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. 2026 May 16;9:100387. doi: 10.1016/j.crpvbd.2026.100387

Molecular identification and characterization of Wolbachia and Cardinium with co-occurrence of Leishmania spp. in Culicoides biting midges (Diptera: Ceratopogonidae) from leishmaniasis-affected areas of Thailand

Sakone Sunantaraporn a, Pathamet Khositharattanakool b,c, Puckavadee Somwang b,c, Pranyu Leemingsawat a,d, Picha Pattrapruettada e, Darlene Ariyaskul e, Tinn Hongboontry e, Chitchanok Cherdchoochart e, Rungfar Boonserm a,d, Thanapat Pataradool a,d, Padet Siriyasatien a,d,⁎
PMCID: PMC13264255  PMID: 42293174

Abstract

Biting midges of the genus Culicoides have been identified as potential vectors for the transmission of species of the Leishmania subgenus Mundinia, the causative agents of autochthonous leishmaniasis in Thailand. Vector competence may be potentially affected by bacterial endosymbionts; however, there is no known correlation between these endosymbionts and Leishmania parasites in Culicoides biting midges. In this study, we aimed to explore the prevalence and association of bacterial endosymbionts and the detection of Leishmania DNA in Culicoides spp. Female midges were captured at five sampling sites in areas with autochthonous leishmaniasis in northern and southern Thailand. Culicoides species were identified using both morphological characteristics and cox1 sequencing. The presence of Wolbachia, Cardinium, and Leishmania DNA in individual midges was molecularly screened targeting the wsp and 16S rRNA genes, and the ITS1 region, respectively. All amplification products were sequenced and subjected to phylogenetic analysis. A total of 593 female midges were collected, comprising 21 species of Culicoides and one species of Culicoides (Trithecoides). The Wolbachia isolates from infected Culicoides spp. were phylogenetically classified into supergroups A, B, and F. Six Wolbachia putative strains belonged to clade Wol-b (Wol-b1 to Wol-b6), two to clade Wol-a (Wol-a1 and Wol-a2), and one to clade Wol-f (Wol-f1). Three Wolbachia strains were identified as wKerlac, wBeva_B, and wCauA. Moreover, both Cardinium groups A and C were identified. It is noteworthy that co-infections between bacterial endosymbionts and Leishmania spp. showed a significant association. To the best of our knowledge, this study provides the first evidence of Wolbachia and Cardinium in Culicoides spp. from leishmaniasis-affected areas in Thailand. Detecting bacterial endosymbionts co-occurring with Leishmania spp. in Culicoides biting midges may suggest a potential, but unconfirmed, antagonistic effect on Leishmania. This provides preliminary data that could inform the development of new vector control strategies for diseases transmitted by Culicoides spp. in Thailand.

Keywords: Culicoides, Leishmania, Wolbachia, Cardinium, Thailand

Graphical abstract

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Highlights

  • •

    First report of Wolbachia and Cardinium endosymbionts in Culicoides spp. from leishmaniasis-affected areas in Thailand.

  • •

    The prevalence of Wolbachia, Cardinium, and co-infection in Culicoides was 29.0%, 14.5%, and 11.47%, respectively.

  • •

    Wolbachia supergroups A, B, and F were identified in Culicoides biting midges.

  • •

    Three Wolbachia putative strains were characterized as wKerlac, wBeva_B, and wCauA.

  • •

    Cardinium groups A and C were identified in Culicoides biting midges.

1. Introduction

Leishmaniasis is a neglected tropical disease caused by obligate intracellular parasites of the genus Leishmania. In Thailand, more than 20 cases of autochthonous leishmaniasis have been documented in both immunocompromised and immunocompetent individuals across the northern and southern regions of the country. The causative agents of autochthonous leishmaniasis in Thailand are Leishmania martiniquensis (Chiewchanvit et al., 2015; Phadungsaksawasdi et al., 2026; Rattanagitpaisan et al., 2026) and Leishmania orientalis (Jariyapan et al., 2018), which have been phylogenetically classified within the subgenus Mundinia (Espinosa et al., 2018). Several studies have reported the detection of Leishmania DNA in phlebotomine sand flies in Thailand. For example, DNA of L. martiniquensis has been detected in several species of sand flies, including Segentomyia gemmae, Se. barraudi, Se. khawi, Phlebotomus stantoni, and Grassomyia indica (Chusri et al., 2014; Sriwongpan et al., 2021; Preativatanyou et al., 2023; Phumee et al., 2024). Furthermore, DNA of L. orientalis was identified in Se. iyegari, Se. gammae, and Se. khawi (Siripattanapipong et al., 2018; Sriwongpan et al., 2021; Phumee et al., 2024). However, non-sand fly vectors, such as biting midges of the genus Culicoides and Forcipomyia, are currently considered possible vectors for the transmission of Leishmania (Mundinia) species (Chanmol et al., 2019; Panahi et al., 2020; Sunantaraporn et al., 2021; Preativatanyou et al., 2024; Tepboonrueng et al., 2025). Recent investigations have successfully demonstrated the development and transmission of species of Leishmania (Mundinia), including L. martiniquensis, L. orientalis, and L. chancei (formerly named Leishmania sp. from Ghana), in C. sonorensis midges (Becvar et al., 2021).

Biting midges of the genus Culicoides are hematophagous insects belonging to the family Ceratopogonidae. It is established that Culicoides-borne pathogens are responsible for significant veterinary and medical diseases, including bluetongue virus (BTV), Schmallenberg virus (SBV), African horse sickness virus (AHSV), epizootic hemorrhagic disease virus (EHDV), and Oropouche virus (OROV) (Sick et al., 2019). A recent study by Fujisawa et al. (2021) demonstrated the presence of bluetongue virus in four species of Culicoides, namely C. orientalis, C. imicola, C. oxstoma, and C. fulvus, collected from ruminant farms in western Thailand. Furthermore, haemosporidian infection in Thai Culicoides biting midges has been reported by Pramual et al. (2021a) and Sunantaraporn et al. (2022). As previously stated, Culicoides spp. can serve as potential vectors of several pathogens for transmission to humans and/or animals.

