Molecular characterization and genetic diversity of Wolbachia endosymbionts in bed bugs (Hemiptera; Cimicidae) collected in Paris

Purpose This study aimed to investigate the genetic diversity of Wolbachia in field-caught bed bug species in Paris areas. Methods The bed bug specimens were captured from various infested localities in Paris and surrounding cities. They belonged to diverse life stages, including egg, nymph, and adult. They were then identified using morphological and molecular approaches. Furthermore, Wolbachia was detected, and its genetic diversity was investigated by conventional PCR of 16S-rRNA and Wolbachia surface protein (wsp) genes. Results A total of 256 bed bug specimens belonging to various life stages [adult (183 specimens), nymph (48), and egg (25)] were captured from seven private apartments, five social apartments, three houses, two immigrant residences, and one retirement home situated in 10 districts of Paris and 8 surrounding cities. They were identified as Cimex lectularius (237 specimens) and C. hemipterus (19) using morphological and molecular approaches. The presence and diversity of Wolbachia were ascertained by targeting 16S-rRNA and wsp genes. Based on molecular analysis, 182 and 148 out of 256 processed specimens were positive by amplifying 16S-rRNA and wsp fragments, respectively. The inferred phylogenetic analysis with 16S-rRNA and wsp sequences displayed monophyletic Wolbachia strains clustering each one in three populations. The median-joining network, including the Wolbachia 16S-rRNA and wsp sequences of C. lectularius and C. hemipterous specimens, indicated a significant genetic differentiation among these populations in Paris areas which was consent with Neighbor-Joining analyses. A phylogenetic analysis of our heterogenic Wolbachia sequences with those reported from other arthropod species confirmed their belonging to supergroup F. Moreover, no difference between Wolbachia sequences from eggs, nymphs, and adults belonging to the same clade and between Wolbachia sequences of C. lectularius and C. hemipterus were observed after sequence alignment. Furthermore, no significant correlation was found between multiple geographical locations (or accomodation type) where bed bugs were collected and the genetic diversity of Wolbachia. Conclusions We highlight a significant heterogeneity within Wolbachia symbionts detected in C. lectularius and C. hemipterus. No correlation between Wolbachia species and bed bug species (C. lectularius versus C. hemipterus), physiological stages (egg, nymph, and adult), and sampling location was recorded in this study.


Introduction
Bed bugs, Cimex lectularius, and C. hemipterus are hematophagous insects feeding almost exclusively on humans.They have a long evolutionary association with humans.They are broadly considered medically important ectoparasites due to the clinical manifestations of bite reactions and the psychological effects elicited by their infestation [1,2].Although they are not regarded as biological vectors of human pathogenic agents, over 45 pathogens, including bacteria, viruses, fungi, and parasites, have been detected in bed bugs [1,3].Since the last three decades, bed bugs have experienced a significant re-emergence, attributed mainly to the increase in travel and their resistance to insecticides [4].Bed bug infestations have become common in private or public settings, including hotels, elder residences, hospitals, and public transportation.
Like many insects, bed bugs depend on their bacterial symbionts to get essential nutrients and extend their adaptive potential to deal with challenging environments [5].
Wolbachia, a Gram-negative alpha-proteobacterium, is the most prevalent endosymbiotic bacterial group, associated with over 60% of insect species [6,7].These intracellular bacteria are transmitted vertically and are essential for various host reproductive processes such as cytoplasmic incompatibility, feminization, male death, parthenogenesis, increased or decreased fitness, and obligate symbiosis [8][9][10][11].Elimination of Wolbachia symbionts result in retarded growth and sterility of the insect host.They express a high genetic diversity, likely due to their interactions with many invertebrate hosts [12,13].They are classified into 18 monophyletic lineage groups from A to T in various arthropods and filarial nematodes.Wolbachia belonging to supergroups C, D, and J are exclusively found in filarial nematodes (Onchocercidae) [14][15][16].Supergroup L contains exclusively plant-parasitic nematodes (Pratylenchidae) [17,18].Supergroup F is a common supergroup for insects and filarioids [19].The latter (T) is a new supergroup recently identified in C. hemipterus from Senegal [20].
The bed bugs' need for regular blood feeding on humans and their residence near the host has resulted in an increased potential for transmission of pathogens, including arboviruses.Fort Morgan virus (FMV), Tonate virus (TONV), and Kaeng Khoi (KKV) viruses (two first viruses belonging to the Alphavirus genus and the latter belonging to Orthobunyavirus) are such arboviruses reported from swallow bugs (Oeciacus vicarius) and bat bugs (Cimex insuetus and Stricticimex parvus) [21][22][23].Despite limited knowledge on the pathogenicity of FMV and KKV viruses, TONV can cause fatal encephalitis in humans [24].Wolbachia can affect the Arboviral life cycle by supporting host factors and strengthening host antiviral mechanisms [25,26].Although the interaction of Wolbachia-arbovirus is less known in bed bugs, accurate species-specific Wolbachia determination is essential, which may help control bed bug populations.Conversely, Wolbachia transmission mode may affect symbiont-host phylogenetic co-variation [27].The increasing number of Wolbachia sequences allows a detailed characterization of their genetic diversity thanks to significant progress in molecular methods over the last three decades.
Despite knowledge of the prevalence of Wolbachia within a wide range of arthropod hosts, including bed bugs, only a few studies analyze the Wolbachia genetic diversity of human bed bug species.This study concentrated on Wolbachia species composition and genetic diversity in bed bugs collected in various districts of Paris areas.

