A survey of Wolbachia infection in brachyceran flies from Iran

Wolbachia is a maternally inherited intracellular bacterium that is considered to be the most plentiful endosymbiont found in arthropods. It reproductively manipulates its host to increase the chances of being transmitted to the insect progeny; and it is currently used as a means of suppressing disease vector populations or controlling vector-borne diseases. Studies of the dissemination and prevalence of Wolbachia among its arthropod hosts are important for its possible use as a biological control agent. The molecular identification of Wolbachia relies on different primers sets due to Wolbachia strain variation. Here, we screened for the presence of Wolbachia in a broad range of Brachycera fly species (Diptera), collected from different regions of Iran, using nine genetic markers (wsp, ftsZ, fbpA, gatB, CoxA, gltA, GroEL dnaA, and 16s rRNA), for detecting, assessing the sensitivity of primers for detection, and phylogeny of this bacterium. The overall incidence of Wolbachia among 22 species from six families was 27.3%. The most commonly positive fly species were Pollenia sp. and Hydrotaea armipes. However, the bacterium was not found in the most medically important flies or in potential human disease vectors, including Musca domestica, Sarcophaga spp., Calliphora vicinia, Lucilia sericata, and Chrysomya albiceps. The primer sets of 16s rRNA with 53.0% and gatB with 52.0% were the most sensitive primers for detecting Wolbachia. Blast search, phylogenetic, and MLST analysis of the different locus sequences of Wolbachia show that all the six distantly related fly species likely belonging to supergroup A. Our study showed some primer sets generated false negatives in many of the samples, emphasizing the importance of using different loci in detecting Wolbachia. The study provides the groundwork for future studies of a Wolbachia-based program for control of flies.


Introduction
Medically important flies belonging to the order Diptera, suborder Brachycera, and the families Sarcophagidae, Calliphoridae, Muscidae, and Fannidae are the vectors of many human and animal pathogens, causing significant nuisance and disease [1][2][3][4][5].These insects have a worldwide spread, especially in the tropical and subtropical regions, and they cause many health problems and damage to livestock each year in those areas [1,6].In human societies, the fly families Muscidae, Calliphoridae, Sarcophagidae, and Fannidae live in close contact with humans [1][2][3][4][5][6], with adult flies and larvae feeding on human and pet feces, decomposing organic matter and spoiling meat [1,7].Moreover, several fly species cause myiasis, which is caused by the hatching and larval growth of the Cyclorrhapha fly in the human or animal body [8][9][10].These flies have developed resistance to several different chemical insecticides, and the use of such insecticides can also contaminate the environment [2].An alternative strategy for controlling the fly population is biological control.Recently, significant progress in our understanding of Wolbachia population ecology and genetics has reinforced the idea of using Wolbachia as a new method in the biological control of medically, veterinary, and agriculturally important arthropods [11][12][13][14].Induction of cytoplasmic incompatibility (CI) by this endosymbiont is the most popular mode of reproductive alteration, in which females that are either uninfected or infected with a different strain of Wolbachia fail to develop viable eggs when cross with Wolbachia-infected males [15,16].Releasing male with Wolbachia suppresses vector population and hence vector-borne diseases [17].Hence, it is essential to find new Wolbachia strains in flies that provide complete incompatibility.
