Investigation of Wolbachia spp. and Spiroplasma spp. in Phlebotomus species by molecular methods

The aim of this study was to determine the presence of Wolbachia spp. and Spiroplasma spp. in natural populations of sand flies in Turkey by molecular methods. A total of 40 Phlebotomus specimens (19 female and 21 male) were used in this study. Genomic DNA from whole sand flies was isolated and Wolbachia spp. infection prevalence was investigated by using Wolbachia gene specific primer sets (wsp and GroEL). In addition, the DNA were analyzed for the presence of Spiroplasma infections utilizing bacterium specific 16 S rDNA PCR-amplification primers. Results of this analysis showed a Wolbachia infection prevalence of 70% (28/40). There was no sex-bias in infection prevalence, being 76% (16/21) and 63% (12/19) in males and females, respectively. Analysis of Spiroplasma infections indicated that 26% (5/19) of female sand flies were positive for infection, while none of the screened males (0/21) were positive. Of the 40 sand fly samples, only 2 were found to be positive for both Wolbachia spp. and Spiroplasma spp. The present study demonstrates the presence of Wolbachia and Spiroplasma infections in the natural sand fly populations in Turkey. This is the first report on Spiroplasma infection in the sand flies from Turkey.

Analysis for the presence of Spiroplasma using 16 S rDNA gene specific primers indicates that 26% (5/19) of female sand flies were positive for infection, while none of the male sand flies sampled (0/21) were positive. Of the 40 sand fly samples analyzed, only two were found to be positive (5%) for both Wolbachia spp. and Spiroplasma spp.
The amplified products were sequenced and the DNA sequence was analyzed by BLASTN analysis. The BLASTN results indicate that the sand fly Wolbachia wsp sequence shares the highest identity with the partial surface protein gene sequences (wsp) of Wolbachia endosymbiont of Bactrocera neohumeralis (100% identity: GenBank Accession Number KC668323) as well as the mosquito Armigeres subalbatus (100% identity: GenBank Accession Number KY523672). However, given the high conservation of this sequence, many different Wolbachia isolates showed 100% identity over the region analyzed. However, it appears that this strain of Wolbachia belongs to the Supergroup A. The sequences of three clones derived from 3 infected sand flies were identical.
The sequencing of the groEl gene of the clones was identical to the partial sequence derived from a Wolbachia endosymbiont of Drosophila melanogaster (100% identity: GenBank Accession Number AE017196) and D. simulans wHa (100% identity: GenBank Accession Number CP003884) based on BLASTN analysis. All clones had identical sequence. Given the high conservation of the groEL gene sequences, the sandfly Wolbachia sequence showed 100% identity with many other insect Wolbachia symbionts. However, many of the characterized Wolbachia strains that showed high similarity belonged to again Supergroup A.
With regards to Spiroplasma PCR-amplification products, the fragments were sequenced and the DNA sequence was subjected to BLAST analysis. The closest resulting match of the putative 16 S rDNA fragment was to the 16 S rRNA gene from an uncultured Spiroplasma sp. clone A9-24 (100% coverage, 100% identity: GenBank Accession Number KT983889.1).
Obtained sequence data were deposited to GenBank under the accession numbers as follows: MH393924 for wsp Wolbachia Phlebotomus, MH393925 for groEL Wolbachia Phlebotomus, and MH393926 for 16 S Spiroplasma Phlebotomus.
The high similarities of these gene products to many of the related sequences in the database make detailed phylogenetic analysis difficult and will require sequence analysis of additional genomic loci with higher levels of sequence variability in these bacteria.

Discussion
In the present study, we tested for the presence of Wolbachia in 40 sand flies using two independent genes, wsp and groEl. Our results showed that twenty-eight out of the 40 sand flies tested were positive for Wolbachia. In addition, 5 out of the 40 sand flies tested were positive for Spiroplasma based on Spiroplasma bacterium specific 16 S rDNA amplification. Interestingly, Spiroplasma infections were only identified in female sand flies suggesting a potential sex specific infection bias. Of the 40 sand fly samples tested, only two were found to be positive for both Wolbachia spp. and Spiroplasma spp.