In recent years, bacterial endosymbionts have received increasing attention for use as an insect vector control strategy, especially to control vector-borne diseases (VBDs) (Floate et al., 2006). Previous studies have shown that Wolbachia endosymbionts (class Alphaproteobacteria) are present in a wide range of insects and filarial nematodes worldwide. Wolbachia strains are well characterized in mosquitoes, particularly within the family Culicidae. In mosquitoes, Wolbachia infection can inhibit the replication of arboviruses, including dengue virus (DENV), chikungunya virus (CHIKV), yellow fever virus (YFV), Zika virus (ZIKV), and West Nile virus (WNV) (Ant et al., 2023), as well as Plasmodium parasites (Qu and Childs, 2025) and filarial nematodes (Setegn et al., 2024). Wolbachia infection can also cause a wide range of abnormal reproductive phenotypes through parthenogenesis, feminization, male killing, and cytoplasmic incompatibility (CI) (Stouthamer et al., 1999). To date, Culicoides midges have been demonstrated to have endosymbiotic bacteria of the genera Wolbachia and Cardinium. A previous study reported evidence of infection by the endosymbiont “Candidatus Cardinium hertigii” (Bacteroidetes group) in various Culicoides species and suggested the existence of a phylogenetically distinct lineage within the Cardinium endosymbiont group (Group C) (Noel and Atibalentja, 2006; Nakamura et al., 2009; Konecka and Olszanowski, 2019). The biological mechanisms of Cardinium bacteria in biting midges are uncertain, while information from different host species suggests that Cardinium-induced reproductive manipulations include parthenogenesis, feminization, and CI (Zchori-Fein et al., 2004; Tarlachkov et al., 2023). However, no studies have investigated the association of Wolbachia and Cardinium endosymbionts in Thai Culicoides species, particularly in leishmaniasis-affected areas.

The present study aimed to investigate the prevalence of Wolbachia and Cardinium endosymbiotic bacteria in field-caught Culicoides spp. collected from five different leishmaniasis-affected areas in northern and southern Thailand. This study provides molecular evidence of the prevalence of Leishmania parasites in Culicoides spp. and examines their association with endosymbiotic bacteria. This information would be valuable for the development of a potentially effective vector control strategy utilizing bacterial endosymbionts in the future.

2. Materials and methods

2.1. Sampling locations and biting midge trapping

Biting midges were collected in the vicinity of the residences of patients diagnosed with autochthonous leishmaniasis in five distinct geographical areas (Fig. 1). Insect samples were collected from three different locations in Songkhla Province where leishmaniasis cases were present, including sampling location 1 (SK1) (6°38′08.7″N, 100°25′35.9″E) and sampling location 2 (SK2) (6°36′14″N, 100°28′57″E) in Sadao District, and sampling location 3 (SK3) (6°44′30″N, 100°41′30″E) in Na Thawi District. The northern region was represented by two sampling locations: Meuang District, Chiang Rai Province (19°51′05.8″N, 99°39′37.1″E), and Wang Nuea District, Lampang Province (19°10′30″N, 99°38′51″E). Four Centers for Disease Control and Prevention (CDC) miniature light traps (25 W bulb) with ultraviolet (UV) light were placed at an approximate height of 1.5 m above the ground and operated from 18:00 to 6:00 h for two consecutive nights at each study site in July and August 2025. The collection bags were cooled on ice for 30 min to anesthetize the insects, then female midges were sorted from other insects by morphological characteristics under a stereomicroscope (Olympus, Tokyo, Japan). In the present study, all collected individuals were in the parous stage, and no gravid specimens were observed. Nulliparous individuals were excluded from the analysis because they have not yet taken a blood meal and are therefore unlikely to have been exposed to infection. Blood-engorged specimens were also excluded because, although parasite detection is feasible in these individuals, the presence of parasite DNA may reflect residual DNA derived from the host blood meal. Female biting midges were preserved in 1.5-ml microcentrifuge tubes containing 80% ethanol and subsequently transported to the Center of Excellence in Vector Biology and Vector-Borne Diseases, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, for further investigation.

Fig. 1.

Fig. 1

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Map of Thailand showing species diversity of Culicoides spp. and their relative abundance at the collection sites of five autochthonous leishmaniasis-affected areas in Chiang Rai (CR), Lampang (LP), and Songkhla (SK1, SK2, and SK3) provinces. Images obtained and modified from Google Earth Pro version 7.3.4.8248 (https://www.google.com/earth/about/).

2.2. Non-destructive DNA extraction from Culicoides spp.

Before DNA extraction, sterilized distilled water was added to remove residual ethanol. Genomic DNA was extracted from individual parous female Culicoides samples using a non-destructive protocol with modifications. Briefly, 100 μl of cell lysis solution (GeneAll, Seoul, Korea) and 10 μl of proteinase K solution were added to each sample and incubated at 50 °C for 16 h. The lysate solution was used to extract DNA according to the instructions using Genti™32 Automated Nucleic Acid Extraction (GeneAll, Seoul, Korea). The concentration and quality of the DNA were measured using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, USA). The whole Culicoides body was stored in a 1.5-ml microcentrifuge tube containing 80% ethanol solution for retrospective further morphological species identification.

2.3. Morphological and molecular identification of Culicoides spp.

Unmounted Culicoides specimens were initially identified based on their wing spot patterns. For samples that were difficult to identify, the head, wings, and genitalia with spermathecae were dissected and removed under a stereomicroscope using a sterile needle in a drop of 0.9% normal saline. The samples were mounted on glass slides using Hoyer’s medium. Culicoides spp. were morphologically identified according to the taxonomic keys by Wirth and Hubert (1989), and the morphology of C. mahasarakhamense was characterized as described by Pramual et al. (2021b). The identification of Culicoides spp. based on morphology was confirmed by sequencing of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. PCR was performed on randomly selected samples of each representative species using primers C1-J-1718 and C1-N-2191 (Dallas et al., 2003), and the PCR amplification protocol was performed as described by Mathieu et al. (2020).

2.4. PCR identification of Wolbachia and Cardinium in field-caught Culicoides spp.

The presence of Wolbachia and Cardinium endosymbionts was assessed through PCR amplification of specific gene fragments. For Wolbachia, a fragment of the wsp (Wolbachia surface protein) gene was amplified using the forward primer wsp81F (5′-TGG TCC AAT AAG TGA TGA AGA AAC-3′) and the reverse primer wsp691R (5′-AAA AAT TAA ACG CTA CTC CA-3′) (Zhou et al., 1998). Detection of Cardinium was performed by amplifying a highly conserved region of the 16S rRNA gene with primers CAR-SP-F (5′-CGG CTT ATT AAG TCA GTT GTG AAA TCC TAG-3′) and CAR-SP-R (5′-TCC TTC CTC CCG CTT ACA CG-3′) (Nakamura et al., 2009). Each PCR reaction master mix was performed in a total volume of 25 μl containing 6 μl of genomic DNA (approximately 5–10 ng), 12.5 μl of Green Hot Start PCR Master Mix Direct-Load 2× (Biotechrabbit, Berlin, Germany), 1 μl of each forward and reverse primer (10 μM), and 4.5 μl of sterile deionized water. The thermal cycling conditions included an initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 1 min, annealing at 55 °C for wsp and 57 °C for 16S rRNA for 1 min, and extension at 72 °C for 1 min, with a final extension step at 72 °C for 5 min. Plasmid DNA containing the specific sizes of the wsp and 16S rRNA genes served as positive controls, while genomic DNA from Culicoides specimens confirmed to be free of Wolbachia and Cardinium, along with sterile deionized water, were included as negative controls. The expected size of the wsp PCR product is approximately 593–644 bp, depending on the Wolbachia strain, while that of the Cardinium PCR product is approximately 595 bp.

