RESEARCH ARTICLE Year : 2021  |  Volume : 58  |  Issue : 4  |  Page : 297-305

Prevalence and transmission potential of Wolbachia in Aedes albopictus population circulating in endemic coastal districts of Odisha, India

Ipsita Mohanty1, Animesha Rath2, Nitika Pradhan2, Barsa Baisalini Panda1, Pradyumna Kishore Mohapatra1, Rupenangshu Kumar Hazra1
1 Regional Medical Research Centre (ICMR), Nalco Square, Chandrasekharpur, Bhubaneswar, Odisha, India
2 Regional Medical Research Centre (ICMR), Nalco Square, Chandrasekharpur; KIIT School of Biotechnology, Kalinga Institute of Industrial Technology, Bhubaneswar, Odisha, India

Date of Submission31-Oct-2018Date of Acceptance12-Mar-2019Date of Web Publication25-Mar-2022

Correspondence Address:
Dr. Rupenangshu Kumar Hazra
Regional Medical Research Centre (ICMR), Nalco Square, Chandrasekharpur, Bhubaneswar 751023, Odisha
India

Source of Support: None, Conflict of Interest: None

Check

DOI: 10.4103/0972-9062.313967

Wolbachia, known for its reproductive manipulation capabilities in insects, are being implemented to control dengue and chikungunya. To understand Wolbachia biology and its utility as a bio-control for vector mosquito’s populations, we investigated its dissemination pattern in field in collected Ae. albopictus along with its maternal transmission efficacy over generations in regions of endemic dengue (DENV) transmission. Field collected Ae. albopictus were subjected to PCR for Wolbachia screening. Overall mean Wolbachia infection frequency in Ae. albopictus was found out to be 87.3% wherein a trend was observed in the pattern of maternal transmission across generations. χ2 for trend revealed a significant variation between Wolbachia infections and non-infections in Ae. albopictus generations. Linear regression analysis revealed the involvement of a strong negative correlation, implying that overall Wolbachia infection tends to decrease in places with high dengue cases.The reduction in Wolbachia infection frequency may be attributed to several environmental factors with the probability of being the cause for endemicity of dengue in the studied areas.This study reports on the transmission efficacy of naturally occurring Wolbachia in successive generations of Ae. albopictus and its correlation with dengue cases in clusters of Odisha, India. Studying the transmission trend of Wolbachia along with transovarial transmission of DENV might be indicative towards the interplay of Wolbachia infection in presence/absence of DENV.

Keywords: bio-control; endemic; mono-infection; superinfection; transfection; Wolbachia

How to cite this article:
Mohanty I, Rath A, Pradhan N, Panda BB, Mohapatra PK, Hazra RK. Prevalence and transmission potential of Wolbachia in Aedes albopictus population circulating in endemic coastal districts of Odisha, India. J Vector Borne Dis 2021;58:297-305
How to cite this URL:
Mohanty I, Rath A, Pradhan N, Panda BB, Mohapatra PK, Hazra RK. Prevalence and transmission potential of Wolbachia in Aedes albopictus population circulating in endemic coastal districts of Odisha, India. J Vector Borne Dis [serial online] 2021 [cited 2022 Mar 26];58:297-305. Available from: https://www.jvbd.org/text.asp?2021/58/4/297/313967   Introduction 

Aedes albopictus, known to be a primary or secondary vector[1],[2] of arboviral diseases like dengue, chikungunya has a strong preference for human blood[3],[4] thereby enhancing disease transmission. The wild populations of Ae. albopictus are naturally infected with a maternally inherited endosymbiotic bacterium Wolbachia pipientis[5],[6]. Wolbachia was first reported in ovaries of mosquitos’ by Wright & Barr in 1980[7]. It is also implicated in parasitic manipulation of its host’s reproductive system[8]. It causes cytoplasmic incompatibility (CI) in its host population. This offers reproductive advantage to the infected females, to get the bacterium maternally transmitted thereby invading host populations. Wolbachia can be exploited for disease control viz it inhibits the growth of other microbes in its’ host[9] decreases the longevity of the host[10],[11]. With the reports of its antiviral effects being published, simultaneous releases of Wolbachia-infected mosquitoes were made to prevent the transmission of dengue viruses (DENV)[12]. However, the correct mechanism underlying these effects remains scantily understood. The ability of Wolbachia to invade a host population and create stable equilibrium is the means for the success of disease control that have a noteworthy impact on the transmission of the disease[13].

