ABSTRACT

Objective

This study aimed to determine the Culicoides species distributed across different districts of İzmir province, reveal their molecular characterization, and assess their vector potential for the transmission of avian haemosporidian parasites.

Methods

The study material comprised 800 female Culicoides specimens collected from Bergama, Ödemiş, Kemalpaşa, and Foça districts between May and August 2016. Following morphological identification, specimens from each identified species underwent molecular analyses. The mt-COI gene region of genomic DNA isolates from the specimens was amplified by polymerase chain reaction (PCR) and subjected to sequence analyses to reveal their molecular characterization and phylogenetic relationships. Haemosporidian DNA was investigated by nested PCR in the gDNA isolates of head/thorax (HTP) and abdomen pools, constituted from specimens separated by species and location. Molecular characterization of identified parasites was performed using sequence analyses.

Results

Morphological identification revealed that C. circumscriptus (39.4%) and C. imicola (33.8%) were the most common species in the research areas, followed by Culicoides sp. (ERU-Izm-Culi1) (9.1%), C. nubeculosus complex (7.6%), C. obsoletus (4.3%), C. gejgelensis (2.3%), C. punctatus (1.9%), and C. newsteadi (1.8%). A total of 175 polymorphic sites were distributed among the COI sequences of the obtained isolates, leading to the detection of 18 different haplotypes. The highest haplotype diversity was observed in C. circumscriptus, C. punctatus, and C. newsteadi. Phylogenetic analyses clustered the characterized haplotypes of Culicoides species into three major groups. Haemoproteus sp. GAGLA05 and H. minutus TURDUS2 lineages were detected in C. circumscriptus HTP genomic DNA isolates, providing evidence of this species’ vector potential for Haemoproteus lineages in the research area.

Conclusion

This study determined the Culicoides species distributed in the İzmir Region using an integrated morphological and molecular diagnostic approach, providing original data for the molecular epidemiology of these important flies. Furthermore, the results suggest the potential importance of C. circumscriptus in the transmission dynamics of Haemoproteus lineages.

Keywords:

Culicoides, molecular characterization, avian Haemosporidians, vector potential, İzmir

INTRODUCTION

Culicoides Latreille, 1809 (Diptera: Ceratopogonidae), also known as biting midges, are the smallest blood-feeding insects with a body length of 1 to 3 mm among other biting insects (1). The genus Culicoides is important due to their ability to act as biological vectors for pathogens of medical and veterinary significance (2). Several nematode and protozoan species, as well as more than 50 arboviruses, have been isolated from various Culicoides species, and their roles in the transmission of veterinary (1, 3, 4) and human pathogens (5) have been reviewed. Currently, the most important economic impact of Culicoides flies is their association with the transmission of diseases such as Blue Tongue virus (BTV), Epizootic Hemorrhagic Disease virus, Schmallenberg virus, and African Horse Sickness virus. These arboviruses are highly significant for ruminants, deer, and ungulates, and have been reported by the World Organization for Animal Health to cause outbreaks (6, 7).

Culicoides flies are the main vectors of Haemoproteus (Haemosporida) species, which are important parasites of birds and can cause lethal pathology in non-adapted birds (8-11). Despite more than 1360 Culicoides species have been known (12), completeness of sporogonic phase of avian Haemoproteus parasites have been shown only in 13 species which indicates those serve as active vectors (13, 14). In addition to about 150 Haemoproteus species identified from birds (15). In a study conducted in Türkiye to investigate haemosporidian parasites in Culicoides species (16), various Haemoproteus lineages were molecularly characterized in specimens of C. nubeculosus comp., C. riethi, C. circumscriptus, C. submaritimus, C. gejgelensis, C. longipennis, C. festivipennis, and C. newsteadi collected from the Sultan Marshes Region. The data obtained from this study (16) provided information on the vectorial potentials of the relevant Culicoides species.

The first records on Culicoides species in Türkiye were provided by Kieffer (17), and data on species diversity have mainly increased after the 1970s. In a review by Dik (18), the number of Culicoides species in Türkiye was reported as 61. Later, Korkmaz et al. (19) identified new species through a comprehensive four-year research conducted in 51 provinces at 104 sampling stations, increasing the total number of species to 72 (20). Limited research has been conducted on the genetic diversity of Culicoides species in Türkiye. Dik et al. (21) characterized the Culicoides species collected from the Southern and Southeastern Anatolian Regions based on the ribosomal ITS-1 gene region. The characterization of Culicoides species collected from Sultan Marshes (16), the Western Black Sea Region, and Konya (22, 23) was conducted based on the mitochondrial cytochrome oxidase subunit I (mt-COI) gene region, and their phylogenetic relationships were determined. Korkmaz et al. (19) characterized the mt-COI gene region of the C. grisescens and C. chiopterus species, that were reported for the first time in Türkiye.

In the current study, we aimed to determine the Culicoides species prevalent in different districts of İzmir province using conventional morphological and molecular methods, to perform molecular characterization of the isolates, and to reveal their vector potentials for the transmission of avian haemosporidian parasites.

METHODS

Study Area and Collection of Culicoides Specimens

The Culicoides specimens comprising the study material were collected from areas in the districts of Bergama, Ödemiş, Kemalpaşa, and Foça, located within the boundaries of İzmir province, between May and August 2016. “EVS Black CO2 Light Trap, 4W Black Light Tube (Bioquip Inc., 2801BL)” and Onderstepoort light traps were set up approximately 1.5-2 meters high, one hour before sunset, and operated until one hour after sunrise in livestock facilities in the corresponding districts. After sampling, the nets of the light traps were stored in a deep freezer to immobilize the caught flies. Non-target flies were separated macroscopically, and the remaining flies were placed in 70% alcohol and transported to the laboratory on ice packs. From each region, 200 samples were randomly selected and included in the study.

