Open Access
Issue
Parasite
Volume 32, 2025
Article Number 2
Number of page(s) 13
DOI https://doi.org/10.1051/parasite/2024082
Published online 22 January 2025

© B. Kyi Soe et al., published by EDP Sciences, 2025

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

Haematophagous arthropods represent a crucial threat due to their role in transmitting life-threatening diseases to wild vertebrates [5]. Nowadays, vector-borne diseases transmitted by the blood-feeding arthropods are of significant global public health concern due to their widespread impact, morbidity, and mortality [30]. Besides human welfare considerations, the arthropod vectors can pose significant challenges for the livestock industry, potentially resulting in significant economic losses [48]. Culicoides biting midges (Diptera: Ceratopogonidae), the most common small blood-feeding insects, are known to transmit viruses, such as Bluetongue virus and Oropouche virus [31, 35].

Regarding parasites, Culicoides biting midges are considered to transmit avian haemosporidians, such as Leucocytozoon spp., Plasmodium spp., and Haemoproteus spp., which are responsible for causing diseases in birds [39, 61]. According to previous research, Culicoides biting midges have been proposed as potential vectors of specific pathogens, with some being linked to the transmission of zoonotic diseases, i.e., leishmaniasis caused by some members of Leishmania parasites in the subgenus Mundinia. For example, experimental infection by Leishmania (M.) orientalis in C. sonorensis has been demonstrated by Chanmol et al. [8], and the capability of biting midges C. sonorensis to establish infection and potential transmission of Leishmania (M.) parasites has been reported by Becvar et al. [3]. Also, C. mahasarakhamense has been reported to contain L. martiniquensis DNA [54]. Next, Leishmania (M.) martiniquensis DNA has been investigated in several Culicoides species, such as C. peregrinus, C. oxystoma, C. mahasarakhamense, C. huffi, C. fordae, and C. fulvus in southern Thailand [50]. In addition, a report on natural infection of C. peregrinus by L. martiniquensis has provided evidence to support the notion of biting midges as a potential vector of leishmaniasis, and two C. peregrinus samples were found to be coinfected with L. martiniquensis and Crithidia sp. [22]. Recently, Leishmania sp. DNA was detected from six Culicoides spp.: C. mahasarakhamense, C. guttifer, C. (Trithecoides) sp., C. jacobsoni, C. oxystoma, and C. orientalis, indicating their important role in the transmission of this zoonotic pathogen [1]. For filarial nematodes, natural infections by Mansonella perstans, M. ozzardi, and M. streptocerca in Culicoides spp., which are responsible for mansonellosis in humans, have been recorded [35]. Also, DNA of Onchocercidae gen. sp. has been detected in C. crepuscularis [31], C. mahasarakhamense [40], and M. perstans in C. milnei [10] through molecular studies, suggesting the potential for these Culicoides species to be a vector of this filarial parasite.

Studies on the parasite infection status and host preference of Culicoides biting midges have become critical to understanding the transmission pathways of zoonotic vector-borne diseases. Determining blood meal remnants in arthropods may outline a comprehensive interaction between ectoparasites and hosts, and their efficiency of pathogen transmission [47]. Culicoides biting midges feed on a wide range of mammals and birds, depending on the abundance and accessibility of hosts [28]. In a previous study, avian DNA was detected in C. mahasarakhamense and C. huffi [54]. In addition, Sunantaraporn et al. [53] reported that not only cow, dog, pig, or avian DNA was found in each engorged midge collected in their study, but also the most prevalent blood meal pattern, mixed host blood DNA (cow and avian), was identified in Culicoides spp. of the subgenus Trithecoides, C. innoxius, C. peregrinus, C. shortti, C. fulvus, C. insignipennis, C. jacobsoni, and C. gemellus. Since the presence of suitable hosts may influence the abundance and distribution of the midges, understanding their habits in endemic areas of diseases, particularly host preference, is beneficial. Thailand is well known for its diverse environment, featuring a variety of ecosystems, such as wetlands, coastal areas, mountainous regions, tropical rainforests, and agricultural lands, which in turn represent a wide range of climate zones. Most farmers in Thailand practice mixed-livestock farming in which cattle, goats, chickens, and dogs are raised, living alongside people in the same residential areas. The occurrence of human blood DNA in several Culicoides species has been reported from several studies in different countries [6, 14, 2326, 46, 49, 57]. Nonetheless, Culicoides are described as opportunistic feeders, and host feeding behavior may be strongly influenced by host availability [35]. For example, Slama et al. [49] have acknowledged that a high percentage of C. imicola blood feeding on humans in central Tunisia can most probably be attributed to the location of the traps in the direct vicinity of human habitats. However, opportunistic host feeding may facilitate pathogen transfer between wild and domestic hosts or even to humans [35].

