INTRODUCTION
Aedes aegypti mosquitoes are of great concern to the public health as they transmit various serotypes of viral pathogens causing dengue and chikungunya in human population1–2. Due to the unavailability of vaccine or an- tiviral treatment, disease prevention is dependent on con- trolling mosquito population. Therefore, it is important to have information regarding bionomics and likewise about genetic structure of mosquitoes in terms of refrac- tory or susceptible vector species. A detailed study on Ae. aegypti population of Southeast Asia, Africa, America and other Latin American countries illustrated that there is a local genetic variation and gene flow among same species, which is responsible for different diseases trans- mission rate3.
In India, Ae. aegypti dispersed in various ecological locations has shown diversity in genetic makeup and dis-
eases transmission potential4–5. Aedes is a prime target for disease surveillance programme6, but still informa- tion regarding its distribution, density, disease transmis- sion rate and seasonal prevalence is limited7–8.
In past few decades, mass rearing of Aedes species in laboratory conditions has been carried out for their iden- tification. Conventionally, mosquito identification is car- ried out based on their morphological characteristics9, but since it is time consuming process and needs professional expertise, only 10% of species have been identified so far throughout the world10–11. However, utilization of spe- cific molecular markers is a better alternate for vector identification as it is faster to perform and more reliable.
In combination with conventional morphological char- acteristics, second internal transcribed spacer (ITS-2) of rRNA12 and mitochondrial cytochrome c oxidase subunit I (mtCOI) gene sequences have been used for species iden- tification13. Due to the high rate of evolution in the mito- chondrial DNA, it is extremely useful for species identi-
Molecular identification of Aedes aegypti mosquitoes from Pilani region of Rajasthan, India
Kuldeep Gupta
1*, Rini Dhawan
1*, Mithilesh Kajla
1, Sanjeev Kumar
1, B. Jnanasiddhy
1, Naveen K.
Singh
1, Rajnikant Dixit
2, Ashish Bihani
1& Lalita Gupta
11Molecular Parasitology and Vector Biology Laboratory, Department of Biological Sciences, Birla Institute of Technology and Science (BITS), Pilani, Rajasthan; 2Host-Parasite Interaction Biology Group, National Institute of Malaria Research, New Delhi, India
ABSTRACT
Background & objectives: Aedes aegypti is the most important vector of dengue virus infection in humans worldwide. Accurate identification and colonization are the essential requirements to understand vector biology as well as its diseases transmission potential. In this study, we have used molecular approaches for the identification of Ae. aegypti mosquitoes that were collected from the Pilani region of Rajasthan, India
Methods: Field collected mosquito larvae were colonized under laboratory conditions. Conserved genetic markers, ITS-2 and mtCOI were used for amplification through species-specific primers to identify the mosquito species/
strain. Sequencing result of this strain was phylogenetically compared with other global strains through MEGA software.
Results: A comprehensive multiple sequence alignment and phylogenetic analysis revealed that COI gene of Ae.
aegypti has extremely low genetic variability with one of the Indian isolate from Thirumala, Andhra Pradesh region (GenBank: HM807262.1). However, in context of different geographical locations, it indicated close similarity with Thailand’s strain and high variability when compared with Madagascar strain. On the other hand, ITS-2 illustrated highest identity with Ae. aegypti of Saudi Arabia (GenBank: JX423807.1) whereas, high divergence was observed from Mayotte, France strain (GenBank: KF135506).
Interpretation & conclusion: The findings suggest that this isolate from Rajasthan is similar to other Asian continent strains possibly due to the same origin. Understanding the vectorial capacity of these geographically distributed mosquito strains will enhance our knowledge to improve existing vector surveillance and control programme.
