First report on the presence of natural Wolbachia population from major malarial vector mosquitoes Anopheles culicifacies s.l., and Anopheles stephensi from Tamil Nadu, India

Wolbachia is an alpha-proteobacteria present in several arthropods. The present study focussed on the identification of Wolbachia in wild malarial vector mosquitoes. This was achieved by molecular identification of Wolbachia from collected mosquitoes. A total of four hundred and eight seven mosquito samples were collected. Morphometric and molecular analysis revealed that they belong to Anopheles culicifacies s.l., (48.25%) and Anopheles stephensi (51.75%). The presence of Wolbachia was identified using 16S rRNA, wsp and FtsZ genes, where nested PCR of 16S rRNA alone was successful and then sequenced. Only seven mosquitoes (1.4%) were positive for Wolbachia. In silico and restriction digestion of 16S rRNA gene product using RsaI enzyme showed that the identified Wolbachia belongs to supergroup B. The prevalence rate of natural Wolbachia was lesser in native malarial vector An. culicifacies s.l. and An. stephensi was about 1.7% and 1.2%, respectively. This is the first report on the presence of Wolbachia in Anopheles culicifacies s.l. and Anopheles stephensi.


Introduction
Wolbachia is an intracellular alpha-proteobacteria found in a wide range of arthropods. It was first discovered by Hertig and Wolbach in 1924 considered to be the abundant endosymbiont found in invertebrates (Hertig & Wolbach 1924) and cause reproductive abnormalities (cytoplasmic incompatibility, male killing, parthenogenesis and feminization) in the host (Werren 1997, Werren et al 2008. This endosymbiotic proteobacteria naturally infect 65% of insect species (Hilgenboecker et al 2008, Werren 1997, Werren et al 1995a including the family

Mosquito collection and taxonomy
Mosquito samples were collected from the 5 different locations along the foothills of the Western Ghats, Southern India (Fig.1). Adults were collected using nets and aspirators.
Mosquitoes were identified initially by taxonomic keys (Christophers 1933, Das et al 1990 and later verified by DNA barcoding.

DNA extraction and species identification
DNA isolation from individual mosquito was carried out using the Qiagen Blood and Tissue kit (Qiagen) with slight modification. The initial lysis step post homogenization in PBS was carried-out with proteinase K and lysis buffer at 56°C for 3 hr. Genomic DNA extracted was subjected to COI (cytochrome C oxidase subunit I) gene amplification using primers reported by Folmer and colleagues (1994) for identification of the mosquito species.

Wolbachia detection by PCR
For Wolbachia detection, three different sets of primers targeting conserved genes namely 16S rRNA gene (Werren & Windsor 2000), Wolbachia surface protein (wsp) gene (Zhou et al 1998) and FtsZ cell cycle gene (Werren & Jaenike 1995) were used for screening. In addition, for low infection detection, a nested PCR using internal primers targeting 412 bp of 16S rRNA gene was used (Shaw et al 2016). Multilocus strain typing (MLST) of Wolbachia was done by targeting five conserved genes gatB, coxA, hcpA, ftsZ and fbpA as described earlier (Baldo et al 2006). The primer details are given in Supplementary Table 1.

Molecular phylogenetic studies
The Wolbachia positive samples were further sequenced. The sequencing was carried out from the PCR products of 16S rRNA (nested PCR) and COI from Wolbachia and Anopheles respectively and, Sanger sequenced (Barcode Biosciences Pvt. Ltd, Bangalore). The contig assembly was done using MEGA7. The assembled sequences were submitted to GenBank, ENA sequences were collected from GenBank to clarify the interspecies relationship. The phylogenetic tree was constructed by maximum likelihood (ML) analysis in MEGA7 software.
The tree inference options were set as follows: Heuristic Method Nearest-Neighbor-Interchange (NNI) with the very strong branch swap filter with 1000 bootstrap replicates, gaps were treated as missing. The number of restriction sites for RsaI was studied using multiple sequence alignment (MSA, Clustal Omega) to find out the supergroups of Wolbachia strains reported from this study.

Results:
Mosquito samples were collected from five different study sites (Fig.1) Table 2). MSA of Wolbachia strains reported the presence of four restriction sites (GTAC) for RsaI in agarose gel electrophoresis (data not shown) and in silico (Fig.2), indicating the isolates belong to supergroup B.
The phylogenetic analysis showed that all the Wolbachia strains reported from this study (Table 3) were grouped under Supergroup B and non-monophyletic (Fig.3). The Wolbachia isolates reported from this study showed high sequence similarity within (99.05% to 99.76%) and also with the reference sequences used in the study. The isolate TS3 (MN268749) formed a separate clade (Clade I) within supergroup B with three other strains reported from Drosophila simulans (NC021084.1) and Drosophila mauritiana (NZ_CP034334 and NZ_CP034335) supported with 65% bootstrap value (Fig.3). It also shares 98.55% sequence similarity with all the three reference sequences (Supplementary Table 2) and diverse (0.005±0.005; Supplementary  Table 3). The genetic diversity within group of supergroup B was less (0.005±0.003) and there was no significant diversity observed within supergroup A (Supplementary Table 3). The genetic diversity between supergroup A and supergroup B was 0.02±0.01 (Supplementary Table 4a).

