The mosquito Aedes albopictus (Skuse), commonly referred to as the Asian tiger mosquito is endemic to Asia-Pacific region. Ecological plasticity and competitive ability have enabled its successful invasion world over. This mosquito species is involved in the transmission of endemic dengue in south east Asia region (SEAR) (Hawley, 1988). It has been reported to be an efficient vector under laboratory conditions (Moncayo et al., 2004; Moutailler et al., 2009; Haddad et al., 2012) and the dengue virus (DENV) has been recovered from wild caught mosquitoes (Tewari et al., 2004; Thenmozhi et al., 2007; Kumari et al., 2011). For instance, it has been documented that Ae. albopictus to be naturally infected with DENV virus during outbreaks in Mexico (Ibanez-Bernal et al., 1997). Similarly Ae. albopictus was observed to be naturally infected with DENV during dengue hemorrhagic fever outbreaks in Singapore (Chan et al., 1971; Chow et al., 1998) and Thailand (Gould et al., 1968). In India isolation of DENV has been reported from the northeast (Reuben et al., 1988), rural settings in Southern region (Tewari et al., 2004) and Northern India (Kumari et al., 2011). Recently, it was implicated in the transmission of CHIKV in Kerala (Kumar et al., 2012). However, in the recent years, Ae. albopictus has been implicated as an important vector for DENV and chikungunya virus (CHIKV) in Central Africa (Paupy et al., 2010) and CHIKV transmission in African and European countries (Bonilauri et al., 2008; Delatte et al., 2008; Pages et al., 2009; Paupy et al., 2011).

 

Prevalence of Wolbachia in Ae. Albopictus was unraveled in Thailand (Wright and Barr, 1980). Subsequently, the prevalence of Wolbachia in a field population of the Wolbachia-superinfected Ae. albopictus was reported (Kittayapong et al., 2002). While in India, Wolbachia infection in Ae. Albopictus population of Pune, Maharashtra was reported, indicating that Wolbachia sp. showed homology with members of A super group (Pidiyar et al., 2003). Their report was based on Wolbachia surface protein (wsp) gene sequence phylogenetic analysis. Wolbachia sp. from Ae. albopictus was highly homologous to the  wAlbA strain and hypothesized that this strain of Wolbachia sp. in the Indian population of Ae. Albopictus to be novel. Later, the prevalence of Wolbachia and its phage WO infection and presence of wAlbA and wAlbB using wsp and orf7 gene primers was studied in Ae. albopictus population in Karnataka and Tamil Nadu, India (Ravikumar et al., 2010). In 2014, through our preliminary observations (Sivan et al., 2014), the presence of Wolbachia endosymbionts was detected in the wild population of Ae. albopictus through 16s rDNA polymerase chain reaction, indicating that a small number of Ae. albopictus were infected with Wolbachia endosymbionts (wAlbA and wAlbB). Further its phylogeny using partial genomic nucleotide sequence of wsp gene was determined in the Andaman and Nicobar Islands.

 

Wolbachia mediated cytoplasmic incompatibility has been recommended as an alternative strategy to introduce and spread transmission interrupting genes into wild insect vector populations in pursuit of transforming vectorial capacity in these populations (Sinkins et al., 1997; Sinkins et al., 2000). However, the long-term objective of accomplishing disease control, depends on the ability of Wolbachia to invade the insect population and maintain a balance between the target insect population that is high enough to influence disease transmission (Turelli and Hoffmann, 1999). Studies on Wolbachia infection frequency in Ae. albopictus is scanty, except for a comprehensive field study in a single population in Thailand, which reported 100% infection prevalence (Kittayapong et al., 2002).

 

Therefore, we undertook a comprehensive field study to understand the actual prevalence and map the Wolbachia endosymbionts (wAlbA and wAlbB). We examined the natural Wolbachia infection prevalence in the vector mosquito, Ae. albopictus in South Andaman district endemic for dengue (Vijayachari et al., 2011; Chaaithanya et al., 2012; Muruganandam et al., 2014). The results show 100% prevalence of Wolbachia super group A and Wolbachia super group B in wild Ae. albopictus. The wsp gene sequence of the Wolbachia strain showed 99% sequence homology with Wolbachia sp. of Ae. albopictus isolated from different settings. We discuss the results keeping in perspective of looking at alternative means of control for Ae. albopictus.