Wolbachia manifests vertical transmission i.e. from mother to offspring[14],[15],[18] and are expected to rapidly spread to fixation once a Wolbachia infection enters a population[16],[17]. In the wild, the high fidelity of maternal transmission of Wolbachia in mosquito species is explained by the fact that females carrying both wAlbA and wAlbB strains can successfully mate with males that are either singly or superinfected[18]. Previous studies reported that Wolbachia disappeared slowly in male mosquitoes when they were grow old[19],[20] suggesting that the female mosquitoes could represent the prevalence of Wolbachia in the field population because of their maternal transmission[21]. Regardless to non-infected females, infected females ensure reproductive advantage due to both CI and a fitness increase associated with Wolbachia infection[22]. The host endosymbiont interactions can be best implicated with the study of both prevalence and incidence of Wolbachia infection frequency. Transmission efficiencies and the phenotypic effects have an impact on the infection frequencies of the host[23]. Our study underpins further biological investigations of effects of native Wolbachia on Ae. albopictus.

In this study, we evaluated the Wolbachia infection frequency along with dengue transmission in wild Ae. albopictus collected from endemic districts of Odisha. To evaluate transovarial transmission of DENV across generation, larval pools of F0 and F7 generation were screened by Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR). With a view to accurately measure Wolbachia frequency, we tried a combination of PCR detection in field collected adults as well as in the laboratory-reared F1 to F7 progeny of these individuals collected from those areas. Our findings also recorded vertical or maternal transmission efficacies of Wolbachia mono- and superinfection across generations of field populations of Ae. albopictus (a competent vector for DENV transmission), first study of its kind in India.

Selection of study areas

Odisha extends from 17.49N latitude to 22.34N latitude and from 81.27E longitude to 87.29E longitude. The coastal plains of Odisha have been recognized for serious arboviral outbreaks as per the previous records of State Government, Health Department, Odisha. Considering the facts above, seven affected districts were surveyed for the study. The detailed coordinates of the studied districts are mentioned in [Table 1]. This study included few clusters from districts Puri, Khordha, Kendrapara, Jagatsinghpur, Balasore, Bhadrak and Ganjamas dengue affected areas and Regional Medical Research Centre campus in Bhubaneswar as the control cluster.

Table 1: Geographic coordinate information of collection sites for adult and larvae in Odisha, India

Click here to view

Sample collection

Larvae and pupae were collected from March 2014 to June 2016 (rainy season) following the protocol developed by WHO[24]. All indoor and outdoor water-holding receptacles were thoroughly screened in domestic, peridomestic and business centres. Various breeding spots with water, viz. discarded tires, earthen pots, cement tanks, tree holes and air coolers were located and searched. Larvae and pupae collected by Pasteur pipette, from each located habitat were transferred into aerated bottles, labelled and brought to the laboratory. Adults collected through mechanical aspirator were also brought to the laboratory.

The epidemiological data of dengue cases from the year 2014–2016 was collected from NVBDCP, Odisha, which was later used for comparative analysis study for Wolbachia infection in Ae. albopictus versus occurrence of dengue incidences.

Laboratory Identification

Adults collected were identified using standard keys[25]. They were separated according to sex and species.

Laboratory processing and DNA amplification

Genomic DNA of adult Aedes was extracted by phenol–chloroform method[26] and stored at -20°C for further use. Species-specific PCR using 18S ribosomal DNA was performed to check the quality of DNA extracted from each mosquito[27]. Initial screening for Wolbachia infection was done by PCR using wsp gene (81F and 691R)followed by subgroup screening using Wolbachia subgroup-specific primers, 328F/691R for wAlbA and 183F/691R for wAlbB[28]. The PCR reaction mixture comprised 0.25 mM dNTPs, 2.5 mM MgCl2, 0.2 mM primers and 1 U Taq DNA polymerase (Himedia) with the following thermal cycler conditions: 95°C for 5 min, followed by 35 cycles of 95°C for 1 min, 55°C for 1 min and 72°C for 1 min, followed by 72°C for 10 min.

