Molecular detection and identification of Wolbachia in three species of the genus Lutzomyia on the Colombian Caribbean coast

Background The hematophagous habits of insects belonging to the genus Lutzomyia (Diptera: Psychodidae), as well as their role as biological vectors of Leishmania species, make their presence an indication of infection risk. In the present study, seven species of Lutzomyia were identified and screened for natural infections with Wolbachia. Methods Collection of sand flies was done in an endemic focus of leishmaniasis on the Colombian Caribbean coast (Department of Sucre, Ovejas municipality). DNA collected from Lutzomyia species was evaluated with PCR for wsp gene amplification to screen for bacterial infection. Results Endosymbiotic Wolbachia was found in three species: Lutzomyia c. cayennensis, Lutzomyia dubitans and Lutzomyia evansi. Two Wolbachia strains (genotypes) were found in Lutzomyia spp. These genotypes were previously unknown in dipteran insects. The wLev strain was found in Lutzomyia dubitans, L. c. cayennensis and L. evansi and the wLcy strain was found only in L. c. cayennensis. Conclusions Genetic analysis indicated that the Wolbachia strains wLcy and wLev belong to the B Supergroup. This study provides evidence of infections of more than one strain of Wolbachia in L. c. cayennensis. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2031-x) contains supplementary material, which is available to authorized users.


Background
Los Montes De María is a region located on the Caribbean coast of Colombia which has been historically considered as a focus of several clinical forms of leishmaniasis [1]. In this region, the municipality of Ovejas (Department of Sucre) is of particular epidemiological interest due to the endemic character of leishmaniasis that is occurring in urban, peri-urban and rural areas there. The diversity of Lutzomyia spp. (vector insects) present in Ovejas is high and most of the species are implicated in leishmaniasis transmission [2,3].
In Latin America, vector control campaigns developed for leishmaniasis have mainly focused on chemical control using synthetic pesticides such as pyrethroids and chlorofluazuron [4]. The use of biological alternatives or their derivatives (bacteria, sex pheromones, entomopathogenic fungi and toxic plants) have also been considered, but few are used by vector control agencies in Colombia [2]. The medical importance of phlebotomine sand flies (particularly those of the Lutzomyia species) points to the need to consider new and more effective control measures, including some that have already been used for the control of other insects transmitting vector-borne diseases. Among such methods is transfection with bacteria of the genus Wolbachia [5].
Bacteria in the genus Wolbachia are intracellular microorganisms belonging to α-proteobacteria (Rickettsia), have maternal inheritance and are commonly found in insect intestines, salivary glands, ovaries and thoraces [6,7]. These bacteria may affect the reproductive capabilities of their hosts through diverse mechanisms, generating effects such as the death of male offspring as well as feminization and cytoplasmic incompatibility (CI) [8]. The pathogenic effect of some phenotypes of Wolbachia is now being evaluated on viruses such as Zika, dengue and chikungunya, as well as on Plasmodium [9,10].
The use of certain strains of Wolbachia is considered to be a promising alternative for decreasing the population density of Lutzomyia species and interfering with the multiplication of parasites and, as a result, Leishmania transmission [11][12][13]. Thus, initial research efforts have been directed toward screening the presence and circulation of Wolbachia strains in these and other vectors [14,15].
In the Americas, only five species of the genus Lutzomyia have been found to have low levels of Wolbachia infection, with strains belonging to the A and B Supergroups: Lu. cruciata in México, Lu. trapidoi and Lu. vespertilionis in Panamá and Lu. whitmani in Brazil. In Colombia, only Lu. shannoni was reported as positive for Wolbachia presence [16][17][18]. Supergroup A, also includes the Wolbachia species detected in Sergentomyia and Phlebotomus [19][20][21]. Currently, genes (16S rRNA, ftsZ, wsp gene) and techniques (Multilocus Sequence Typing technique MLST) are being used to validate the identification and phylogeny of strains of Wolbachia [22].
Partial wsp gene sequences exhibited informative characters useful in the identification of Wolbachia strains detected in Lutzomyia spp. The wsp gene has evolved at a much faster rate than any previously reported gene in Wolbachia [19][20][21][22]. Due to this reason, its nucleotide variability facilitates the division into Subgroups and Groups in a consistent manner [22]. The nucleotide variability of the wsp gene and the combination of different primers in PCR reactions is an approach that enables a fast assigning of unknown strains to a particular group, due to its specificity and lack of cross-reactions.
The aim of the present study was molecular detection and identification of the endosymbiont Wolbachia in natural populations of Lutzomyia species found in the municipality of Ovejas on the Colombian Caribbean coast, as well as an analysis of the gene sequence coding for the main surface protein of endosymbiotic Wolbachia (wsp).