 

The study was undertaken in South Andaman district. The District of South Andaman comprises of three tehsils viz., Port Blair, Ferrargunj and Little Andaman. Each Tehsil is divided into villages. This district predominantly is forest covered spanning an area to about 2613.53 km², followed by rural area (340.37 km²), while the urban area is limited to about 26.10 km². The area is inhabited by tribal community and mainlanders who have settled for administrative and business purposes. Major crops of south Andaman are predominantly banana (Musa paradisiaca), arecanut (Areca catechu), sugarcane (Saccharum officinarum), mango (Mangifera indica), sapota (Achras sapota), ginger (Zingiber officinale), paddy (Oryza sativa), coconut (cocus nucifera) and papaya (Carica indica). It has been reported that competent vectors viz. Ae. aegypti and Ae. albopictus are well established and prefer to breed in domestic and peri-domestic locations in water holding receptacles (Shriram and Sehgal, 1999; Shriram et al., 2008; Shriram et al., 2009; Vijayachari et al., 2011) in south Andaman district. The climate may be described as normal. It is always warm and tempered by pleasant sea breezes; very hot during summer. The climate is highly humid (Relative humidity about 83%) with a warm temperature ranging between 22ºC and 34ºC. The area receives heavy rainfall from May to January, influenced by both southwest (May to October) and Northeast monsoon (November to January or April). However, the heaviest rainfall is experienced during the months of May-September and the peak rainfall is during the month of September (554 mm). In other months rainfall is generally low with February (37.38 mm) being the driest month. The average annual rainfall is about 3253 mm. Monthly mean maximum temperature ranged from 27.8ºC to 34.1ºC. The monthly mean minimum temperature ranges between 22ºC to 25ºC. March and April are the hottest months with the daily mean maximum temperature of 30.6ºC-34.1ºC During the relatively cooler months of November, December, January and February the daily mean maximum temperature ranged between 29.9ºC-32.5ºC and daily mean minimum temperature was about 22ºC-25ºC.

 

The entire district of South Andaman could be  classified into four topographies viz. (1) densely built urban- an area thickly populated, composed of mainly residential and commercial buildings; (2) Low vegetation coverage-an area thinly populated composed of short vegetation, mostly grass; (3) medium vegetation coverage/fringe area –an area composed of short sized vegetation, shrubs or small trees that are spatially scattered and sparsely populated (4) high vegetation coverage-a forested area, with sparse human habitation  composed mainly of tall vegetation/trees by visual review of the satellite imagery (Figure 1). Over the last decade, rapid urbanization is evident in and around the capital town of Port Blair.

 

Entomological survey was carried out in 29 locations encompassing 4 heterogeneous landscapes of South Andaman district (Figure 1a, 1b) by household visit for the presence of Ae. albopictus species from August 2012–October 2012. A trunk road connects Port Blair (south Andaman district) and Mayabunder (north and middle Andaman District) (Figure 1a). This trunk road traverses through the study area of south Andaman district. In order to understand the actual field prevalence of Wolbachia in Ae. albopictus larval infestation surveys were carried out along the trunk road. One arbitrary point was identified from the urban Port Blair on the trunk road, following the road; the survey team stopped at every 3 km, geo-referenced the site with GPS unit (Garmin), and inspected all sides of the road, with an area of 1.5 km radius. A similar step was followed in Little Andaman Tehsil (Figure 1b). No specific consents were necessary for these survey locations vis a vis field surveys since the survey per se did not involve endangered or protected species. The insect collectors systematically examined the domestic water holding containers (metal drums, plastic drums, cement tanks, plastic pots) and peri-domestic water collections (discarded tins, flower vases, discarded tires, receptacles which could hold water, tree holes etc.). Larvae and/or pupae were sampled using pipettes and sweep nets. Field collected larvae and pupae were transferred to a plastic container and brought to the Centre’s laboratory for raising them to adults. Emerged adults were confirmed as Ae. albopictus using standard taxonomic keys (Barraud, 1934).

 

Twenty emerged Ae. albopictus from each sampling location, or the total number of Ae. albopictus emerged from each of the sampling location were used for detection of Wolbachia endosymbiont. Whenever, the emerged Ae. albopictus were<20, the available mosquitoes were used for testing. They were then stored in individual tubes containing isopropanol at -20°C for undertaking molecular assays. In order to assess the prevalence of Wolbachia sex wise in Ae. albopictus, a fixed number of larval samples were collected and emerged to adult from different sampling locations for PCR based screening.

 

DNA extraction was carried out from individual mosquito sample using DNA Extraction Solution kit (Genie, Bangalore, India), following manufacturer’s instructions and subjected to PCR amplification using Wolbachia specific primers to amplify the wsp gene (650 bp) using primers wsp81F [5’-TGG TCC AAT AAG TGA TGA AGA AAC-3’] and wsp 691R [5’-AAA AAT TAA ACG CTA CTC CA-3’] (Pidiyar et al., 2003). Detection of Wolbachia species was carried out by PCR using the super group specific wsp primers for super group A; (136A; F5’- TGAAATTTTACCTCTTTTC-3’ and 691R; 5-’AAAAATTAAACGC TACTCCA-3’) and for super group B (81F; 5’-TGG TCCAATAAGTGATGAAGAAAC-3’ 522R; 5’-ACCAGCTTTT GCTTGATA-3’) following an earlier reported protocol (Zhou et al., 1998).