Wolbachia was first observed and isolated by Marshall Hertig and S. Burt Wolbach in the reproductive tissue of Culex pipiens mosquitoes [18]; it is an obligate intracellular gram-negative bacterium and is classified in class Alphaproteobacteria, order Rickettsiales [18,19].This bacterium is transmitted vertically from the female insect to the next generation through the cytoplasm of the egg and can also be horizontally transmitted to other species [20,21].Wolbachia is a widespread intracellular bacterial symbiont that has been isolated from various insect species, ticks, and nematodes [20][21][22].The Wolbachia genus has only one species, which has recently, based on gene sequence information, been classified into 21 major clades of Wolbachia known as 'supergroups' (A-F, H-Q, and S-W), with each group being classified into dozens of different subgroups, often based on analysis of the bacterial surface protein (wsp) [23][24][25][26][27][28][29].Supergroups A and B are known to exclusively infect arthropods.The presence of this bacterium in the insect reproductive tissue is the result of changes in the host reproductive system that include cytoplasmic incompatibility (CI), thelytokous parthenogenesis, male feminization, and male killing [3,[30][31][32][33].It is also reported that Wolbachia can play important roles in insect speciation and local adaptation [34].
It is known that different primer sets vary in their capability to detect Wolbachia in their hosts [35].Different molecular markers such as 16s rRNA, ftsZ and wsp have been widely used to detect and to study the phylogenetics of Wolbachia [28,36], however, several investigations have shown the false negative for ftsZ [21,37,38] or false positive results for 16s rRNA and wsp markers in discriminating between supergroups A and B which can lead to unreliable results in bacterial detection when these primer sets are used alone [39].Besides, detection of recombination both between and within Wolbachia genes [40] suggests that a single-locus approach to strain characterization may be misleading [41].
Wolbachia has previously been detected in some Brachyceran flies, for example in Hydrotea spinigena, Musca domestica, and Haematobia irritans irritans of Muscidae and Sarcophaga dux of Sarcophagidae family [3,38].In Iran, there are some reports regarding the detection of Wolbachia in limited specific arthropod groups, but, so far, no general surveys of Wolbachia distribution among arthropods have been conducted [42].For example, infection of Wolbachia has been detected in some species of Phlebotomus [22,[43][44][45][46], in cockroaches [47], and in some species of Diptera-in Culicidae [48,49] and in Coleoptera (Staphilinidae) [50,51].However, the presence of Wolbachia in different fly species has not yet been studied.In the present study using nine different genetic markers, we show the incidence of Wolbachia infection in medically important flies of the suborder Brachycera (and one species of Nematocera), originating from a vast geographical area of Iran.