Maternal transmission efficacy of Wolbachia

Field collected larvae and pupae were reared to adulthood for generation studies. Field-collected adult female mosquitoes from coastal districts of Odisha were randomly selected, fed blood, and then transferred to an individual cage for oviposition. After egg laying, female mosquito were screened by PCR to confirm the Wolbachia infection. Successive generations of mosquitoes starting from F1 to F7 were maintained under laboratory conditions (28±2°C and 70±10% RH). Random samples of adult females from each generation was taken and screened for Wolbachia infection. The immature stages collected from five dengue endemic districts namely Balasore, Bhadrak, Jagatsinghpur, Kendrapara and Ganjam were selected for measuring the maternal transmission efficacy taking a dengue free locale (Regional Medical Research Centre, Bhubaneswar) as a control site.

Viral RNA isolation and RT-PCR

To study the transovarial transmission of DENV across generation, pools of larvae were initially screened at F0 generation and later screened again at F7 generation. The extracted RNA were stored at -80°C till further processing. DENV positive RNA sample was taken as positive control for RT-PCR. DENV detection and typing in pools was carried out by RT-PCR kit (Qiagen, Hilden, Germany) targeting the C-prM region[29] with slight modifications in two steps[30] that involved the synthesis of viral cDNA from viral RNA by using the dengue complex-specific primers followed by nested PCR using serotype specific primers.

Data analysis

The percentage of variability between Wolbachia infections and non-infections in Ae. albopictus generations that were maintained in laboratory from populations of the aforementioned studied dengue prone districts (p<0.05) were tested using χ2 for trend (Extended Mantel-Haenszel). Linear regression analysis was performed to compare the trend of Wolbachia infections in Ae. albopictus against the incident of dengue cases in five of our studied clusters. Further Pearson correlation coefficient was calculated at p=0.05 to estimate the level of relation among the two.

Non-parametric statistical Kruskal Wallis one-way ANOVA was used to assess the statistical differences for Wolbachia infection across generations in the affected areas, and p values < 0.05 were considered statistically significant. Pair-wise comparison of mean infection for Wolbachia was done using the post-hoc test based on two-tailed t test assuming equal variances. All the calculations were performed with the statistical package of Microsoft XLSTAT 2007.

  Results 

On initial screening for the vectors in the studied districts it was found that Ae. albopictus is prevalent in all the districts and it outnumbered Ae. aegypti. Further it was found that all the studied districts have very high number of dengue cases [Figure 1]. This prevalence of Ae. albopictus along with occurrence of dengue cases led to the further study on Ae. albopictus as the vector of importance. The epidemiological data of dengue cases (2014–2016) was used for comparative analysis study for Wolbachia infection in Ae. albopictus versus occurrence of dengue incidences [Figure 2].

Figure 1: (a) Map of India highlighting the location of Odisha state. (b) Map of Odisha showing the study areas (coastal belt) (c) Composition of the mosquito population in percentage collected from the study areas.

Click here to view

Figure 2: Comparative distribution of Ae. albopictus and Ae. aegypti in the studied districts along with the incidences of cumulative dengue cases for the year 2014–2016.

Click here to view

Sex related Wolbachia infection frequency of Ae. albopictus population

Out of 1295 Aedes sampled from 27 clusters, 323 Ae. albopictus were screened for Wolbachia infection using wsp universal and later using, wAlbA and wAlbB subgroup specific gene primers. Samples screened consisted of 71 males and 252 females of Ae. albopictus wherein the overall infection was found out to be 87.3%, among which 78.6% were found to be superinfected with both wAlbA and wAlbB supergroups and only 8.7 % were found to be infected with wAlbB. The percentage of males and females among the infected individuals were 18.6% and 68.7% respectively [Figure 3]. Surprisingly, none of the individuals were found to be infected with wAlbA.

Figure 3: Showing male and female Wolbachia infection and non-infection rate in field collected Ae. albopictus.