Phlebotomine survey, processing and identification
Sand flies were collected in peri-urban environments in the municipality of Ovejas (75°13'E; 9°31'N; 277 m above sea level) during an entomological survey performed between February 21 and 27, 2013. This location is classified as a tropical dry forest ecosystem. Collection was done using CDC white light traps, located indoors and near homes, overnight, between 17:00 and 06:00 h. Shannon traps were also used for collection near homes. Additionally, diurnal collection using a mouth aspirator was done in the vicinity of nocturnal trapping sites. Collected specimens were kept dry in 1.5 ml vials and transported to the laboratory with dry ice. Once at the laboratory, they were kept at −20°C. The head and last three abdominal segments were removed from the specimens in order to perform taxonomic identification following the Young & Duncan classification system [23]. The thorax and remaining abdominal segments were stored at −20°C until DNA extraction.

Pool formation and DNA extraction
Following taxonomic identification, males and females were separated by species in groups with a variable number of individuals (6 to 10) in 1.5 ml Eppendorf tubes. The formation of groups in this way is justified by differences in the abundance of species in the study area, which complicates statistical interpretation regarding Wolbachia infection rates, but increases the success of molecular detection of bacteria found in natural populations of Lutzomyia in the conditions encountered. In addition, the samples were all collected at the same time.
DNA extraction was done according to the high salt concentration protocol [24]. The quality of DNA (260/ A280 ratio) and concentrations was analysed by Spectrophotometry (Thermo Scientific™ NanoDrop, Wilmington, USA). Additionally, a partial fragment of the cytochrome c oxidase subunit 1 (cox1) gene was amplified ( Fig. 1) and the spacer region (ITS) between the 23S and 16S ribosomal gene (Fig. 1), in order to evaluate the quality of DNA present, as well as the absence of PCR inhibitors.
PCR, cloning and DNA fragment sequencing for Wolbachia wsp gene Primers wsp81F (5'-TGG TCC AAT AAG TGA TGA AGA AAC-3') and wsp691R (5'-AAA AAT TAA ACG CTA CTC CA-3') were used to amplify a partial fragment (600 bp) of the gene coding for the main surface protein of endosymbiotic Wolbachia (wsp) (Fig. 1) [25]. The reaction mix used to detect Wolbachia included 80 ng of sample DNA according to the conditions previously described [26,27]. High fidelity Taq DNA Polymerase (Thermo Scientific, Wilmington, USA) was employed, as well as a conventional thermocycler (BIOMETRA). As a PCR positive control, DNA from ten Aedes (Stegomyia) aegypti larvae (kindly donated by the insectary of the PECET group) infected under laboratory conditions with a reference strain of Wolbachia (Supergroup A, strain wMel) were included (Fig. 1). As a PCR negative control, ultrapure water and DNA of Aedes (= Stegomyia) aegypti without Wolbachia was included (Fig. 1).
Wsp gene amplicons were ligated into JET1.2 vectors (Thermo Scientific) and then transformed into DH5α Escherichia coli. At least five independent clones were sequenced for each positive sample involved in detecting Wolbachia strains to generate consensus sequences for further analysis, as well as to mitigate the potential of a mixed infection in the pools [27]. Clones with the partial products of wsp were verified by sequencing in both directions using universal primers from Macrogen Inc., Korea. For each assay, a negative control (no DNA) as well as a positive control (control PCR product by the cloning kit) was included.