Further investigation on the host preference of various species of Culicoides biting midges is needed. The aims of the present study, therefore, are to (i) investigate the most dominant biting midge species in selected mixed livestock farming areas in Chiang Mai and Nakhon Si Thammarat provinces, Thailand, (ii) identify blood parasites in the wild-caught biting midges, and (iii) determine host preference via blood meal analysis of the midges. These findings may contribute to an understanding of the role of Culicoides biting midges in the transmission of parasitic infections. Importantly, this knowledge could offer a roadmap to predicting possible vector-parasite specificity, planning future research on prevention, and improving existing control strategies.

Materials and methods

Ethics statement

The design and methodology of this research was approved by the animal research ethics committee of Chulalongkorn University Animal Care and Use Protocol (CU-ACUP) under COA No. 037/2566, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.

Study period, location, and insect trapping

The present study was carried out between February 2024 and July 2024. In this study, three districts (San Sai, Doi Saket, and Hang Dong) in Chiang Mai province were involved as the northern sample collection area, while three districts (Na Bon, Nopphitam, and Tha Sala) in Nakhon Si Thammarat province were included as the southern sample collection area (Table 1, Fig. S1). The two study areas (northern and southern parts of Thailand) are in different climatic zones: Chiang Mai is characterized by a tropical savannah climate, and Nakhon Si Thammarat has a tropical rainforest climate. Annual rainfall is 1,185 mm and 2,381 mm in Chiang Mai and Nakhon Si Thammarat, respectively (https://en.climate-data.org/asia/thailand/). Farming practices in the study areas are categorized as mixed-livestock farming systems, in which people live near their farms. At each location, sampling took place over three days. A total of six trapping localities from the two study areas were selected, according to urban natural environments at the sample collection sites. Following verbal permission from the house owner, insects were collected using light traps (25 W bulb) with ultraviolet (UV) light, as shown in Figure 1. Briefly, at each site, a total of 6 light traps were set up approximately 1.5 m above ground level for three consecutive nights/month. The operating hours of the traps were from 6 pm to 6 am. The collected biting midges were subsequently sent to the laboratory of the Center of Excellence in Vector Biology and Vector-Borne Disease Research Unit, Department of Parasitology, Faculty of Medicine, Chulalongkorn University.

thumbnail Figure 1

Examples of sample collection sites. A: Cattle pen nearby forest; B–D: Mixed-livestock farming with cattle, chickens, and dogs nearby farm owners’ houses; E: Semi-intensive farming practice with cattle grazed nearby pasture.

Table 1

Details of collection sites in Chiang Mai and Nakhon Si Thammarat provinces, Thailand.

Morphological identification, DNA extraction, and molecular identification of midges