Click here to view

Distribution of Wolbachia in Ae. albopictus population in Odisha

Wolbachia was detected among all the Ae. albopictus population from the 27 clusters of seven affected districts in the coastal Odisha, India. The detailed infection frequency of the samples recorded district wise is listed in [Table 2]. The percentage of Wolbachia AB superinfection ranged from 72.4% in Balasore to 93.1% in Kendrapara. The lowest and highest Wolbachia B monoinfection percentages are 4.2% in Kendrapara and 15.2% in Balasore. The overall infection increased gradually across Balasore to Kendrapara ranging from 87.6% to 97.3%. Variable infection prevalence (40-100%) was observed among localities. The two extreme values (40 and 100) have been observed only in few populations (Soro of Balasore displayed lower extreme value of 40% whereas Khaira of Balasore, Biridi of Jagatsinghpur, Pattamundai of Kendrapara and Kodolo of Ganjam displayed the extreme value of 100%). All other localities have infection prevalence ranging between 46% and 99%.

Table 2: Wolbachia infected Ae. albopictus collected from seven dengue endemic districts of Odisha, India.

Click here to view

Maternal transmission efficacy of Wolbachia

The overall Wolbachia infection in laboratory reared field collected Ae. albopictus across F1 to F7 from Balasore and Bhadrak districts decreased from 92.1% and 97.2% to 85.5% and 87.7% respectively. An increase in Wolbachia infection in the laboratory reared Ae. albopictus was observed from populations of Jagatsinghpur, Kendrapara and Ganjam districts. This generated a trend in the pattern of maternal transmission of Wolbachia infection status across generations from the studied areas. The Wolbachia infection status from the control site had shown a trend similar to that of populations from Jagatsinghpur, Kendrapara and Ganjam districts. In Bhadrak, the percentage of Wolbachia superinfection AB decreased from 84.2% to 62.6% whereas monoinfection increased from 7.8% to 22.9% across F1 to F7. The reverse was observed for Kendrapara where Wolbachia superinfection AB increased from 87.1% to 99.2% whereas monoinfection B decreased from 6.5% to 4.02% across F1 to F7. None of the generations witnessed Wolbachia A strain monoinfection. The pattern of Wolbachia infection (B and AB) in percentage in Ae. albopictus of dengue endemic population clusters and control site in successive generations is shown in [Figure 4].

Figure 4: Showing Wolbachia (B and AB) infection % in female Ae. albopictus in successive generations in the studied clusters.

Click here to view

Incidence of transovarial transmission for dengue virus

Transovarial transmission was confirmed as evident from the larval pools of F0 and F7 [Table 3].

Data analysis

χ2 test for trend or Cochran-Armitage test for trend revealed a significant variation between Wolbachia infections and non-infections in Ae. albopictus generations that were maintained in laboratory from populations of the aforementioned studied dengue prone districts ( p<0.05). However no significant variation was observed between Wolbachia infected and non-infected populations in generations maintained from dengue free locale (p>0.05), though the laboratory conditions maintained were the same for all sets of populations collected [Table 4]. All the areas showed an insignificant p value (p>0.05) on comparing Wolbachia B monoinfection with Wolbachia AB superinfection.

Table 4: χ2 for trend for mean of number of Wolbachia infected samples and Wolbachia non-infected samples

Click here to view

Linear regression analysis for the pattern of overall Wolbachia infection against the positive dengue cases in the studied clusters, revealed the involvement of a strong negative correlation (Pearson correlation coefficient = -0.9469, p=0.05) implying that the overall Wolbachia in fection in laboratory reared Ae. albopictus reared in laboratory across generations tend to decrease in the places with higher number of dengue cases [Figure 5].

Figure 5: Linear regression graph for overall Wolbachia infection against the positive dengue cases.

Click here to view

Furthermore, Kruskal Wallis one-way ANOVA revealed a significant variation (p<0.05) in mean infections among F1 to F7 generations for all sets of populations. On conducting a post-hoc ANOVA, i.e. Tukey’s HSD test, a significant difference was observed for mean Wolbachia infection across generation in all the studied clusters (absolute mean difference > calculated Tukey’s HSD value).

  Discussion 

The present study demonstrated widespread dissemination of Wolbachia in screened population of Aedes i.e. Ae. albopictus in regions of endemic dengue transmission. Our study revealed that Wolbachia is native to Ae. albopictus field populations in India and are predominantly superinfected with two strains (walbA and walbB) whereas the other vector Ae. aegypti is devoid of Wolbachia. The study has shown a decline in Wolbachia infection through maternal transmission in some population as well as an incline in Wolbachia infection through maternal transmission in other population. Besides maternal transmission efficacy of Wolbachia, reports do exist where geographical populations are strong predictors affecting Wolbachia infection rate and pattern[31].

