Molecular Detection of Filarial and Avian Haemosporidian Parasites in Culicoides Biting Midges (Diptera: Ceratopogonidae) from Laos

Simple Summary

Insect-borne diseases are major threats to human and animal health. Understanding which blood-feeding insects carry parasites is essential for assessing disease risk and developing effective control strategies. In this study, we screened biting midges of the genus Culicoides from Laos for parasites using molecular methods. Overall, 59 of 237 female specimens (25%) were infected with at least one parasite. The detected parasites belonged to the groups Onchocercidae (filarial nematodes), Leucocytozoon spp., Plasmodium sp., and Haemoproteus spp. Several abundant Culicoides species (C. actoni, C. mahasarakhamense, C. huffi, and C. guttifer) are known to feed on livestock, including chickens, cattle, and water buffalo, which are potential hosts of these parasites. Our findings provide the first molecular evidence of parasite infections in Culicoides spp. from Laos and identify several species that may contribute to parasite transmission in livestock. These results provide a foundation for future studies on the epidemiology and control of insect-borne diseases in the country.

Abstract

Species of Culicoides Latreille (Diptera: Ceratopogonidae) are important biting pests and vectors of pathogens affecting humans and animals. However, information on Culicoides–parasite associations in Laos is lacking. We used molecular methods to screen 237 specimens representing 27 Culicoides species from Laos for filarial nematodes, avian haemosporidians, Trypanosoma spp., and Leishmania spp. Overall, 59 specimens representing 19 Culicoides species were positive for at least one parasite. Only filarial nematodes and avian haemosporidians were detected. Filarial parasites were found in four Culicoides species (C. actoni, C. guttifer, C. huffi, and C. mahasarakhamense). Analysis of 12S rRNA sequences revealed at least two filarial taxa, one closely related to Onchocerca lienalis and the other to an unidentified onchocercid previously detected in Culicoides. Avian haemosporidians were more prevalent. Leucocytozoon spp. were detected in 40 specimens from 18 Culicoides species, with cyt b sequences identifying Leucocytozoon caulleryi and an unidentified Leucocytozoon sp., the latter accounting for 39 of the 40 positive specimens. Plasmodium juxtanucleare was detected in 27 specimens (11.4%), whereas Haemoproteus spp. were found in only two specimens (0.8%), which represented two distinct lineages previously reported from wild birds. No Trypanosoma or Leishmania DNA was detected. Culicoides mahasarakhamense and C. huffi had the highest infection frequencies. Their high abundance and documented feeding on livestock, particularly chickens and cattle, suggest that they may play important roles in pathogen transmission and should be prioritized for future vector surveillance in Laos.

1. Introduction

Biting midges of the genus Culicoides Latreille (Diptera: Ceratopogonidae) comprise approximately 1360 described species worldwide [1,2]. Many species are important pests and biological vectors that transmit a wide range of pathogens, including viruses, bacteria, protozoa, and filarial nematodes, to humans and other vertebrates [3]. Major diseases transmitted by Culicoides spp. include Oropouche fever and mansonellosis in humans, bluetongue disease in ruminants, African horse sickness in equids, and leucocytozoonosis in domestic poultry and wild birds [3]. Outbreaks of these diseases can result in substantial economic losses to the livestock industry. For example, bluetongue disease is estimated to cause annual global economic losses of approximately USD 3 billion [4]. Likewise, the 2020 outbreak of African horse sickness in Thailand affected more than 2700 horses and resulted in the deaths of 565 animals [5,6]. These examples highlight the substantial medical, veterinary, and economic importance of Culicoides spp. worldwide.

Birds are also affected by several groups of vector-borne blood parasites, particularly avian haemosporidians of the genera Plasmodium, Haemoproteus, and Leucocytozoon, which are globally distributed and infect a wide diversity of wild and domestic birds. Their prevalence and diversity can vary considerably among host species and geographic regions and can be influenced by host age, environmental conditions, and habitat characteristics [7,8,9,10,11]. Although many infections are subclinical, avian haemosporidians can negatively affect host health and, in some circumstances, cause severe or fatal disease, particularly in susceptible hosts [12] (Yoshimoto et al., 2021). Culicoides spp. are particularly important vectors of Haemoproteus spp. of the subgenus Parahaemoproteus and have also been implicated in the transmission of Leucocytozoon spp. in birds. Consequently, understanding associations between biting midges and avian blood parasites is important for assessing their potential effects on poultry production, captive birds, and wild bird populations.