Identification of different species of Culicoides was performed. First, males were separated from females. Thereafter, species identification of female specimens was conducted using a taxonomic key, based on morphological characteristics such as head features (including the palp and antenna) and wing patterns [59]. Briefly, the wings of biting midges were examined, and the following information was recorded: size, length, either dark or pale spots on the entire wing with their particular locations, wing color, and situation of the microtrichia. For each species, classification of female insects was carried out based on their physiological stage: parous (empty abdomen with remnants of burgundy pigment), engorged (abdomen full of blood), gravid (abdomen containing eggs), and nulliparous (empty abdomen without blood remnants) [26]. The wings of representative insects were dissected, and images were taken. The remaining insect bodies were subjected to DNA extraction. Genomic DNA was extracted using a genomic DNA extraction kit (Thermo Fisher Scientific Inc., Waltham, MA, USA), according to the manufacturer’s instructions. In this study, 1–30 parous females from each species (depending on the number of collected parous females) were randomly selected and individually used for DNA extraction. To identify Culicoides species through molecular techniques, the primer pair targeting the mitochondrial cytochrome c oxidase subunit 1 gene (COI) was used [13], following the PCR protocol described by Harrup et al. [16].

Molecular identification of Leishmania and trypanosomatids

For Leishmania spp., the internal transcribed spacer 1 (ITS1) [51] and 3′ untranslated region (3′UTR) of Leishmania HSP70-type I (HSP70-I) [19, 44] were used, with DNA sequencing for species identification. For Trypanosoma spp. and Crithidia spp., small subunit ribosomal RNA (SSU rRNA) gene was targeted for amplification with the primer pair reported by Noyes et al. [36], following the PCR protocol described by Srisuton et al. [52] and Kaewmee et al. [22].

Molecular identification of Leucocytozoon spp., Plasmodium spp., and Haemoproteus spp.

In order to identify Leucocytozoon spp., Plasmodium spp., and Haemoproteus spp. DNA, the extracted genomic DNA samples were amplified by nested PCR (nPCR). Briefly, nPCR targeting the cytochrome b (cyt b) gene was conducted using the outer primer pairs described by Hellgren et al. [17]. For nested PCR, the inner primer pairs previously reported by Bensch et al. [4] were used to amplify Plasmodium and Haemoproteus species.

Molecular identification of filarial nematodes

For filarial nematode identification, PCR amplification targeting the mitochondrial 12S ribosomal RNA (12S rRNA) gene was carried out using the primer pairs described by Morales-Hojas et al. [34], following the PCR protocol described by Pramual et al. [40].

Blood-meal molecular analysis

Total genomic DNA was extracted from blood meal remnants from the 29 engorged female Culicoides using a DNA extraction and purification kit (Thermo Fisher Scientific Inc.), as per the manufacturer’s protocol. The extracted genomic DNA samples were stored at −20 °C until molecular identification. The universal vertebrate primers for amplification of the mitochondrial cytochrome b (cyt b) gene were used to identify host blood meal [42] with the following PCR cycling procedure, with some modifications: initial denaturation at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 1 min, followed by final extension at 72 °C for 10 min, with a total volume of 25 μL reaction mixture. The list of the primers used in the present study is shown in Table 2.

Table 2

List of primers used for PCR amplification and sequencing.

DNA sequencing

The amplified products were purified using a GeneJET PCR Purification kit (Thermo Fisher Scientific Inc.) and sent for Sanger sequencing at the sequencing service of Macrogen Inc., Seoul, South Korea. Here, we sequenced DNA from (i) insects that were found to be positive for parasites by molecular methods, and (ii) engorged female insects only.

Phylogenetic analyses

The resulting nucleotide sequences of the mitochondrial cytochrome b (cyt b) gene of blood meal remnants, and cyt b genes of haemosporidian parasites (Leucocytozoon spp. and Plasmodium spp.) were checked using a BLASTn search tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Reference sequences were retrieved from GenBank to conduct phylogenetic analyses in order to determine genetic similarity and diversity. Briefly, multiple sequence alignment was performed with Molecular Evolutionary Genetics Analysis (MEGA), software version 11, and the genetic inference was analyzed by the maximum likelihood method using IQ-TREE software https://iqtree.org/ [27, 45]. A 1,000 repetition bootstrapping test was used to evaluate the confidence of the branching pattern [11], in which the bootstrap values ≥70% were taken as an indication of support [18]. Evolutionary distances were computed using the code to find the best model. The phylogenetic tree was visualized using FigTree, version 1.4 [43].