Key words Aedes aegypti; COI; colonization; ITS-2; phylogenetic analysis; Pilani; Rajasthan
*Authors made equal contribution.
fication and analyzing the evolutionary processes14. These reliable molecular markers are essential for molecular systematic15 as well as species barcoding/DNA array tech- nologies4, 16. In this study, we also examined different DNA sequences of ITS-2 and mtCOI gene from globally distributed Aedes species. The identified Indian Aedes mosquito species showed significant variation in the DNA sequence when compared with Ae. aegypti from other geographical region. Our comparative analysis of pre- colonization and post-colonization (post ~50 cyclic colo- nies) verifies that establishment of an isofemale line with unique genetic makeup was successful.
MATERIAL & METHODS Mosquito sampling and colony establishment
Aedes aegypti larvae were collected during the summer of 2010 as described earlier17–18 from various locations of Pilani, a small town situated in Shekhawati region of Rajasthan, India (28°22' N 75°36' E). The collect- ing sites majorly included semi-permanent groundwater pits in the open areas, fields and water storage containers filled with stagnant rainwater. The collected larvae were morphologically identified and continuously rearedin labo- ratory condition since then, as discussed before17–18. Swiss albino mice (Mus domesticus) were used for feeding the mosquitoes according to the approved guidelines of Insti- tutional Animal Ethical Committee (IAEC/RES/15/01).
Pure line maintenance
The larvae brought from field to the laboratory were allowed to undergo the developmental cycle till the adult mosquitoes emerged. The Ae. aegypti adult females and males were identified morphologically with the help of pictorial keys for the identification of mosquitoes (Diptera:
Culicidae) associated with dengue virus transmission guide, published by Magnolia Press18. Out of the above identified mosquitoes, 20 self-mated adult female mosqui- toes were blood fed and kept individually in separate cages.
Each isolated gravid female was allowed to lay the eggs in water cups after two days of blood feeding. The laid eggs were allowed to hatch and continue the whole life cycle.
Larvae from five of these individual colonies were further characterized at molecular levels. Out of these five molecu- larly characterized mosquito colonies, only one was se- lected for propagation and named as AePL-2.
DNA extraction from larvae
Genomic DNA was extracted from the IV instar lar- vae using method as described by Gupta and Preet19 for initial screening. Precisely, 10 IV instar Aedes larvae were
grinded in 100 μl of the lysis buffer (100 mM Tris-HCl, pH 8.0; 0.5% SDS; 50 mM NaCl; 100 mM EDTA) and treated with 5 μl of proteinase K (20 mg/ml) for 1 h at 55°C. To this cell lysate 5 μl of RNase was added (10 mg/
ml) and kept for 30 min at 37°C in water bath. The suspen- sion was extracted twice with an equal volume of phenol- chloroform, and DNA was precipitated by the addition of pre-chilled isopropanol. Concentration of DNA sample was determined by a double-beam UV-Vis spectropho- tometer (JASCO, V-630, Japan) at 260 nm. Purity of DNA was analyzed by measuring absorbance ratios A260/A280 for protein contamination. Further, the sample was run on a 0.8% agarose gel and visualized under UV light using a Gel documentation system, Syngene, India.
PCR amplification and cloning
ITS-2 sequences of Ae. aegypti from different world isolates, available at NCBI database, were aligned to re- trieve the common conserved region and designing the primers. The forward primer: AeITS-2-Fwd: 5'-ATCAC- TCGGCTCGTGGATCG-3' and the reverse primer AeITS-2-Rev: 5'-ATGCTTAAATTTAGGGGGTAGT-3' are located in the 5' end of 5.8S region and 3' end of the 28S region, respectively (Fig. 1a).
For mtCOI, the oligonucleotides were used from already published4 primers sequence AeCOI-Fwd: 5'- GATTTGGAAATTGATTAGTTCCTT-3' and AeCOI- Rev: 5'-AAAA ATTTTAATTCCAGTTGGAACAGC-3' (Fig. 1b). Nuclear ITS-2 and mtCOI regions were ampli- fied through PCR reactions in a total volume of 50 μl per reaction. Each reaction tube contained 50 ng of genomic DNA, 1U/μl of Taq DNA polymerase, 1 μl of 10 mM dNTP mix, 5 μl of 10 x PCR buffer having 2.5 mM MgCl2 and 1 μl of each 20 pmol of forward and reverse primers.