To date, 71 Culicoides species have been recorded from Laos [13,14]. Among these, adult females of at least four species (C. fulvus Sen and Das Gupta, C. guttifer (Meijere), C. peregrinus Kieffer, and C. oxystoma Kieffer) are recognized vectors of medically and veterinary-important pathogens [3]. Several additional species reported from Laos are considered potential vectors because they have been found carrying pathogenic organisms. In neighboring Thailand, for example, avian haemosporidians (Leucocytozoon spp. and Plasmodium spp.) and Trypanosoma spp. have been detected in C. huffi Causey and C. mahasarakhamense [15,16,17,18]. Bluetongue virus has also been detected in C. fulvus, C. orientalis Macfie, and C. oxystoma [19]. Furthermore, the human pathogenic protozoa Leishmania martiniquensis Desbois, Pratlong, and Dedet and L. orientalis Bates and Jariyapan, which cause leishmaniasis, have been detected in several Culicoides species, including C. actoni Smith, C. arakawae (Arakawa), C. fulvus, C. guttifer, C. innoxius Sen and Das Gupta, C. jacobsoni Macfie, C. mahasarakhamense, C. orientalis, C. oxystoma, C. peregrinus Kieffer, C. shortti Smith and Swaminath, and C. sumatrae Macfie [20,21,22]. Several of these potential vector species, particularly C. peregrinus, C. mahasarakhamense, and C. oxystoma, are abundant in Laos. Blood-meal analyses have shown that these species commonly feed on domestic animals (including chickens), water buffaloes, and cattle, respectively [14], suggesting that their high abundance and feeding preferences may facilitate pathogen transmission to economically important livestock. Despite this potential risk, there is a lack of information on pathogen infections in Culicoides populations from Laos, limiting our understanding of disease ecology and hindering the development of effective surveillance and control strategies.

In the present study, we employed PCR-based molecular screening to detect haemosporidians, Trypanosoma spp., Leishmania spp., and filarial nematodes in Culicoides spp. collected from Laos. Although molecular detection of pathogen DNA in hematophagous insects does not, by itself, demonstrate vector competence [23,24], it provides valuable baseline information for identifying candidate vector species and prioritizing them for subsequent investigations of vector competence and pathogen transmission [25]. Because some closely related Culicoides species are morphologically similar and difficult to distinguish based solely on morphological characteristics [16], we used an integrative approach combining morphological and molecular methods for species identification.

2. Materials and Methods

2.1. Culicoides Specimens and Species Identification

Female Culicoides specimens collected during our previous survey of biting midges in Laos [14] were used for molecular screening of parasites. The specimens were collected between August 2024 and October 2025 from six provinces of Laos: Vientiane, Borikhamxai, Houaphan, Khammouan, Xaisomboun, and Xiangkhoang (Figure 1). Sampling was conducted during this period, which encompassed the rainy seasons, when Culicoides populations are generally more abundant, thereby increasing the likelihood of obtaining sufficient specimens for parasite screening. Detailed information on sampling methods and collection sites has been reported previously [14]. We included all Culicoides species for which female specimens were available from our collections. For species collected at more than one sampling location, specimens from each available location were selected to provide broader geographic representation. Specimens were initially identified morphologically using the taxonomic keys and species descriptions of Culicoides spp. from Laos [13], Southeast Asia [26], and the Culicoides spp. Wing Atlas [27]. The morphological parts of the specimens (heads, wings, and legs) retained for species identification were preserved in 80% ethanol and stored at −20 °C in the Insect Biodiversity Laboratory, Department of Biology, Faculty of Science, Mahasarakham University, Thailand, for future reference and study. For specimens that tested positive for parasites, species identifications were further confirmed by DNA barcoding of the mitochondrial cytochrome c oxidase subunit I (COI) gene.

Figure 1.

Map showing the geographical location of Laos (white area in the top panel) and the Culicoides sampling areas in six provinces of Laos (bottom panel). The map of Laos was retrieved from the Humanitarian Data Exchange (HDX) (https://data.humdata.org/dataset/cod-ab-lao) (accessed on 18 March 2026), and topographic data were obtained from the EarthEnv project (https://www.earthenv.org/topography) (accessed on 18 March 2026).

2.2. DNA Extraction, Parasite Detection, and Sequencing

DNA was extracted from the whole individual adult female specimens using the GF-1 Nucleic Acid Extraction Kit (Vivantis Technologies Sdn. Bhd., Subang Jaya, Malaysia) according to the manufacturer’s instructions. Extracted DNA was stored at −20 °C until further analysis. For molecular confirmation of Culicoides species, a fragment of the mitochondrial COI gene was amplified using the universal primers LCO1490 and HCO2198 [28] following the PCR protocol described by Tangkawanit et al. [29].