2.5. Detection of Leishmania DNA in Culicoides spp.

The presence of Leishmania DNA was detected using a conventional PCR assay targeting the internal transcribed spacer 1 (ITS1) region of the ribosomal DNA cluster. Amplification was performed with the forward primer LeF (5′-TCC GCC CGA AAG TTC ACC GAT A-3′) and the reverse primer LeR (5′-CCA AGT CAT CCA TCG CGA CAC G-3′) (Spanakos et al., 2008). PCR reactions were carried out using the Green Hot Start PCR Master Mix Direct-Load 2× (Biotechrabbit, Berlin, Germany) according to the manufacturer’s instructions. The PCR cycling conditions followed the protocols previously described by Srisuton et al. (2019) and Sunantaraporn et al. (2021). DNA extracted from Leishmania martiniquensis culture (MHOM/TH/2012/CULE1) served as the positive control, while male Culicoides and sterilized deionized water were used as the negative control.

2.6. DNA cloning and sequencing

The PCR products for Leishmania spp., Wolbachia, and Cardinium were inserted into the pGEM-T Easy Vector (Promega, Madison, WI, USA) using a rapid DNA ligation kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. The plasmid DNA was transformed into Escherichia coli (DH5α) competent cells, and the transformants were screened by blue-white colony selection using a colony PCR assay. Three to five positive clones, presumed to contain the desired genes, were cultured in Luria-Bertani (LB) broth, with the addition of ampicillin (100 mg/ml). The chimeric DNA was extracted using the GeneAll® Exprep™ plasmid purification kit (GeneAll, Seoul, Korea) following the manufacturer’s instructions. The extracted DNA was then sent to a commercial service at Macrogen Inc., South Korea, for Sanger DNA sequencing.

2.7. Phylogenetic tree construction and haplotype network

All nucleotide sequences were subjected to manual editing and trimming prior to alignment using the Clustal W multiple alignment function in BioEdit sequence alignment editor v.7.2.5 (Hall, 1999). The consensus nucleotide sequences were compared to previously published sequences in the GenBank database using the Basic Local Alignment Search Tool (BLAST) online in the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The phylogenetic relationships were reconstructed using the maximum likelihood (ML) method, based on the model with the lowest Bayesian Information Criterion (BIC) scores, as implemented in MEGA 11 (Tamura et al., 2021). The reliability of the phylogenetic tree was estimated based on 1000 bootstrap pseudoreplicates. The Wolbachia putative strain prediction was evaluated based on the analysis of the wsp sequences using the DnaSP software version 6 (Rozas et al., 2017). A Templeton-Crandall-Sing (TCS) network was constructed using PopART v.1.7 (Leigh and Bryant, 2015).

2.8. Statistical analysis

Species richness (the number of species in the study areas) and species relative abundance (the number of samples per species/total number of samples × 100) for each site were calculated. Prevalence was calculated as the proportion of PCR-positive samples relative to the total number of individuals examined for each Culicoides species, study site, and infection status (single infection and co-infection). Prevalences are presented as percentages with corresponding 95% confidence intervals (95% CI). The associations between the detection of Leishmania DNA and locations, Culicoides species, and endosymbiont presence were assessed using binary logistic regression. A P-value of < 0.05 was defined as statistically significant. All statistical analyses were performed using SPSS v.29 (IBM Corp., Armonk, NY, USA).

3. Results

3.1. Species composition of Culicoides biting midges

A total of 593 specimens of Culicoides, representing 21 species of Culicoides and one species of Culicoides (Trithecoides), were collected from five collection sites across Songkhla (SK1, SK2, and SK3) in the southern region and Chiang Rai and Lampang in the northern region of Thailand. Species richness of Culicoides spp. demonstrated the lowest richness observed in SK1 (5 species) and the highest in Lampang (16 species). Overall species richness across all sampling locations was 21 species. The relative abundance of Culicoides spp. differed across sampling locations. Lampang contributed the high relative abundance of Culicoides samples (36.76%; 218/593), followed by Chiang Rai (26.98%; 160/593). In southern Thailand, SK2 showed the highest relative abundance (17.87%; 106/593), followed by SK3 (13.49%; 80/593) and SK1 (4.89%; 29/593). Culicoides peregrinus was the most frequent species (n = 127), accounting for 21.4% of all collected samples, followed by C. guttifer (n = 78), C. orientalis (n = 59), C. oxystoma (n = 48), and C. shortti (n = 48). The Culicoides species composition varied among sampling sites, with distinct dominant species observed at each location. In SK1, C. oxystoma was the most abundant species (n = 11; 37.9%). For SK2, C. peregrinus predominated (n = 85; 80.2%), whereas SK3 was dominated by C. guttifer (n = 49; 61.3%). Culicoides tainanus (n = 35; 21.9%) and C. orientalis (n = 34; 21.3%) were mainly found in Chiang Rai. In addition, C. shortti (n = 42; 19.3%) was the most frequently collected species in Lampang (Table 1 and Fig. 1).

Table 1.

Species of Culicoides collected from five locations with leishmaniasis cases in three provinces of Thailand.

Species Study site
 Total
Songkhla
 Chiang Rai Lampang
SK1 SK2 SK3
C. actoni 0 0 0 0 3 3
C. arakawae 0 0 1 0 0 1
C. fulvus 0 0 0 0 4 4
C. guttifer 0 3 49 0 26 78
C. huffi 2 0 4 8 2 16
C. innoxius 0 0 3 1 11 15
C. insignipennis 0 1 0 10 5 16
C. jacobsoni 0 0 0 31 2 33
C. liui 0 0 0 7 0 7
C. mahasarakhamense 3 7 7 0 25 42
C. malayae 0 0 0 4 0 4
C. orientalis 1 3 2 34 19 59
C. oxystoma 11 7 0 6 24 48
C. palpifer 0 0 7 0 9 16
C. parahumeralis 0 0 2 0 1 3
C. peregrinus 12 85 4 0 26 127
C. shortti 0 0 0 6 42 48
C. sumatrae 0 0 0 1 11 12
C. tainanus 0 0 0 35 0 35
C. tamada 0 0 1 0 0 1
C. tenuipalpis 0 0 0 0 3 3
C. (Trithecoides) sp. 0 0 0 17 5 22
Total 29 106 80 160 218 593
Relative abundance 4.89 17.87 13.49 26.98 36.76
Species richness 5 6 9 10 16 21
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Note: The calculation of species richness was based on all identified species, except for C. (Trithecoides) sp.