It was found that all Wolbachia strains in Ae. aegypti showed ~100% maternal transmission thereby inducing high levels of CI[32],[33],[34]. The fitness costs along with the ability of two Wolbachia strains to occupy mosquito population were experimented in semi-field cage conditions. wMelPop strain invaded at a slower rate than wMel resulting in lower fecundity of infected females, (~60%) relative to non-infected wildtype and wMel infected mosquitoes[34]. On being released into the wild near Cairns in north Queensland in Australia, the wMel infected mosquitoes resulted in near fixation within a few months[12].

A great variability of Wolbachia infection was observed in our study populations of Ae. albopictus with a mean prevalence of 87.3%. This variability may be attributed to the differences in the genetic backgrounds of Wolbachia or differences in the host carrying the endobacterium. Several environmental parameters like temperature, diet and larval density have also been anticipated, besides host-Wolbachia interactions that contribute to the variability in infection[45],[46]. These parameters might have provided an advantage to the fitness of Ae. albopictus populations for persistence of Wolbachia whose infection might differ locally or with time depending on their colonization dynamics.

The phenotypic effects induced upon the host and consistent transmissions are the major factors on which infection dynamics of vertically transmitted symbiont rely. The stable maintenance of Wolbachia in the natural populations of Ae. albopictus may provide a direct benefit to the host. The frequent intraspecific horizontal transfers of Wolbachia in the natural population might be the cause of this phenomenon[35]. Since, Wolbachia are known to be mutualist in insects[36] they provide advantageous effects to their native hosts, such as an increase of host survival[37] fecundity under nutritional stress[38], or protection against pathogens[39],[40].

Studies on D. simulans have demonstrated that maternal transmission rates of Wolbachia may shift in response to environmental conditions that impact larval development[41]. In Balasore and Bhadrak, the overall infection pattern of Wolbachia in Ae. albopictus decreased gradually across F1 to F7 generations reared in laboratory whereas a reverse pattern was observed in Jagtsinghpur, Kendrapada and Ganjam. Another important fact observed from this study is the pattern of Wolbachia AB superinfection and B monoinfection. Superinfection AB followed a decreasing trend across generation in Bhadrak and Balasore whereas it followed an increasing trend across generation in Ganjam, Jagatsinghpur and Kendrapara. Monoinfection B followed a pattern of increased infection across generation in Balasore and Bhadrak while following a decreasing trend in Ganjam, Jagatsinghpur and Kendrapara. The pattern of Wolbachia infection in the control location also followed the same trend as Ganjam, Jagatsinghpur and Kendrapada. Despite the trends of infection pattern being affected by the strain type across generation, the overall infection increased across Balasore to Kendrapara. Along with the above observation, complete absence of Wolbachia A was observed in the present study. The inefficient transmission of Wolbachia A infection may be the reason behind its complete abolishment from the population[42].

The peculiarity in the pattern of Wolbachia infection across generation and clusters was further correlated with the occurrence of dengue positive cases in the studied districts. The transovarial transmission study of DENV was carried out with an objective to identify the probable role of DENV in the infection pattern of Wolbachia across generation. It was observed that Wolbachia superinfection AB is gradually decreasing and Wolbachia monoinfection B is increasing in places with high number of dengue cases i.e. Balasore, while vice versa is observed in places with low number of dengue cases i.e. Ganjam. This observation in particular may lead to be the predictive role of Wolbachia monoinfection B being the major strain inhibiting the replication of virus for the spread of dengue. The above observation has been previously supported by Bian et al, 2010[43]. The gradual decrease in Wolbachia superinfection AB and gradual increase in Wolbachia monoinfection B, affects the overall infection status of Ae. albopictus across the studied clusters. Balasore witnessing highest number of dengue cases comprised the lowest percentage of Wolbachia infection (87.6%) while Ganjam witnessing lowest number of dengue cases comprised the highest percentage of Wolbachia infection (97.3%).