Filarial parasites were detected by amplifying an approximately 550 bp fragment of the 12S rRNA gene using the primers 12SOvC and 12SOvB with PCR conditions described by Morales-Hojas et al. [30]. DNA extracted from a filarial parasite previously detected in Culicoides sp. from Thailand [17] was used as a positive control. Haemosporidian parasites were screened using the nested PCR protocol of Hellgren et al. [31]. The first-round PCR employed the primers HaemNFI and HaemNR3 to amplify a fragment of the mitochondrial cytochrome b (cyt b) gene. The second-round PCR used the primers HaemFL and HaemR2L for the specific detection of Leucocytozoon spp., whereas the primers HaemF and HaemR2 [32] were used to detect Plasmodium and Haemoproteus. PCR conditions followed Jumpato et al. [33]. DNA extracted from haemosporidian parasites previously detected in black flies (Simulium spp.) from Thailand [27] was used as positive controls. Detection of Leishmania was performed by amplifying an approximately 379 bp fragment of the internal transcribed spacer 1 (ITS1) region of the ribosomal RNA gene using the primers LeF and LeR [28] under the PCR conditions described by Spanakos et al. [34]. Trypanosoma was detected using the primers S-762 and S-763 [35], which amplify an approximately 2000 bp fragment of the small subunit ribosomal RNA (SSU rRNA) gene. PCR amplification followed the protocol described by Thaijarern et al. [36]. DNA extracted from Trypanosoma sp. previously detected in Simulium spp. from Thailand [36] was used as positive controls. For all PCR reactions, a negative control containing sterile distilled water instead of template DNA was included to monitor for potential contamination.

PCR products were visualized by electrophoresis on 1% agarose gels stained with Novel Juice (GeneDirex, Taoyuan, Taiwan, China). Successfully amplified products were purified using the GF-1 AmbiClean (Gel & PCR) Kit (Vivantis Technologies Sdn. Bhd., Subang Jaya, Malaysia) and sequenced bidirectionally at ATCG Company Limited (Thailand Science Park, Pathum Thani Province, Thailand) using the same primers employed for PCR amplification.

2.3. Data Analysis

Sequence chromatograms (Supplementary Data S1) were inspected manually and edited using the Edit/View Sequence function in MEGA version X [37]. A total of 24 COI sequences of Culicoides spp. were generated in this study (GenBank accession nos. PZ798657-PZ798680). In addition, six 12S rRNA sequences of filarial parasites (GenBank accession nos. PZ815588-PZ815593) and 69 cyt b sequences of haemosporidian parasites (GenBank accession nos. PZ809999-PZ810067) were obtained. No Leishmania or Trypanosoma DNA was detected in any of the Culicoides specimens examined.

Species identification of Culicoides spp. based on COI barcodes was performed using the Identification Engine of the Barcode of Life Data System (BOLD database (https://boldsystems.org/), employing the Animal Species-Level Library (Public + Private) and the Rapid Species Search option [38]. Parasite sequences were identified by comparison with sequences available in GenBank using the Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (accessed on 3 March 2026). Phylogenetic analyses were performed using both Neighbor-Joining (NJ) and Maximum Likelihood (ML) methods implemented in MEGA version X [37] to determine the genetic relationships of parasite sequences detected in Culicoides spp. from Laos with reference sequences retrieved from GenBank based on BLAST searches.

For filarial parasites, a total of 33 sequences, including the six sequences generated in this study, were included in the phylogenetic analyses. Haemosporidian parasites were analyzed separately for Leucocytozoon spp. and Haemoproteus spp. The analyses included 40 and 2 sequences generated in this study, together with 40 and 30 reference sequences retrieved from GenBank for Leucocytozoon spp. and Haemoproteus spp., respectively. Phylogenetic analyses were not performed for Plasmodium because all detected in Culicoides spp. from Laos were P. juxtanucleare.

Neighbor-Joining trees were constructed using the Kimura two-parameter (K2P) model, and node support was assessed with 1000 bootstrap replicates. Maximum Likelihood analyses were performed using the General Time Reversible model with a proportion of invariant sites and gamma-distributed rate variation among sites (GTR + I + G). Branch support was estimated using the ultrafast bootstrap method with 1000 replicates.

3. Results

3.1. DNA Barcoding of Culicoides Species Positive for Parasites

A total of 237 adult female specimens representing 27 Culicoides species were included in this study (Table 1). In total, 59 Culicoides specimens tested positive for at least one parasite (Table 1). COI sequences of 25 specimens of Culicoides from Laos were reported previously [8], whereas 24 additional sequences (GenBank accession nos. PZ798657-PZ798680) were generated in the present study. Identification using the Barcode of Life Data System (BOLD) showed that 21 of the 24 newly generated sequences were congruent with morphological identification and included C. huffi (n = 11), C. mahasarakhamense (n = 9), and C. peregrinus (n = 1). Three specimens showed discrepancies between morphological and molecular identifications. Two specimens morphologically identified as C. mahasarakhamense were assigned by DNA barcoding to C. arakawae and C. thurmanae. Likewise, one specimen morphologically identified as C. huffi was identified as C. similis based on its COI sequence.