Morphological Identification

Identification of the Culicoides specimens was made based on wing morphology using a combination of established and more recent diagnostic keys (24-26). Examinations were conducted under a digital camera attachment stereomicroscope (Olympus SZX-16). The imaging of the wing morphologies of Culicoides species was performed using CellSens Standard 1.13 software (Olympus) under the camera attachment stereomicroscope.

Genomic DNA Extraction

Morphologically identified Culicoides specimens were separated individually from the collection material preserved in 70% ethyl alcohol for genotyping and phylogenetic analyses. The remaining specimens were pooled in groups of ten (10 samples/pool) to investigate the presence of Haemosporidian parasites, including females, according to species and collection area. To demonstrate the vector potential of Culicoides specimens, the heads/thoraxes (HTP) (sporozoite development) and abdomens (AP) (haemosporidian generations acquired through blood feeding) of each specimen were dissected separately and placed in separate sterile microcentrifuge tubes and pooled accordingly, resulting in 88 pools each for HTP and AP. Genomic DNA isolation was performed using the GeneJET Genomic DNA Purification Kit (Thermo Scientific) following the manufacturer’s instructions.

Amplification of Culicoides Mt-COI and Avian Haemosporidian Mt-CYTB Gene Regions

Genomic DNA extracts isolated from individual female Culicoides specimens were subjected to polymerase chain reaction (PCR) analysis using primers C1-J-1718M (F) (5’-GGAGGATTTGGAAATTGATTAGT-3’) and C1-N-2191M (R) (5’-CAGGTAAAATTAAAATATAAACTTCDGG-3’), which amplify a 523 bp partial fragment of the mt-COI gene of the Culicoides genus (27). The reaction mixture was prepared to a final volume of 25 µL. It contained 10X PCR buffer, 2.5 mM MgCl₂, 100 nM of each primer, 200 µM of each dNTP, 2.5 U Taq DNA polymerase, and 10-20 ng template DNA. The thermal cycling protocol was as follows: Initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, and extension at 72 °C for 30 s, with a final extension step at 72 °C for 7 min.

The genomic DNA isolates obtained from Culicoides HTP and AP were analyzed by Nested PCR targeting a 524 bp portion of the mt-CYTB gene region of haemosporidian parasites. In the first step of the Nested PCR analysis, universal primers HaemNFI (5’-CATATATTAAGAGAATATGGAG-3’) and HaemNR3 (5’-ATAGAAAGATAAGAAATACCATTC-3’) were used for haemosporidians. In the second step, Leucocytozoon-specific primers HaemFL (5’-ATGGTGTTTTAGATACTTACATT-3’) and HaemR2L (5’-CATTATCTGGATGAGATAATGGIGC-3’), and Plasmodium/Haemoproteus-specific primers HaemF (5’-ATGGTGCTTTCGATATATGCATG-3’) and HaemR2 (5’-GCATTATCTGGATGTGATAATGGT-3’) were used (28, 29). The reaction mixture for both primer sets was prepared to a final volume of 20 µL, containing 10X PCR buffer, 2.5 mM MgCl₂, 100 nM of each primer, 200 µM of each dNTP, and 2.5U Taq DNA polymerase. The PCR products were subjected to electrophoresis on a 1.5% agarose gel (10 µL), visualized and analyzed using the CLP Gel Documentation System and Gene Snap from Syngene analysis program (UVP INC Uplant, CA).

Sequence and Phylogenetic Analyses

The amplicons of mt-COI from individual Culicoides specimens and haemosporidian mt-cytb from pools were subjected to gel purification (using the High Pure PCR Product Purification Kit, Roche) for sequence analyses. Sequencing was utilized (Macrogen Europe) bi-directionally with the primers used in the PCR (the second primer pair for haemosporidian mt-CYTB). Chromatograms for forward and reverse reads were processed with DeNovo Assembly in Geneious R10 (30) software to obtain consensus sequences. The molecular characterizations of the identified isolates were achieved by aligning the sequences with the published sequences of the isolates using BLASTn algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the MalAvi Haemosporidian lineage sequence database (http://mbio-serv2.mbioekol.lu.se/Malavi/), and data sets were created for phylogenetic analyses. The mt-COI gene sequences of the identified Culicoides isolates in the study were recorded in the GenBank database with accession numbers MF594384-MF594401; and the mt-CYTB sequences of haemosporidian isolates were recorded with accession numbers MF594402-MF594403. DnaSP 5.10.01 (31) software was used to determine DNA polymorphism and haplotype diversity in the characterized Culicoides isolates. Intra- and inter-specific genetic differences were performed using the Kimura (32) two-parameter (K2P) distance model in MEGA 7 software (33). Bayesian inference (BI) analyses were applied to determine the phylogenetic relationships of Culicoides species and haemosporidian lineages. jModelTest v.0.1.1 (34) was used to determine the best-fit substitution model for sequence evolution, and the models with the lowest Akaike Information Criterion values were used to construct the phylogenetic trees. BI analyses were performed using the MrBayes (35) plugin in Geneious R10 (30) software. Markov Chain Monte Carlo scans were run for 1,100,000 generations with four chains, and tree sampling was performed every 200 generations with the first 100,000 trees discarded as “burn-in”.