DENV transmission is limited in Ae. aegypti transfected with Wolbachia from D. melanogaster [11] thereby crashing local populations of Ae. aegypti[44]. Several factors may be responsible for incompetence of native Wolbachia against DENV. But when the same Wolbachia strain (wAlbB) from Ae. albopictus is transfected to Ae aegypti, it limits the DENV transmission[22]. The native endosymbiont in Ae. albopictus is also unable to affect the replication of DENV[45]. This may further attribute to its reduced or insufficient density in Ae. albopictus[46] or the strains harboured in Ae albopictus are non-virulent[42]. However, there is a need to study the partiality of wAlbB to different hosts. In particular the host, endobacterium and virus association studies are needed to make a concrete conclusion.

Exploring symbiotic microorganisms in biocontrol management programs, have gained an upsurging interest in the recent past[34],[47],[48],[49]. Our purpose of considering dengue prone and dengue control site was to assess the infection status of Wolbachia in laboratory reared Ae. albopictus along with the vision of its transmission pattern in subsequent generations along with screening for DENV positive pools. As evident from the observations of the correlation study considering overall Wolbachia infection in Ae. albopictus and number of dengue positive cases in the studied clusters it could be summarized that places composing higher Wolbachia infection rate witnessed lower number of dengue cases and vice versa. This observation could mark a remarkable milestone in elucidating the relation of Wolbachia infection with dengue. Our control site did not harbour DENV (no clinical cases reported and none of the mosquito pools were positive) but had Wolbachia which increased at a very slow rate in the following generations. Along with these, further studies at molecular level can help in arriving at a robust conclusion in the near future.

There has been a substantial effort in demonstrating that Wolbachia-inducing CI can be used as a tool for vector population control[50]. High CI-inducing wAlbB strain from Ae. albopictus can be transfected to Ae. aegypti to assess modification and rescue capabilities of different strain combinations towards a bidirectional CI-based biological control strategy. It is anticipated that the spread of Wolbachia in a given vector population can ultimately lead to a high prevalence of infection, along with greater frequencies of producing viable progenies by infected adults. This can be especially useful in the development of a gene-drive like system where the wild-type competent vector populations are replaced with obstinate populations. The employment of these techniques could thus make Wolbachia a useful biological weapon to curb the menace of dengue in Odisha. Though there was a variance in the trend for the Wolbachia infection frequency in areas covered under our study, longer duration with large sampling time points are required for a conclusive remark.

In an attempt to modify the vector competence of Ae. albopictus populations, the capacity of Wolbachia for population invasion may be harnessed to push the genes for blocking parasite transmission into natural populations of arthropod disease vectors[13],[41],[42] . The success of Wolbachia as a gene-driving mechanism is critically dependent on the efficiency of its maternal transmission under field conditions. This parameter has only been measured in Drosophila[16] and no data are available for other species except for those of Ae. aegypti transfected with wMel from Drosophila[17]. In addition, the transmission efficiencies of Wolbachia infections under field conditions have also not been well studied.

This study is first to report on transmission efficacy of naturally occurring Wolbachia in successive generations of Ae. albopictus and its correlation with dengue cases in clusters of Odisha, India. The study also determined the prevalence and effect of naturally occurring Wolbachia in Ae. albopictus and its utility in dengue control strategy. Studying the transmission trend of Wolbachia along with transovarial transmission of DENV. might be an indictive towards the interplay of Wolbachia infection in presence/ absence of DENV. The reduction in Wolbachia infection frequency may be attributed by several environmental factors with the probability of being the cause for endemicity of dengue in the studied areas.

  Acknowledgements 

This work is funded by Lady Tata Memorial Trust, Mumbai, India and Indian Council of Medical Research, New Delhi.

Conflict of interest: None

 