Table 1.

Number of female specimens of each Culicoides species and parasites detected in Laos.

Subgenus/Species	n (Bloodfed)	Filarial Parasite (n)	Heamosporidian Parasite (n)
			Haemoproteus	Leucocytozoon	Plasmodium
Avaritia Fox
C. actoni Smith	18 (11)	Onchocerca sp. (1)	-	Leucocytozoon sp. (1)	-
C. brevipalpis Delfinado	3 (2)	-	-	Leucocytozoon sp. (1)	-
C. fulvus Sen & Das Gupta	5 (0)	-	-	Leucocytozoon sp. (1)	-
C. hui Wirth & Das Gipta	2 (0)	-	-	-	-
C. jacobsoni Macfie	2 (0)	-	-	-	-
C. orientalis Macfie	5 (1)	-	-	Leucocytozoon sp. (1)	-
Hoffmania Fox
C. innoxius Sen & Das Gupta	1 (1)	-	-	-	-
C. peregrinus Kieffer	39 (36)	-	-	Leucocytozoon sp. (1)	P. juxtanucleare (1)
C. sumatrae Macfie	6 (0)	-	-	Leucocytozoon sp. (2)	-
Meijerehelea Wirth & Hubert
C. arakawae (Arakawa)	2 (1)	-	-	Leucocytozoon sp. (1)	P. juxtanucleare (2)
C. guttifer de Meijere	7 (5)	Onchocerca sp. (2)	-	Leucocytozoon sp. (1)	P. juxtanucleare (4)
C. mahasarakhamense Pramual et al.	25 (16)	Onchocerca sp. (1)	-	Leucocytozoon sp. (9)	P. juxtanucleare (10)
				L. caulleryi (1)
Monoculicoides Khalaf
C. homotomus Khalaf	6 (0)	-	-	-	-
Remmia Glukhova
C. oxystoma Kieffer	17 (12)	-	-	-	-
Trithecoides Wirth & Hubert
C. flavescens Macfie	18 (0)	-	-	Leucocytozoon sp. (2)	-
C. fordae Lee	3 (0)	-	-	-	-
C. palpifer Das Gupta & Ghosh	9 (1)	-	-	Leucocytozoon sp. (2)	-
C. parahumeralis Wirth & Hubert	10 (3)	-	-	Leucocytozoon sp. (1)	-
Subgenus unplaced, Clavipalpis species group
C. clavipalpis Mukerji	4 (3)	-	-	Leucocytozoon sp. (2)	P. juxtanucleare (1)
C. huffi Causey	26 (13)	Onchocerca sp. (2)	-	Leucocytozoon sp. (7)	P. juxtanucleare (9)
C. similis Carter, Ingram & Macfie	1 (1)	-	Haemoproteus sp. (1)	-	-
Subgenus unplaced, Ornatus species group
C. pampangensis Delfinado	2 (0)	-	-	Leucocytozoon sp. (1)	-
Subgenus unplaced, Shermani species group
C. geminus Macfie	12 (7)	-	-	-	-
C. thurmanae Wirth & Hubert	1 (0)	-	Haemoproteus sp. (1)	Leucocytozoon sp. (1)	-
Subgenus unplaced, Shortti species group
C. shortti Smith & Swaminath	20 (12)	-	-	Leucocytozoon sp. (2)	-
Unknown species group
Culicoides sp. 1	3 (0)	-	-	Leucocytozoon sp. (2)	-
Culicoides sp. 2	1 (1)	-	-	Leucocytozoon sp. (1)	-
Total	237 (126)	Onchocerca sp. (6)	Haemoproteus sp. (2)	Leucocytozoon sp. (39) L. caulleryi (1)	P. juxtanuleare (27)

Phylogenetic analyses based on NJ and ML methods resolved similar tree topologies; therefore, only the latter is presented (Figure 2). All species are monophyletic with strong bootstrap support (>99%) except for C. palpifer, as one specimen separated from the main clade.

Figure 2.

Maximum likelihood tree based on 102 mitochondrial cytochrome oxidase I gene sequences, including 59 from Laos (24 generated in this study (blue)), that were infected with parasites (bold), representing 21 Culicoides species. For each species, the country of origin and NCBI GenBank accession number are provided. Bootstrap values for ML and NJ analyses are shown above or near the branches.

3.2. Filarial Parasites in Culicoides spp. from Laos

Filarial parasites were detected in six specimens representing four Culicoides species: C. actoni (n = 1), C. guttifer (n = 2), C. huffi (n = 2), and C. mahasarakhamense (n = 1) (Table 2). Comparison of the 12S rRNA sequences with those available in GenBank revealed that five sequences (one from C. mahasarakhamense and two each from C. guttifer and C. huffi) shared >99% sequence identity with an unidentified Onchocercidae previously reported from C. mahasarakhamense in northeastern Thailand [11]. The remaining sequence, detected in C. actoni, was most closely related (98% sequence identity) to Onchocerca lienalis (GenBank accession no. AY462925) from cattle in the UK (Table 2).