Abbreviations: SK1 and SK2, Sadao District; SK3, Na Thawi District.

3.2. Detection of Wolbachia and Cardinium endosymbionts in Culicoides spp.

Conventional PCR was used to amplify the Wolbachia surface protein (wsp) gene in several Culicoides spp. Overall, a single Wolbachia infection was detected in 18 Culicoides species, with a prevalence of 29.01% (95% CI: 25.49–32.79%). In Songkhla, the prevalence exhibited variation among the collection sites, with 34.48% (95% CI: 19.85–52.74%), 21.70% (95% CI: 14.86–30.52%), and 12.50% (95% CI: 6.74–21.69%) recorded at SK1, SK2, and SK3, respectively. In northern Thailand, the prevalence of Wolbachia infection was 31.88% (95% CI: 25.14–39.46%) in Chiang Rai and 35.78% (95% CI: 29.71–42.34%) in Lampang.

The presence of Cardinium infection was detected through the amplification of the 16S rRNA gene. Among the 593 specimens examined, 86 (14.50%, 95% CI: 11.89–17.57%) were found to be positive for Cardinium. In Songkhla, the prevalences were 3.45% (95% CI: 0–18.63%), 10.38% (95% CI: 5.74–17.79%), and 48.75% (95% CI: 38.11–59.51%) at sampling locations SK1, SK2, and SK3, respectively. In northern Thailand, the prevalence of Cardinium infection was 1.25% (95% CI: 0–4.73%) in Chiang Rai and 15.14% (95% CI: 10.95–20.53%) in Lampang. Cardinium infection was most frequently detected in C. guttifer and C. mahasarakhamense.

Furthermore, co-infection with Wolbachia and Cardinium endosymbionts was observed in several Culicoides species, with an overall prevalence of 11.47% (95% CI: 9.14–14.30%). Co-infections were identified at three collection sites: SK3 in Songkhla (13.75%, 95% CI: 7.68–23.14%), Chiang Rai (7.50%, 95% CI: 4.23–12.77%), and Lampang (20.64%, 95% CI: 15.78–26.52%) (Table 2).

Table 2.

Detection of endosymbionts and Leishmania parasite in Culicoides spp. from northern and southern Thailand.

Location/Culicoides spp. N Molecular detection of endosymbionts
 Leishmania detection Prevalence (95% CI) (%)
Single infection
 Co-infection
 Single infection
 Co-infection
 Leishmania
W C W + C W C W + C
Songkhla (SK1)
C. huffi 2 0 0 0 0 0 0 0 0
C. mahasarakhamense 3 1 1 0 0 33.33 (5.63–79.76) 33.33 (5.63–79.76) 0 0
C. orientalis 1 0 0 0 0 0 0 0 0
C. oxystoma 11 6 0 0 0 54.55 (27.99–78.75) 0 0 0
C. peregrinus 12 3 0 0 0 25.00 (8.27–53.85) 0 0 0
Subtotal 29 10 1 0 0 34.48 (19.85–52.74) 3.45 (0–18.63) 0 0
Songkhla (SK2)
C. guttifer 3 0 3 0 0 0 100 (38.25–100) 0 0
C. insignipennis 1 0 0 0 0 0 0 0 0
C. mahasarakhamense 7 0 6 0 0 0 85.71 (46.65–99.47) 0 0
C. orientalis 3 0 0 0 0 0 0 0 0
C. oxystoma 7 1 0 0 2 14.29 (0.53–53.35) 0 0 28.57 (7.56–64.76)
C. peregrinus 85 22 2 0 7 25.88 (17.70–36.15) 2.35 (0.14–8.68) 0 8.24 (3.79–16.29)
Subtotal 106 23 11 0 9 21.70 (14.86–30.52) 10.38 (5.74–17.79) 0 8.49 (4.35–15.54)
Songkhla (SK3)
C. arakawae 1 0 0 0 0 0 0 0 0
C. guttifer 49 1 37 8 3 2.04 (0–11.69) 75.51 (61.78–85.53) 16.33 (8.25–29.30) 6.12 (1.48–17.15)
C. huffi 4 1 0 0 0 25.00 (3.41–71.09) 0 0 0
C. innoxius 3 0 0 0 0 0 0 0 0
C. mahasarakhamense 7 0 2 3 2 0 28.57 (7.56–64.76) 42.86 (15.75–75.02) 28.57 (7.56–64.76)
C. orientalis 2 1 0 0 0 50.00 (9.45–90.55) 0 0 0
C. parahumeralis 2 1 0 0 0 50.00 (9.45–90.55) 0 0 0
C. peregrinus 4 3 0 0 0 75.00 (28.91–96.59) 0 0 0
C. tamada 1 1 0 0 0 100 (16.75–100) 0 0 0
C. palpifer 7 2 0 0 0 28.57 (7.56–64.76) 0 0 0
Subtotal 80 10 39 11 5 12.50 (6.74–21.69) 48.75 (38.11–59.51) 13.75 (7.68–23.14) 6.25 (2.36–14.15)
Total (Songkhla) 215 43 51 11 14 20.00 (15.18–25.88) 23.72 (18.51–29.86) 5.12 (2.78–9.03) 6.51 (3.83–10.72)
Chiang Rai
C. jacobsoni 31 9 0 1 3 29.03 (15.94–46.75) 0 3.23 (0–17.58) 9.68 (2.56–25.69)
C. orientalis 34 11 0 2 3 32.35 (19.04–49.25) 0 5.88 (0.65–20.07) 8.82 (2.29–23.72)
C. tainanus 35 11 0 2 3 31.43 (18.45–48.08) 0 5.71 (0.62–19.57) 8.57 (2.21–23.13)
C. huffi 8 5 1 0 1 62.50 (30.38–86.51) 12.50 (0.11–49.22) 0 12.50 (0.11–49.22)
C. innoxius 1 0 0 0 0 0 0 0 0
C. insignipennis 10 5 0 0 2 50.00 (23.66–76.34) 0 0 20.00 (4.59–52.06)
C. liui 7 3 0 4 2 42.86 (15.75–75.02) 0 57.14 (24.98–84.25) 28.57 (7.56–64.76)
C. malayae 4 0 0 3 0 0 0 75.00 (28.91–96.59) 0
C. sumatrae 1 0 0 0 0 0 0 0 0
C. oxystoma 6 2 0 0 0 33.33 (9.25–70.43) 0 0 0
C. shortti 6 0 0 0 0 0 0 0 0
C. (Trithecoides) sp. 17 5 1 0 1 29.41 (12.99–53.43) 5.88 (0–28.92) 0 5.88 (0–28.92)
Total 160 51 2 12 15 31.88 (25.14–39.46) 1.25 (0–4.73) 7.50 (4.23–12.77) 9.38 (5.67–14.99)
Lampang
C. actoni 3 1 0 1 1 33.33 (5.63–79.76) 0 33.33 (5.63–79.76) 33.33 (5.63–79.76)
C. fulvus 4 0 1 1 1 0 25.00 (3.41–71.09) 25.00 (3.41–71.09) 25.00 (3.41–71.09)
C. guttifer 26 0 16 10 3 0 61.54 (42.48–77.63) 38.46 (22.37–57.52) 11.54 (3.18–29.80)
C. huffi 2 1 0 0 0 50.00 (9.45–90.55) 0 0 0
C. innoxius 11 5 0 2 1 45.45 (21.25–72.01) 0 18.18 (3.99–49.85) 9.09 (0–39.91)
C. insignipennis 5 4 0 0 1 80.00 (35.96–97.97) 0 0 20.00 (2.03–64.04)
C. jacobsoni 2 2 0 0 0 100 (29.02–100) 0 0 0
C. mahasarakhamense 25 2 9 12 5 8.00 (1.09–26.10) 36.00 (20.16–55.57) 48.00 (30.03–66.50) 20.00 (8.41–39.58)
C. orientalis 19 9 0 6 2 47.37 (27.33–68.30) 0 31.58 (15.16–54.20) 10.53 (1.70–32.63)
C. oxystoma 24 12 2 2 1 50.00 (31.43–68.57) 8.33 (1.16–27.00) 8.33 (1.16–27.00) 4.17 (0–21.87)
C. peregrinus 26 11 2 3 3 42.31 (25.52–61.08) 7.69 (1.02–25.26) 11.54 (3.18–29.80) 11.54 (3.18–29.80)
C. shortti 42 24 0 4 5 57.14 (42.19–70.89) 0 9.52 (3.21–22.62) 11.90 (4.73–25.46)
C. parahumeralis 1 0 0 0 0 0 0 0 0
C. sumatrae 11 3 0 1 1 27.27 (9.20–57.11) 0 9.09 (0–39.91) 9.09 (0–39.91)
C. tenuipalpis 3 0 0 1 0 0 0 33.33 (5.63–79.76) 0
C. palpifer 9 1 3 1 0 11.11 (0.00–45.67) 33.33 (11.73–64.91) 11.11 (0–45.67) 0
C. (Trithecoides) sp. 5 3 0 1 0 60.00 (22.91–88.40) 0 20.00 (2.03–64.04) 0
Total 218 78 33 45 24 35.78 (29.71–42.34) 15.14 (10.95–20.53) 20.64 (15.78–26.52) 11.01 (7.46–15.91)
Grand total 593 172 86 68 53 29.01 (25.49–32.79) 14.50 (11.89–17.57) 11.47 (9.14–14.30) 8.94 (6.88–11.52)
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Abbreviations: N, no. of tested; SK1 and SK2, Sadao district; SK3, Na Thawi district; W, Wolbachia, C, Cardinium.