Fly collection and identification
Adult fly specimens were collected from various field sites covering the northern, western, southeastern, and central regions of Iran, from 2016 to 2021.Samples were obtained directly using insect netting over plants or garden beds, or traps already designed by Akbarzadeh et al [52] using liver baits including chicken (Gallus gallus domesticus), fish (Oncorhynchus mykiss), and beef (Bos indicus) based on their local availability, from urban, semi-urban, and rural settings in 18 different districts (Fig 1).Physically large fly (more than 0.5 cm in length) https://doi.org/10.1371/journal.pone.0301274.g001specimens were kept dry in silica (large samples) whereas small specimens were preserved in 70% ethanol and stored at -20˚C until identification and DNA extraction.Specimens were identified according to the parameters of Crosskey and Lane [53], James [54], Zumpt [55] and Parchami-Araghi [56].For sarcophagid flies, male specimens were morphologically identified to species level, based on the comparative morphology of male genitalia, whereas females were recorded as Sarcophaga sp. because it is not possible to identify them to species level morphologically.
DNA extraction and PCR.Whole bodies of small flies were homogenized individually in squash buffer [57], whereas larger flies were dissected in 1X PBS, and leg/s were used for homogenization and DNA extraction.Although reproductive tissues harbor more Wolbachia, it was more complex to retrieve DNA from the tiny reproductive tissues of dried specimens preserved for long time than legs.Total genomic DNA was extracted using the Collins method and kept at -20˚C for further molecular investigations [57].In case of a lack of proper DNA sample (poor DNA quality and quantity), the Qiagen DNA extraction kit was used, following the protocols recommended by the manufacturer (Qiagen, Hilden, Germany).Briefly, each specimen was homogenized in a special lysate buffering condition followed by QIAamp spin procedure which allows binding of the DNA to the QIAamp membrane upon centrifugation.Then the bound DNA was washed in two centrifugation steps and the purified DNA was eluted from the QIAamp spin column using a buffered solution.The DNA was eluted in 50-100μl buffer (based on the specimen size) and subsequently stored at 4˚C.All samples were measured using a NanoDrop and quantified by spectrophotometer (Thermo Scientific™ Nano-Drop™ One, MA, USA) at 260nm and diluted to a final concentration of 10 to 50 ng genomic DNA per μl.Barcode region of mitochondrial COI gene amplification served as an internal control to monitor the efficiency of both the DNA extraction and amplification [58].
To test the sensitivity of different primer sets, flies were screened for the presence of Wolbachia using standard PCR against nine different loci.Initial screening started with the primers wsp81F and wsp691R (Table 1) to amplify 632 bp of partial sequence of the wsp gene of Wolbachia [20,59,60].Screening was followed by using other loci: ftsZ, fbpA, gatB, CoxA, gltA, GroEL, 16s rRNA, and dnaA, using the primers and thermal program shown in Tables 1 and 2. Control DNA samples were prepared using double distilled water (ddH 2 O) as negative and previously confirmed adult females of either the Culex pipens mosquito [48] or the Phlebotomus papatasi sand fly [43] as positive controls, infected respectively with the wPip or wPap strains of Wolbachia.PCR amplifications were performed in an automatic thermocycler (Eppendorf, Germany) in a total volume of 25 μl containing 2-5 μl (~0.5 μg) of genomic DNA, 12.5 μL of Taq DNA Polymerase 2x Master Mix RED, Ampliqon (Denmark), 1 μL of each primer (10 mM), supplemented with ddH 2 O. PCR products were electrophoresed on 1% agarose gels with a voltage of 85 v and time less than 30 minutes, and the size of each PCR product was estimated using a 100-base pair (bp) molecular marker (Sina-Clon, Iran), visualized under a UV transilluminator.Samples with expected PCR product sizes as indicated in Table 1 and lack of PCR products were considered as positive and negative to Wolbachia respectively.For samples that did not amplify, conventional PCRs were repeated.
To perform phylogenetic analyses and to confirm the PCR results and, thereby, the Wolbachia infection status, a subset of 13 PCR products were sequenced.The PCR products with a clean, sharp, and single band with no smears and the expected sizes (Table 1) were selected for sequencing.The positive PCR products of 16s rRNA (n = 4), gatB (n = 3), GroEL (n = 2), wsp (n = 1), gltA (n = 1), dnaA (n = 1), and CoxA (n = 1) genes were purified and sequenced bidirectionally by the Sanger analysis (Sinuhebiotech, Iran) using the same PCR primers used for PCR amplification.The sequences acquired in this study were edited and assembled using Chromas [61] software to construct consensus sequences by matching the forward and reverse sequences of the same PCR products to obtain an actual sequence which approximately occurs in the specimen genome.The sequence data were then analyzed using the NCBI Blast database (Nucleotide collection) [62].Wolbachia classification was performed based on the identity score of the obtained sequences with the available GenBank sequences through the BLAST algorithm and phylogenetic analysis.Moreover, the sequences were compared with the data from the Wolbachia MLST database (https://pubmlst.org/bigsdb?db=pubmlst_Wolbachia_seqdef).All the sequences obtained were submitted to GenBank database [63].The consensus high-confidence sequences were aligned with a subset of other corresponding Wolbachia sequences that were available in GenBank, using multiple-sequence alignments available in CLUSTAL Omega [64].

