552

Tropical Biomedicine 30(3): 552–558 (2013)

Research Note

Genetic analyses of Ribosomal loci of Anopheles minimus

species from North east India

Dutta, P.*, Khan, S.A., Topno, R., Chowdhury, P., Baishya, M., Prakash, A., Bhattacharyya, D.R. andMahanta, J.Regional Medical Research Centre, ICMR (NE Region), Dibrugarh, Assam, India*Corresponding author email: duttaprafulla@yahoo.comReceived 19 March 2013; received in revised form 19 May 2013; accepted 28 May 2013

Abstract. Anopheles minimus is one of the major vectors for transmission of malaria diseasein north eastern (NE) region of India. The minimus species complex of Minimus subgroup ofMyzomyia series of anophelines were studied in malaria affected states- Assam and ArunachalPradesh (AP) of NE India. Ribosomal DNA markers- second internal transcribed spacer (ITS2)and third domain (D3) of 28S gene were used to characterize An. minimus species. Sequencehomogeneity was observed in D3 sequences of An.minimus specimens throughout both thestates. However, a transversion in ITS2 sequence of single specimen collected from Assam-Meghalaya border areas illustrates possibility of intra population polymorphism in ITS2sequence within the geographical region.

Anopheles minimus sensu lato (s.l.)Theobald 1901, of Minimus subgroup ofMyzomyia Series is recognized as animportant malaria vector species in Orientalregion. Anopheles minimus comprises ofthree sibling species, namely An. minimus

(formerly species A), Anopheles harrisoni

Harbach & Manguin (formerly sp. C) andAnopheles yaeyamaensis Somboon &Harbach (formerly sp. E). In India (latitude-22° 00' N and longitude- 77° 00' E), An.

minimus s.l. is considered as one of themajor malaria vectors in North-eastern (NE)region (latitude- 21o58' N to 29o30’N andlongitude- 88o3' E to 97o30' E) (Prakash et

al., 2004). Anopheles minimus had beenpreviously incriminated as vectors in tworeportedly malaria endemic states of NEregion viz. Assam (latitude- 24° 8’N to 28° 2'N and longitude- 89° 42' E to 96° E) andArunachal Pradesh (AP) (latitude- 26o30’Nto 29o 30’N and longitude- 91o 30' E to 97o

30’E) (Rao, 1984; Dutta & Baruah, 1987; Dutta

& Mahanta, 1995; Prakash et al., 2004). Thephysiography of NE region is very muchsimilar with neighbouring southeasterncountries like China, Thailand and Vietnamwhere An. minimus is a major vector (Chenet al., 2011). Thus the objectives of presentstudy were molecular identification andgenetic diversity of An. minimus s.l. basedon ribosomal markers- ITS2 and D3.

Mosquito collections in human dwellingswere carried out from 17 sites of Assamand 8 sites of AP during 2008-2011 usingCenters for Disease Control (CDC) miniaturelight traps (Table 1). Mosquitoes weremorphologically identified using standardanopheline identification keys (Das et al.,1990). Genomic DNA was extracted by usingFTA Classic card nucleic acid extractiontechnology (Whatman) (Mohanty et al.,2007). Extracted DNA was subjected to AlleleSpecific PCR (ASPCR) based on Phuc et al.(2003). Ribosomal markers- ITS2 and D3were used to study genetic diversity. ITS2 and

553

Tab

le 1

. M

ole

cula

r co

nfi

rmed

An

. m

inim

us s

pec

ies

at d

iffe

ren

t ge

ogr

aph

ical

lo

cali

ties

Sl.

No.

Are

a o

f co

llec

tio

nE

colo

gica

lTy

pe

of

coll

ecti

on

Lat

itu

de

An

op

hel

es C

oll

ecte

dA

n m

inim

us

terr

ain

Lo

ngi

tud

eid

enti

fied

1.

Tora

jan

, D

ibru

garh

Fo

oth

ills

Cat

tles

hed

& H

um

an d

wel

lin

gs27

.3 N

, 95

.4E

Cat

tles

hed

-71

Hu

man

dw

elli

ng-

59–

2.

Lez

ai,

Dib

ruga

rhP

lain

sH

um

an d

wel

lin

gs27

.4N

, 94

.8E

60

–

3.

Mar

gher

ita,

Dib

ruga

rhP

lain

sH

um

an d

wel

lin

gs27

.2N

, 95

.6E

95

–

4.

Bok

o, K

amru

pF

oo

thil

lsC

attl

esh

ed &

Hu

man

dw

elli

ngs

25.9

N,

91.2

EC

attl

esh

ed-7

5 H

um

an d

wel

lin

gs-6

84

5.

Sona

pur,

Kam

rup

Fo

oth

ills

Cat

tles

hed

& H

um

an d

wel

lin

gs26

.1N

, 91

.9E

Cat

tles

hed

-24

Hu

man

dw

elli

ngs

-12

2

6.