In previous studies using similar PCR-based molecular methods, the prevalence of Wolbachia in both Old and New world sand fly species has been investigated. Ono et al. 13 has reported that 27% of sand flies (Lutzomyia spp. and Phlebotomus spp.) were infected with Wolbachia. Benlarbi and Ready 14 determined that in Phlebotomus perniciosus, 60.3% were positive for the B group wPrn strain of Wolbachia and in P. papatasi, 81.7% were positive for the A group wPap strain of Wolbachia. The A-group strain of Wolbachia (wPap) was also found in P. papatasi by PCR amplification by Parvizi et al. 15 . Matsumoto et al. 16 found Wolbachia sp. in pooled sand fly samples (P. perniciosus, P. mascittii, P. ariasi, Phlebotomus sp., Sergentomyia minuta) in Marseille, France. The presence of Wolbachia was detected in three of 20 sand fly species, including Lutzomyia trapidoi, in which it frequently co-occurred with Leishmania parasites 17 . Parvizi et al. 18,19 determined presence of Wolbachia pipientis in P. perfiliewi transcaucasicus, P. mongolensis and P. caucasicus. Bordbar et al. 20 found that 65.5% of Iranian sand flies were positive for Wolbachia based on wsp gene amplifications. Vivero et al. 21 detected Wolbachia in Lutzomyia species in Colombian Caribbean coast. Da Rocha et al. 22 were found that Wolbachia pipientis was positive 2.5% in Lutzomyia species. Li et al. 23 determined Wolbachia (8.6%) and Spiroplasma (2.3%) in Phlebotomus chinensis in Henan and Sichuan collections from China, respectively.
In the present study, the prevalence of Wolbachia spp. was found to be 70% in sand flies analyzed from Antalya province of Turkey along the Mediterranean. The rate of infection prevalence found in this study is similar to the results reported in Iranian sand flies by Benlarbi and Ready 14 and Bordbar et al. 20 . Observed differences in prevalence of Wolbachia spp. may be associated with variation in the geographical locations and the species of sand flies sampled 13 .
Work by Aksoy and Rio 24 and Rio et al. 25 describe the potential role of symbionts, including Wolbachia, as a novel tool for control of parasitic diseases. Wolbachia is responsible for inducing a number of reproductive modifications that enable its spread and maintenance in natural insect populations. The most common effect is cytoplasmic incompatibility 8,9,13 . In one study, it has been shown that a natural Wolbachia infection in sand flies causes cytoplasmic incompatibility, leading to embryonic mortality in them 18,20 . Wolbachia also causes induction of parthenogenesis in female artropods, thus insect populations can be negatively impacted upon infection with Wolbachia 8,18 . In addition, introduction of new Wolbachia infections into Drosophila as well as mosquitoes have been shown to induce the host immune system to make them resistant to pathogenic viruses and parasites [26][27][28] . For these reasons, it has been suggested that Wolbachia infections can prevent the transmission of parasites in infected insects. If Wolbachia infections in sand flies also confirm increased resistance to Leishmania parasites or to viruses, it can be desirable to incorporate this endosymbiont as a biocontrol agent against sand fly-transmitted diseases.
Much of the research on Spiroplasma has focused on Drosophila species. Drosophila melanogaster one of the most commonly used model organisms, naturally harbours Spiroplasma 2,3,29-31 . The prevalence of male killing Spiroplasma was found to vary between 0% and 17.7% in different Drosophila species 31 . In this study we determined Spiroplasma positivity rate as 26% in female sand flies analyzed from the southern coast of Turkey. None of the male sand flies were positive for Spiroplasma. This observation suggests that in sand flies Spiroplasma may employ a male killing drive strategy although additional prevalence and functional data are required to support this hypothesis.