Phylogenetic analysis
For phylogenetic analysis, we combined the Wolbachia sequences obtained in this study with a subset of the representative sequences of Wolbachia from different supergroups, available in GenBank (Table 3).All the DNA sequences (S1 Fig) used for alignment were cut to obtain a consistent region.The data were aligned (S2 Fig) , and the Neighbor-Joining (NJ) algorithm distance analysis using the Kimura 2-parameter model [65] was employed to construct a phylogenetic tree with bootstrap analysis of 1000 replicates in MEGA 7 software [66].

Statistical analysis
The data obtained for sensitivity of different primer sets were analyzed with the aid of the oneway ANOVA test.All analyses were performed using the Graph Pad Prism 5.0 program, with a significance level of 5%.

Ethic statement
This study followed the guidelines of the institutional ethical committee (Tehran University of Medical Sciences, TUMS).The protocols were approved by TUMS ethical committee under registry IR.TUMS.SPH.REC.1398.052.

Results
In  1).The remaining species had less than a three percent frequency rate.
Out of 1202 brachyceran flies, a total of 838 fly specimens (70%), including 363 males (69.1% of total males) and 475 females (70.2% of total females), were screened for Wolbachia in the 22 species.The results showed that out of the 838 specimens screened, only 66 specimens (7.88%), belonging to six species: Fannia canicularis, Fannia sp., Hydrotaea armipes, Pollenia sp., Desmometopa varipalpis, and Delia platura, were positive for Wolbachia (Table 5).The other 772 (92.12%) screened specimens, belonging to the remaining 16 species, were negative for the Wolbachia genome using the nine different primer sets.Also, the six moth fly (sink fly) specimens were free of Wolbachia.
In this study nine pairs of primers were used, to test the sensitivity of different primer sets as well as to increase the chance of detecting different Wolbachia strains in the fly specimens: 16s rRNA, gatB, wsp, ftsZ, fbpA, CoxA, gltA, GroEL, and dnaA.PCR-amplified fragments were identical in length to the expected sizes (632 bp for wsp, 650-700 bp for ftsZ, 450-500 bp for GroEL, 450-500 bp for CoxA, 450-500 bp for fbpA, 535 bp for dnaA, 693 bp for gatB, 450-500 bp for gltA, and 450-500 bp for 16s rRNA) (Table 1).Of the different primer sets, the 16s rRNA, gatB, wsp, ftsZ, and dnaA primers in order were more sensitive in detecting Wolbachia infections than the other four primer sets, which were used for standard PCR.We found that, within the infected species, a mean of 53.0% specimens were positive for the 16s rRNA, 52.0% for gatB, 35.1% for wsp, 34.5% for the ftsZ, and 30% for the dnaA primer set (Fig 3 , Table 5).However, the differences between these five primer sets were not statistically significant (p>0.05,one-way ANOVA test).

Sequence and phylogenetic analysis
The Wolbachia infection was confirmed by sequencing a subset of PCR-positive samples, as the Blasted sequences had several hits to Wolbachia.The Blast search showed the sequences obtained in this study had 98.36-100% similarities to the closest GenBank sequences of 16s rRNA, gltA, wsp, dnaA, GroEL, gatB, and CoxA genes of Wolbachia strains (Table 7).
The sequences obtained in this study have been submitted to GenBank with the Accession Numbers (GenBank IDs: OR793855-OR793858 correspond to 16s rRNA gene of Delia platura, Hydrotaea armipes, Fannia canicularis, and Pollenia sp.respectively, and OR865320-OR865322 relate to gatB gene of Hydrotaea armipes, Pollenia sp., and Fannia canicularis respectively, OR865323 relates to wsp gene of Hydrotaea armipes, and OR865324-OR865328 correspond respectively to coxA, gltA, groEL (n = 2), and dnaA genes of Pollenia sp.. To confirm the Blast search results, phylogenetic analysis was performed using the sequences of Wolbachia 16s rRNA, wsp, and gatB genes obtained from this study, in combination with the 16s rRNA, wsp, and gatB representatives of various Wolbachia supergroups retrieved from the GenBank database.All the phylogenetic analysis inferred from the three genes showed that the Wolbachia strains infecting the Iranian Brachycera flies were clustered with the A supergroup.The phylogenetic tree inferred from the 16s rRNA gene is shown in  The 16s rRNA gene is the most important marker in identifying and characterizing new species or strains of bacteria and has been widely used in Wolbachia species determination and identification [76,84].This analysis showed that the Wolbachia strains infecting the Iranian Brachycera flies are associated with the strains of supergroup A and are quite distinct from the other 16 supergroups compared in this analysis.
Wolbachia strain MLST analysis showed that the Wolbachia strain detected in H. armipes matched to Wolbachia strains of supergroup A hosted by 15 different insect species across 13 different genera, mostly from Hymenoptera (ants) and Lepidoptera orders.Same analysis against the gatB sequences from H. armipes, Pollenia sp., and F. canicularis provided evidence for presence of similar Wolbachia strains of supergroup A in three ant species of Pheidole obtusospinosa, Azteca sp., and Wasmannia sp.all belonged to Formicidae (Hymenoptera).No Wolbachia MLST sequences with definite supergroup were available for the remaining (coxA, gltA, groEL, and dnaA) gene sequences.