Table 2.

The top hit sequence from the NCBI GenBank BLAST search results for the 12S rRNA sequences of filarial parasites detected in Culicoides species from Laos.

Culicoides Species	n	Top Hit (% Similarity)	Accession Number	Host	Country
C. actoni	1	Onchocerca lienalis (98%)	AY462925	Cattle	United Kingdom
C. guttifer	2	Onchocercidae sp. (99–100%)	OQ658729	C. mahasarakhamense	Thailand
C. huffi	2	Onchocercidae sp. (99%)	OQ658729	C. mahasarakhamense	Thailand
C. mahasarakhamense	1	Onchocercidae sp. (99%)	OQ658729	C. mahasarakhamense	Thailand

Neighbor-Joining (NJ) and Maximum Likelihood (ML) analyses produced similar phylogenetic topologies; therefore, only the ML tree is presented (Figure 3). The phylogeny resolved two major clades. Clade I comprised Onchocerca species, including O. lienalis, O. gutturosa, O. fasciata, O. jakutensis, O. gibsoni, O. ochengi, O. volvulus, O. lupi, O. flexuosa, and Onchocerca sp. The filarial parasite detected in C. actoni was placed within this clade and clustered with O. lienalis from several geographic regions. Clade II consisted of unidentified Onchocercidae detected in five specimens from Laos (two each from C. guttifer and C. huffi and one from C. mahasarakhamense), which formed a strongly supported cluster (98% bootstrap support) with unidentified Onchocercidae previously detected in C. mahasarakhamense from Thailand. Additional members of this clade included unidentified Onchocercidae reported from Culex spp. in Germany and Senegal, Culex pipiens in Hungary, and Filarioidea sp. from Aegolius acadicus in Canada.

Figure 3.

Maximum likelihood tree based on the mitochondrial 12S rRNA gene sequences of six filarial parasites detected in Culicoides species in Laos (blue) and closely related sequences retrieved from GenBank. Bootstrap values (1000 replicates) for ML and NJ are above the branches. Names of vertebrate hosts or invertebrate vectors and country of origin (if available in GenBank) are listed following the accession number.

3.3. Haemosporidian Parasites in Culicoides spp. from Laos

Haemosporidian parasites were detected in 55 specimens representing 21 Culicoides spp. The most frequently detected parasites were Leucocytozoon spp. (40 of 55 positive specimens), followed by Plasmodium spp. (27 of 55), whereas Haemoproteus spp. were detected in only two specimens. Mixed infections were detected in 14 specimens, including 13 coinfections with Leucocytozoon spp. and Plasmodium spp. and one coinfection with Leucocytozoon sp. and Haemoproteus sp.

3.3.1. Leucocytozoon spp.

Leucocytozoon spp. were the most frequently detected parasite, occurring in 40 specimens representing 19 Culicoides species (Table 1 and Table 3). Culicoides mahasarakhamense had the highest number of positive specimens (n = 10), followed by C. huffi (n = 7). The remaining species each contained one or two infected individuals. Sequence comparisons of the cyt b gene showed that 39 of the 40 sequences shared >98% identity with unidentified Leucocytozoon sp. previously detected in domestic chickens (Gallus gallus) from Malaysia and Culicoides sp. from Thailand (Table 3). The remaining sequence, detected in C. mahasarakhamense, shared >99% identity with Leucocytozoon caulleryi previously reported from chickens in Thailand. NJ and ML analyses produced similar phylogenetic trees; therefore, only the ML tree is shown (Figure 4). Two major clades were recovered. Most Leucocytozoon sequences from Laos clustered with unidentified Leucocytozoon sp. previously reported from chickens and hematophagous insects, including black flies (Simulium) and biting midges, from neighboring countries. The second clade consisted of L. caulleryi detected in C. mahasarakhamense together with reference sequences from chickens in Southeast Asia.

Table 3.

The top hit sequence from the NCBI GenBank BLAST search results for the cyt b sequences of Leucocytozoon detected in Culicoides species from Laos.