Statistical Analysis

Statistical analysis is not required for the data available in this study.

RESULTS

Culicoides Species and Distribution

The distribution of the morphologically identified Culicoides specimens by species, collection area, and month are shown in Table 1. A total of eight Culicoides species were identified in the research areas, and their distribution rates are shown in Figure 1. Generally, the most common species were C. circumscriptus (39.4%) and C. imicola (33.8%), while the species with the lowest distribution rates were C. punctatus (1.9%) and C. newsteadi (1.8%).

A unique Culicoides lineage was identified that exhibited a similar wing morphology to the species in the Silvaticulicoides subgenus (36, 37), and was genetically closer to the species in this group. This lineage was characterized as Culicoides sp. ERU-Izm-Culi1. The specimens of Culicoides sp. ERU-Izm-Culi1 were detected in Bergama and Ödemiş districts in July and August samples (Table 1), and the overall distribution rate was determined to be 9.1% (Figure 2).

Sequence and Phylogenetic Analyses of Culicoides Specimens

Genomic DNA isolates of the specimens identified by morphological identification were amplified with the relevant primers for the mt-COI gene region, and target amplicons of 553 bp were obtained. The morphological diagnosis results were confirmed by aligning the sequences of the relevant isolates with reference isolates in GenBank using the BLASTn algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and the molecular characterization of the isolates was provided. The isolates belonging to the species whose mt-COI gene region sequences and haplotype characterization were determined were given in Table 2 with GenBank accession numbers. One hundred seventy five polymorphic regions were found among the mt-COI sequences of the identified species, revealing 18 different haplotypes, and the average genetic diversity among haplotypes was found to be 25.3%±2.4. The haplotype diversity was determined to be highest in C. circumscriptus, C. punctatus, and C. newsteadi (Table 2). ERU-Izm-Culi1 was found to be genetically closer to species under the Silvaticulicoides subgenus, showing the highest genetic similarity of 92.9% with C. subfasciipennis.

The phylogenetic relationships of the identified isolates in this study along with reported isolates from various regions of Türkiye and the world, were shown in Figure 3. The mt-COI sequences formed three major clusters on the phylogenetic tree, containing monophyletic species, and this resolution was highly supported by posterior probabilities (0.9-1.0). The first major cluster was divided into two subclusters. The first subcluster included C. nubeculosus, C. gejgelensis, C. riethi, Culicoides sp. ERU-Izm-Culi1, and C. subfasciipennis species, and the phylogenetic relationship between the species was moderately supported by posterior probability (0.58). The second subcluster was formed by C. circumscriptus, and the phylogenetic relationship of the isolates belonging to this species was highly supported by posterior probability (1.0). The second major cluster consisted of haplotypes of C. punctatus, C. newsteadi-1, and C. newsteadi-2 species, which were highly supported by posterior probability (0.92). The third major cluster included C. obsoletus and C. imicola species, and the phylogenetic relationship of the isolates belonging to these species was highly supported by posterior probability (1.0).

Identification, Distribution, and Molecular Characterization of Haemosporidian Parasites in Culicoides Specimens

The molecular analysis results of haemosporidian parasites in Culicoides specimens, which were collected during the study and diagnosed by morphological and molecular identifications, are provided in Table 3 for HTP and AP samples. An agarose gel image including the amplicons from some positive samples is given in Figure 4. Of the 88 AP samples examined, 9 (10.22%) were found to be infected with Plasmodium/Haemoproteus species or lineages, and 7 (7.95%) of the 88 HTP samples were also found to be infected with these parasites. No positivity for Leucocytozoon sp. was found in any of the samples.

The haemosporidian parasite species or lineages identified by sequence analysis along with their GenBank accession numbers, are given in Table 4. The GenBank accessions were provided only for isolates identified in the head/thorax pools as an indicator of their vector potential. Of the seven positive isolates from C. circumscriptus HTP, four belonged to the Haemoproteus sp. GAGLA05 lineage, and three belonged to the H. minutus TURDUS2 lineage. All of the positive AP isolates were characterized by Plasmodium lineages, and this result could be related to infected avian blood in their abdomens. The latest finding provided evidence of the widespread occurrence of various Plasmodium lineages, especially the P. relictum SGS1, in avian populations in the study area.