Results

Culicoides species found in the mixed-livestock farming areas

In the present study, a total of 6,578 Culicoides spp., 170 males and 6,408 females (5,275 parous, 1,092 nulliparous, 12 gravid, and 29 engorged) were collected, comprising 15 species of six subgenera (Hoffmania, Meijerehelea, Remmia, Avaritia, Haemophoructus, and Trithecoides), and two groups (Clavipalpis and Shortti). These species included C. asiana (2.6%), C. innoxius (20.5%), C. mahasarakhamense (13.2%), C. arakawae (15.3%), C. peregrinus (20.9%), C. oxystoma (7.6%), C. palpifer (4.4%), C. huffi (3.1%), C. shortti (2.5%), C. guttifer (2.9%), C. fulvus (2.8%), C. orientalis (1.1%), C. fordae (2.3%), C. insignipennis (0.2%), and Culicoides spp. (unknown) (0.6%) depending upon the characteristics of wing patterns (Table 3). Geographically, C. arakawae 18.7% (716/3829), C. mahasarakhamense 18.1% (692/3829), C. peregrinus 17.5% (672/3829), and C. innoxius 15.6% (599/3829) were the most dominant Culicoides spp. in Chiang Mai, while C. innoxius 27.3% (752/2749) and C. peregrinus 25.6% (705/2749) were the most dominant in Nakhon Si Thammarat; therefore, C. peregrinus and C. innoxius seemed to be widely spread in both the southern and northern parts of Thailand. Characteristics of wing patterns of 15 representative Culicoides species identified from the current study are shown in Figure 2.

thumbnail Figure 2

Characteristics of wing patterns of 15 Culicoides species identified from this study. Scale bar = 200 μm.

Table 3

Total number of Culicoides species captured in Chiang Mai and Nakhon Si Thammarat provinces, Thailand, February–July 2024.

Parasite infection status in the wild-caught biting midges

A total of 738 biting midges (parous) were used to screen for parasite infection status. As per the results, no genomic DNA samples of Leishmania spp., trypanosomatids, and filarial nematodes were detected. Fifteen haemosporidian mitochondrial cyt b sequences from five species of biting midges, i.e., C. arakawae (6.7%), C. mahasarakhamense (46.7%), C. oxystoma (13.3%), C. guttifer (20%), and C. fulvus (13.3%) were investigated. The DNA of Leucocytozoon spp., Le. caulleryi, and P. juxtanucleare parasites was detected in 13, 1, and 1 Culicoides sample(s), respectively. Identified parasite species in selected Culicoides biting midges (parous) collected from Chiang Mai and Nakhon Si Thammarat provinces are shown in Table 4. Resulting haemosporidian sequences were determined for sequence similarity compared to other global sequences. One sequence produced here (accession no. PQ287341) showed high similarity (100.00%) with one sequence identified as P. juxtanucleare (accession No. LC550059), another (accession No. PQ287327) showed 99.81% similarity with one sequence identified as Le. caulleryi (accession No. OK181451), whereas 13 other sequences (accession Nos. PQ287328PQ287340) showed similarities (95.90% to 100.00%) with sequences identified at the genus level, namely Leucocytozoon (Fig. 3).

thumbnail Figure 3

Phylogenetic construction involving 15 sequences from the current study and 56 reference sequences (23 sequences of Plasmodium species and 33 sequences of Leucocytozoon species) retrieved from GenBank. These sequences were analyzed using the maximum likelihood method with the TIM+F+G4 model under 1,000 bootstrap replicates using IQTree software. Tree with branch lengths indicating number of substitutions per site. The sequences from the present study are written in red, whereas the green-dot circles refer to samples from Chiang Mai province (northern) and orange-dot circles refer to samples from Nakhon Si Thammarat province (southern), Thailand.

Table 4

Molecular identification of parasites in selected Culicoides biting midges (Parous) collected from Chiang Mai and Nakhon Si Thammarat provinces, Thailand, February–July 2024.