Culicoides Species	n	Top Hit (% Similarity)	Accession Number	Host	Country
C. actoni	1	Leucocytozoon sp. (99%)	KT290945	Chicken	Malaysia
C. arakawae	1	Leucocytozoon sp. (99%)	KT290945	Chicken	Malaysia
C. brevipalpis	1	Leucocytozoon sp. (99%)	KT290945	Chicken	Malaysia
C. clavipalpis	2	Leucocytozoon sp. (99%)	KT290945	Chicken	Malaysia
C. flavescens	2	Leucocytozoon sp. (99%)	KT290945	Chicken	Malaysia
C. fulvus	1	Leucocytozoon sp. (98%)	KT290945	Chicken	Malaysia
C. guttifer	1	Leucocytozoon sp. (99%)	KT290939	Chicken	Malaysia
C. huffi	1	Leucocytozoon sp. (99%)	KT290939	Chicken	Malaysia
C. huffi	4	Leucocytozoon sp. (99%)	KT290945	Chicken	Malaysia
C. huffi	2	Leucocytozoon sp. (99%)	OP227015	Culicoides sp.	Thailand
C. mahasarakhamense	9	Leucocytozoon sp. (99–100%)	KT290939, KT290944, KT290945, OP227015	Chicken, Culicoides sp.	Malaysia, Thailand
C. mahasarakhamense	1	Leucocytozoon caulleryi (99%)	LC713400	Chicken	Thailand
C. orientalis	1	Leucocytozoon sp. (99%)	KT290945	Chicken	Malaysia
C. palpifer	2	Leucocytozoon sp. (99%)	KT290945	Chicken	Malaysia
C. pampangensis	1	Leucocytozoon sp. (99%)	KT290945	Chicken	Malaysia
C. parahumeralis	1	Leucocytozoon sp. (99%)	KT290945	Chicken	Malaysia
C. peregrinus	1	Leucocytozoon sp. (99%)	KT290945	Chicken	Malaysia
C. shortti	2	Leucocytozoon sp. (99%)	KT290945	Chicken	Malaysia
C. sumatrae	2	Leucocytozoon sp. (99%)	KT290945	Chicken	Malaysia
C. thurmanae	1	Leucocytozoon sp. (99%)	KT290945	Chicken	Malaysia
Culicoides sp. 1	2	Leucocytozoon sp. (99%)	KT290945	Chicken	Malaysia
Culicoides sp. 2	1	Leucocytozoon sp. (99%)	KT290945	Chicken	Malaysia

Figure 4.

Maximum likelihood tree based on the mitochondrial cytochrome b gene sequences of 40 Leucocytozoon specimens detected in Culicoides species in Laos (blue) and closely related sequences retrieved from GenBank. Bootstrap values (1000 replicates) for ML and NJ are above the branches. Names of vertebrate hosts or invertebrate vectors and country of origin (if available in GenBank) are listed following the accession number.

3.3.2. Plasmodium

Plasmodium was detected in 27 specimens representing six Culicoides species (Table 4). The highest numbers of positive specimens were found in C. mahasarakhamense (n = 11) and C. huffi (n = 9), followed by C. guttifer (n = 3), C. arakawae (n = 2), C. clavipalpis (n = 1), and C. peregrinus (n = 1). All cyt b sequences were identified as Plasmodium juxtanucleare, sharing >99% sequence identity with isolates previously reported from domestic chickens (Table 4).

Table 4.

The top hit sequence from the NCBI GenBank BLAST search results for the cyt b sequences of Plasmodium detected in Culicoides species from Laos.

Culicoides Species	n	Top Hit (% Similarity) (Accession No.)	Accession Number	Host	Country
C. arakawae	2	P. juxtanucleare (99%)	KU248841	Chicken	Thailand
C. clavipalpis	1	P. juxtanucleare (99%)	KU248842	Chicken	Thailand
C. guttifer	3	P. juxtanucleare (99%)	KU248841	Chicken	Thailand
C. huffi	9	P. juxtanucleare (99%)	KU248841	Chicken	Thailand
C. mahasarakhamense	10	P. juxtanucleare (99%)	KU248842	Chicken	Thailand
	1	P. juxtanucleare (99%)	KU248841	Chicken	Thailand
C. peregrinus	1	P. juxtanucleare (99%)	KU248842	Chicken	Thailand

3.3.3. Haemoproteus spp.

Haemoproteus spp. were detected in two specimens, one each from C. similis and C. thurmanae. The sequence obtained from C. similis was identical to a Haemoproteus lineage previously reported from the spotted munia (Lonchura punctulata) in India [39]. The sequence from C. thurmanae showed 97% similarity to an unidentified Haemoproteus species detected in Culicoides sp. from Germany [40] (Table 5). NJ and ML analyses produced similar phylogenetic relationships; therefore, only the ML tree is presented (Figure 5). The two Haemoproteus spp. sequences from Laos were placed in separate clades, each clustering with lineages previously detected in a variety of avian hosts.

Table 5.

The top hit sequence from the NCBI GenBank BLAST search results for the cyt b sequences of Haemoproteus spp. detected in Culicoides species from Laos.