Fig. 1a:Schematic representation of Ae. aegypti rDNA gene with the position of forward and reverse primers and the size of amplified PCR product.
Fig. 1b:Partial gene organization of Ae. aegypti mtDNA for COI, COII and COIII genes. Region between two arrowheads represents the fragment used for PCR amplification.
'
The PCR conditions were single cycle of pre-denatur- ation at 95°C for 3 min followed by 35 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 45 sec and final extension 72°C for 10 min. Amplification product of ITS- 2 (313 bp) and COI (714 bp) were purified using Qiaquick PCR purification kit (Qiagen Cat No. 28104) and used for direct sequencing while ITS-2 PCR product was used for cloning. The purified PCR product of ITS-2 was cloned using PCR-II TOPO TA-Vector® (Invitrogen Cat No.
K46001-01) and transformed to TOP10F' DH5-α® Es- cherichia coli cells following the standard protocol pro- vided with the kit. Plasmid was isolated using Qiaprep Spin Miniprep Kit (Qiagen Cat No. 27104) and sequenced through vector specific M13 universal primers. The ob- tained forward and reverse sequences were assembled using gene tool 4.1.
Sequence and phylogenetic analysis
Sequencing data were analyzed using freely avail- able Chromas software and confirmed through blast search. ITS-2 and COI sequences of Ae. aegypti mosquito isolates submitted by others were retrieved from the NCBI.
DNA sequences were aligned using Clustal omega plat- form20. Sequence divergences among individual species were determined using the Kimura two-parameter (K2P) distance model. Average evolutionary divergence was estimated using the Neighbour-Joining (NJ) method in MEGA 5.121. The average evolutionary divergence was estimated as a number of base substitutions per site by averaging over all sequence pairs within and between each group. All results were based on the pair wise analysis of known sequences of ITS-2 and COI from other reported isolates (Tables 1, 2 and 3). All positions containing gaps and missing data were eliminated from the dataset (com- plete deletion option). Phylogenetic tree was constructed by MEGA 5.1 using NJ method with 1000 bootstrap value and partial deletion option.
RESULTS
Identification of Aedes species and sequence analysis Laboratory adapted adult mosquitoes were morpho- logically examined under microscope for their unique features as described in the key guides for Aedes mosqui- toes18. For molecular identification, ITS-2 region was amplified using gDNA isolated from established pure- line mosquito colonies AePL-1 to AePL-5. Amplifica- tion of 313 bp fragment in each case suggested that every pure line colony of mosquito belongs to same species (Fig. 1c). To get the terminal gene sequence, PCR prod- uct of finally selected AePL-2 was used for cloning and
Table 2. NCBI retrieved sequences of COI of Ae. aegypti from India
Variants Accession number
Ennore, Tamil Nadu 1 DQ424949
Agricultural farm, Tamil Nadu AB907183
Puducherry AY729987
Mamulapusi, Odisha HM807269
Thiruvananthapuram, Kerala HM807268
Thirumala, Andhra Pradesh P7 HM807267
Thirumala, Andhra Pradesh P6 HM807266
Thirumala, Andhra Pradesh P5 HM807265
Thirumala, Andhra Pradesh P4 HM807264
Thirumala, Andhra Pradesh P3 HM807263
Thirumala, Andhra Pradesh P2 HM807262
Puthur, Andhra Pradesh P1 HM807261
Rajasthan, India KP121340
Table 3. NCBI retrieved sequences of COI of Ae. aegypti from different geographical locations of world
Variants Accession number
Cambodia JQ926688
Bolivia JQ926679
Brazil JQ926703
Cameroon JQ926702
Ivory Cost JQ926694
France HQ688296
Guinea JQ926700
Madagascar HQ688298
Martinique JQ926696
Mexico JQ926698
Portugal KF909122
Tanzania JQ926704
Thailand JQ926692
USA JQ926684
Venezuela JQ926701
Vietnam JQ926687
Table 1. NCBI retrieved ITS-2 sequences of Ae. aegypti from different geographical locations of world
Variants Accession number
Ryukyu, Japan AB548800
Cajamarca, Peru AY512665
Hosta, Russia HE820724
Saudi Arabia JX423807
Mayotte, France KF135506
Uganda M95126
Rajasthan, India KJ862124
sequencing (Fig. 1c, lane 2). From the 313 bp of cloned sequence, 28S rDNA and 5.8S rDNA regions were ex- tracted and 204 bp ITS-2 region was used for analysis (Fig. 1a). To strengthen the genetic analysis, mtCOI re- gion was amplified using AePL-2 gDNA. The expected 714 bp PCR product of COI gene was sent for sequenc- ing at Delhi University, India (Figs. 1b and d). The ITS- 2 and COI sequences were checked for their nucleotide identity through BLASTN analysis which confirmed that selected pure-line belongs to Ae. aegypti. Complete ITS-2 sequence and partial COI sequences have been
reported to NCBI (GenBank) with accession number KJ862124 and KP121340 respectively. Thus, in combi- nation with morphological characteristics of the field collected mosquito, molecular markers based analysis further confirmed the identity of the mosquito species as Ae. aegypti.