Man

ash

, B

arp

eta

Fo

rest

Cat

tles

hed

& H

um

an d

wel

lin

gs26

.7N

, 91

.0E

Cat

tles

hed

-63

Hu

man

-33

2

7.

Tam

ulpu

r N

alba

riF

oo

thil

lsC

attl

esh

ed &

Hu

man

dw

elli

ngs

26.6

N,

91.5

EC

attl

esh

ed-2

6 H

um

an-1

41

8.

Koi

lam

ari,

Lakh

impu

rF

ore

stC

attl

esh

ed &

Hu

man

dw

elli

ngs

27.3

N,

94E

Cat

tles

hed

-6 H

um

an-3

–

9.

Bhi

tori

pam

, La

khim

pur

Pla

ins

Cat

tles

hed

& H

um

an d

wel

lin

gs27

.2N

, 93

.9E

Cat

tles

hed

-9 H

um

an-7

–

10

.K

oh

ora

, G

ola

ghat

Fo

oth

ills

Hu

man

dw

elli

ngs

26.5

N,

93.4

E3

40

–

11

.B

ok

akh

at,

Go

lagh

atP

lain

sC

attl

esh

ed &

Hu

man

dw

elli

ngs

26.6

N,

93.6

EC

attl

esh

ed-3

3 H

um

an-2

62

12

.T

itab

or,

Jo

rhat

Pla

ins

Cat

tles

hed

& H

um

an d

wel

lin

gs26

.5N

, 94

.1E

Cat

tles

hed

-289

Hu

man

-178

–

13

.So

ru A

mso

i, N

agao

nF

oo

thil

lsH

um

an d

wel

lin

gs26

N,

92.1

E4

1

14

.G

oal

par

a, K

amru

pP

lain

sC

attl

esh

ed &

Hu

man

dw

elli

ngs

26.1

N,

90.6

EC

attl

esh

ed-1

9 H

um

an-9

–

15

.H

amre

n, K

arbi

Ang

long

Fo

oth

ills

Cat

tles

hed

& H

um

an d

wel

lin

gs25

.8N

, 92

.5E

Cat

tles

hed

-34

Hu

man

-23

2

16

.H

affl

on

g,F

oo

thil

lsC

attl

esh

ed &

Hu

man

dw

elli

ngs

25.1

N,

93.0

EC

attl

esh

ed-2

3 H

um

an-1

2

17

.C

hir

ang,

Fo

oth

ills

Cat

tles

hed

& H

um

an d

wel

lin

gs26

.6N

, 90

.3E

Cat

tles

hed

-30

Hu

man

-19

18

.B

and

ard

ewa,

A

PF

ore

stC

attl

esh

ed &

Hu

man

dw

elli

ngs

27.1

N,

93.8

EC

attl

esh

ed-7

Hu

man

-43

19

.P

iong

, A

PF

oo

thil

lsC

attl

esh

ed &

Hu

man

dw

elli

ngs

27.5

N,

95.9

EC

attl

esh

ed-2

01 H

um

an-1

841

20

.K

imin

, A

PF

oo

thil

lsC

attl

esh

ed &

Hu

man

dw

elli

ngs

27.3

N,

93.9

EC

attl

esh

ed-9

3 H

um

an-6

21

5

21

.N

amsa

i, A

PF

oo

thil

lsC

attl

esh

ed &

Hu

man

dw

elli

ngs

27.6

N,

95.8

EC

atte

shed

-65

Hu

man

-28

22

.K

huns

a, A

PF

oo

thil

lsC

attl

esh

ed &

Hu

man

dw

elli

ngs

26.9

N,

95.5

EC

attl

esh

ed-3

1 H

um

an-2

3

23

.R

uksi

n, A

PF

oo

thil

lsC

attl

esh

ed &

Hu

man

dw

elli

ngs

28.0

N,

95.3

EC

attl

esh

ed-1

4 H

um

an-6

24

.B

halu

kpon

g, A

PF

oo

thil

lsC

attl

esh

ed &

Hu

man

dw

elli

ngs

27N

, 92

.5E

Cat

tles

hed

-15

Hu

man

-76

25

.A

mpe

n, M

iao,

AP

Fo

oth

ills

Hu

man

dw

elli

ngs

27.4

N,

96.2

E1

64

37

554

D3 region were amplified based on Walton et

al. (1999) and Singh et al. (2004) respectivelywith modifications. Amplified PCR productswere purified by Invitrogen PureLinkTM PCRpurification kits. Purified products were thensequenced in both directions. Sequencingwas outsourced to Anshul Biotech, India.Both forward and reverse direction of eachsequence was checked and edited manuallyusing BioEdit Sequence Alignment EditorSoftware. ITS2 and D3 sequences werealigned using default parameters inClustal W software. Edited sequenceswere submitted to NCBI GENBANK andrespective accession numbers wereobtained. Sequences inferences were doneby using Maximum Likelihood method basedon Tamura- Nei model. Evolutionary analyseswere conducted in Molecular EvolutionaryGenetics Analysis (MEGA) version 5.0.