Discussion
This is the first report on Wolbachia infection in Iranian Brachycera flies.Here, we used a standard PCR technique and showed that the infection rate in Iranian fly species is about 27.3%, which is consistent with previous reports estimating that at least 20% of all insect species are infected with Wolbachia [21,87].Those estimates resulted from numerous Wolbachia screens, using standard PCR techniques, in which several species were tested for infection.Another study, reporting much higher infection rates (76%) [37], used a 'long PCR' method that is more sensitive to low concentrations of Wolbachia molecules.A later report by Hilgenboecker et al ( 2008) estimated that strains of the genus Wolbachia are naturally present in 66% of all insect species worldwide [87], which is more than twice what we found in the Iranian Brachyceran fly species.However, in the systematic review by Ina ´cio da Silva et al on mosquitoes of the Order Diptera, Wolbachia was detected in only 30% of the mosquito species investigated [88], which agrees with our finding.Discrepancies between reported infection rates in insects are, mainly or partly, a function of the number of specimens screened for Wolbachia.The problem arises in studies using a distinct species, or an only a few individuals per species.If a single specimen is infected, the species is rightly categorized as infected.However, if only one, or a few, uninfected individuals are tested, this can result in this species being defined as uninfected.The problem increases when infection rates are low, as the likelihood of detecting infection in this species would clearly Evolutionary analysis was conducted on 1000 bootstrap replications [85], using the Neighbor-Joining method [86].Evolutionary analyses were conducted in MEGA7 [66].GenBank ID, host species, and supergroup are indicated against the strains.The bootstrap values are shown in percentages on the internal nodes.Rickettsia sp. was used as an outgroup.https://doi.org/10.1371/journal.pone.0301274.g004have been low if only an individual specimen had been examined.It has been suggested that studies in which more than 100 individuals per species are tested incline to be biased towards infected species [87].The sample size of some of the species screened in this study was high enough to detect Wolbachia; for example in M. domestica (475 specimens) and C. vicinia (180 specimens) more than 100 specimens were analysed, while L. sericata (n = 67), Sarcophaga sp.(n = 49), Ch. albiceps (n = 43) Muscina stabulans (n = 17), Ch. marginalis (n = 14), and Ch.megacephala (n = 13) numbered between 10-100 specimens.Although insect species differs in Wolbachia infection status and frequency across space and time, in this study, in most cases the specimens were collected from different geographical regions (Table 1).Therefore, the lack of Wolbachia detection in some species probably reflects the small sample sizes for these species, rather than the absence of Wolbachia.
In this study, we did not detect Wolbachia infection in certain species of the Muscidae family such as M. domestica, Mu. stabulans, and St. calcitrans.However, some species, such as M. domestica, had previously been found to be Wolbachia-positive by studies performed in Iran, Thailand, USA, Singapore, Denmark, and Brazil [3,[89][90][91], and Wolbachia-negative by others in different geographical regions [92], suggesting a variable infection rate and/or limitations of the Wolbachia detection methods employed.In this study, we were not able to study connection between the time of collection of samples because all the fly specimens were collected in warm seasons (spring or summer) when flies are active, however, no obvious connection were observed between Wolbachia infection and geographic origin.
All the Wolbachia strains detected in the Iranian flies belong to supergroup A, which, together with supergroup B, is the most common supergroup found in arthropods [80].Interestingly, the 16s rRNA gene sequences of Wolbachia strains were found in four diverse species D. platura, H. armipes, F. canicularis, and Pollenia sp.(except for one DNA singleton mutation) and the gatB gene sequences in three species H. armipes, Pollenia sp., and F. canicularis were identical.The presence of a single supergroup in different fly species suggests a pattern of horizontal transmission.Supergroup A has been detected in several different dipteran species, including members of the Muscidae family: Hydrotea spinigena [3], Hydrotea aenescens [93], Musca autumnalis [94], Haematobia irritans irritans [38,92,95]; members of the Calliphoridae family: Protocalliphora sialia and Pollenia rudis [96,97]; members of the Fannidae: Fannia canicularis, F. scalaris and F. pusio [93]; member of the Glossinidae: Glossina spp.[98,99], and a member of the Sarcophagidae family: Sarcophaga dux [3].In addition to supergroup A, other Wolbachia supergroups have been reported in dipteran insects, for example supergroups A and B in sand flies [22]; A, B, C, D, and F in mosquitoes [48,88]; A and B in Sarcophaga spp.[3]; A, B, and F in Hippoboscidae [100]; and A and B in Culicoides spp.(Ceratopogonidae) [101].