The present study was performed to investigate the presence or absence of reproductive associated endosymbiotic bacteria in sand flies in Turkey. Molecular studies in Turkey have previously reported that Wolbachia infection was detected in Trissolcus species, Culex pipiens and Phlebotomus spp [32][33][34] . Further studies are needed to determine the extent and the potential biological significance of Wolbachia spp. and Spiroplasma spp. infections in sand flies in Turkey.

Materials and Methods
This project was carried out to investigate the prevalence of Wolbachia spp. and Spiroplasma spp. in Phlebotomus flies by using molecular methods. A total of 40 Phlebotomus individuals (19 females and 21 males) were used in this study. Sand flies were collected from Antalya, in south of Turkey (with an altitude of 20 m, 36° 13′-36° 34′ N latitudes and 32° 15′-32° 38′ E longitudes), which has general characteristics of Mediterranean climate in August 2014 (Fig. 1). Sand flies were caught and identified with the help of the relevant resources 33,35 . Genomic DNA was extracted from whole sand flies using the DNeasy Blood and Tissue Kit (Qiagen, Valencia CA) according to the manufacturer's instructions. The concentration of the extracted DNA was detected using a Nanodrop 2000 spectrophotometer and keep at −20 °C for further molecular experiments.
Genomic DNA integrity was tested by standard Polymerase Chain Reactions (PCR) amplification using primers against sand fly beta-tubulin (forward primer 5′ GCC TCC TGG TAT TGT TGG T 3′, reverse primer 5′ CTC ACG CAG CAG ATG TTC 3′, product size 490 bp). PCRs were performed in a total volume of 25 µl using 1 µl of DNA template and GoTaq Green Master Mix (Promega, Madison, WI, USA) following the manufacturer's instructions. Thermocycling conditions were as follows: 94°C for 3 minutes; 40 cycles of 94 °C for 30 seconds, 55 °C for for 30 seconds, and 72 °C for 30 seconds; and a final step at 72 °C for 10 minutes.
Samples were then tested for the presence of Wolbachia using primers for Wolbachia wsp (forward primer 5′ AGT TGA TGG TAT TAC CTA TAA G 3′ reverse primer 5′ TGA CTT CCG GAG TTA CAT CAT AAC 3′, product size 410 bp) and Wolbachia GroEL (forward primer 5′ TTT GAT CGC GGT TAT C 3′ and reverse primer 5′ AGA TCT TCC ATC TTG ATT CC 3′, product size 410 bp) genes. Nuclease free water was used as negative control and female tsetse fly DNA (Glossina morsitans morsitans) from the lab line maintained at Yale University insectarium was used as a positive control. PCR reactions were performed using the following conditions: denaturation at 94 °C for 3 minutes, followed by 38 cycles consisting of at 94 °C for 30 seconds, at 50 °C for 30 seconds, and at 72 °C for 30 seconds; and a final extension for 10 minutes at 72 °C.
In addition, the DNA samples were analyzed for the presence of Spiroplasma utilizing primers against 16S rDNA (forward primer 5′ GGG TGA GTA ACA CGT ATC T 3′ and reverse primer 5′ CCT TCC TCT AGC TTA CAC TA 3′, product size 1000 bp). Nuclease free water was used as negative control and female tsetse fly DNA (Glossina fuscipes fuscipes) obtained from the field in Uganda was used as a positive control. PCR reactions were performed using the following conditions: denaturation at 94 °C for 3 minutes, followed 38 cycles consisting of at 94 °C for 30 seconds, at 55 °C for 30 seconds, and at 72 °C for 1.30 seconds; and a final extension for 10 minutes at 72 °C.
The resulting PCR products were analyzed by agarose gel-electrophoresis on a 1% gel, stained with ethidium bromide and recorded using the Gel Doc EQ quantification analysis software (Bio-Rad, Image Lab Software Version 4.1).
The amplified DNA fragments of the correct size were excised from the agarose gel and extracted using the Monarch DNA Gel Extraction Kit (New England Biolabs, Inc.). The purified PCR products were ligated into pGEM-T Vector (Promega, Madison, WI) and recombinant plasmids were used to transform competent