  References 
1.Tsetsarkin KA, Weaver SC. Sequential adaptive mutations enhance efficient vector switching by chikungunya virus and its epidemic emergence. PLoSPathog 2011; 7(12): e1002412.  
    2.Tsetsarkin KA, Chen R, Sherman MB, Weaver SC. Chikungunya virus: evolution and genetic determinants of emergence. CurrOpinVirol2011;1(4): 310-317.  
    3.Ponlawat A, Harrington LC. Blood feeding patterns of Aedes aegypti and Aedes albopictus in Thailand. J Med Entomol 2005; 42(5):844-9.  
    4.Delatte H, Desvars A, Bouetard A, Bord S, Gimonneau G, Vourc’h G, et al. Blood-feeding behavior of Aedes albopictus, a vector of chikungunya on La Reunion. Vector Borne Zoon Dis 2010; 10(3):249-58.  
    5.Sinkins SP, Braig HR, O’Neill SL. Wolbachia superinfection and the expression of cytoplasmic incompatibility. Proc Biol Sci 1995;261(1362): 325-30.  
    6.Kittayapong P, Baimai V, O’Neill SL. Field prevalence of Wolbachia in the mosquito vector Aedes albopictus. Am J Trop Med Hyg 2002; 66(1): 108-11.  
    7.Wright JD, Barr AR. The ultrastructure and symbiotic relationships of Wolbachia of mosquitoes of the Aedesscutellaris group. J Ultrastr Res 1980; 72(1): 52-64.  
    8.Werren JH, Baldo L, Clark ME. Wolbachia: master manipulators of invertebrate biology. Nat Rev Microbiol 2008; 6(10): 741-751.  
    9.Moreira LA, Iturbe-Ormaetxe I, Jeffery JA, Lu G, Pyke AT, Hedges LMet al. A Wolbachia symbiont in Aedes aegypti limits infection with dengue, Chikungunya, and Plasmodium. Cell 2009; 139(7): 1268-1278.  
    10.Rasgon JL, Styer LM, Scott TW. Wolbachia-induced mortality as a mechanism to modulate pathogen transmission by vector arthropods. J Med Entomol2003;40(2):125-32.  
    11.Yeap HL, Mee P, Walker T, Weeks AR, O’Neill SL, Johnson P et al. Dynamics of the “Popcorn” Wolbachia Infection in Outbred Aedes aegypti Informs Prospects for Mosquito Vector Control. Genetics 2011; 187(2): 583-595.  
    12.Hoffmann AA, Montgomery BL, Popovici J, Iturbe-Ormaetxe I, Johnson PH, Muzzi Fet al. Successful establishment of Wolbachia in Aedes populations to suppress dengue transmission. Nature 2011; 476(7361): 454-457.  
    13.Turelli M, Hoffmann AA. Microbe-induced cytoplasmic incompatibility as a mechanism for introducing transgenes into arthropod popuations. Insect Mol Biol 1999; 8(2): 243-255.  
    14.Bennett GM, Pantoja NA, O’Grady PM. Diversity and phylogenetic relationships of Wolbachia in Drosophila and other native Hawaiian insects. Fly2012; 6(4): 273-83.  
    15.Nikoh N, Tanaka K, Shibata F, Kondo N, Hizume M, Shimada M et al. Wolbachia genome integrated in an insect chromosome: evolution and fate of laterally transferred endosymbiont genes. Genome Res 2008; 18(2):272-280.  
    16.Turelli M, Hoffmann AA. Rapid spread of an inherited incompatibility factor in California Drosophila. Nature 1991; 353(6343): 440-442.  
    17.Turelli M, Hoffmann AA. Cytoplasmic incompatibility in Drosophila simulans: dynamics and parameter estimates from natural populations. Genetics 1995;140(4): 1319-1338.  
    18.Kittayapong P, Baisley KJ, Sharpe RG, Baimai V, O’Neill SL. Maternal transmission efficiency of Wolbachia superinfections in Aedes albopictus populations in Thailand. Am J Trop Med Hyg2001; 66(1): 103-107.  
    19.Tortosa P, Charlat S, Labbé P, Dehecq JS, Barré H, Weill M. Wolbachia age-sex-specific density in Aedes albopictus: a host evolutionary response to cytoplasmic incompatibility? PLoS One 2010; 5(3): e9700.  
    20.Mohanty I, Rath A, Swain SP, Pradhan N, Hazra RK. Wolbachia Population in Vectors and Non-vectors: A Sustainable Approach Towards Dengue Control. CurrMicrobiol2019 ;76(2): 133-143.  
    21.Zhang D, Zhan X, Wu X, Yang X, Liang G, Zheng Z et al. A field survey for Wolbachia and phage WO infections of Aedes albopictus in Guangzhou City, China.Parasitol Res2014; 113(1): 399-404.  