Materials and methods
The Parasitology Department of Avicenne hospital (Bobigny, France) is a reference center for ectoparasitic diseases in Paris.The present study was conducted between April and June 2021 on the patients referred for suspected bed bug infestation.Participation in this study was voluntarily based on the participants' decision.Before bed bug collection, the participants were convinced about this study's goal and verbally agreed to bed bug collection.Moreover, absolute privacy was assured to inhabitants of inspected locations.

Bed bug collection
Bed bug samples were collected alive using a handheld vacuum cleaner (Dyson V7 trigger) and entomological forceps from various infested locations in Paris areas.They included private houses, apartments, social complexes, immigrant residences, and retirement home.The samples were transferred to the laboratory in plastic pots with folded papers.The gathered samples were then identified under a stereo microscope (Olympus SZ61, Yokohama, Japan) according to the identification keys of Usinger [28] and Walpole [29].All the specimens were labeled and kept at -20˚C for further molecular analysis.

DNA extraction, PCR amplification, and species assignation
The caught bed bugs' DNA was extracted individually using Chelex 10% (Bio-Rad, CA, USA).The processed specimens were undergone for species identification by conventional PCR targeting the cytochrome oxidase 1 (COI) gene [30].
The extracted specimens were then subjected to conventional PCR amplifications targeting two genes to assess the genetic diversity of Wolbachia symbionts within and between bed bug populations.The first PCR batch was performed by amplification of 1500 bp of 16S-rRNA using forward (16suF: 5 0 -GAGTTTGATCCTGGCTCAG-3 0 ) and reverse (16suR: 5 0 -GTTACCTTGTTA CGACTT-3 0 ) primers [12].The PCR reactions were performed under the following conditions: initial denaturation at 94˚C for 1 min, followed by 35 cycles of 95˚C for 30 s, annealing at 57.5˚C for 40 s, 72˚C for 30 s, and a final extension at 72˚C for 8 min.The second PCR of 600 bp fragments of the Wolbachia surface protein (wsp) gene was performed using forward (81F: 5 0 -TGGT CCAATAAGTGATGAAGAAAC-3 0 ) and reverse (wsp691R: 5 0 -AAAAATTAAACGCTACTCCA-3 0 ) primers [13].The PCR program was carried out with an initial denaturation at 95˚C for 5 min, followed by 35 cycles of 94˚C for 40s, 45˚C for 40s, 72˚C for 1 min, with a final extension of 72˚C for 5 min.Negative and positive controls were included in each PCR batch.The amplicons were analyzed using electrophoresis on 1.5% agarose gel containing ethidium bromide.

