Molecular characterization of Wolbachia infection in bed bugs (Cimex lectularius) collected from several localities in France

Wolbachia symbionts are maternally inherited intracellular bacteria that have been detected in numerous insects including bed bugs. The objective of this study, the first epidemiological study in Europe, was to screen Wolbachia infection among Cimex lectularius collected in the field, using PCR targeting the surface protein gene (wsp), and to compare obtained Wolbachia strains with those reported from laboratory colonies of C. lectularius as well as other Wolbachia groups. For this purpose, 284 bed bug specimens were caught and studied from eight different regions of France including the suburbs of Paris, Bouches-du-Rhône, Lot-et-Garonne, and five localities in Alpes-Maritimes. Among the samples, 166 were adults and the remaining 118 were considered nymphs. In all, 47 out of 118 nymphs (40%) and 61 out of 166 adults (37%) were found positive on wsp screening. Among the positive cases, 10 samples were selected randomly for sequencing. The sequences had 100% homology with wsp sequences belonging to the F-supergroup strains of Wolbachia. Therefore, we confirm the similarity of Wolbachia strains detected in this epidemiological study to Wolbachia spp. reported from laboratory colonies of C. lectularius.


Introduction
Bed bugs are blood-sucking insects and human ectoparasites, and have re-emerged over the last two decades worldwide. In economically advanced countries, they are a serious public health issue that still threaten many people. They may cause problems in housing facilities, public facilities, and residential complexes. They are therefore an important risk factor that cannot be neglected by the tourism industry, the housing industry, and public institutions.
Bed bug infestations have been reported to have physical and psychological effects in humans. Despite isolation of several pathogens, found in the bed bug body, they have not been confirmed as a vector of pathogens to humans [18]. Recently, Salazar et al. [32] and Leulmi et al. [15] reported the vectorial competence of C. lectularius for Trypanosoma cruzi and Bartonella quintana transmissions in laboratory experiments. In addition, bed bug infections by Burkholderia multivorans have been reported by Saenz et al. [28], who screened different bed bug populations in the United States.
A wide range of symbiotic microorganisms are associated with insect hosts that mainly reside in their guts, body cavities, or cells [4,13,19]. Some obligate symbionts with a mutualistic nature -like Buchnera in aphids and Wigglesworthia in tsetse -have been reported to contribute to host fitness [9,22], whereas some other symbionts such as Wolbachia are often parasitic causing negative effects in their hosts [24]. The latter seem to be obligate commensal symbionts and facultative parasites that are of importance due to their roles in forces driving the evolutionary transition into mutualism [8,10].
Wolbachia are maternally inherited rickettsia-like bacteria (Gram-negative alpha-proteobacteria), harbored by a wide range of arthropods and are mainly found in the host gonads [5,23,26,35]. These intracellular bacteria were first reported in 1924 in the reproductive tissues of Culex pipientis mosquitos [11]. Since this discovery, several papers have reported the presence of these bacteria in the host tissues of many different arthropod species including bed bugs. This presence and association of Wolbachia in hosts is mainly considered as a commensal or somewhat parasitic relationship. The Wolbachia infection often leads to host reproductive phenotypes, such as cytoplasmic incompatibility, feminization, male-killing, anti-virus properties, parthenogenesis, increased or decreased fitness, and sperm-egg incompatibility, which consequently results in unfertilized eggs [6,8,16,20,34,37]. In addition to the roles mentioned, Wolbachia have been shown to occupy bacteriocytes (specialized cells that constitute a pair of organs known as bacteriomes) and to be essential for bedbug growth and reproduction via provisioning of B vitamins [14,21]. Moreover, Wolbachia increase male bed bug fitness by increasing the egg laying rate of the female after mating. The bacterium also leads to an increase in female host fitness by increasing egg hatch and development success, increasing offspring body size, and reducing fitness costs (wounding and infection) associated with traumatic insemination [13].
Several Wolbachia strains have been reported from different arthropod species. They have been classified in eight major supergroups from A to H. Wolbachia species belonging to A, B, and E groups infect diverse arthropods; C and D infect nematodes; G infects spiders and H infects termites, whereas F infects arthropods and nematodes [3,21,25,27,[29][30][31]34]. Infections with Wolbachia species of F supergroup seem to be prevalent in the Cimicinae subfamily (Cimex and Oeciacus genera). The species appears to be monophyletic, suggesting that Wolbachia symbionts were first introduced to a primitive insect population and then diverged dependently, along with the insect hosts in this subfamily.
Despite this knowledge on the spread of Wolbachia infection among different arthropod hosts including bed bugs, the contribution rate of Wolbachia in field collected bed bug populations has not been studied sufficiently in Europe.
Therefore, the aim of the present study was to screen the prevalence of Wolbachia sp. that colonize naturally field collected populations of Cimex lectularius using a PCR-based survey, and to compare obtained Wolbachia strains with those that have been reported from laboratory colonized C. lectularius as well as other Wolbachia groups.