Discussion:
The prevalence of Wolbachia has been reported in several arthropods including the orders Coleoptera, Diptera, Hemiptera, Homoptera, Hymenoptera, Lepidoptera andOrthoptera (de Oliveira et al 2015, Werren et al 1995a). Among the Diptera, Wolbachia is been reported in several Culicidae including Aedes, Culex andCoquillettidia (de Oliveira et al 2015, Ricci et al 2002). Novel Wolbachia infection in Anopheles species was least reported (de Oliveira et al 2015, Kittayapong et al 2000, Ricci et al 2002. In this study, we report the occurrence of natural markers to study either genome-wide divergence rate or strain identification. However in our study, MLST amplification was unsuccessful and, it might be due to low infection density or primer sequence divergence or both. Werren and colleagues (1995b) have reported 16S rRNA can be used to distinguish between A and B supergroup due to the absence of RsaI restriction site in Wolbachia belonging to group A. Interestingly, Pourali and colleagues (2009) have shown the presence of RsaI restriction site in supergroup A; however they have shown there was more RsaI site in B than A.
We have performed in silico RsaI restriction site search in 16S rRNA gene on reported Wolbachia supergroup A (EU096232, NC_002978.6, NC_012416, KP089991), supergroup B (NC_021984, CAGB01000162, MH967031) and our isolates; supergroup A has two restriction sites for all strains except KP089991 (Wolbachia from An. coluzzii (Buck et al 2016)) that has three restriction sites, which is possible due to recombination; our isolates and supergroup B shows four restriction sites (Fig.2).
The 16S rDNA phylogeny shows all the Wolbachia isolates from this study belongs to  Table 3) and clades of supergroup B was less (Supplementary Table 4b). This shows that the nucleotide substitution or recombination was minimum in 16S rRNA gene.
Diversity of Anopheles mosquitoes and Wolbachia prevalence: The genus Anopheles belongs to the family Culicidae that comprises of 465 species further divided into seven subgenera. Anopheles is one among the subgenera consists of 182 species (Harbach 2004, Harbach 2013. The molecular phylogeny of Anopheles was limited to lower level classification respective to malarial vector and, morphologically defined groups found to be monophyletic (Harbach 2004). Similarly in our study, An. culicifacies and An.
stephensi was first identified morphologically; later identified by COI gene amplification and, observed to be monophyletic within their respective species.
To till date, the prevalence of Wolbachia has been reported in 20 species of wild Chrostek and Gerth (2019) pointed out that true symbiosis has to be established by demonstrating intercellular bacterial cells and intraovarian transmission. To till date, the available techniques are limited and thus addressing the above said factors in wild mosquitoes is quite a challenge, but the future might hold a better way to prove the above said factors in elucidating true symbiosis of Wolbachia in wild mosquitoes.

Conclusion:
The current study has shown Wolbachia for the first time in Anopheles mosquitoes namely An.
culicifacies and An. stephensi from Tamil Nadu, India. Nested 16S rRNA PCR amplification is 1 helpful in identification than wsp, FtsZ and MLST loci genes. The prevalence is lesser compared to other mosquitoes, which may be due to inhibition by native microbiota, host and Wolbachia interaction, and/or inhibition by endogenous gene product that result from LGT; these factors will be analyzed in our future research.    s  s  e  t  F  ,  V  a  u  t  r  i  n  D  ,  S  o  l  i  g  n  a  c  M  .  1  9  9  2  .  M  o  l  e  c  u  l  a  r  i  d  e  n  t  i  f  i  c  a  t  i  o  n  o  f  W  o  l  b  a  c  h  i  a  ,  t  h  e  a  g  e  n  t  o  f  c  y  t  o  p  l  a  s  m  i  c   i  n  c  o  m  p  a  t  i  b  i  l  i  t  y  i  n  D  r  o  s  o  p  h  i  l  a  s  i  m  u  l  a  n  s  ,  a  n  d  v  a  r  i  a  b  i  l  i  t  y  i  n  r  e  l  a  t  i  o  n  w  i  t  h  h  o  s  t  m  i  t  o  c  h  o  n  d  r  i  a  l  t  y  p  e  s  .   P  r  o  c  e  e  d  i  n  g  s  .  B  i  o  l  o  g  i  c  a  l  s  c  i  e  n  c  e  s   2  4  7  :  1  6  3  -8   S  a  w  a  s  d  i  c  h  a  i  S  ,  C  h  a  u  m  e  a  u  V  ,  D  a  h  T  ,  K  u  l  a  b  k  e  e  r  e  e  T  ,  K  a  j  e  e  c  h  i  w    The phylogeny was inferred from the nucleotide dataset of COI gene by using the ML method. The sequences from this study were represented as green diamond. The tree with the highest log likelihood (-1429.44) is shown. The analysis involved 19 nucleotide sequences.

Reference:
There were a total of 396 positions in the final dataset. Scale bar 0.05 represents nucleotides substitution per position.