 

The PCR products of wAlbA (379 bp) and wAlbB (501 bp) using 328F/691R and 183F/691R primers of the wsp gene were then subjected to cycle sequencing.For each sequencing reaction, 50 ng of purified PCR product was mixed with a reaction mixture containing 2.5X sequencing buffer, 5X big dye terminator and 20 mM of either forward/reverse primers of wAlbA and wAlbB genes in two separate sets of reaction. Cycle sequencing parameters used were: 96°C for 1 min followed by 25 cycles of 96°C for 10 sec, 50°C for 5 sec, and 60°C for 4 min.

 

The cycle sequencing products were then purified by EDTA/ethanol precipitation. The dried pellet was resuspended in 10 ml of Hi-Di-formamide, and sequenced in an automated DNA sequencer (ABI 3130, Applied Biosystems, Foster City, Ca, USA) using performance optimized polymer 7 following the manufacturer’s instructions. The nucleotide sequences obtained after sequencing were edited and analyzed by the Seqscape software (Applied Biosystems, Foster City, Ca, USA). Multiple sequence alignments and phylogenetic analysis were performed using partial wsp gene sequences of the mosquito isolates obtained from Orissa and other regions in Mega 5 software (Tamura et al., 2011). The phylogenetic tree was constructed by using Maximum-likelihood method using Tamura Nei model of Mega 5 software. The robustness of each node was estimated using 1000 bootstrap replications under the Nearest-Neighbor Interchange procedure, with input genetic distance determined under the Maximum-likelihood substitution model. Three PCR products each from four landscapes were screened for Wolbachia A and Wolbachia B respectively, constituting 24 DNA sequences.

 

We developed a GIS based map of the Aedes sampling locations and prevalence of Wolbachia in Ae. albopictus in south Andaman using ArcGIS software.

 

A total of 1441 individuals of Ae. albopictus were collected from 29 sampling locations encompassing three tehsils (four heterogeneous landscapes) in south Andaman district. Of these 472 individuals of Ae. albopictus were screened for the presence of Wolbachia endobacterium by PCR (Table 1). Our PCR results showed that 100% of the mosquitoes screened were positive for Wolbachia A&B. Prevalence of Wolbachia type A and B were similar (100%) in both sexes and different ecological locations. Prevalence and distribution of Wolbachia infection in Ae. albopictus in 29 sampling locations spread over the district of South Andaman is depicted in the Figure 1a and 1b.

 

The wsp gene obtained was used for analyzing the phylogenetic relationships with other known sequences retrieved from NCBI databases. Three samples from each sampling location were subjected to sequencing. The sequence representing Wolbachia wsp gene was submitted to GenBank (accession no. KU056918). The wsp gene sequence of Wolbachia collected in the present study showed homology with known sequences obtained from the NCBI database. Phylogenetic analyses of wsp gene sequences of Wolbachia generated from Ae. albopictus mosquitoes of South Andaman showed the circulation of both wAlbA and wAlbB strains. The phylogenetic tree confirmed the co-infection of wAlbA and wAlbB in Ae. albopictus mosquitoes collected from different sampling locations. Phylogenetic analysis revealed that the present Wolbachia isolate is closely related with Wolbachia from main land India and elsewhere. Phylogenetic analysis showed 100% nucleotide sequence homology of wAlbB species with other wAlbB species isolated from different geographical regions of India and elsewhere. The phylogenetic analysis showed 96% nucleotide sequence homology of wAlbA species with other wAlbA species isolated from different geographical regions of India and elsewhere (Figure 2). The pairwise genetic distance was found to be 0.000 among other Wolbachia B isolated from different parts of India and elsewhere in the world. The pairwise genetic distance was found to be 0.000 among other Wolbachia A isolated from within the Andaman and Nicobar archipelago indicating no genetic variability in the populations within group A. However low genetic variability was observed with Wolbachia A isolated from different part of India and elsewhere in the world with genetic distance ranging from 0.006 to 0.1029.

PCR based methodologies have enabled to understand the Wolbachia distribution and diversity (Werren, 1997). We used polymerase chain reaction for detecting Wolbachia among Ae. albopictus, and determined its field prevalence and phylogenetic relationship with other known species using the partial nucleotide sequence of Wolbachia surface protein (wsp) gene. In continuation to our preliminary observations on the prevalence of Wolbachia endosymbionts (wAlbA and wAlbB) in the population of Ae. albopictus in Andaman and Nicobar Islands (Sivan et al., 2014), we provide information on the actual prevalence and phylogeny of Wolbachia in wild populations of this mosquito species in south Andaman district, endemic for dengue. Further GIS based information on sampling locations and depiction of the Wolbachia across heterogenous landscapes are discussed.