Culicoides Species	n	Top Hit (% Similarity)	Accession Number	Host	Country
C. similis	1	Haemoproteus sp. (100%)	MF565826	Lonchura punctulata	India
C. thurmanae	1	Haemoproteus sp. (97%)	KF192999	Culicoides sp.	Germany

Figure 5.

Maximum likelihood tree based on the mitochondrial cytochrome b gene sequences of two Haemoproteus spp. specimens detected in Culicoides species in Laos (blue) and closely related sequences retrieved from GenBank. Bootstrap values (1000 replicates) for ML and NJ are above the branches. Names of vertebrate hosts or invertebrate vectors and country of origin (if available in GenBank) are listed following the accession number.

Coinfections involving filarial and haemosporidian parasites were detected in two specimens. One specimen of C. guttifer was simultaneously infected with an unidentified Onchocercidae and Plasmodium juxtanucleare. One specimen of C. mahasarakhamense harbored an unidentified Onchocercidae together with Leucocytozoon sp. and P. juxtanucleare. Coinfection with Leucocytozoon and Plasmodium was the most common pattern, occurring in 13 of the 55 haemosporidian-positive specimens, whereas only one specimen exhibited coinfection with Leucocytozoon and Haemoproteus.

4. Discussion

Accurate species identification is fundamental for downstream biological studies, particularly those involving disease epidemiology and insect vectors [41,42,43]. In this study, we demonstrate that an integrative taxonomic approach combining morphological identification with DNA barcoding is essential for accurately assessing the biodiversity of Culicoides biting midges. Three specimens showed discrepancies between morphological and molecular identification. One specimen identified morphologically as C. huffi was assigned to C. similis based on COI sequences, whereas the other two specimens identified as C. mahasarakhamense were molecularly identified as C. arakawae and C. thurmanae. Such discrepancies may result from insufficient genetic variation in the molecular marker or errors in morphological identification [44,45]. Numerous studies have demonstrated that COI sequences provide reliable species discrimination in Culicoides [16,46,47,48,49]. Therefore, limited resolution of the COI marker is unlikely to explain these discrepancies. Instead, morphological misidentification is the most plausible explanation. Adult Culicoides spp. are extremely small (1–3 mm) and are easily damaged during collection, particularly the wings, which provide many of the diagnostic characters used for species identification. The wing patterns of C. huffi closely resemble those of C. similis. Similarly, wing patterns of C. mahasarakhamense are very similar to C. arakawae and C. thurmanae. Therefore, increasing the likelihood of misidentification. Consequently, C. similis and C. thurmanae are reported here for the first time from Laos. Following the checklist of Howarth [13], which documented 66 Culicoides species in Laos, and our previous study that added five new country records, the discovery of these two additional species increases the known Culicoides fauna of Laos to 73 species.

Biting midges of the genus Culicoides are recognized vectors of a wide range of pathogens, including viruses, protozoa, and filarial nematodes [3]. Our findings indicate that several Culicoides species in Laos are candidate vector species of both filarial nematodes and haemosporidian parasites. Filarial nematodes were detected in four species (C. actoni, C. mahasarakhamense, C. huffi, and C. guttifer). Based on cyt b sequences, these parasites were separated into two major groups. The parasite detected in C. actoni shared more than 98% sequence similarity with several members of the genus Onchocerca. Phylogenetic analysis further placed this parasite within the O. lienalis clade. Additional studies are required to determine its species identity, although it is likely to represent a bovine Onchocerca species circulating among cattle. Onchocerca lienalis is transmitted by black flies, with Simulium jenningsi and S. ornatum recognized as its principal vectors [50]. The detection of a bovine Onchocerca species in C. actoni is consistent with the known feeding preference of this biting midge for water buffalo [14].

The second filarial lineage detected in Laos was genetically most similar to an unidentified onchocercid previously reported from C. mahasarakhamense in Thailand [17]. Phylogenetic analyses showed that parasites detected in C. huffi, C. guttifer, and C. mahasarakhamense from Laos formed a clade together with those unidentified onchocercid parasites previously reported from mosquitoes and C. mahasarakhamense in Thailand. Although the vertebrate hosts remain unknown, the predominantly avian feeding habits of these Culicoides species [14,51] indicate that these parasites may infect birds. More than 160 filarioid species have been reported from birds [52], three of which are transmitted by Culicoides species (C. stilobezzioides and C. travisi) [52,53]. Future studies examining filarial parasites in birds from areas where infected Culicoides occur would help clarify the transmission cycle and host associations of these parasites.