Host preference of the biting midges

Host blood meal identification was conducted in a total of 29 engorged Culicoides biting midges, including C. peregrinus (n = 7), C. innoxius (n = 6), C. guttifer (n = 6), C. mahasarakhamense (n = 4), C. arakawae (n = 3), C. oxystoma (n = 1), and unknown Culicoides sp. (n = 2; accession Nos. PQ287342PQ287343) (Table 3). Three different vertebrate hosts, i.e., cattle (Bos indicus), chickens (Gallus gallus), and humans (Homo sapiens), were determined as preference hosts. In the study areas, C. peregrinus, C. innoxius, and C. mahasarakhamense fed on cattle with 58.6% (17/29) (n = 17; accession Nos. PQ287344PQ287360), whereas C. arakawae, C. oxystoma, and C. guttifer were found to feed on chickens with 34.5% (10/29) (n = 10; accession Nos. PQ287361PQ287370). Interestingly, we found two unknown Culicoides sp. that fed on humans with 6.9% (2/29) (n = 2; accession Nos. PQ287371PQ287372). None of the samples with parasite DNA showed coinfection, and no Culicoides specimens were detected for mixed-host blood meal DNA. The relationships of the investigated blood meal remnants in seven Culicoides species are shown in Figure 4.

thumbnail Figure 4

Host preference analysis of 29 engorged females of seven different Culicoides species.

Discussion

This study provides information about the most dominant biting midge species in selected mixed-livestock farming areas in Chiang Mai and Nakhon Si Thammarat provinces, Thailand. In addition, the molecular occurrence of parasites in five Culicoides species is shown, and the host preference of wild-caught biting midges was determined. In our study, C. arakawae and C. mahasarakhamense were investigated as the most dominant Culicoides species, followed by C. peregrinus and C. innoxius in Chiang Mai, whereas C. innoxius and C. peregrinus in Nakhon Si Thammarat province. So far, C. mahasarakhamense, C. peregrinus, and C. innoxius have been investigated as the most dominant Culicoides in different areas of eastern, northern, and southern Thailand [14, 39, 40, 53, 54]. Therefore, our study revealed common findings of dominant Culicoides species similar to the above-mentioned studies. Three species of Culicoides, including C. mahasarakhamense, C. peregrinus, and C. innoxius are expected to be widely distributed and are probably the most dominant Culicoides species in Thailand, suggesting that the local transmission capabilities of the biting midges for vector-borne pathogens should be considered. In addition, all the Culicoides samples were collected near mixed-livestock farms with a particular distance (10–30 m) from the farm owner’s house in the current study. Therefore, these dominant species appeared to be capable of pathogen transmission in nature as well [7].

In our samples, no genomic DNA samples of Leishmania spp., trypanosomatids, and filarial nematodes were detected. This may be due to variations in insect species and sampling regions. Despite some Culicoides species facilitating parasite development in laboratory studies, this may not occur under natural conditions where transmission dynamics are influenced by ecological and host-related factors [54]. In our study, DNA of haemosporidians, Leucocytozoon sp., and P. juxtanucleare was detected in C. mahasarakhamense, C. oxystoma, C. guttifer, and C. fulvus, which is similar to previous studies in Thailand [35, 39, 46, 53]. Interestingly, Le. caulleryi DNA was detected for the first time in Thailand inside the biting midge C. arakawae in the present study. This is consistent with a previous study on the experimental infection of C. arakawae by Le. caulleryi to prove that the insect species is a vector of this parasite [60]. In addition to this, Le. caulleryi has been diagnosed by histopathology and molecular examination in chickens in South Korea, as the causal agent of chicken leucocytozoonosis [29]. Therefore, we should be aware of Le. caulleryi infection in chickens in Thailand. Further studies on the prevalence of Le. caulleryi infection in chickens in Thailand should be carried out, and the role of C. arakawae as a natural vector of this parasite should be investigated through the presence of the parasite inside this vector. Although Culex and Ochlerotatus mosquitoes have been described as avian Plasmodium vectors [12], the DNA of P. juxtanucleare has been detected in C. mahasarakhamense, as reported by Pramual et al. [39]. Therefore, the role of C. mahasarakhamense as a vector of Plasmodium species in Thailand should be further investigated.