 

In the recent past Wolbachia have attracted the interest of several scientists (Dobson et al., 2004; Hoffmann et al., 2011; Mousson et al., 2012; Blagrove et al., 2013), with the widespread distribution and effects on host, consequences on evolutionary processes and in looking for ways to utilize for controlling vector mosquitoes that spread human pathogens. When a vector population comprises of individuals both uninfected and infected with same or different Wolbachia spp, three possibilities arise in their mating viz. the first possibility is compatible and produce viable offspring, the second possibility is that of being incompatible in both directions resulting in infertile egg in other words commonly referred to as bidirectional cytoplasmic incompatibility (CI)  and the third situation being incompatible in one direction with reciprocal cross being fertile, referred to as unidirectional CI. These characteristics are being examined with the perspective of developing strategies to bring about improvements in the control of vector mosquitoes. Therefore information on natural infection of Wolbachia in mosquitoes is central for assessing the usefulness of developing Wolbachia based vector control intervention strategies.

 

In the current study which is based on the PCR based genotyping of Wolbachia infection in Ae. albopictus mosquitoes of south Andaman, indicates widespread infection in the populations screened. Our field investigations revealed 100% infection and superinfection of two types of Wolbachia strains i.e. wAlbA and wAlbB in Ae. albopictus mosquito population of south Andaman district. These observations indicate that the dynamics of Ae. albopictus could be modulated for transmission of arboviral pathogens in South Andaman. Elsewhere, it is reported that two different strains of Wolbachia perhaps contribute to success in maternal transmission of Wolbachia in wild populations of this mosquito species (Kittayapong et al., 2001). The results obtained from this study are identical with that observed in Thailand (Kittayapong et al., 2002) and in agreement with studies that suggested both wAlbA and wAlbB strains in natural female Ae. albopictus mosquitoes are common and almost fixed in a population and hence can be vertically transmitted efficiently (Dobson et al., 2004; Hoffmann et al., 2011).

 

Studies undertaken on only few samples of Ae. albopictus in Karnataka (Ravikumar et al., 2010), indicate infection with AB group of Wolbachia. It has been reported that wAlbA in Pune population of Ae. albopictus represents a novel strain of Wolbachia sp (Pidiyar et al., 2003). On the other hand, a quite recent study by Das et al., (2014) indicated that Ae. albopictus field populations of Orissa were mostly superinfected with wAlbA and wAlbB strains and that Wolbachia superinfection (100%) was distinct in the coastal plain areas as compared to other physiography. However, in the present study 100% infection and superinfection of two types of Wolbachia strains i.e. wAlbA and wAlbB in Ae. albopictus mosquito population across heterogeneous landscapes of south Andaman district is evident, suggesting that it could be genetically and biologically exclusive than the other populations, the reasons for which is beyond the scope of study. This attribute probably could have implication on the vectorial capacity of this mosquito species. Besides, in the current study it was also observed that 100% of males and females of Ae. albopictus populations were infected with Wolbachia.

 

Phylogenetic analysis of Wolbachia spp based on the fast evolving wsp gene showed that the present Wolbachia isolate was closely related with Wolbachia from different geographical locations of India, USA, China, France, United Kingdom and Taiwan. As opined by Turelli and Hoffmann (1999), data on natural infection prevalence is central to evaluate the potential use of Wolbachia as a means to alter insect vector populations. To that extent the present study brings out the actual infection prevalence of Wolbachia endosymbiont in the Ae. albopictus population from this region of the country, which provides us an opportunity to look for alternative means of controlling this vector species.

 

The authors thank Dr I.P. Sunish, Scientist ‘C’, Regional Medical Research Centre (ICMR), Port Blair, India, for providing useful suggestions on the manuscript. The authors acknowledge the assistance rendered by Mr Murthy, Deputy Conservator of Forests, & Mr Raja, Forester, Van Sadan/Department of Forests, A & N administration, Port Blair for developing GIS based map of sampling locations and Wolbachia prevalence. Technical assistance rendered by the staff of the Division of Medical Entomology and Vector Borne Diseases is gratefully acknowledged.

 

Arun Sivan carried out the field work, participated in the analysis of data and writing the manuscript. A N Shriram designed the study and drafted the manuscript. Debdutta Bhattacharya sequenced PCR products and participated in analysis. P. Vijayachari provided technical and administrative support. All authors reviewed the report and approved the final manuscript.

 

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