3.3. Phylogenetic relationships and Wolbachia putative strain identification

Partial wsp sequences were successfully obtained from 52 Wolbachia-infected Culicoides samples, representing all morphologically identified species found to harbor Wolbachia endosymbionts. BLAST analysis revealed that all wsp sequences were identical to those of Wolbachia bacterial endosymbionts, with sequence similarities ranging from 91.43% to 100% (Supplementary Table S1). Phylogenetic analysis of these Wolbachia-positive sequences revealed their allocation to three major Wolbachia supergroups: Supergroup A (n = 10), Supergroup B (n = 36), and Supergroup F (n = 6). In one sample from C. guttifer, a co-infection was observed with Wolbachia Supergroup B (isolate MGSS28-26) and Supergroup F (isolate MGSS28-28). According to phylogenetic analysis, the majority of wsp sequences were assigned to Wolbachia Supergroup B and clustered into six putative strains: Wol-b1, Wol-b2, Wol-b3, Wol-b4, Wol-b5, and Wol-b6. Two putative strains, Wol-a1 and Wol-a2, belonged to Wolbachia Supergroup A, and one putative strain, Wol-f1, was classified within Supergroup F (Fig. 2).

Fig. 2.

Fig. 2

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Phylogenetic relationships among Wolbachia supergroups inferred from partial wsp sequences. The maximum likelihood phylogenetic tree was generated using the Kimura 2-parameter model with gamma distribution (K2+G) and includes representative Wolbachia reference sequences retrieved from the GenBank database.

Haplotype network analysis of the newly characterised Wolbachia strains and 35 strains previously reported by Zhou et al. (1998) demonstrated that the wsp sequences clustered into three Wolbachia putative strains, which is consistent with previous classifications. The first cluster corresponded to the wKerlac strain (Wolbachia of Kerria lacca) from Wol-b4 and comprised endosymbionts from 10 Culicoides samples: C. peregrinus (n = 3), C. guttifer (n = 3), C. mahasarakhamense (n = 2), C. oxystoma (n = 1), and C. huffi (n = 1) collected from Songkhla, and one each, C. shortti (LP54) and C. mahasarakhamense (LP82), from Lampang. Wolbachia from several additional Culicoides species exhibited close genetic similarity to wKerlac, differing by only 1–2 base pairs (Fig. 3). These species included C. malayae (CRP29) and C. orientalis (CRP64) from Chiang Rai, and C. mahasarakhamense (MGSS26) and C. peregrinus (T1SS49) from Songkhla. The second cluster, corresponding to the wBeva_B strain (Wolbachia of Bicyclus evadne) from Wol-b3, was identified in C. orientalis (n = 2) and C. tainanus (n = 2) from Chiang Rai, and in six Culicoides spp. including C. oxystoma, C. guttifer, C. huffi, C. actoni, C. peregrinus, and C. insignipennis collected from Lampang. The third cluster, wCauA strain (Wolbachia of Cadra cautella) from Wol-a2, was identified in C. guttifer from Songkhla. Other Wolbachia variants detected in this study exhibited close genetic relationships to strains previously reported from a range of different insect hosts (Fig. 3).

Fig. 3.

Fig. 3

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Genetic diversity and relationships among putative Wolbachia strains inferred from partial wsp sequences using a TCS haplotype network.

3.4. Phylogenetic relationships of Cardinium endosymbionts

A total of 52 Cardinium-positive samples from 16 Culicoides spp. were randomly selected for 16S rRNA sequencing. BLAST analysis of these sequences against the GenBank database revealed 97.98–99.83% similarity with “Candidatus Cardinium hertigii” (GenBank: KR026922) previously reported in Culicoides williwilli. Additionally, eleven 16S rRNA sequences from multiple Culicoides species showed 98.65–98.99% similarity with the Cardinium endosymbiont of Microzetorchestes emeryi (GenBank: MG889459) (Supplementary Table S2).