Identity of Wolbachia strains and their positions in phylogroups
The wsp gene obtained from Wolbachia were sent for sequencing (Macrogen, Korea) and the results were compared to previously identified sequences using the basic local alignment search tool (BLASTN) (https:// www.ncbi.nlm.nih.gov/) and edited with Bioedit v.7.2.5 [28] in order to obtain detected consensus sequence for every Lutzomyia species. This was also made with gene sequences of Wolbachia, which were available in the National Center for Biotechnology Information (NCBI) database and Wolbachia MLST database (http://pubmlst.org/wolbachia/). The nucleotide alignment reading framework reported by O'Neill (ftp://ftp.ebi.ac.uk/pub/ databases/embl/align/; Access Number DS42468) was considered, which suggests starting the analysis by translating the sequences to amino acids as a guide to align the DNA sequences of the wsp gene [27].
Alignments of sequences of wsp genes obtained in Lutzomyia and reported in GenBank (Additional file 1) were performed using the Clustal W and Muscle algorithms incorporated in MEGA 6. Verification of recombination events and the presence of chimeras was performed with RDP4 (Recombination Detection Program version 4) software, using all sequences of wsp obtained in this study in order to ensure the accuracy of nucleotide variability with respect to previously reported sequences in GenBank (Additional file 1). Patterns of genetic divergence (nucleotide composition, number of haplotypes, variable sites) and K2P genetic distances were evaluated using Bioedit v.7.2.5 and DNAsp 5.0 software.
All aligned sequences (= haplotypes) of wsp genes obtained in this study and reported in GenBank were exported using MEGA software. Description codes include the following abbreviations for species: Lev, Lutzomyia evansi; Lcy, Lutzomyia c. cayennensis and Luduv, Lutzomyia dubitans followed by the letters ov, which refer to the place where they were collected in Colombia (ov, municipality of Ovejas) and numbers corresponding to specimens with the same sequence.
Subsequently, the identities and relationships of the Wolbachia strains obtained in our study was determined by performing a phylogenetic inference analysis using the Bayesian method (number of generations = 1,000,000) with the MrBayes 3.0 software under the substitution model GTR + G (number of estimated parameters k = 139; Akaike information criterion (AIC) = 7807.8819); with jModeltest 2.1.4 software [29]; and Phyml 3.0 software [30]. All of the sequences obtained in the present study (KR907869-KR907874) were submitted to GenBank (Additional file 1).

PCR amplification of the HSP-70 N Leishmania gene in female groups
A PCR test was done to screen Leishmania infection in females of Lutzomyia. The primers used were HSP70-  (5'-GGA CGC CGG CAC GAT TKC T-3') and HSP70-R617 (5'-CGA AGA AGT CCG ATA CGA GGG A-3'), which amplify a 593 bp partial segment of the HSP-70 N gene (coding for Heat shock protein 70) [31]. PCR testing was done following the conditions and thermal profile described by Fraga et al. [31]. As a positive control, DNA from Leishmania panamensis (reference strain UA140) and Leishmania braziliensis (reference strain UA 2903), which was kindly provided by the PECET group of the Universidad de Antioquia, was included.