According to the phylogenetic analyses, four unnamed Leucocytozoon sp. detected in the current study were genetically related to Leucocytozoon sp. found in biting midges (Culicoides sp., C. fulvus, C. oxystoma, C. mahasarakhamense, and C. guttifer), chickens (Gallus gallus), and black flies (Simulium spp.) previously reported in Thailand [21, 38, 41, 53]. In addition, a Le. caulleryi sequence detected in the present study showed a strong genetic relationship with Le. caulleryi extracted from chickens (Gallus gallus) in Thailand [9]. Moreover, P. juxtanucleare detected in C. mahasarakhamense from our study belonged to a clade of P. juxtanucleare that has been reported to infect chickens (Gallus gallus) and biting midges of C. mahasarakhamense, C. guttifer, and C. huffi in Thailand and Myanmar [39, 53, 55, 58]. Further experimental investigation is needed to demonstrate the role of biting midges as competence vectors of the above haemosporidian parasites.

Determining host blood DNA in arthropod vectors is crucial to better understand the transmission of vector-borne pathogens. Globally, molecular identification of blood meals in Culicoides biting midges has been done in Germany [2], Spain [32], France [15], Tunisia [49], Serbia [57], Romania [56], India [25], and the United States [33]. According to the results, C. peregrinus, C. innoxius, and C. mahasarakhamense preferred to feed on cattle, whereas C. arakawae, C. oxystoma, and C. guttifer were found to feed on chickens. Culicoides peregrinus and C. innoxius are known to primarily feed either on cattle or on at least two mammalian hosts, such as cattle and sheep [20, 25]. However, a C. mahasarakhamense with a cattle blood meal was detected for the first time in this study. This may be caused by opportunistic feeding, as C. mahasarakhamense has been reported feeding on chickens, and avian parasites have been identified within it [39]. The presence of a cattle blood meal in C. mahasarakhamense underscores the need for further investigations of their feeding behavior and host specificity. Furthermore, C. arakawae, C. oxystoma, and C. guttifer were found to feed on chickens rather than other hosts. Similarly, C. arakawae is thought to be a common pest on poultry farms, where they primarily feed on chicken blood [20]. In addition, C. oxystoma is reported to show host preference in a wide range of vertebrates [23]. To the best of our knowledge, this is the first study detecting chicken DNA in C. oxystoma in Thailand. Previously, chicken DNA has been detected in several Culicoides species, including C. guttifer in Thailand [14, 20, 54]. In some parts of Thailand, C. oxystoma, C. imicola, C. brevitarsis, C. peregrinus, and C. guttifer have been reported with canine DNA [23, 53], even though canines are considered a less preferred host for biting midges. However, no canine DNA was detected in this study.

Although cattle and chickens have been considered major sources of feeding for biting midges, recently, human DNA has been found in C. oxystoma, C. imicola, and C. brevitarsis in Thailand [14, 23]. Interestingly, in this study, two unknown Culicoides sp. were found to feed on humans, suggesting that more anthropophilic Culicoides species remain to be discovered. Importantly, anthropophilic Culicoides species are likely to inhabit nearby human residences with year-round banana and plantain cultivation, since this environment provides a highly favourable setting for their development [10]. In the current study, the trapping locations were close to residential areas (10–30 m), making it easy for the insects to feed on the available hosts, i.e., cattle (6–15) and chickens (>20– > 60 in some places). However, the study failed to detect any canine DNA. This may be due to the small number of dogs at the sampling sites. Apart from the influence of host availability near the traps, host-switching behavior in Culicoides biting midges can occur if feeding is interrupted, which may play a significant role in their capacity for pathogen transmission [37, 53]. Environmental factors such as temperature, humidity, and the presence of specific vegetation can alter the availability and attractiveness of hosts [54].