Leucocytozoon spp. were the most frequently detected haemosporidian parasites in Culicoides spp. from Laos. Based on cyt b sequences, two taxa were recognized: Leucocytozoon caulleryi and an unidentified Leucocytozoon species. Among all known Leucocytozoon species, only L. caulleryi has been established to be transmitted by Culicoides, with C. arakawae serving as its principal vector [54,55]. In the present study, L. caulleryi was detected in C. mahasarakhamense, a species closely related to C. arakawae [10]. This finding suggests that C. mahasarakhamense may serve as a potential vector of L. caulleryi in Laos. The unidentified Leucocytozoon lineage has also been detected in Culicoides spp. and black flies (Simuliidae) from both Laos [56] and Thailand [33,57], and occurs frequently in domestic chickens [58]. Nevertheless, detection of parasite DNA in hematophagous insects alone does not demonstrate vector competence or natural transmission [23,24]. Experimental transmission studies are therefore needed to determine whether this parasite is biologically transmitted by Culicoides spp.

Plasmodium sp. was the second most common haemosporidian parasite detected in Culicoides spp. Comparison of cyt b sequences with those available in GenBank indicated that all positive specimens belonged to Plasmodium juxtanucleare, an avian malaria parasite commonly infecting chickens and other birds. Because mosquitoes are the recognized biological vectors of Plasmodium spp., the presence of P. juxtanucleare DNA in Culicoides spp. may well reflect parasite acquisition during blood feeding rather than active transmission. Nevertheless, several Culicoides species, including C. festivipennis, C. kibunensis, C. pictipennis, C. simulator, and C. truncorum, have been experimentally shown to transmit the avian malaria lineage CulPlas1, whereas C. festivipennis can also transmit CulPlas2 [59]. Although our findings do not provide evidence that Culicoides spp. transmit P. juxtanucleare in Laos, they indicate that further investigations into the vector competence of local Culicoides species are warranted.

Species of Haemoproteus are primarily transmitted by Culicoides spp. [60,61]. Two specimens, one C. similis and one C. thurmanae, were infected with unidentified Haemoproteus species. Phylogenetic analyses suggested that these parasites represent two distinct taxa. The Haemoproteus lineage detected in C. similis clustered with unidentified lineages previously detected in a variety of wild birds and shared an identical cyt b sequence with a parasite reported from the spotted munia (Lonchura punctulata) in India [39], indicating that they are likely the same species. In contrast, the parasite detected in C. thurmanae formed a separate clade with unidentified Haemoproteus lineages infecting diverse wild birds. These findings suggest that C. similis and C. thurmanae are candidate vectors of these avian Haemoproteus parasites.

Neither Leishmania nor Trypanosoma was detected in Culicoides specimens investigated in this study. The lack of detection may reflect a low prevalence of these parasites in the sampled areas, the number and geographic coverage of specimens examined, temporal variation in parasite circulation, and limited contact between the sampled Culicoides spp. and infected vertebrate hosts. In addition, although Trypanosoma and Leishmania DNA has been detected in some Culicoides spp. elsewhere [18,20,21,22], the vector competence and epidemiological importance of most Culicoides spp. for these parasites remain uncertain. Thus, our negative results should not be interpreted as evidence that these parasites are absent from Culicoides populations in Laos.

5. Conclusions

This study demonstrates that Culicoides species in Laos harbor a diverse assemblage of parasites, including filarial nematodes of the genus Onchocerca and haemosporidian parasites belonging to the genera Leucocytozoon, Plasmodium, and Haemoproteus. Although molecular detection of parasite DNA in hematophagous insects does not confirm vector competence or natural transmission [23,24], it does provide valuable information for identifying candidate vector species and prioritizing future epidemiological investigations. Widespread and abundant species such as C. mahasarakhamense, C. huffi, and C. guttifer deserve particular attention in future vector competence studies. Our results also highlight the importance of integrating morphological identification with DNA barcoding to achieve accurate species identification. Such an integrative approach is especially critical in studies of vector–parasite associations, where reliable identification of both vectors and parasites forms the foundation for understanding disease transmission.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/insects17090963/s1, Data S1.

Author Contributions

Conceptualization, visualization, R.M., K.I. and P.P.; investigation, R.M., W.W., C.J., S.N., K.I. and I.T.; methodology, R.M., W.W., C.J., S.N. and I.T.; data curation, R.M., W.W. and P.P.; visualization, C.J. and P.P.; writing—original draft, R.M., W.W. and P.P.; writing—review and editing, W.W., C.J., S.N. and I.T.; supervision, K.I. and P.P.; project administration, P.P.; funding acquisition, P.P. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Animal Care and Use Committee, Mahasarakham University (approval number: IACUC-MSU-045-009/2025 on 26 May 2025) for studies involving animals.

Data Availability Statement

The sequences generated in this study were deposited in NCBI GenBank under accession numbers PZ798657-PZ798680, PZ809999-PZ810067, and PZ815588-PZ815593. All other data and materials supporting this article are reported within the paper.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This study was funded by Mahasarakham University.