Wolbachia (wsp gene) infection
As expected, all PCR fragments of the wsp gene were approximately 600 bp in size, and were obtained from three species: Lu. dubitans, Lu. c. cayennensis and Lu. evansi. Among these three sand fly species, seven pools were positive for Wolbachia (Fig. 1, Table 1). Low relative infection rates were found in Lu. dubitans and Lu. c. cayennensis (3 positive pools; 8.5% for both species) ( Table 1). In Lu. evansi (1 positive pool; 2.8%), only one group was positive. It worth noting that Wolbachia was present in both sexes of Lutzomyia, particularly in Lu. dubitans (males, 5.7%; females, 2.8%) and Lu. c. cayennensis (males, 5.7%; females, 2.8%) ( Table 1), while in Lu. evansi Wolbachia was detected only in males. Lutzomyia rangeliana, Lu. trinidadensis, Lu. gomezi and Lu. atroclavata were all negative for Wolbachia. The positive control strain wMel successfully amplified in all PCR assays of the wsp gene for Wolbachia and the negative controls showed no PCR products.
Wolbachia identity based on comparisons with previous sequences and assignation of phylogroups using wsp gene sequences Based on DNA sequences, the presence and identity of Wolbachia in Lu. dubitans, Lu. evansi and Lu. c. cayennensis was determined. Nucleotide variability analysis based on fragments of 523 bp, showed only 15 variable sites among wsp sequences of Wolbachia obtained from Lutzomyia species (Fig. 2). In the Bayesian inference, 59 partial sequences of the Wolbachia wsp gene were included from strains related to arthropods, which are located in supergroups A and B, representing 24 groups with 57 previously detected strains from a wide number of insects (Additional file 1). Five haplotypes (HP) of the wsp gene (HP1 to HP5) were found in this study, which were described with short codes that allow the location of Wolbachia genotypes to be determined in relation to the species in which they were detected and that facilitate locating them in the tree created with all the sequences by Bayesian inference (Fig. 3).
The percentage divergence based on alignment, which includes a large number of available sequences, suggests that wsp gene sequences from Wolbachia present considerable intra-and inter-genic variation. This can be summarized as follows: between sequences of the same strain there is 0.4% variation; between strains of the same group there is 1-2.1% variation; between strains of different groups located in the same supergroup there is 1.9-2.7% variation; and between strains of different supergroups there is 13.4-25.5% variation ( Table 2). These percentages are consistent with the established ranges   Table 2 Values of genetic distances K2P and percent of sequence identity based on alignment of the wsp gene among strains of Wolbachia in the Leva, Con, Unif and Pern groups (Supergroup B) and some strains (WNiv and wWhi) of Supergroup A The superscripts indicate the percent similarity between the sequences and were determined only among some strains representing different levels of variation: within the same strain, between strains of the same group, between strains of different groups, among strains from different supergroups for the separation of strains and current assignment of Wolbachia groups [27]. Phylogenetic relationships estimated by Bayesian Inference analysis (including 449 bp in the final alignment) grouped the strains wLev and wLcy in a new group called "wLeva" (branch support of 0.97), located in the Supergroup B, and based on the robustness of clade posterior probability (0.71) with respect to Supergroup A (Fig. 3). The Leva group has a close phylogenetic relationship (0.98) with the Dei, Crag, Unif, and Prn groups (Fig. 3).