Out of 2582 anophelines collected from17 sites in Assam and 8 sites in AP, only 76were confirmed by AS-PCR to be An.

minimus from 12 sites (Assam- 6 and AP-6).Molecular characterization based on ITS2and D3 region of An.minimus was carriedout. Two specimens each representing all12 sites was further processed for sequenceanalysis. Interestingly, in ITS2 amplification,an AS-PCR confirmed minimus species from

Sonapur area of Assam-Meghalaya bordershowed amplification at 585 bp, whereasother ITS2 sequences showed amplificationproduct at 562 bp. ITS2 amplicons weresequenced, edited with 5.8S and 28S bordersdefined region, actual length of ITS2sequences was determined to be 370 bp. Thetree generated with nucleotide sequenceswith highest log likelihood (-485.7631) isshown (Figure 1). 100% homogeneity with nointraspecific variations among the sequenceswas observed. However, the single specimenof An. minimus collected from Sonapurarea of Assam- Meghalaya border (GenBankaccession no. JQ046375) showed variationsfrom rest of An. minimus species. Thisparticular specimen had a transversion(T-A) at nucleotide position 4 (Figure 2).The sequence showed 100% similarity witha sequence from China (Gen Bank accessionno: AF 416783.1). D3 region of confirmedspecies were amplified showing amplifi-cation at 375bp. Phylogeny analysis basedon D3 region for An. minimus from allsequences revealed no differences. Oursubmitted sequences showed 100%homology with sequences previouslyreported from the region (GenBank accessionno. EF221771.1).

Figure 1. Phylogeny of An. minimus based on ITS2. Phylogenetic analysis is basedon ITS2 region of rDNA. Maximum Likelihood phylogeny was constructed basedon Tamura- Nei model

555

Figure 2. Alignment of ITS2 sequences. Dots (.) indicate identity of nucleotides within the alignment

556

Figure 2. Cont'd

557

Figure 3. Phylogeny of An. minimus based on D3. Phylogenetic analysis is based on D3 region ofrDNA. Maximum Likelihood phylogeny was constructed based on Tamura- Nei model

We studied genetic diversity of An.

minimus species of mosquitoes from twostates- Assam and AP of NE India coveringalmost all malaria endemic areas. SinceITS2 sequences are likely to be fixed withinthe species and vary between closelyrelated species due to high intraspecifichomogeneity and interspecific variability,ITS2 region of rDNA of ASPCR confirmedAn. minimus was amplified. ITS2 sequencesof specimens in our collected sites werecompletely identical with sequences of thatpreviously recorded from China, Thailand andother parts of India (Prakash et al., 2004)(Figure 1). However, a single specimenshowed a single base substitution(Transversion at site 4) (Figure 2) in ITS2sequence from Sonapur area of Assam-Meghalaya border. This variation showed adistinct population structure. Larger numberof samples from the area is needed tosequence to determine extent and nature ofany distinct population structure since thisbit of information has potential implicationsfor malaria control in malaria endemic PHCof Sonapur (NIMR, 2012). The sequenceanalysis of An. minimus species showed100% homology in 28S- D3 rDNA (Figure 3).Thus, it can be stated that no intra- specificvariations were found among the specimens.It has been observed in the present study thatour submitted sequences showed similaritywith previously submitted sequences from theregion and also with the sequences from

China, Thailand and Cambodia. However, thesequences showed variations with sequenceof Vietnam and placed in different clades.Distant geographical locations may be thepossible reason for diversity of sequences.D3 sequence homogeneity remains over alocal geographical scale. The molecularphylogenetic analysis of An. minimus

species from these two states showedhomogeneity in D3 regions. These similaritiesshowed that there is lack of intra specificvariations at the loci throughout thegeographical range of Assam and AP states.

Acknowledgements. This work wassupported by the Indian Council of MedicalResearch (ICMR), Ministry of Health andFamily Welfare; Government of India. Theauthors are thankful to Mr. Niranjan K.Baruah, Mr. Pobitra Doloi, Mr. Robin C. Doloi,Mr. Lakhyajit Borah and Dr. (Ms.) Jani Borahfor their excellent assistance. Authors alsoappreciate piece of advice put forward byNilanju P. Sarma, Sweta Singh and Devojit K.Sarma of Malariology Division, RMRC duringthe experimental work.

Conflict of Interest:

None to declare.

Funding Source:

This work was supported by the IndianCouncil of Medical Research, New Delhi,India.

558

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