Sequence variation of Ae. aegypti
To compare the genetic diversity among Ae. aegypti of laboratory colonized mosquitoes with other isolates from different geographical locations, 204 bp fragment ITS-2 was examined. Since, no ITS-2 sequence of Indian isolate was available, comparison was made with isolates from different geographical locations of the world (Table 1). Sequence analysis with other global isolates indicated highest identity to Ae. aegypti of Saudi Arabia origin (GenBank: JX423807.1) and lowest to Mayotte, France, isolate (Fig. 2a). Amongst 204 nucle- otides of ITS-2, nine polymorphic sites (4.4%) were ob- served in all six global isolates. The number of base differences per site from averaging over all sequence pairs was 0.02 in total 178 positions. Analyses were con- ducted using the K2P model obtained by 1000 bootstrap replicates.
For the COI analysis, a fragment of 609 bp was se- lected. A total of 12 isolates of Indian origin (Table 2) and 16 isolates from different countries (Table 3) were compared with this isolate of Rajasthan. Unlike ITS-2, less genetic difference was observed within Indian iso- late of Andhra Pradesh as well as other globally reported isolates. In total, 19 polymorphic sites (3.1%) were found when we compared Rajasthan COI sequence with other Indian isolates. However, 36 polymorphic sites (5.9%) were observed with the worldwide isolates of Ae. aegypti (Figs. 2b and c). The numbers of base differences per site from averaging over all sequence pairs were 0.011 and 0.014, respectively. Interestingly, laboratory colonized Ae. aegypti COI gene showed only one difference at 51
Fig. 1c:Molecular characterization of morphologically identified individual mosquito (AePL 1-5); gel image showing the desired band size (313 bp) of ITS-2. The DNA marker SM0331 (Fermentas) was used to determine the size of PCR product in the gel.
Fig. 1d:PCR amplification of COI gene (714 bp) of the Ae. aegypti mosquito pure-line (AePL-2).
Fig. 2a:Comparison of ITS-2 of Indian Ae. aegypti with world isolates showing nine nucleic acid polymorphic sites.
[ 111 1 ]
[ 2 2 2 2 3 11 5 5 ]
[ 2 6 8 9 7 0 6 0 4 ]
#India_ITS–2 TGCGCGACC
#SaudiaArab_ITS–2 - - - -
#Russia_ITS–2 C - - - -
#Japan_ITS–2 C - - - -
#France_ITS–2 CTAAGCGTT
#Uganada_ITS–2 C - - - -
#Peru_ITS–2 C - - - -
Fig. 2b:Indian Ae. aegypti showing 19 variable nucleic acid polymorphic sites for COI gene when compared with Rajasthan isolates.
Fig. 2c:Comparison of Indian Ae. aegypti COI gene with worldwide isolates showing 36 variable nucleic acid polymorphic sites.