The phylogenetic analysis classified the Cardinium sequences into two major groups, designated as Group A and Group C. The majority of sequences (n = 41) clustered within Cardinium Group C, which was further divided into two subgroups (Fig. 4). Subgroup C1 comprised 22 Cardinium sequences detected in 9 Culicoides spp., including reference sequences from Cardinium previously reported in various Culicoides species. Subgroup C2 contained 19 sequences identified in six Culicoides spp. from the present study (Fig. 4). Notably, eleven Cardinium-positive samples were phylogenetically classified for the first time within Cardinium Group A (Fig. 4).

Fig. 4.

Fig. 4

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Phylogenetic relationship of Cardinium endosymbionts based on partial 16S rRNA sequences. The maximum likelihood tree was constructed using the Kimura 2-parameter model with gamma distribution (K2+G) and includes Cardinium sequences from Culicoides spp. obtained in this study and reference sequences from the GenBank database.

3.5. Detection of Leishmania DNA in Culicoides spp.

A total of 593 extracted DNA samples were subjected to molecular analysis in order to determine the presence of Leishmania DNA in Culicoides spp. samples. The results demonstrated the presence of Leishmania DNA in 53 (8.94%, 95% CI: 6.88–11.52%) Culicoides samples collected from Chiang Rai (n = 15, 9.38%, 95% CI: 5.67–14.99%), Lampang (n = 24, 11.01%, 95% CI: 7.46–15.91%), and Songkhla (n = 14, 6.51%, 95% CI: 3.83–10.72%). No Leishmania DNA was detected at the SK1 sampling site in Songkhla (Table 2).

The BLAST analysis of positive Leishmania samples based on ITS1 sequences revealed that a single infection of L. martiniquensis occurred in 45 samples from all sampling sites. In contrast, a single infection with L. orientalis was identified in two C. orientalis samples collected in Lampang. Co-infection of L. martiniquensis + L. orientalis was demonstrated in six samples from two sampling sites from Lampang (four samples of C. guttifer, C. shortti, C. peregrinus, and C. fulvus) and Chiang Rai (two samples of C. jacobsoni and C. tainanus) (Supplementary Table S3).

3.6. Co-occurrence of Leishmania parasites and endosymbionts in Culicoides spp.

The binary logistic regression analysis showed that neither sampling location nor Culicoides species was significantly associated with Leishmania detection in either crude or adjusted models. Although higher crude odds were observed in Chiang Rai (OR = 1.49, 95% CI: 0.69–3.17) and Lampang (OR = 1.78, 95% CI: 0.89–3.53) compared with Songkhla, these associations were not significant after adjustment. No significant associations were also detected between Leishmania detection and individual Culicoides species. While Culicoides mahasarakhamense showed a higher crude odds ratio (OR = 2.20, 95% CI: 0.60–8.12), this was not retained in the adjusted model (adjusted OR = 0.82, 95% CI: 0.19–3.62, P = 0.799).

In contrast, endosymbiont infections were significantly associated with Leishmania detection after adjustment. Wolbachia-positive samples had higher odds of Leishmania detection than negative samples (adjusted OR = 3.51, 95% CI: 1.52–8.12, P = 0.003). Similarly, Cardinium positivity was associated with increased odds (adjusted OR = 4.21, 95% CI: 1.15–15.38; P = 0.030), despite a non-significant crude association. Co-infection with Wolbachia and Cardinium showed a strong association, with markedly higher odds of Leishmania detection compared with non-co-infected samples (adjusted OR = 17.70, 95% CI: 6.53–48.03, P < 0.001) (Table 3). A tripartite network analysis demonstrating the associations among Culicoides species, bacterial endosymbionts, and Leishmania spp. detection is presented in Fig. 5. The data on positive Leishmania DNA and associated endosymbionts, including both single infections and co-infections of Wolbachia and Cardinium, are presented in Supplementary Table S3.

Table 3.

Association between Leishmania detection and sampling location, species of Culicoides, and endosymbiont infections.

Variable Leishmania-positive (n, %) Leishmania-negative (n, %) Crude OR (95% CI) Adjusted OR (95% CI) P-value
Location
Songkhla 14 (6.5) 201 (93.5) 1 (reference)
Chiang Rai 15 (9.4) 145 (90.6) 1.49 (0.69–3.17) 1.56 (0.50–4.89) 0.448
Lampang 24 (11.0) 194 (89.0) 1.78 (0.89–3.53) 0.90 (0.37–2.14) 0.806
Culicoides speciesa
C. oxystoma 4 (8.3) 44 (91.7) 1 (reference)
C. orientalis 5 (8.5) 54 (91.5) 1.02 (0.26–4.02) 0.63 (0.14–2.83) 0.550
C. shortti 5 (10.4) 43 (89.6) 1.28 (0.32–5.09) 1.14 (0.26–4.89) 0.862
C. guttifer 6 (7.7) 72 (92.3) 0.92 (0.24–3.43) 0.37 (0.08–1.74) 0.208
C. mahasarakhamense 7 (16.7) 35 (83.3) 2.20 (0.60–8.12) 0.82 (0.19–3.62) 0.799
C. peregrinus 9 (7.1) 118 (92.9) 0.84 (0.25–2.86) 1.09 (0.29–4.07) 0.894
Other species 17 (8.9) 174 (91.1) 1.07 (0.34–3.35) 0.74 (0.20–2.68) 0.645
Wolbachia
Negative 35 (8.3) 386 (91.7) 1 (reference)
Positive 18 (10.5) 154 (89.5) 1.29 (0.71–2.34) 3.51 (1.52–8.12) 0.003∗∗
Cardinium
Negative 47 (9.3) 460 (90.7) 1 (reference)
Positive 6 (7.0) 80 (93.0) 0.73 (0.30–1.77) 4.21 (1.15–15.38) 0.03∗
Wolbachia + Cardinium co-infection
Negative 33 (6.3) 492 (93.7) 1 (reference)
Positive 20 (29.4) 48 (70.6) 6.21 (3.31–11.66) 17.70 (6.53–48.03) <0.001∗∗∗
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Note: ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

a

Six Culicoides species were included in the analysis for individual comparisons.

Fig. 5.

Fig. 5

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An alluvial diagram illustrating the associations between Culicoides species, bacterial endosymbionts, and Leishmania spp.