DISCUSSION

The molecular genotyping and DNA barcoding studies of blood-sucking insects with medical and veterinary importance such as mosquitoes, biting midges, and black flies continue to gain importance in many regions of the world (38-41), while genetic studies in Türkiye have been limited (21, 22, 42, 43). The Culicoides genus is important because it contains many species that are biological vectors for numerous pathogens of medical and veterinary importance (1, 4, 5, 36). In the province of İzmir, due to its climatic characteristics (high temperature and humidity), the circulation of BTV is continuous in the field because Culicoides activity continues for a long period of the year. Therefore, it has been reported that BTV disease causes significant losses in the livestock sector in the province of İzmir every year (44). In this study, field surveys conducted in the regions of Bergama, Ödemiş, Kemalpaşa, and Foça districts of İzmir between May and August revealed that those regions were suitable ecosystems for the development of Culicoides species. As a result of the morphological and molecular diagnoses of Culicoides generations collected from the sampling areas, it was determined that C. circumscriptus and C. imicola were the most frequently encountered species during the study period, representing 39.4% and 33.8% of the total collected specimens, respectively. In particular, it is known that C. imicola is one of the most effective vectors of BTV in many geographies, especially in Southern Europe (45, 46), and it may play a role in the transmission dynamics of BTV observed in the research areas in the current study. Studies on populations sampled from Türkiye (47) and Italy (48) have suggested that C. circumscriptus, which was identified as the most frequently encountered species in our study, could also potentially contribute to the transmission dynamics of BTV. In research conducted in the last 10 years (21, 22, 47, 49-54), both species have been reported as widespread in Southern and Southeastern Anatolia, Central and Western Black Sea, Central Anatolia, Western Inner Anatolia, and the Marmara Region in Türkiye. The other species C. nubeculosus complex (7.6%), C. obsoletus (4.3%), C. gejgelensis (2.3%), C. punctatus (1.9%), and C. newsteadi (1.8%), which have a more limited distribution in our study, were found in various regions of Türkiye such as Southern and Southeastern Anatolia, Central and Western Black Sea, Central Anatolia, Western Inner Anatolia, and the Marmara Region (21, 22, 47, 49-54). A unique Culicoides lineage was identified in Bergama and Ödemiş districts. This lineage showed similarities to members of the Silvaticulicoides subgenus based on wing morphology and was genetically closer to the species in this group based on mt-COI sequence characterization. The wing morphologies of these specimens were consistent and symmetrical across all samples. Furthermore, sequence analysis of a representative number of specimens confirmed the genetic similarity within this group. There was no genetic heterogeneity among the mt-COI sequences of samples belonging to this Culicoides lineage, and this group was designated as Culicoides sp. ERU-Izm-Culi1.

Based on the phylogenetic analysis of the mt-COI gene sequences of the isolates identified in our study, the characterized haplotypes were monophyletically placed in three major clusters. Low genetic diversity (0.2±0.2%) was identified between C. nubeculosus ERU-Izm-C.nube1-2 haplotypes within the first sub-cluster of the first major cluster, with ERU-Izm-C.nube1 haplotype being 100% identical to isolates from Sweden (JQ620127) and France (KP969017, 55), and ERU-Izm-C.nube2 haplotype being 100% identical to isolates from France (KP969029, KF178273, 55, 56). The ERU-Izm-C.rie1 haplotype, identified within the C. nubeculosus complex and molecularly characterized as belonging to C. riethi, formed a cluster showing high identity (99.6% and 99.4% respectively) with isolates reported from Sultan Marshes, Türkiye (MF105774, 16) and Sweden (JQ620237). This isolate also showed 99.1% identity with other C. riethi isolates reported from Sultan Marshes, Türkiye (MF105772, MF105773, 16). The C. gejgelensis ERU-Izm-C.gejg1 haplotype showed 100% identity with isolates reported from Sultan Marshes, Türkiye (MF105763, 16) and Slovakia (KJ624086, 37), and formed a cluster together. The mt-COI sequences of the specimens characterized as Culicoides sp. ERU-Izm-C.culi1 showed 100% identity to each other, thus representing the same haplotype. The ERU-Izm-C.culi1 haplotype showed the highest genetic similarity (92.9%) with C. subfasciipennis isolates reported from Sweden (JQ620237, JQ620238), and formed a cluster together. This Culicoides lineage was closer to the species in the Silvaticulicoides subgenus with more than 7% genetic differences than to other Culicoides species. Therefore, the related Culicoides lineage may be an unidentified new species, and further studies are needed for species identification. The isolates of C. circumscriptus in the second sub-cluster of the first major cluster were found to form three separate groups. The ERU-Izm-C.circ1 haplotype showed high identity (average 97.3%) and formed a cluster with isolates from Sweden and Sultan Marshes, Türkiye (JQ620051, MF105754, 16). One isolate each from Spain (HM241854), Slovakia (KJ624071, 37), and Sultan Marshes, Türkiye (MF105755, 16) were included in the second group. A genetic difference of 0.4±0.3% was identified between the ERU-Izm-C.circ2 and ERU-Izm-C.circ3 haplotypes, and these two haplotypes formed the third cluster within the relevant group.

The second major group was formed by haplotypes belonging to C. punctatus, C. newsteadi-1, and C. newsteadi-2 species. The clustering of the C. newsteadi-1 and 2 species were made according to the classification of Pages et al. (25). A genetic difference of 0.3±0.2 was detected between the ERU-Izm-C.punc1-3 haplotypes of C. punctatus, and these haplotypes exhibited mean similarities of 99.5%, 99.7%, 99.4% and 99.6% to C. punctatus isolates from Sultan Marshes, Türkiye (MF105768-70, 16), Western Black Sea (KU754181, 22), England (AM236733, 24), and Denmark (JF766326, 57), respectively. Phylogenetic analysis of C. newsteadi revealed two separate groups, one of which was close to C. punctatus. ERU-Izm-C.news1-3 haplotypes were found in the first phylogenetic group, with the ERU-Izm-C.news1 haplotype grouping with the Western Black Sea isolates (KU754164, KU754167, 22) with 100% identity. The ERU-Izm-C.news2,3 haplotypes showed the highest identity with ERU-Izm-C.news1 and again with the Western Black Sea isolates (KU754164, KU754167, 22) (avg. 99.7%). The characterized isolates were also found to be on average 99.5% identical to the C. newsteadi isolate reported from Italy (AM236746, 24) in the same phylogenetic group. Isolates from the Western Black Sea (KU754168, KU754169, 22) and Sultan Marshes, Türkiye (MF105766, 16) comprised the second phylogenetic group of C. newsteadi, and this group showed an average genetic difference of 19.5±2.7% with the first phylogenetic group of C. newsteadi.