Genetic diversity analysis and phylogenetic reconstruction
PCR products were then subjected to bidirectional DNA sequencing with the same primer pairs used for amplification.The sequences were edited, aligned, and compared with homologous sequences from GenBank using the BLAST (Basic Local Alignment Search Tool) (www.ncbi.nlm.nih.gov/BLAST).Strains were assigned to species level, based on �99% homology to GenBank sequences.All nucleotide sequences of Wolbachia were deposited in GenBank with the assigned accession numbers NK65843901 to NK65844051.Sequence alignment was performed with the BioEdit v7.0.0 software [31], and the phylogenetic analysis was carried out using MEGA v.6 software [32].The inferred phylogenetic trees of Wolbachia species (identified in this study) and homonym sequences from GenBank were constructed using the neighbor-joining (NJ) method and bootstrap values determined by 1000 replicates.To display the genetic relationships within Wolbachia populations, the median-joining algorithms were implemented using NETWORK v.5 software [33].

Results
A total of 256 bed bug specimens were captured from various human dwellings.The latter includes seven private apartments, five social apartments, three houses, two immigrant residences, and one retirement home situated in 10 districts of Paris and eight surrounding cities.The gathered bed bugs belonged to diverse life stages, including adults (183 specimens), nymphs (48), and eggs (25).The highest sample number was from the 19 th district of Paris (41 specimens), followed by the 18 th (40), and the lowest number of specimens was collected in the Neuiy-Sur-Seine district (2).Morphological identification of adult specimens disclosed the presence of 2 species, C. lectularius (237 specimens) and C. hemipterus (19).Immature life stages and adult specimens underwent molecular identification using conventional PCR targeting the mitochondrial COI gene [30].BLAST analysis of COI gene sequences revealed �98% identity with counterpart sequences of C. lectularius (e.g., KR035952) and C. hemipterus (e.g., MN915235) in GenBank.Information on the bed bugs' origin, species composition, and their positivity rate with Wolbachia sp are given in Table 1.
Based on molecular analysis, 182 and 148 out of 256 bed bug specimens collected were positive for Wolbachia sp.revealed by amplifying the 16S-rRNA and wsp fragments, respectively.The acquired sequences were identified at the species level,based on identity equal to or more than 99% compared with homologous 16S-rRNA (e.g., AY316361, OP730740) and wsp (e.g., KR706527, QXIM01000003) sequences collected in GenBank.The inferred NJ phylogenetic trees constructed by 16S-rRNA and wsp sequences using the p-distance substitution model and supported by bootstrap displayed the presence of three monophyletic populations for each analysis separately (Figs 1A and 2A).
Furthermore, the clustering displayed by the median-joining network agreed well with the topology of the phylogenetic trees generated by NJ analysis.The median-joining network, including the Wolbachia sequences of C. lectularius and C. hemipterous specimens processed in the present study, indicated a significant genetic differentiation among these populations in Paris areas with three populations for 16S-rRNA and wsp (Figs 1B and 2B).
Further unrooted phylogenetic analysis of 16S-rRNA sequences of Wolbachia sp.identified in this study and homologous counterparts of other arthropods and nematodes collected in GenBank confirmed that all our Wolbachia specimens belonged to supergroup F (Fig 3).

High genetic diversity within and between Wolbachia taxa associated with diverse arthropod and nematode species was depicted by global network analysis of the 16S-rRNA sequences (Fig 4).
The distributions of 16S-rRNA and wsp haplotypes within Wolbachia in Paris areas processed in the present study are shown in S2 File.