4. Discussion

The present study investigates the prevalence of two bacterial endosymbionts, Wolbachia and Cardinium, in Culicoides biting midges collected from affected areas of autochthonous leishmaniasis in northern and southern Thailand. The overall Wolbachia infection rate was 29.0%, indicating a moderately high prevalence in natural populations. This finding contrasts with previous reports showing lower Wolbachia prevalence rates in Culicoides species collected in Spain (6.2%) (Pagès et al., 2017) and Australia (6.4%) (Mee et al., 2015). The prevalences of Wolbachia detected in this study are closely aligned with those previously observed in the USA (22.0%) (Covey et al., 2020). Wolbachia infection was detected in multiple Culicoides species, including C. mahasarakamense, C. peregrinus, C. oxystoma, C. guttifer, C. jacosoni, and C. orientalis. These species have also been previously identified as Leishmania-positive in areas with a documented history of leishmaniasis, as reported by Sunantaraporn et al. (2021), Songumpai et al. (2022), Ampol et al. (2024), and Promrangsee et al. (2024). As demonstrated in a previous report by Pagès et al. (2017), Wolbachia has been identified in C. imicola, C. obsoletus (s.l.), and C. pulicaris (s.l.), which are considered Palaearctic vectors of bluetongue and Schmallenberg diseases. Low-level Wolbachia infection was identified in 10 Culicoides species from Australia (Mee et al., 2015). The Wolbachia infection was identified in C. crepuscularis, C. debilipalpis, C. edeni, C. haematopotus, C. insignis, C. venustus, and C. sonorensis from the USA (Covey et al., 2020).

The 16S rRNA, ftsZ, and wsp genes have been frequently employed as genetic markers for the classification of the eight supergroups (A to H) of Wolbachia (Augustinos et al., 2011; Wang et al., 2014; Glowska et al., 2015). The most common Wolbachia supergroups in arthropods are A and B (Breeuwer et al., 1992; Werren et al., 1995). Phylogenetic analysis revealed that the Wolbachia isolates detected in Culicoides spp. in the present study were genetically clustered into supergroups A, B, and F. The majority of our samples were phylogenetically separated into multiple clades within Supergroup B. Wolbachia sequences previously detected in Australian and Spanish Culicoides spp. were segregated into two distinct clades within Supergroup B (Mee et al., 2015; Pagès et al., 2017). The Wolbachia Supergroup F has been identified in filarial nematodes, cat fleas, termites, and bed bugs (Driscoll et al., 2020; Zimmermann et al., 2021; Chebbah et al., 2023; Sinha et al., 2023). It was a notable finding of this study that some Wolbachia isolates from Culicoides spp. were detected in the Supergroup F. It is noteworthy that co-infection with supergroups B and F was observed in C. guttifer in Songkhla.

The overall infection rate of Cardinium was 14.5%, detected across multiple Culicoides species, indicating a moderate prevalence in this study. Previous investigations have reported varying prevalences of Cardinium infection in different regions, including Australia (25.5%), Japan (16.0%), and Spain (1.1%) (Nakamura et al., 2009; Mee et al., 2015; Pagès et al., 2017). Previously, several reports have identified the Cardinium endosymbiont in relevant Palaearctic Culicoides species: C. imicola, C. pulicaris, and C. punctatus. A study by Pagès et al. (2017) has demonstrated the presence of Cardinium infection in a wide range of C. obsoletus (s.l.), C. festivipennis, C. flavipulicaris, C. haranti, C. maritimus, C. minutissimus, C. newsteadi, C. punctatus, and C. sahariensis. Cardinium infections were observed in several vector species, including C. imicola and the Pulicaris species complex (C. pulicaris, C. bysta, C. newsteadi, and C. punctatus) (Pilgrim et al., 2021). The most prevalent Culicoides species infected with Cardinium was C. guttifer (94.87%; 74/78), followed by C. mahasarakhamense (78.57%; 33/42). High Cardinium prevalence in Culicoides species is likely influenced by several factors. First, species-specific variation plays an important role, as previous studies have shown that certain species, such as C. imicola and members of the C. pulicaris complex, often exhibit moderate to high infection rates, whereas others, including the C. obsoletus species group, may display low or undetectable levels of infection (Pilgrim et al., 2021). Three Culicoides species (C. arakawae, C. ohmorii, and C. peregrinus) exhibited a 100% Cardinium infection rate, as all examined individuals tested positive (Nakamura et al., 2009). Secondly, environmental factors have been implicated, with evidence suggesting that conditions such as land surface temperature are strongly associated with infection prevalence. In particular, higher temperatures during early developmental stages may promote increased infection frequencies in adult midges (Morag et al., 2012, 2013). Thirdly, habitat-related differences may also contribute to variation in prevalence. For instance, Culicoides populations collected near livestock premises have been reported to exhibit significantly higher Cardinium infection rates compared to those from more natural habitats, indicating that host-associated ecological factors may influence the distribution and maintenance of this endosymbiont (Pagès et al., 2017). In this study, the observed Cardinium prevalence may be shaped by a combination of habitat-related and species-specific factors. Notably, C. guttifer and C. mahasarakhamense were both collected from livestock-associated environments, specifically chicken coops (Jomkumsing et al., 2021; Sunantaraporn et al., 2022; Tepboonrueng et al., 2026), which may promote higher infection frequencies due to shared ecological conditions and increased opportunities for symbiont persistence and transmission. Accordingly, these species represent the most suitable candidates for examining the influence of Cardinium on vectorial capacity (Pilgrim et al., 2021). The present study demonstrates the occurrence of co-infections with Wolbachia and Cardinium in several Culicoides species, with a moderate prevalence of 11.47%. Previous studies have reported substantially higher co-infection rates in other insect groups, such as planthoppers, where a prevalence of 32.7% has been observed (Nakamura et al., 2009). However, to date, co-infection of these endosymbionts has not been reported in Culicoides biting midges. A phylogenetic analysis of Cardinium based on 16S rRNA sequences revealed the existence of seven distinct groups: A, B, C, D, E, F, and G (Tarlachkov et al., 2023). In the present study, the majority of Cardinium-positive samples were grouped in Group C, which contained Cardinium endosymbionts detected in diverse Culicoides species from regions worldwide. Furthermore, the 16S rRNA sequences of the Cardinium strains were genetically grouped into two subgroups within Group C (C1 and C2). Furthermore, this is the first report of Cardinium being classified into Group A in Culicoides spp. A previous report has indicated that Cardinium Group A has been identified in several insect groups, including hemipterans, hymenopterans, opilionids, mites, and spiders (Noel and Atibalentja, 2006).