In the third major cluster, C. obsoletus and C. imicola are present. There is a genetic difference of 0.6±0.4% between the ERU-Izm-C.obs1 and ERU-Izm-C.obs2 haplotypes of C. obsoletus. The ERU-Izm-C.obs1 haplotype is identical to the isolate reported from Spain (DQ162814, 57) and is grouped accordingly. The ERU-Izm-C.obs2 haplotype has the highest similarity of 99.8% with the isolates from Spain (DQ162814, 57) and Slovakia (KJ624104, 36). C. imicola has a genetic difference of 0.4±0.3% between the haplotypes ERU-Izm-C.imic1 and ERU-Izm-C.imic2. The ERU-Izm-C.imic1 haplotype is 100% identical to the isolates from Greece (KT027174, 58), while the ERU-Izm-C.imic2 haplotype has a 99.6% similarity with the same isolates. Furthermore, the characterized haplotypes have shown similarities of 99.4% and 98.9% with the isolate reported from the Konya Region of Türkiye (KU175173).

In this study, as various researchers have indicated (59, 60), to determine the infective and infected Culicoides specimens with haemosporidian parasites in the research area, pools were formed by dissecting the head-thorax (potentially infective) and abdomen (potentially infected) of each fly diagnosed at the species level. As is known, infective stage sporozoites are transmitted through the salivary glands of the female fly during feeding. The salivary glands extend towards the esophagus on the lateral sides of the thorax (61). Fertilization and oocyst development (non-infective stage) take place in the mid-gut part (abdomen) of the fly. It has been expressed in various studies (59, 60, 62) that head-thorax pool (HTP) positivity can be considered an indicator of potential vector competence. Indeed, in our study, sequence analyses of the positive isolates obtained from HTP confirmed the presence of Haemoproteus parasites that Culicoides species are known to vector. In contrast, the sequences obtained from abdomen pools (AP) showed sequences of Plasmodium parasites, which are likely not vectored by Culicoides, possibly due to infected blood intake.

The Haemoproteus sp. GAGLA05 lineage identified in C. circumscriptus has been recorded in the blood of the Eurasian jay (Garrulus glandarius) belonging to the Passeriformes order (63), and parallel to our study, it was also isolated from C. circumscriptus in the Sultan Marshes Region of Türkiye (16). The H. minutus TURDUS2 lineage has been found to be common in avian communities, reported from 17 bird species belonging to eight families in the Passeriformes order and one bird species in the Psittaciformes order (MalAvi: http://mbio-serv2.mbioekol.lu.se/Malavi/). Additionally, the TURDUS2 lineage has been reported in mosquito species such as Cx. pipiens (64) and in Culicoides species including C. festivipennis, C. kibunensis (65), and C. pictipennis (66). Our study has revealed the first record of the potential vector status of C. circumscriptus for the possible transmission of the H. minutus TURDUS2 lineage. The Plasmodium sp. CXPIP10 lineage isolated and characterized from the abdominal pools of C. circumscriptus in our study has been reported in MalAvi records as a potential vector by various Culex species (67, 68). The Plasmodium sp. CXPIP23 lineage isolated from C. obsoletus has been reported in the Little Bittern (Ixobrychus minutus) belonging to the Ciconiiformes order in the Sultan Marshes Region, Türkiye (16) and potential mosquito vectors Cx. pipiens (16) and O. caspius (62). The P. relictum SGS1 lineage identified in AP from C. gejgelensis, C. imicola, and C. obsoletus is a common lineage in birds and has been isolated from numerous bird species in 11 orders. Plasmodium sp. YWT4 isolated from C. imicola and C. gejgelensis AP and Plasmodium sp. SYCON02 from C. obsoletus AP have been reported in a limited number of bird species in the Passeriformes order up to the present day (MalAvi: http://mbio-serv2.mbioekol.lu.se/Malavi/). The results obtained in this study have revealed the prevalence of the relevant Plasmodium lineages in avian communities in the İzmir Region and that Culicoides species feed on infected hosts.

CONCLUSION

Culicoides species that are prevalent in the İzmir Region have been morphologically and molecularly characterized, and unique epidemiological data have been provided based on molecular ecology regarding the vector potential of these species for avian haemosporidian parasites. The identification of species, such as C. imicola, known as the active vector for the BTV, highlights the need for control and management strategies for this disease in the context of host-pathogen-vector. Furthermore, the results of this study have shown that molecular-based techniques, in conjunction with morphometric analyses, can reliably determine species diversity of Culicoides in different geographies in Türkiye, and can also reveal cryptic or sibling species in epidemiological context. Considering the different biogeographical characteristics in Türkiye, there is a need for detailed studies on Culicoides species and the diseases they transmit based on molecular epidemiology.

Ethics

Ethics Committee Approval: No need to get ethics approval due to the material used in the study were insect specimens.

Informed Consent: Not required.

Acknowledgments

The authors would like to thank the Erciyes University Scientific Research Projects Unit, which supported this study.

Authorship Contributions

Concept: H.Y., Z.Ö., Design: H.Y., Z.Ö., Data Collection or Processing: H.Y., Z.Ö., A.Y., Analysis or Interpretation: H.Y., Z.Ö., Literature Search: H.Y., Z.Ö., A.Y., Writing: H.Y., Z.Ö., A.Y.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: This research was financially supported by Scientific Research Projects Coordination Unit of Erciyes University with the PhD thesis project coded TDK-2016-6397.