Discussion
This study reports a natural infection of endosymbiotic Wolbachia in natural populations of Lu. dubitans, Lu. c. cayennensis and Lu. evansi for the first time from the peri-urban environment of a leishmaniasis focus transmission on the Caribbean coast of Colombia.
Different studies with similar sample sizes (between 141 and 547 individuals) and grouping of individuals by species (10-100) have been developed, and determine infection rates [32]. We decided not to do calculations infection rates from DNA Lutzomyia groups because we consider that the prevalence of Wolbachia may be low and poorly estimated. For this reason, we only emphasize on infected species and characterization of genetic haplotypes.
Lutzomyia evansi and Lu. dubitans were found to be infected with Wolbachia by a strain named wLev, while Lu. c. cayennensis was infected with both strains of Wolbachia (wLcy and wLev). This is consistent with the presence of these insect species in a uniform ecological region (similar collection localities). Regarding Lu. c. cayennensis, there exists a possibility that Wolbachia infected this species more than once, which would explain the presence of two different strains. In some studies, some Wolbachia strains belonging to different subgroups or groups have been observed to infect the same host species [33].
The groupings based on Wolbachia wsp gene sequences included in this study were well supported and consistent with those previously reported for Supergroups A and B [34]. The Wolbachia strains wLev and wLcy reported in this study appear to be included as a group in Supergroup B, which is common in arthropods. Wolbachia strains wLev and wLcy show close relationships to the Prn, Con and Unif groups of Supergroup B [12]. Proximity to the group Prn is highlighted, because the wPrn strain was found in the host Ph. pernisiosus [12]. In contrast, strains wLcy and wLev located in this group do not appear to show a close relationship to Wolbachia strains in group Whi (Lu. whitmani and Lu. shannoni), which are detected in species of the subfamily Phlebotominae, even though they have a closely related host and a similar continental distribution [23]. Interestingly, some strains of Supergroup B (wPip, WBoL and wVul) have phenotypes associated with feminization of males, as well as mortality and cytoplasmic incompatibility [35]. Each of these reproductive alterations are advantageous to Wolbachia as they are correlated to an increase in infected females. This group of strategies is called reproductive parasitism [36].
The species Lu. evansi, Lu. dubitans and Lu. c. cayennensis were found positive for Wolbachia infection both by PCR and by sequencing of the wsp gene, that enables a fast assigning of unknown strains to a particular group [37]. These three species have a history of natural infection by species of Leishmania [1,3]. However, in this study, Leishmania was not detected in them. The prevalence of natural infections with Leishmania in sand flies is low. The process of simultaneous identification of Leishmania and Wolbachia can be complicated and needs to be initially standardized under laboratory conditions. Other researchers have reported differences in the sensitivity of different molecular markers and conventional tests (PCR, RFLP, isozyme patterns, hybridization with DNA probes) for the detection, diagnosis and identification of Leishmania species [37]; and they propose that exploring the possibility of viewing promastigotes by the dissection of digestive tracts and the implementation of more variants of PCR with genus-specific primers would be beneficial. Also it is necessary to indicate that the absence of Wolbachia and Leishmania in Lutzomyia species may be influenced by the sampling scheme (spot scouting) and the size of the analyzed sample, which reduces the possibility of detecting positive DNA of Leishmania. Identification of species of Leishmania from vectors has also been constrained by the need to isolate the parasite from one or more of the small proportion of sand flies that are normally found to be infected, ranging from 0.001 to 2.26% for Leishmania transmission [37].
It is desirable to advance our understanding of the biology and spread of Wolbachia bacteria in relation to Leishmania infection, given the fact that different studies show the impact of these bacteria in host-parasite interactions with a potential use in reducing the risk of infectious diseases caused by parasites and transmitted to humans by insects [38]. Many invertebrates are infected by Wolbachia, and the bacteria's success may be credited to the diverse phenotypes (mutualism or reductive parasitism) that result from infection. The persistence of the Wolbachia infections and phenotype estimation in natural populations of Lutzomyia in the municipality of Ovejas, are determinants to make strong correlations of the role of Wolbachia on the development of Leishmania. Another area of study, may include the introduction of Wolbachia in Lutzomyia evansi (main vector and abundant species in the Caribbean coast) and its interaction with Leishmania.
Additionally, it has been found that the presence of some strains of Wolbachia in mosquitoes can regulate the expression of genes involved in the immune responses, resulting in inhibition of the replication, multiplication, or resistance to the proliferation of viruses, parasites, and microfilariae [39]. In this sense, Wolbachia can also be visualized as a microorganism for biological control, that is based on the substitution of the microbiome of the vector by microorganisms that affect vector's pathogen load. Replacement microbiota may represent unmodified microbial species that normally do not colonize a particular vector species, or genetically engineered symbiotic bacteria [40]. A vector's microbiome can be altered either through the stable "conversion" of vector populations in the wild or by introducing the desirable microbiota through bait stations [40,41], which allows for a continuous modification of vector populations.

Conclusions
Our study represents a significant advance in the understanding of natural infections of Wolbachia in Lutzomyia.