This study identified Wolbachia putative strains using a TCS haplotype network, which illustrates the genealogical relationships among strains detected in host populations. Genetic variation (mutations) in Wolbachia surface protein (wsp) gene was analyzed to determine the relatedness among strains, infer ancestral lineages, and assess their distribution across different host species. The Wolbachia endosymbiont networks in Culicoides biting midges were grouped according to the Wolbachia putative strains previously described by Zhou et al. (1998) based on wsp sequences. In this study, three Wolbachia putative strains were identified as wKerlac (Wolbachia of Kerria lacca), wBeva_B (Wolbachia of Bicyclus evadne), and wCauA (Wolbachia of Cadra cautella). The effects of Wolbachia infection are diverse and include inhibiting arbovirus, malaria, and filarial nematode development, as well as inducing various reproductive abnormalities (Stouthamer et al., 1999). The results of this study suggest that the putative Wolbachia strains identified may play a role in determining sexual characteristics and the sex ratio of insect populations (Sasaki et al., 2005; Vashishtha et al., 2011; Duplouy and Brattström, 2018). In the present study, Wolbachia infection was classified solely based on the wsp gene. The separation of the clades into distinct Wolbachia Supergroup B sequences indicated a high degree of divergence, consistent with the expectation that the strain would be novel. To accurately characterize the novel Wolbachia strain, a multi-locus sequence typing (MLST) analysis should be conducted using the gatB, coxA, hcpA, fbpA, and ftsZ genes (Baldo et al., 2006). This approach will facilitate a more precise delineation of the Wolbachia strains.

Detection of Leishmania parasites was carried out using an ITS1-PCR assay, revealing an overall prevalence of 8.94%. Although Leishmania DNA was identified using this molecular approach, these results do not confirm vector competence. A key limitation of PCR-based detection is its inability to distinguish between viable parasites and residual DNA originating from recent blood meals. Therefore, further studies, such as parasite isolation, cultivation, and experimental infection assays involving Leishmania and their suspected vectors, are necessary to evaluate their potential for transmission.

This study demonstrated the co-occurrence of Leishmania parasites and endosymbionts, exhibiting distinct patterns of Wolbachia and Cardinium infection. Additionally, it revealed the presence of double infections with Wolbachia and Cardinium. A research publication by Zhao et al. (2013) suggested that multiple infections of Cardinium and Wolbachia affect host age through a combination of CI shared in the spider mite (Tetranychus phaselus). Nevertheless, the function of Cardinium infection in arthropod fitness remains uncertain. In Culicoides biting midges, Cardinium infection in C. imicola did not affect the survival rate under laboratory conditions (Morag et al., 2013). The presence of Wolbachia supergroups A and B in sympatric infections was identified in Leishmania-positive samples. However, Wolbachia Supergroup F and co-infection with any strains were not identified in Leishmania-positive samples. Azpurua et al. (2010) demonstrated the occurrence of Leishmania and Wolbachia in Lutzomyia trapidoi. In contrast, previous reports of Wolbachia-positive sand flies and Leishmania DNA have demonstrated the absence of co-infection between the two organisms in sand flies (Lozano-Sardaneta et al., 2022). Although the impact of Wolbachia on pathogen development, including viruses, filarial nematodes, and malaria, has been examined in various insect species, there is currently no evidence of Wolbachia infection or a study of host-symbiont-pathogen interactions, particularly in Leishmania parasites and their bacteria in biting midges.

The present investigation demonstrates that endosymbiotic bacteria, including single infections with Wolbachia and Cardinium, as well as co-infection with both, are significantly associated with Leishmania DNA detection. We hypothesized that the presence of endosymbionts would be associated with negative detection of Leishmania DNA. However, both single and co-infections with Wolbachia and Cardinium were detected in samples that were either positive or negative for Leishmania DNA. These findings indicate no clear association between endosymbiont presence and Leishmania DNA detection in Culicoides spp. These findings highlight the complexity of tripartite associations among hosts, symbionts, and pathogens (Schinkel et al., 2024). Further experimental and longitudinal studies are required to clarify the mechanical basis of these relationships and to determine whether endosymbionts directly affect Leishmania development or transmission.

5. Conclusions

To the best of our knowledge, this is the first report of Wolbachia and Cardinium endosymbionts in field-collected Culicoides spp. from Leishmania-affected areas in Thailand. In this study, Wolbachia belonging to supergroups A, B, and F were identified, and putative strains including wKerlac, wBeva_B, and wCauA were characterized. Additionally, Cardinium of groups A and C was detected. Co-occurrence of these bacterial endosymbionts with Leishmania parasites was also demonstrated. These findings provide further evidence for the tripartite interactions among hosts, symbionts, and pathogens, with potential implications for vector control strategies. However, the functional roles and properties of Wolbachia and Cardinium in influencing vector fitness and pathogen transmission require further investigation.

Ethical approval

The study protocol was approved by the Animal Research Ethics Committee of Chulalongkorn University Animal Care and Use Protocol (CU-ACUP), Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (COA No. 2391008).

CRediT authorship contribution statement

Sakone Sunantaraporn: Conceptualization, Methodology, Investigation, Data curation, Formal analysis, Validation, Writing – original draft, Writing – review & editing. Pathamet Khositharattanakool: Methodology, Investigation. Puckavadee Somwang: Methodology, Investigation. Pranyu Leemingsawat: Methodology, Validation. Picha Pattrapruettada: Methodology, Data curation. Darlene Ariyaskul: Methodology, Data curation. Tinn Hongboontry: Methodology, Data curation. Chitchanok Cherdchoochart: Methodology, Data curation. Rungfar Boonserm: Methodology, Investigation. Thanapat Pataradool: Methodology, Validation. Padet Siriyasatien: Conceptualization, Project administration, Supervision, Methodology, Data curation, formal analysis, Funding acquisition, Writing – review & editing.

Statement on the use of AI-assisted technologies

During the preparation of this work the authors used Grammarly (https://www.grammarly.com/) to correct grammatical errors and improve readability. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.

Funding

This research project was supported by Chulalongkorn University, the Second Century Fund (C2F).

Declaration of competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.

Acknowledgments

This research was supported by the Second Century Fund (C2F) of Chulalongkorn University, Thailand. The author would like to thank the staff of the Center of Excellence in Vector Biology and Vector-Borne Diseases, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, for providing laboratory facilities and technical assistance.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.crpvbd.2026.100387.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1
mmc1.pdf (234.4KB, pdf)

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary files. The newly generated sequences were submitted to the GenBank database under the accession numbers PX884007-PX884058 (Wolbachia), PX845917-PX845968 (Cardinium), PX856299-PX856349 (L. martiniquensis), and PX856350-PX856357 (L. orientalis).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Multimedia component 1
mmc1.pdf (234.4KB, pdf)

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its supplementary files. The newly generated sequences were submitted to the GenBank database under the accession numbers PX884007-PX884058 (Wolbachia), PX845917-PX845968 (Cardinium), PX856299-PX856349 (L. martiniquensis), and PX856350-PX856357 (L. orientalis).

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