERSTLLSPAYSRGLTGWMACMDFLDKHWDAYGNLNGGFGGTNPLDDAIRGMSSYANNEEQENRLLALVAK Musca_domestica ISSLGCSQNEKQLKTYLESSIDKDNKLRNQERITILSPVYSRGSVGLMATIDFLDEHWDEYGNLNTGFGGTNPLNDALGGMSLYVVDTEQEKRLLALVDK Clustal Consensus :.:***: *: . *:: . * :*

signature sequences in multiple APN orthologs. The

APN gene belonging to different orders was found to be

AT rich except Diptera. Dipterans APN gene was found

to be GC rich and at third codon position high GC con- 610 620 630 640 650 660 670 680 690 700

710 720 730 740 750 760 770 780 790 800

810 820 830 840 850 860 870 880 890 900

::* ..*.:. * :.** * *.:: :** : .:: . *. * *:

tent was observed. The presence of high GC content in

Diptera clearly indicates that dipteran APN gene is a bi-

ased gene. The Leucine residues are present in highest

frequency in the amino acid composition of protein of ....|....|....|....|....|....|....|....|....|....|....|....|....|....|. ...........................| Drosophila_persimilis VKTNGPEYVNDDLSTVVDSRVKANLDWLAVHRDPLISWLAAYNTVKDNGSPALTTSLATILASALTLLLWQLF-- Drosophila¬_pseudoobscura VKTNGPEYVNDDLSTDIDSRVKDNLDWLAVHREPLISWLAAYNTVKDNGSPALTTSLATILASALTLLLWQLF-- Drosophila_sechellia VKAEGPSYINDDLSTTIDSRITFNFAWLEVNRDPVLNWIEDS--FRENGSPSLTSSITTILVSAMALLLFRLY-- Drosophila_simulans VKAEGPSYINDDLSTTIDSRITSNFAWLEVNRDPVLNWIEDS--FRENGSPSLTSSITTILVSAMALLLFRLY-- Drosophila_yakuba VKAEGPSYVNDDLSDTIDSRITSNFAWLEVNRDPVLNWIEDS--LRENGSPSLTASITTILVSAMALLLFRL--- Drosophila_ananassae VQSKENGYLQDDLSSTVNSRIQSNFDWLDTNRDPVISWVNSH--ISGSGSSGLTVSLATVLISAMALLLSKLY-- Drosophila_willistoni VKAHGPAYVNDDLSEIITARIDANFAWLNEHRDSVINWLVSYR---TSGSSALTTSLTTFMAAALTLLLCKVF-- Drosophila_grimshawi INGNGIEHVNDDIDTAVNRNIQANFDWLNVHRDPVINWLIAYR----SGTAPLSASIFATLTALITLFLCKFS-- Drosophila_mojavensis VK--DSDYVPATLQASVDTNVEANMAWLTSNRGPIISWVTSFR----NGSPALTASVLAMALCLFVAMFF----- Drosophila_virilis VK--GSEFVSDTLQDSVDTTIAANMEWLSTNREPIISWVTGFR----NGSPAMTASVLALALCFLAAKLF----- Drosophila_erecta VK--NSEYVPSTLQASVDSNVNANYAWLASNRDPLMSWVAGFR----SGSSALSASIVAILACLLAAKLF----- Drosophila_melanogaster VK--GSEYVPSTLQDSVDSNVNANYAWLASNRDPLMTWVAGFR----SGSSALNASILAILACLLAAKLF----- Stomoxys_calcitrans VK--NSVHASANLEEVVEANFKTNRDWLKINRDPLMSWLISYR----SNGGATLAISTLSMVS--VILLVVSRLF Musca_domestica VK--SSEHVSSSLEASVKYNIQSNFDWLKSNRDPLINWFQSFN IKSGASAVISSSSMAISTLLLLVISRLF Clustal Consensus :: . :. : . * ** :* .::.*. :

Figure 2. Amino acids sequence alignment of APN in non-mosquitoes (Diptera). Signal peptide was underlined at N terminal region. Yellow represents GAMEN motif, Blue NEGFA conserved regions, red represents conserved Cys residues.

each species, accounting for 9% of total amino acids

and cysteine residues are least abundant, 1% in the

APN protein of insects. A significant high synonymous

codon usage bias has been shown by mosquito APN

genes belonging to the order Diptera (preferentially

using one or two synonymous codons) as compared to

other species.

Acknowledgement: MDU Rohtak, MRU PGIMS represents HEXXH, Zn binding region, green represents

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