Phylogenetic analysis of Ae. aegypti
To know the phylogenetic relationships amongst Ae.
aegypti isolates, ITS-2 and COI sequences were aligned with their respective counterparts from different region.
Indian isolates of Ae. aegypti have never been identified using both COI and ITS-2 together; this is the first report where the species is identified using both nuclear and mitochondrial molecular markers. ITS-2 sequences of seven global Ae. aegypti isolates were selected for con- structing evolutionary tree by NJ method using K2P model with 1000 bootstrap value. As expected from sequence analysis, phylogeny showed a close proximity with Saudi Arabia strain and significant diversity with France iso- late (Fig. 3a).
In case of COI, the evolutionary divergence among Indian strains of Ae. aegypti and global strains was ana- lyzed separately by using phylogenetic tree. Phylogenetic tree with Rajasthan COI (GenBank: KJ862124) revealed close resemblance with Andhra Pradesh P2 isolate. How- ever, globally it comes in first clade along with Thailand, Brazil and Martinique isolates, which is also according to the lesser sequence variability that exists among these continental species of subtropical and tropical origin (Figs. 3b and c).
[ 11 2 2 2 3 3 3 3 3 4 4 4 4 4 4 4 ] [ 5 6 8 9 0 1 2 0 3 5 6 8 11 2 2 2 3 6 ] [ 1 3 3 2 4 6 5 3 9 4 0 4 7 8 3 4 6 5 5 ]
#Rajasthan_COI ATGGGTCATG CTAAACCAC
#Andhra_P1_COI T-AAACTG-A T C - - - T
#Andhra_P2_COI T - - - - - - - -
#Andhra_P3_COI T-AAACTG-A T C - - - T
#Andhra_P4_COI T-AAA-TG-A - C - - - T
#Andhra_P5_COI T-AAA-TG-A - C - - - T
#Andhra_P6_COI T-AAA-TG-A - C - - - T
#Andhra_P7_COI T-AAA-TG-A T C - - - T
#Kerala_COI T-AAA-TG-A - C - - - T
#Odisha_COI T-AAA-TG-A - C - - - T
#Puducherry_COI TGAAACTGCA T C - - - T A - -
#Tamilnadu1_COI T - AAACTG - A T C - - - T
#Taminadu2_COI T - AAA - TG - A T- T T- A G G -
[ 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 4 444555]
[ 4 5 5 6 7 9 0 3 4 5 8 9 9 0 0 1 2 2 5 7 9 0 2 4 5 6 6 8 3 367279]
[ 6 2 1 4 6 5 9 2 8 7 9 3 2 8 1 4 6 0 5 8 9 1 3 1 2 4 0 9 4 1 254831]
#India_Rajasthan_COI GGAGTCATTC GGGGAGTCCC TAACTGCATT TCGGCT
#Bolivia_COI - A T A - T - - - - - A A - - - - - - - - - T- - T-
#Brazil_COI - - T - - - - - - - - - - - - - - - A - -
#Cambodia_COI - - T - - - - - A A - - A - - T - - - G - - AT - C - - T - - - -
#Cameroon_COI - - T - - - - - A A - - A - - T - - - G - - AT - C - - T - - - -
#Guinea_COI - - T - - - - - A A - - A - - - - - - G - - AT- C A C T - - - -
#France_COI - A T A - T - - - - - A A - - A - - - - - - - - - T- T- -
#Vietnam_COI - A T - - - - - A A - - A - - T - - - G - - AT - C - - T - - - -
# Côte d’Ivoire_COI - - T - - - - - A A - - A - - - - - - - AT G C - - T - - - -
#Madagascar_COI AAT-C-GCCA -AAAGACTTT C G - T C A - - - - -T-A-C
#Portugal_COI - - T - - - - - A A - - A - - T - - - G - - AT - C - - T - - - -
#Martinique_COI - - T - - - - - - - - - - - - - - - A - -
#Mexico COI - - T - - - - - - A - - - - - - - - - - - A - -
#Tanzania_COI - - T - - - - A A A - - A - - - - - - - - - T- AT-
#Thailand_COI - - T - - - - - - - - - - - - - - A - - -
#USA_COI - - T - - - - - A A - - A - - T - - - G - - AT - C - - T - - - - -
#Venezuela_COI - - T - - - - - A A - - A - - T - - - G - - AT - C - - T - - - - -
nucleotide position with Indian isolate of Ae. aegypti from Thirumala, Andhra Pradesh region (GenBank:
HM807262.1), while other isolates of Andhra Pradesh showed high genetic variation.