Various molecular techniques, such as PCR with specific and/or degenerate primers, PCR with various markers such as multilocus sequencing Typing (MLST), quantitative PCR (qPCR), metabarcoding, Loop Mediated Isothermal Amplification (LAMP), and Next Generation Sequencing (NGS), have been used for Wolbachia detection: each method shows variable specificity and sensitivity and each has certain advantages and limitations that can influence the detection of different Wolbachia-derived molecules and/or a true Wolbachia infection [22,44,68,102,103].In this study, conventional PCR was used, with nine primer sets against nine different target genes of Wolbachia genome.Our results show that using various loci increases the chance of Wolbachia detection in the specimens.Also, using several target genes avoids the problem of integration of Wolbachia genes into the host's genome, and provides a true measure of Wolbachia infection [99].Consequently, we recommend that researchers should precisely select more than one method, because the evaluation of Wolbachia using a single method limits the inference of exact Wolbachia infection rates.However, compared with conventional PCR, qPCR and NGS techniques are more expensive.
Although Wolbachia infection has been reported in some species of Calliphoridae and Sarcophagidae, the results of this study, and available data in the literature [90,104], show that the species of these two families are not, or are only rarely, infected with Wolbachia.Here, we showed that only one species (Pollenia sp.), out of six screened species of Calliphoridae, was infected with Wolbachia, and none of the six screened species of Sarcophagidae were infected.In contrast, based on the results of this study and other searches, species of the Fannidae family are usually found to be infected with Wolbachia [93].
Results revealed Wolbachia infection in six species of Fannia canicularis, Fannia sp., Hydrotaea armipes, Pollenia sp., Desmometopa varipalpis, and Delia platura.Some of these species are potential vectors for several important animal and human pathogens like viruses and bacteria, intermediate hosts of an eye worm, or have been associated with myiasis in humans [105, 106 and references herein].However, further studies need to find any connection between the pathogens particularly the viruses and the Wolbachia strains found in these species.
Results of this study showed that most medically important brachyceran flies such as M. domestica were uninfected with Wolbachia.However, it is possible to establish stable Wolbachia infections in wild uninfected flies through trans-infection and thereby selectively infecting uninfected flies with specific Wolbachia strains and releasing infected males to the entire population.It could make flies sterile, and infertile, with reduced longevity leading to suppress population [107].Moreover, it seems the presence of Wolbachia in a target insect species does not hinder control efforts because the natural infection can be either removed by antibiotic treatment (46) or kept making co-infected host [108].Currently, three strains of Wolbachia (wAlbB, wMel and wMelPop) have been successfully injected into adults and pupae of Buffalo flies (Haematobia irritans exigua) and the closely-related horn flies (Haematobia irritans irritans).The bacterially-induced reduced fitness, such as decreased and delayed adult emergence, reduced longevity, and reduced fecundity in Wolbachia-infected flies, suggest the potential of the wMel or wMelPop strains for use in Wolbachia-based biocontrol programs for suppressing Buffalo fly populations [109].It is worth mentioning that high fitness costs for released males are detrimental to insect control with Wolbachia.Alam et al. [110] showed that Wolbachia caused strong CI in Wolbachia-free females of tsetse flies when mated with Wolbachia-infected males.Wolbachia wMel and wAlbA strains of supergroup A can induce strong CI in mosquitoes [111].The Wolbachia strains that were found in this study belong to supergroup A and could be used as biocontrol agents to reduce fly-borne diseases if the strains could induce strong CI in target species.Wolbachia strain can be transinfected to new host by embryo microinjection [112,113].The suppression of harmful fly populations through CI induced by the bacterium has been used to suppress the populations of a few medically important mosquitoes such Aedes aegypti in USA [114] and Aedes albopictus in China [115].