Conclusion

Overall, our study provides current knowledge on the most abundant Culicoides biting midges in Chiang Mai and Nakhon Si Thammarat provinces. Out of 15 species, C. mahasarakhamense, C. arakawae, C. oxystoma, C. guttifer, and C. fulvus might be potential vectors of haemosporidians. Three vertebrate host blood DNA types (cattle, chicken, and humans) were detected from seven Culicoides species. Nonetheless, our study highlighted that many Culicoides species remain to be investigated, and cattle are at risk in mixed-livestock farming practices, as they are the targeted hosts of biting midges. Until now, parasitic diseases transmitted by Culicoides biting midges have been overlooked. Understanding the habitual actions of these insects in accepting different geographical regions could be beneficial, and more surveillance research should be performed.

Funding

This study was funded by Thailand Science, Research and Innovation Fund Chulalongkorn University (HEAF67300089) and the Second Century Fund (C2F), Chulalongkorn University.

Conflicts of interest

The authors declare that they have no competing interests.

Authors contributions

N.J. and B.K.S.: study design and methodology; S.K., C.M. and N.J.: field work; S.K., U.P., C.M., N.T. and N.J.: sample dissection, microscopic and molecular identification; B.K.S. and N.J.: data analyses; B.K.S. and N.J.: writing original draft; B.K.S., D.G., M.D.U., P.S., P.A.B. and N.J.: writing - review & editing. All authors read and approved the final version of the manuscript.

Supplementary material

thumbnail Figure S1:

Map showing the 6 sampling sites with geographical boundaries: 3 sites in Chiang Mai; San Sai, Doi Saket, and Hang Dong, and 3 sites in Nakhon Si Thammarat; Nopphitam, Tha Sala, and Na bon. The map was drawn using QGIS version 3.34.

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Cite this article as: Kyi Soe B, Kaewmee S, Mano C, Pattanawong U, Tipparawong N, Siriyasatien P, Gatherer D, Urbaniak MD, Bates PA & Jariyapan N. 2025. Molecular detection of parasites and host preference in wild-caught Culicoides biting midges (Diptera: Ceratopogonidae) in Chiang Mai and Nakhon Si Thammarat Provinces, Thailand. Parasite 32, 2. https://doi.org/10.1051/parasite/2024082.

All Tables

Table 1

Details of collection sites in Chiang Mai and Nakhon Si Thammarat provinces, Thailand.

Table 2

List of primers used for PCR amplification and sequencing.

Table 3

Total number of Culicoides species captured in Chiang Mai and Nakhon Si Thammarat provinces, Thailand, February–July 2024.

Table 4

Molecular identification of parasites in selected Culicoides biting midges (Parous) collected from Chiang Mai and Nakhon Si Thammarat provinces, Thailand, February–July 2024.

All Figures

thumbnail Figure 1

Examples of sample collection sites. A: Cattle pen nearby forest; B–D: Mixed-livestock farming with cattle, chickens, and dogs nearby farm owners’ houses; E: Semi-intensive farming practice with cattle grazed nearby pasture.

In the text
thumbnail Figure 2

Characteristics of wing patterns of 15 Culicoides species identified from this study. Scale bar = 200 μm.

In the text
thumbnail Figure 3

Phylogenetic construction involving 15 sequences from the current study and 56 reference sequences (23 sequences of Plasmodium species and 33 sequences of Leucocytozoon species) retrieved from GenBank. These sequences were analyzed using the maximum likelihood method with the TIM+F+G4 model under 1,000 bootstrap replicates using IQTree software. Tree with branch lengths indicating number of substitutions per site. The sequences from the present study are written in red, whereas the green-dot circles refer to samples from Chiang Mai province (northern) and orange-dot circles refer to samples from Nakhon Si Thammarat province (southern), Thailand.

In the text
thumbnail Figure 4

Host preference analysis of 29 engorged females of seven different Culicoides species.

In the text
thumbnail Figure S1:

Map showing the 6 sampling sites with geographical boundaries: 3 sites in Chiang Mai; San Sai, Doi Saket, and Hang Dong, and 3 sites in Nakhon Si Thammarat; Nopphitam, Tha Sala, and Na bon. The map was drawn using QGIS version 3.34.

In the text

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