Discussion
In the present study, field collected St . albopicta from different parts of the Kerala with distinct breeding sites were subjected to PCR amplification of WSP gene to determine the prevalence of this intracellular bacteria and further amplified using strain specific primers to confirm the strain. Also, selected amplified sequences will then subjected to phylogenetic analysis.
The study revealed higher percentage of Wolbachia infection; 93% of the 105 studied samples emphasizing the natural infection ofWolbachia in St . albopicta in Kerala, South India. The two Wolbachia groups found were genetically distant and there was no gene flow between wAlbA and wAlbB . RatherWolbachia in higher densities would cover the way for exploiting this association for controlling vector mosquitoes of important arboviral diseases. The study was aimed at identification ofWolbachia strains in field collected St. albopicta from different breeding sites in Kerala. A total of 105 Samples were collected from 3 different areas with different breeding sites, areca nut planation in Kasaragod (North Kerala), rubber and pineapple plantation in Kottayam (Central Kerala) and tyres and containers in Thiruvananthapuram (South Kerala). The purified products were subjected to PCR amplification (shown in Fig 3, 4, 5, 6, 7 and 8 ), usingWSP general primers (81F/ 691R). High prevalence and distribution of Wolbachia strains are currently being reported from different countries in Asian region including Korea and Malaysia (Park etal., 2016; Joanne et al., 2015). The importance of such baseline studies play a vital role, mainly due to the initiative on global campaign on eliminate dengue using Wolbachia as a biological control measure (Eliminatedengue.com, 2016). Similarly in India, studies in different states showed Wolbachia infection and more recently a focus on natural infection with genotyping of its strains in St. albopicta are reported (Park et al., 2016; Joanne et al., 2015). However, an earlier report by Pidiyar (2003) in Pune showed single infection with super group A forming a separate group within wAlbA strain.
The present study focused on identification of Wolbachia strains in St . albopicta from different breeding sites in Kerala. The findings of this study concur with other published results showing 93% positive Wolbachia infection. The A and B superinfection rate among positives sample was found to be 95%, and single strain infection of wAlbA and wAlbB was 5% and 3% respectively. Moreover, the negative or uninfected samples were recorded from Thiruvananthapuram (4 samples) where major breeding sources, mostly man made habitats. The samples may have been feeding from natural or synthetic bactericide containing food source causing theWolbachia in them to perish in the wild. Besides, negative samples may indicate maternal transmission leakage of Wolbachia(Park et al., 2016). Future studies should take this into consideration specifically studies focused on prevalence ofWolbachia in an area. In addition five samples among 31 positive samples from Thiruvananthapuram showed single infection withwAlbA strain; 16% within the study site samples. Similarly in Kottayam 9% of the samples were singly infected with wAlbBstrain. Generally single infection points to incomplete cytoplasmic incompatibility, Armbruster et al., 2003 conducted a study focused on 18 different populations and found 100% positive infection ofWolbachia , hence more extensive sampling has to be conducted within study sites before conclusions can be deciphered.
Phylogenetic analysis of Wolbachia strains based on the fast evolving WSP gene showed that the prevalent Wolbachiaisolate was closely related with Wolbachia strains reported from different geographical locations of India, such as Pune and Odisha (Daset al., 2014). Further analysis suggested that there was no difference between the strains observed from the field collected mosquitoes from different breeding sites, except for minor difference from two sites having single infection; in Thiruvananthapuram and Kottayam. The two strains belonging to group A and B, in St. albopicta ; wAlbA and wAlbB respectively, were found to be genetically distant (K2P=0.301) with no gene flow. There can be further evolutionary strain within the groups.
Furthermore, evolutionary theory predicts that, due to maternal inheritance, Wolbachia should evolve in a mutualistic fashion to increase the fitness of infected females (Turelli, 1994). Overall the effect of Wolbachia infection on insect fitness can be mutualistic, parasitic or neutral, or even a combination. As forSt. albopicta , a laboratory studies of the mosquitoWolbachia -infected females lived longer and had more offspring than their uninfected counterparts (Dobson et al., 2004, Dobsonet al. , 2002) but these benefits are offset by an increased rate of death in infected larvae (Islam and Dobson, 2006). In addition intraspecific transfer of Wolbachia strains into St. albopicta was demonstrated by Xi et al., (2006) and these strains as well as native strains in St. albopicta is known to modulate arboviral infection specifically CHIKV (Moussonet al., 2012, 2010; Xi et al., 2006). Further, fewSt. aegypti samples; 20 were subjected to PCR amplification
using WSP general primers (81 forward and 691 reverse), to determine if Wolbachia infection is present. But all the 20 samples including 8 males and 12 females were found to be uninfected with Wolbachia.