References

Mellor PS, Boorman J, Baylis M.Culicoides biting midges: their role as arbovirus vectors. Annu Rev Entomol. 2000; 45: 307-40.

Harrup LE, Bellis GA, Balenghien T, Garros C.Culicoides Latreille (Diptera: Ceratopogonidae) taxonomy: current challenges and future directions. Infect Genet Evol. 2015; 30: 249-66.

Borkent A. The biting midges, the Ceratopogonidae (Diptera), biology of disease vectors, 2nd ed. Elsevier, Burlington, MA: USA; 2004: 113-126.

Meiswinkel R, Venter GJ, Nevill EM. Vectors:Culicoides spp. In: Coetzer JAW, Tustin RC, editors. Infectious Diseases of Livestock. Cape Town: Oxford University Press; 2004: 93-136.

Carpenter S, Groschup MH, Garros C, Felippe-Bauer MF, Purse B.Culicoides biting midges, arboviruses and public health in Europe. Antiviral Res. 2013; 13: 102-13.

OIE. OIE-Listed diseases, infections and infestations in force in 2014, updated: 1st January 2014, accessed: 17th January 2014. Available at: http://www.oie.int/animal-health-in-the-world/oie-listed-diseases-2014/.

Elbers AR, Meiswinkel R, van Weezep E, van Oldruitenborgh-Oosterbaan MM, Kooi EA. Schmallenberg virus inCulicoides spp. biting midges, the Netherlands, 2011. Emerg Infect Dis. 2013; 19: 106-9.

Valkiūnas G. Avian malaria parasites and other haemosporidia. Boca Raton: CRC Press; 2005.

Atkinson CT.Haemoproteus. In: Atkinson CT, Thomas NJ, Hunter BC, editors. Parasitic diseases of wild birds. Ames: Wiley-Blackwell; 2008. p.13-35.

Donovan TA, Schrenzel M, Tucker TA, Pessier AP, Stalis IH. Hepatic hemorrhage, hemocoelom, and sudden death due toHaemoproteus infection in passerine birds: eleven cases. J Vet Diagn Invest. 2008; 20: 304-13.

Ortiz-Catedral L, Brunton D, Stidworth MF, Elsheikha HM, Pennycott T, Schulze C, et al.Haemoproteus minutus is highly virulent for Australasian and South American parrots. Parasit Vector. 2019; 12: 40.

Borkent A. World species of biting midges (Diptera: Ceratopogonidae). New York: American Museum of Natural History; 2019.

Bukauskaitė D, Iezhova TA, Ilgūnas M, Valkiūnas G. High susceptibility of the laboratory-reared biting midgesCulicoides nubeculosus to Haemoproteus infections, with review on Culicoides species that transmit avian haemoproteids. Parasitology. 2019; 146: 1-9.

Bernotienė R, Žiegytė R, Vaitkutė G, Valkiūnas G. Identification of a new vector species of avian haemoproteids, with a description of methodology for the determination of natural vectors of haemosporidian parasites. Parasites & Vectors. 2019; 12: 307.

Iezhova TA, Dodge M, Sehgal RN, Smith TB, Valkiunas G. New avianHaemoproteus species (Haemosporida: Haemoproteidae) from African birds, with a critique of the use of host taxonomic information in hemoproteid classification. J Parasites. 2011; 97: 682-94.

Yıldırım A, Dik B, Düzlü Ö, Gürsoy Ergen A. Sultan sazlığı ekosisteminde kanatlı Haemosporidialarının moleküler ekolojisi. TÜBİTAK 1001 Projesi, Proje no: 114 O 646, 2017.

Kieffer JJ. Chironomides d’Afrique et d’Asie conservés au Museum National Hongrois de Budapest. Ann Hist Nat Mus Nat Hung. 1918; 16: 31-136.

Dik B.Culicoides (Diptera: Ceratopogonidae) vektörlükleri ve mücadelesi. İçinde: Özbel Y (editör), Vektör artropodlar ve mücadelesi. Türkiye Parazitoloji Derneği: İzmir; 2017. s.46-122.

Korkmaz C, Alten B, Erol U, Deniz A. Updated checklist ofCulicoides Latreille (Diptera: Ceratopogonidae) of Türkiye with ten new records. J Vector Ecol. 2021; 46: 124-36.

Turgut F. Morphological characteristics of twoCulicoides Latreille biting midges (Diptera, Ceratopogonidae) with a new record for Turkish fauna. Trakya Univ J Nat Sci. 2023; 23: 3-8.

Dik B, Muz D, Muz MN, Uslu U. The geographical distribution and first molecular analysis ofCulicoides Latreille (Diptera: Ceratopogonidae) species in the Southern and Southeastern Türkiye during the 2012 outbreak of bovine ephemeral fever. Parasitol Res. 2014; 113: 4225-32.

Yildirim A, Dik B, Duzlu O, Onder Z, Ciloglu A, Yetismis G, et al. Genetic diversity ofCulicoides species within the Pulicaris complex (Diptera: Ceratopogonidae) in Türkiye inferred from mitochondrial COI gene sequences. Acta Trop. 2019; 190: 380-8.

Şevik M, Oz ME. Detection of Peste des petits ruminants virus RNA inCulicoides imicola (Diptera: Ceratopogonidae) in Türkiye. Vet Ital. 2019; 55: 173-9.

