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: [email protected] 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
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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
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.
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