Materials and methods
Cimex lectularius specimens were collected from four different parts of France, including hotel and individual apartments from 2010 to 2012. They were caught from the suburbs of Paris (Saint-Ouen), Bouches-du-Rhône (Aix-en-Provence), Lot-et-Garonne (Agen), and Alpes-Maritimes (Nice, Cannes, Le Cannet, Beausoleil, and La Roquette-sur-Siagne) ( Fig. 1). All samples within infested structures were collected from a single room. All bed bugs (except the egg stage) were hand-removed by entomological tweezers during inspections, transferred immediately into the labeled microtubes containing ethanol 96%, and stored at À20°C for further molecular analysis.
DNA was extracted using Qiagen's DNeasy extraction kit (Valencia, CA, USA) according to the manufacturer's protocol. Moreover, the purity and concentration of extracted DNA was measured using a NanoDrop spectrophotometer (BioTek Synergy 2, USA). The samples were then screened for the presence of Wolbachia species. For this purpose, specific primers for wsp, 81F (F: 5 0 -TGG TCC AAT AAG TGA TGA AGA AAC-3 0 ) and wsp 691R (R: 5 0 -AAA AAT TAA ACG CTA CTC CA-3 0 ), were used for 600 bp fragment amplification of Wolbachia surface protein (wsp) by polymerase chain reaction [36]. A pair of positive (Wolbachia positive samples previously sequenced for confirmation) and negative (DNA free distilled water) controls were used in each PCR run. Following amplification, the samples were fractionated via horizontal submerged gel electrophoresis using 1.5% agarose gels with DNA size markers (Promega PCR markers), and obtained DNA fragments were visualized by ethidium bromide staining before sequencing. PCR products were sequenced in both directions directly, using the wsp 81F and wsp 691R primers used for DNA amplification. The sequences were then edited using Staden package software and compared to homologous sequences in GenBank thanks to the nucleotide Basic Local Alignment Search Tool (BLAST: www.ncbi.nlm.nih.gov/BLAST) in order to identify the samples at the species level. Strains were considered as identified at the species level when their sequence showed !99% homology with a sequence deposited in GenBank. Sequences were also aligned with BioEdit v7.0.0 software [12] for comparison.

Results
In the present study, the prospected samples were demonstrated to have suitable DNA quality using Nanodrop spectrometry and electrophoresis gel running. In total, 284 bed bug specimens were captured from eight sites: Aixen-Provence, Saint-Ouen, Nice, Beausoleil, Le Cannet, Cannes, La Roquette-sur-Siagne, and Agen. They were screened to detect the Wolbachia wsp gene. The largest number of samples was from Nice (96 samples) followed by Aixen-Provence (89 samples), and the lowest number of samples analyzed in this study was from La Roquette-sur-Siagne (4 samples). Among all samples, 166 were adults and the remaining samples (117) were considered nymphs (Fig. 2, Table 1).
Based on the results, 47 out of 118 nymphs (40.2%) and 61 out of 166 adults (36.7%) were found positive in wsp PCR screening (Fig. 2, Table 1). Among positive samples, PCR products of 10 samples were selected randomly to perform sequencing. These samples carried Wolbachia DNA according to the BLAST process. The sequences obtained from this study have been deposited in GenBank under Accession Numbers KR706518 to KR706527. They are identical or highly similar (100% homology) to several Wolbachia sequences from C. lectularius deposited in GenBank, including isolates from Australia (AB475130, AB475131), Japan (AB475128, AB475129), and USA (DQ842459).