Nolan DV, Carpenter S, Barber J, Mellor PS, Dallas JF, Mordue Luntz AJ, et al. Rapid diagnostic PCR assays for members of theCulicoides obsoletus and Culicoides pulicaris species complexes, implicated vectors of bluetongue virus in Europe. Vet Microbiol. 2007; 124: 82-94.

Pages N, Munoz-Munoz F, Talavera S, Sarto V, Lorca C, Núñez JI. Identification of cryptic species ofCulicoides (Diptera: Ceratopogonidae) in the subgenus Culicoides and development of species-specific PCR assays based on barcode regions. Vet Parasitol. 2009; 165: 298-310.

Mathieu B, Cêtre-Sossah C, Garros C, Chavernac D, Balenghien T, Carpenter S, et al. Development and validation of IIKC: an interactive identification key forCulicoides (Diptera: Ceratopogonidae) females from the Western Palaearctic region. Parasites & Vectors. 2012; 5: 137.

Dallas JF, Cruickshank RH, Linton YM, Nolan DV, Patakakis M, Braverman Y, et al. Phylogenetic status and matrilineal structure of the biting midge,Culicoides imicola, in Portugal, Rhodes and Israel. Med Vet Entomol. 2003; 17: 379-87.

Hellgren O, Waldenström J, Bensch S. A new PCR assay for simultaneous studies ofLeucocytozoon, Plasmodium, and Haemoproteus from avian blood. J Parasitol. 2004; 90: 797-802.

Bensch S, Hellgren O, Pérez-Tris J. MalAvi: A public database of malaria parasites and related haemosporidians in avian hosts based on mitochondrial cytochrome b lineages. Mol Ecol Resour. 2009; 9: 1353.

Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, et al. Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012; 28: 1647-9.

Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics. 2009; 25: 1451-2.

Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980; 16: 111-20.

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013; 30: 2725-9.

Posada D. jModelTest: phylogenetic model averaging. Mol Biol Evol. 2008; 25: 1253-6.

Huelsenbeck JP, Ronquist F. MRBAYES: bayesian inference of phylogenetic trees. Bioinformatics. 2001; 17: 754-5.

Ramilo DW, Nunes T, Madeira S, Boinas F, da Fonseca IP. Geographical distribution ofCulicoides (Diptera: Ceratopogonidae) in mainland Portugal: presence/absence modelling of vector and potential vector species. PLoS One. 2017; 12: e0180606.

Sarvašová A, Kočišová A, Halán M, Delécolle JC, Mathieu B. Morphological and molecular analysis of the genusCulicoides (Diptera: Ceratopogonidae) in Slovakia with five new records. Zootaxa. 2014; 3872: 541-60.

Carvalho LPC, Costa GDS, Pereira Júnior AM, de Paulo PFM, Silva GS, Carioca ALPM, et al. DNA Barcoding of genusCulicoides biting midges (Diptera: Ceratopogonidae) in the Brazilian Amazon. Acta Trop. 2022; 235: 106619.

Gopurenko D, Bellis G, Pengsakul T, Siriyasatien P, Thepparat A. DNA barcoding ofCulicoides Latreille (Diptera: Ceratopogonidae) from Thailand reveals taxonomic inconsistencies and novel diversity among reported sequences. J Med Entomol. 2022; 59: 1960-70.

Ortega-Morales AI, Hernández-Triana LM, Chan-Chable RJ, Garza-Hernández JA, González-Álvarez VH, Ruiz-Arrondo I, et al. DNA barcoding of mosquitoes from the Pantanos de Centla biosphere reserve, Southeastern Mexico. J Am Mosq Control Assoc. 2021; 37: 198-207.

Dähn O, Werner D, Mathieu B, Kampen H. Large-scale cytochrome C Oxidase Subunit I gene data analysis for the development of a multiplex polymerase chain reaction test capable of identifying biting midge vector species and haplotypes (Diptera: ceratopogonidae) of theCulicoides Subgenus Avaritia Fox, 1955. Genes (Basel). 2024; 15: 323.

Onder Z, Yildirim A, Duzlu O, Arslan MO, Sari B, Tasci GT, et al. Molecular characterization of black flies (Diptera: Simuliidae) in areas with pest outbreaks and simuliotoxicosis in Northeast Anatolia Region, Türkiye. Acta Trop. 2019; 199: 105149.

Inci A, Yildirim A, Duzlu O, Onder Z, Ciloglu A, Seitz G, et al. Genetic diversity and identification of Palearctic black flies in the subgenus Wilhelmia (Diptera: Simuliidae). J Med Entomol. 2017; 54: 888-94.

Anon. İzmir Bornova Veteriner Kontrol Enstitüsü Kayıtları; 2016.

Goredo M, Catalani M, Federici V, Portanti O, Marini V, Mancini G, et al. Vector species ofCulicoides midges implicated in the 2012-2014 Bluetongue epidemics in Italy. Vet Ital. 2015; 51: 131-8.

Goredo M, Meiswinkel R, Federici V, Di Nicola F, Mancini G, Ippoliti C, et al. The “Culicoides obsoletus group” in Italy: Relative abundance, geographic range, and role as vector for Bluetongue virus. Vet Ital. 2016; 52: 235-24.

Yavru S, Dik B, Bulut O, Yavru N, Kale M, Bulut A. NewCulicoides vector species for BTV transmission in Central and Central west of Anatolia. Annu Res Rev Biol. 2018; 27: 1-9.