    22.Dobson SL, Rattanadechakul W, Marsland RJ. Fitness advantage and cytoplasmic incompatibility in Wolbachia single- and superinfected Aedes albopictus. Heredity 2004; 93(2): 135-142.  
    23.Narita S, Shimajiri Y, Nomura M. Strong cytoplasmic incompatibility and high vertical transmission rate can explain the high frequencies of Wolbachia infection in Japanese populations of Coliaseratepoliographus (Lepidoptera: Pieridae). Bull Entomol Res2009; 99(4): 385-391.  
    24.WHO (1975) Manual on Practical Entomology in Malaria Part- II, Methods Tech. Offset Publication13 World Health Organization, Geneva.  
    25.Barraud PJ (1934) The fauna of British India, including Ceylon and Burma pp 217-246 in Diptera, V. (Ed.), Family Culicidae, Tribe Megharini and Culicini. Taylor and Francis, London.  
    26.Coen E, Strachan T, Dover G. Dynamics of concerted evolution of ribosomal DNA and histone gene families in the melanogaster species subgroup of Drosophila. J Mol Bio 1982; 158(1): 17-35.  
    27.Das B, Swain S, Patra A, Das M, Tripathy H K, Mohapatra N,et al. Development and evaluation of a single-step multiplex PCR to differentiate the aquatic stages of morphologically similar Aedes (subgenus: Stegomyia) species. Trop Med Int Health 2012; 17(2): 235-243  
    28.Zhou W, Rousset F, O’ Neil SL. Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences. Proc R Soc Lond B 1998; 265(1395): 509-515.  
    29.Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol 1992; 30(3): 545-551.  
    30.Das B, Das M, Dwibedi B, Kar SK, Hazra RK. Molecular investigations of dengue virus during outbreaks in Orissa state, Eastern India from 2010 to 2011. Infect Genet Evol2013;16: 401-410.  
    31.Ahmed MZ, Araujo-Jnr EV, Welch JJ Kawahara AY. Wolbachia in butterflies and moths: geographic structure in infection frequency. Front Zool 2015; 12: 16.  
    32.Xi Z, Khoo CCH, Dobson SL. Wolbachia establishment and invasion in an Aedes aegypti laboratory population. Science 2005; 310(5746):326-28.  
    33.McMeniman CJ, Lane RV, Cass BN, Fong AW, Sidhu M, Wang YFet al. Stable introduction of a life-shortening Wolbachia infection into the mosquito Aedes aegypti.Science2009;323(5910): 141-144.  
    34.WalkerT, Johnson PH, Moreira LA, Iturbe-Ormaetxe I, Frentiu FD, McMeniman CJ et al. The wMelWolbachia strain blocks dengue and invades caged Aedes aegypti populations. Nature 2011;476(7361): 450-453.  
    35.Morrow JL, Frommer M, Royer JE, Shearman DCA, Riegler M. Wolbachia pseudogenes and low prevalence infections in tropical but not temperate Australian tephritid fruit flies: manifestations of lateral gene transfer and endosymbiont spill over? BMC EvolBiol 2015;15: 202.  
    36.Zug R, Hammerstein P. Bad guys turned nice? A critical assessment of Wolbachia mutualisms in arthropod hosts. Biol Rev CambPhilos Soc 2015; 90(1): 89-111.  
    37.Fry AJ, Rand DM. Wolbachia interactions that determine Drosophila melanogaster survival. Evolution 2002;56(10): 1976-1981.  
    38.Brownlie JC, Johnson KN. Symbiont-mediated protection in insect hosts. Trends Microbiol 2009;17(8): 348-354.  
    39.Hedges LM, Brownlie JC, O’Neill SL, Johnson KN. Wolbachia and virus protection in insects. Science 2008; 322(5902): 702.  
    40.Osborne SE, Leong YS, O’Neill SL, Johnson KN. Variation in antiviral protection mediated by different Wolbachia strains in Drosophila simulans. PLOS Pathog2009; 5(11): e1000656.  
    41.Hoffmann AA, Turelli M. Cytoplasmic incompatibility in insects pp 42-80 O’Neill S.L, Hoffmann, A.A., Werren, J.H., (eds) Influential Passengers. Oxford University Press New York.  
    42.Sinkins SP, Braig HR, O’Neill SL. Wolbachia pipientis: bacterial density and unidirectional cytoplasmic incompatibility between infected populations of Aedes albopictus. Experiment Parasitol 1995;81(3): 284-291.