Discussion
In recent years, several investigations have concentrated on Wolbachia infection among different arthropod hosts. These studies are important for several reasons: (i) Wolbachia species are important for studies targeting evolutionary processes due to their widespread distribution among different host populations, (ii) Wolbachia species can be used in studies focusing on biological and cellular processes because they lead to changes in the development and mitotic processes in their hosts, and (iii) due to the potential role of Wolbachia species in reducing host ability to reproduce and consequently decreasing the population size, they are an important part of biological control of vector/pest insect populations [2,7,17]. These reasons have thus encouraged researchers to investigate the occurrence and prevalence of Wolbachia in several different insects, including bed bugs.
For this purpose, studies conducted to survey Wolbachia infection among bed bug populations have used samples from nature as well as from laboratories. Rasgon and Scott [25] identified Wolbachia symbionts in laboratory colonized cliff swallow bugs (Oeciacus vicarious Horvath) and common bed bugs (Cimex lectularius) by PCR amplification and sequencing using Wolbachia-specific 16S rDNA and FtsZ genes. Their phylogenetic analyses indicated that Wolbachia infections in these two cimicid hosts form a monophyletic group, and the Wolbachia strains detected belong to the F clade. In the same way, Baldo et al. [1] carried out a multilocus sequence typing (MLST) study on 42 laboratory-reared bed bug specimens harboring single infection with different Wolbachia pipientis strains, using a specific set of markers, e.g. wsp, FtsZ, and 16S. Their results demonstrated that the Wolbachia strains isolated from their prospected Cimex lectularius belonged to the F supergroup. Four years later, Hosokawa et al. [14] characterized the bacterial symbionts of laboratory bed bug samples including two Japanese (TUA and TIH) and two Australian samples (SYDW and SYDL). They isolated Wolbachia species from each bed bug specimen individually and conducted PCR, cloning, and sequencing of a bacterial 16S rRNA gene fragment. They reported complete similarity for Wolbachia strains isolated from Japanese and Australian bed bugs.
All of the mentioned studies were carried out on bed bug samples from laboratory colonies. Endosymbiotic Wolbachia bacteria have previously been shown to infect laboratory colonies of the common bed bug, Cimex lectularius, but there is little information on the extent of infection in natural populations. One of the studies on the prevalence of Wolbachia infection among field collected samples was carried out by Sakamoto and Rasgon [29] on 39 bed bug samples from the United States and Zambia. They concluded that Wolbachia infections were prevalent in the populations with an infection rate of 83%-100% and there were no significant differences in infection frequency between geographic regions, between sexes, or between life stages (adult versus nymph). Recently, Siddiqui and Raja [33] assayed Cimex lectularius populations from different regions of India for Wolbachia infection using a PCR-based analysis. They reported an infection rate of over 75% for their prospected samples.
To our knowledge, the present study is the first epidemiological investigation carried out in France, as well as Europe, on field collected bed bugs in order to detect and identify Wolbachia strains and prevalence among samples that come from different regions of France.
The total rate of Wolbachia infection of bed bugs in this study is calculated as 38.2% which is lower than rates reported by Sakamoto and Rasgon [30] and by Siddiqui and Raja [33]. This value is, however, an underestimate compared to infection rates reported previously for C. lectularius. To date, no study has been conducted on Wolbachia infection in C. lectularius in France. However, analysis with more bed bug samples in terms of quantity and diversity from different biological (male/female, nymph/adult) and physiological (unfed/blood fed, parous/nulliparous) stages will enhance  findings and provide a more accurate estimate of the Wolbachia infection rate in C. lectularius in France. Also, among positive cases, 10 samples were selected and sequenced randomly. The obtained sequences were edited, aligned, and blasted with GenBank database sequences to identify Wolbachia species. These sequences were identified as Wolbachia sp. and had 100% and 99% homology with other already known wsp sequences reported from Cimex lectularius (DQ842459 from the United States) and Cimex japonicas (AB508952 from Japan), respectively, which belong to the F supergroup strains of the Wolbachia genus. These sequences also had more than 95% homology with Wolbachia pipientis bacterium as a type strain of the Wolbachia genus, demonstrating the genetic variations and subdivision of the Wolbachia genus.

Conclusion
This is the first epidemiological study of this kind carried out in France as well as Europe. This study allowed us to identify the prevalence of Wolbachia infection among several bed bug populations collected from different localities and to compare our findings with previously reported results in the literature. Our results demonstrate the occurrence of Wolbachia infection in both nymphs and adults of C. lectularius, although the infection rates were not similar. Based on sequencing results obtained from partial DNA amplification of the wsp gene, there was 100% homology with Wolbachia wsp sequences (previously associated with C. lectularius), reported from other regions, such as the United States and Japan.