Discussion
Bed bugs are regarded as one of the most critical human ectoparasites with a global geographical distribution; the investigation of Wolbachia, a symbiont of bed bugs, as a non-chemical biological method in controlling bed bugs has received significant attention in recent years.They were the subject of some investigations indicating the high prevalence of this symbiont in various bed bug populations worldwide [34,35].Nevertheless, the genetic diversity of these symbionts, which may impact their application in bed bug control, has not been thoroughly addressed.One of the few studies on Wolbachia's presence in bed bugs was conducted by Hypsa and Aksoy [8], in which two different inherited bacterial symbionts from the ovary tissue of C. lectularius were characterized by targeting the 16S-rRNA gene.They included Wolbachia and BEV (bacterial endosymbiont of the leafhopper, Euscelidius variegatus).A phylogenetic study of Wolbachia symbionts in Oeciacus vicarious (cliff swallow bug) and C. lectularius targeting 16S-rRNA and FtsZ genes revealed a monophyletic group belonging to the clade F [34].In another investigation performed on the C. lectularius populations of five North American regions (California, Connecticut, Florida, New York, and Toronto, Canada) and one African region (Macha, Zambia), a Wolbachia prevalence of 83-100% with no significant difference in Wolbachia frequency between geographic regions, between sexes, or between life stages (adult versus nymph) was reported [36].Determination of the whole genome of Wolbachia in C. lectularius (wCle) indicated its similarity to the genomes of insect-associated facultative Wolbachia strains [10].A recent study on the C. hemipterus of Senegal highlighted the diversity of Wolbachia genotypes and presented W. massiliensis as a type strain of newly discovered supergroup T [20].In this study, a high level of Wolbachia prevalence (71.1% of processed specimens) was detected, which was significantly more than what we reported in our previous article (~38%) from other regions of France [37].This is the first report of Wolbachia prevalence in Paris areas' bed bug populations and the second one, besides our previous article, in France.No difference between Wolbachia sequences from eggs, nymphs, and adults belonging to the same clade was observed after sequence alignment using BioEdit.This can be explained by the fact that Wolbachia is a maternally inherited bacterium with no significant genetic difference between Wolbachia of a female bed bug and its descendants [38,39].In addition, no difference between Wolbachia sequences of C. lectularius and C. hemipterus was recognized.Despite the genetic diversity of Wolbachia strains identified in various bed bug populations of infested locations, no significant correlation was found between multiple geographical locations where bed bugs were collected and the genetic diversity of Wolbachia.Furthermore, high heterogeneity of Wolbachia 16S-rRNA and wsp sequences was observed regrouped in three populations with no hybrid or genetic recombination.These findings are consent with investigations performed by Schuler et al. [40] and Webb et al. [41] on the Belonocnema treatae (gall wasp) and Polyrhachis (spiny ants), in which no strict association between Wolbachia diversity and geography of studied insect hosts was reported.Supergroup F is a phylogenetic clade composed of some arthropod and filarial-infecting strains of Wolbachia [42,43].Supergroup T, characterized recently as a new Wolbachia strain associated with C. hemipterus from Senegal, is closely related to supergroups F and S of insects and supergroup D of filarial nematodes (Fig 3) [20].A global phylogenetic analysis of our 16S-rRNA sequences and Wolbachia sequences reported from diverse nematodes and arthropods collected from GenBank demonstrated that high heterogenic Wolbachia sequences detected in this study belonged to supergroup F (Figs 3 and 4).These findings highlight the genetic diversity of bed bugs Wolbachia symbionts in the framework of the Wolbachia strains belonging to 18 supergroups reported from other arthropods and nematodes.There is growing evidence that various Wolbachia strains carry blocking effects in diverse arthropod species [44].The genetic diversity of Wolbachia may therefore have potential application in biologic control of bed bugs and the pathogenic agents reported to harbor by them.In the recent years, pest control programs are developing that rely on the driving ability of artificial Wolbachia infections that reduce pathogen transmission [45,46].A highly enough release of insects' infected individuals beyond threshold infection frequencies is reported to be successful in population replacement [45].

Conclusions
We highlight Wolbachia prevalence in 71.7% of processed specimens belonging to various life stages (eggs, larva and adult) and bed bug species (C.lectularius and C. hemipterous).No significant correlation was found between various geographical locations or life stages and genetic diversity of Wolbachia.The phylogenetic reconstruction using 16S-rRNA and wsp sequences demonstrated high heterogeneity and the occurrence of three monophyletic populations.Further global analysis of 16S-rRNA revealed that these sequences belonged to the supergroup F of Wolbachia.These findings complete our knowledge of the Wolbachia symbionts in bed bugs and their competency in the biological control of these ectoparasites.

Fig 1 .
Fig 1. A) Neighbor-joining (NJ) tree reconstructed from the 16S-rRNA sequences of Wolbachia isolates detected in various life stages (egg, larva & adult) of bed bug specimens we collected (beginning with AVC) and sequences from GenBank, B) Median-joining network for the 16S-rRNA sequences of C. lectularius and C. hemipterus specimens processed in this study.https://doi.org/10.1371/journal.pone.0292229.g001

Fig 2 .
Fig 2. A) Neighbor-joining (NJ) tree reconstructed from the wsp sequences of Wolbachia isolates detected in various life stages (egg, larva & adult) of bed bug specimens we collected (beginning with AVC) and sequences from GenBank, B) Median-joining network analysis of wsp sequences for the same specimens.https://doi.org/10.1371/journal.pone.0292229.g002