Foxi C, Meloni G, Puggioni G, Manunta D, Rocchigiani A, Vento L, et al. Bluetongue virus detection in newCulicoides species in Sardinia, Italy. Vet Re. 2019; 184: 621.

Dik B, Kuçlu Ö, Öztürk R.Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) species in the Western Black Sea region of Türkiye, new records for the Turkish fauna. Turk J Vet Anim Sci. 2017; 41: 228-37.

Reyhan E. Güney Doğu Anadolu Bölgesinin Gaziantep, Kahramanmaraş ve Kilis illerindeCulicoides (Diptera: Ceratopogonidae) türlerinin yayılışı, Yüksek Lisans Tezi, Gaziantep Üniversitesi Fen Bilimleri Enstitüsü: Gaziantep; 2018.

Turgut F, Kilic AY. The Ceratopogonidae (Insecta: Diptera) fauna of the Central Black Sea region in Türkiye. Turk Zool Derg. 2015; 39: 1071-89.

Muz D, Dik B, Muz MN, Öztürk R. Tekirdağ iliCulicoides (Diptera: Ceratopogonidae) faunasının mavi dil virus ve Schmallenberg virus varlığı yönünden araştırılması. Namık Kemal Üniversitesi Araştırma Projesi: 2016, Proje no: NKUBAP.00.23.AR.14.08.

Turgut F. Samsun iliCulicoides Latreille, 1809 (Diptera: Ceratopogonidae) faunasına katkılar. GBAD. 2018; 7: 14-20.

Turgut F. Contributions toCulicoides Latreille, 1809 (Diptera: Ceratopogonidae) fauna of Sinop province. SAUJS. 2018; 22: 1683-93.

Hadj-Henni L, De Meulemeester T, Mathieu B, Depaquit J, Augot D. Taxonomic assessment ofCulicoides brunnicans, C. santonicus and C. vexans (Diptera: Ceratopogonidae) in France: Implications in systematics. Infect Genet Evol. 2015; 33: 324-31.

Augot D, Ninio C, Akhoundi M, Lehrter V, Couloux A, Jouet D, et al. Characterization of two cryptic species,Culicoides stigma and C. parroti (Diptera: Ceratopogonidae), based on barcode regions and morphology. J Vector Ecol. 2013; 38: 260-5.

Lassen SB, Nielsen SA, Skovgård H, Kristensen M. Molecular differentiation ofCulicoides biting midges (Diptera: Ceratopogonidae) from the subgenus Culicoides Latreille in Denmark. Parasitol Res. 2012; 110: 1765-71.

Pagès N, Sarto I Monteys V. Differentiation ofCulicoides obsoletus and Culicoides scoticus (Diptera: Ceratopogonidae) based on mitochondrial cytochrome oxidase subunit I. J Med Entomol. 2005; 42: 1026-34.

Ishtiaq F, Guillaumot L, Clegg SM, Phillimore AB, Black RA, Owens IP, et al. Avian haematozoan parasites and their associations with mosquitoes across Southwest Pacific Islands. Mol Ecol. 2008; 17: 4545-55.

Njabo KY, Cornel AJ, Bonneaud C, Toffelmier E, Sehgal RN, Valkiūnas G, et al. Nonspecific patterns of vector, host and avian malaria parasite associations in a central African rainforest. Mol Ecol. 2011; 20: 1049-61.

James AA, Rossignol PA. Mosquito salivary glands: parasitological and molecular aspects. Parasitol Today. 1991; 7: 267-71.

Inci A, Yıldırım A, Njabo KY, Duzlu O, Biskin Z, Ciloglu A. Detection and molecular characterization of avianPlasmodium from mosquitoes in central Türkiye. Vet Parasitol. 2012; 188: 179-84.

Mata VA, da Silva LP, Lopes RJ, Drovetski SV. The strait of gibraltar poses an effective barrier to host-specialised but not to host-generalised lineages of avian Haemosporidia. Int J Parasitol. 2015; 45: 711-9.

Martinez-de la Puente J, Munoz J, Capelli G, Montarsi F, Soriguer R, Arnoldi D, et al. Avian malaria parasites in the last supper: identifying encounters between parasites and the invasive Asian mosquito tiger and native mosquito species in Italy. Malaria J. 2015; 14: 32.

Synek P, Munclinger P, Albrecht T, Votypka J. Avian haemosporidians in haematophagous insects in the Czech Republic. Parasitol Res. 2013; 112: 839-45.

Bobeva A, Zehtindjiev P, Bensch S, Radrova J. A survey of biting midges of the genusCulicoides Latreille, 1809 (Diptera: Ceratopogonidae) in NE Bulgaria, with respect to transmission of avian haemosporidians. Acta Parasitol. 2013; 58: 585-91.

Ferraguti M, Martinez-de la Puente J, Munoz J, Roiz D, Ruiz S, Soriguer R, et al. AvianPlasmodium in Culex and Ochlerotatus mosquitoes from Southern Spain: effects of season and host-feeding source on parasite dynamics. PLoS One. 2013; 8: e66237.

Kim KS, Tsuda Y. AvianPlasmodium lineages found in spot surveys of mosquitoes from 2007 to 2010 at Sakata wetland, Japan: do dominant lineages persist for multiple years? Mol Ecol. 2012; 21: 5374-85.

Molecular Characterization of Culicoides Species and Their Vector Potentials for Haemosporidia Infections in the İzmir Region of Türkiye