Conclusion
The results of this study showed that, on average, the rate of Wolbachia infection in the Iranian Brachycera flies is 27.3%.Also, gene sequence-based similarity analysis and phylogenetic study suggest that these flies harbor only Wolbachia supergroup A. The study's limitations include detection method, sample size for some species, and lack of complete spatial and temporal sampling.More comprehensive sampling over time and space helps increase diversity of brachyceran fly species, and better view of their infections to Wolbachia.Such data will be fundamental to developing and employing diverse strategies that use Wolbachia to suppress fly populations and/or to decrease the public health burden of different fly-borne pathogens.

Fig 4 .
Fig 4.  The 16s rRNA gene is the most important marker in identifying and characterizing new species or strains of bacteria and has been widely used in Wolbachia species determination and identification[76,84].This analysis showed that the Wolbachia strains infecting the Iranian Brachycera flies are associated with the strains of supergroup A and are quite distinct from the other 16 supergroups compared in this analysis.Wolbachia strain MLST analysis showed that the Wolbachia strain detected in H. armipes matched to Wolbachia strains of supergroup A hosted by 15 different insect species across 13 different genera, mostly from Hymenoptera (ants) and Lepidoptera orders.Same analysis against the gatB sequences from H. armipes, Pollenia sp., and F. canicularis provided evidence for presence of similar Wolbachia strains of supergroup A in three ant species of Pheidole obtusospinosa, Azteca sp., and Wasmannia sp.all belonged to Formicidae (Hymenoptera).No Wolbachia MLST sequences with definite supergroup were available for the remaining (coxA, gltA, groEL, and dnaA) gene sequences.

Fig 4 .
Fig 4. 16s rRNA gene phylogeny based on 300-400 bp of 31 Wolbachia strains from 17 supergroups, showing the position of the Wolbachia strains isolated from Hydrotaea armipes, Fannia canicularis, Pollenia sp. and Delia platura identified in this study (black circle).Evolutionary analysis was conducted on 1000 bootstrap replications[85], using the Neighbor-Joining method[86].Evolutionary analyses were conducted in MEGA7[66].GenBank ID, host species, and supergroup are indicated against the strains.The bootstrap values are shown in percentages on the internal nodes.Rickettsia sp. was used as an outgroup.

Table 5 . The sensitivity rate of different primer sets used for detection of Wolbachia in six brachyceran fly species, as determined by PCR assay against nine loci.
The rate of positivity for each primer set is shown in the relevant cell.