DISCUSSION
Ae. aegypti is an important disease vector causing large number of deaths every year, and therefore, justi- fies the attention that has been given to its genetic diver- sity. Wide combinations of genetic markers are used to examine population structure, genetic differentiation and gene flow of the species. These are, therefore, an essen- tial component of vector-borne diseases management strategies as the geographic origins of mosquito popula- tions have epidemiological significance. This has been shown by several studies where association among the geographic origin of vectors with traits such as vector competence and insecticide resistance has been estab- lished22. Following the same approach, sequences of modern molecular markers of COI and ITS-2 of Ae.
aegypti from Pilani region, Rajasthan have been phylo- genetically analysed and compared with their counter- parts in India and the world. The results revealed that COI region of the Rajasthan isolate exhibits a striking polymorphism in 51st position (from A to T, Figs.
2b and c) when compared with other Indian and global isolates. Position 183,192 and 204 has ‘G’ in Rajasthan isolate, whereas other isolates of India have ‘A’ in all three positions except the Thirumala Andhra Pradesh P2 isolate (Fig. 2b). This suggested that mosquitoes collected even from one region have variations, which may be responsible for different rate of infection. However, it warrants further investigations. A similar study was carried in mainland Australia where 2 COI haplotypes have been identified in Ae. aegypti whereas in Thursday Island (only 39 km away from mainland) only one COI haplotype was observed. Interestingly, the Thursday Island haplotype revealed enhanced vector competence23. Isolates of Brazil, Martinique and Thailand also had ‘G’
in similar position as in Rajasthan isolate (Fig. 2c). It was also seen in case of Madeira Island (Portugal); the same COI haplotype has also been reported from America, Africa and Asia24.
On the other hand, ITS-2 region showed close homology with the isolates from Saudi Arabia. This close relationship can be attributed to the similar climatic conditions and arid environment that led to the similar evolutionary event within two mosquito strains for their successful adaptation. The present study is the first report on genetic analysis of Ae. aegypti from northwest- ern India. Sequence analysis of ITS-2 and COI from morphologically identified Aedes confirmed the identity of the species that was subsequently colonized in the laboratory. The purpose of colonization of mosquitoes will aid in the comprehension of the complexity of vector
Fig. 3a:Evolutionary relationship of Ae. aegypti based on the aligned region of ITS-2 sequences.
Figs. 3 (b–c): Phylogenetic tree deriving the relationship of COI sequence from Indian isolate and world isolates, respectively. The details of sequence used for analysis are shown together with their GenBank accession numbers in Tables 2 and 3.
competence. Correct vector identification is crucial as- pect for the designing of strategies that could effectively manage the vector-borne diseases.
ACKNOWLEDGEMENTS
This study was supported by the research grant from Aditya Birla Group and Department of Biotechnology (DBT), India. KD and MK acknowledge basic scientific research fellowship from University Grant Commission, New Delhi, India. RD pays gratitude to SERB-DST, New Delhi, India for the continuous monetary support. The authors are obliged to the Birla Institute of Technology and Science, Pilani, Rajasthan, for providing required facilities and logistic support for the unhindered conduc- tance of research.
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Correspondence to: Dr Lalita Gupta, Molecular Parasitology and Vector Biology Laboratory, Department of Biological Sciences, Birla Institute of Technology and Science (BITS), Pilani–333 031, Rajasthan, India.
E-mail: lalitagupta@yahoo.com; lalita@pilani.bits-pilani.ac.in Received: 23 June 2015 Accepted in revised form: 27 January 2016
