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Article

Trypanosomatids in Bloodsucking Diptera Insects (Ceratopogonidae and Simuliidae) Wild-Caught at Raptor Bird Nests in Temperate Forests

by
Margarita Kazak
*,
Rasa Bernotienė
*,
Rimgaudas Treinys
and
Dovilė Bukauskaitė
Nature Research Centre, Akademijos St. 2, 08412 Vilnius, Lithuania
*
Authors to whom correspondence should be addressed.
Diversity 2023, 15(5), 692; https://doi.org/10.3390/d15050692
Submission received: 6 April 2023 / Revised: 16 May 2023 / Accepted: 17 May 2023 / Published: 21 May 2023
(This article belongs to the Special Issue Diversity of Wildlife Pathogens)
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Abstract

:
Trypanosomatids are a huge group of vertebrate, invertebrate and plant parasites that can cause severe illnesses in hosts. Although avian trypanosomes are widely spread throughout the world, information about their transmission and vector–host relationships is still scarce. This study aimed to investigate Trypanosoma prevalence in bloodsucking Diptera collected at tree-nesting raptor birds’ nests. Insects were collected in temperate forests of Eastern-Central Europe, in Lithuania, using UV light traps hung near common buzzard (Buteo buteo) and lesser spotted eagle (Clanga pomarina) nests at about 15 m height above the ground. A total of 1248 Culicoides (Ceratopogonidae) females and 3 blackflies (Simuliidae) were collected and tested for the presence of trypanosomatids using PCR-based methods. The blood of 85 nestlings, belonging to three different species (Buteo buteo, Clanga pomarina and Haliaeetus albicilla) was collected and tested using the same methods. We found that 11.1% of the investigated insects (one Simulium female and Culicoides biting midges belonging to five species) were infected with Trypanosoma parasites (Trypanosoma sp., T. bennetti group, T. avium) and monoxenous trypanosomatids (Crithidia sp., Obscuromonas sp.). Only one common buzzard nestling was found to be infected with Trypanosoma avium. The phylogenetic relationships of detected parasites were determined. Our findings supplement information on the ornithophilic behavior of Culicoides females, potential vector species of avian Trypanosoma, and produce some new information on the detection of monoxenous trypanosomatids (Crithidia sp. and Obscuromonas sp.) in Culicoides.

Graphical Abstract

1. Introduction

Trypanosomatids are a diverse group of plant, invertebrate and vertebrate parasites mostly known by the genera Trypanosoma and Leishmania that cause severe diseases in humans [1]. Although approximately one fifth of all Trypanosoma species has been described from birds [2,3], avian trypanosomes are not so well studied because of their low economic importance as parasites of domestic birds [4]. Nevertheless, some Trypanosoma species might be pathogenic and affect the growth and fitness of intensively infected individuals [5]. Data from recent studies have shown that many birds are heavily infected with Trypanosoma parasites [6]. For example, studies from Czech Republic showed that 78% of Accipiter nisus females and 69% of males were infected with Trypanosoma parasites [7]. It was detected that passerine birds of some species, for example Phylloscopus trochilus and Sylvia curruca, could be infected with trypanosomes up to 33.3% and 32.0%, respectively, in Lithuania [8], and the prevalence of avian trypanosomes was up to 82.7% in Cyanomitra olivacea birds in Africa [6].
The transmission of avian trypanosomes is carried out by bloodsucking arthropods—dipterous insects (blackflies (Simuliidae), mosquitoes (Culicidae), biting midges (Culicoides, Ceratopogonidae), hippoboscids (Hippoboscidae), sandflies (Psychodidae)) and arachnids such as mites (Dermanyssidae) [9,10,11,12,13,14]. However, vectors of most of the described avian trypanosomes remain unknown [6,10]; although the parasite could be detected in an insect, its competence as a vector in many cases had not been proved as in the case of other kinetoplastid parasites [15].
In addition to Trypanosoma infections, dipterous insects may be infected with other monoxenous trypanosomatids, such as Crithidia, Herpetomonas, Sergeia and others, which complete their development only in insects and do not need a vertebrate hosts [1,16].
To date, there have been various studies on trypanosomes infection in raptor birds, and Trypanosoma avium, T. bennetti and T. corvi were found [7,17,18,19]. Moreover, nestlings of common buzzard (Buteo buteo) and sparrowhawk (Accipiter nisus) have been studied for Trypanosoma infections and the results showed that phylogenetically unrelated parasites which share the same vectors tend to have similar distributions within host populations [20]. The most likely vectors of raptor bird Trypanosoma parasites may be simuliids [21]. Trypanosoma parasites have been screened in biting midges attacking buzzard and sparrowhawk, with negative results [22], but it was proved experimentally that Culicoides nubeculosus were highly susceptible to parasites of the Trypanosoma bennetti group, and T. bennetti-infected Culicoides spp. have been found in wild [4].
The aim of this study was to investigate Trypanosoma prevalence in bloodsucking dipteran (Ceratopogonidae and Simuliidae), which were collected from raptor bird’s nests located in mature tree canopies. We used a molecular approach to test collected insects, not only for avian Trypanosoma infections, but for the prevalence of monoxenous trypanosomatids as well. Common buzzard, lesser spotted eagle (Clanga pomarina) and white-tailed eagle (Haliaeetus albicilla) nestling blood was collected and tested for the presence of Trypanosoma parasites using the same methods.

2. Materials and Methods

2.1. Investigation of Nestlings

We searched for the raptors’ (common buzzard, lesser spotted eagle and white-tailed eagle) nests year-round in the forests of the eastern Lithuania and checked for occupancy during May–July, 2020–2021. We climbed to the successful nests with broods consisting of well-feathered nestlings and transported nestlings (the age 4–7 weeks) to the ground for blood sampling. The blood from nestlings (n = 85) from nests of three bird species (common buzzard (9 nests), lesser spotted eagle (37 nests) and white-tailed eagle (17 nests)) was collected from the brachial vein using a syringe. Three small drops of blood were used to make smears, which were air dried, fixed with absolute methanol and stained with 10% Giemsa solution, as described in [23]. The remaining blood was stored in SET buffer for molecular analysis and was kept at −20 °C at the laboratory.

2.2. Insect Collection

Biting midges were collected using UV light traps in temperate forests of eastern-central Europe, in eastern Lithuania (Figure 1). In June–July 2020 and 2021, we climbed to on average 15 m height above the ground to nests with broods of raptor birds of two species—common buzzard and lesser spotted eagle—and attached UV light traps to the branches. Biting midges were collected on thirteen nights (two nights in June 2020, seven nights in June and four nights in July 2021). Traps were operated 5–7 h before sunset until 5–7 h after sunrise. Biting midges were collected in a container with tap water supplemented with a drop of liquid soap. Collected insects were transported to the laboratory the same day as collection.

2.3. Insect Identification

Freshly collected Culicoides were investigated under a stereoscopic microscope SMZ-171-TLED (Motic, Spain), and only parous females were selected for further investigation due to the presence of visible burgundy-colored pigment in the subcutaneous cells of the abdomen, which is the indication that at least one gonotrophic cycle had occurred [24], which means that these females had had bloodmeal and could have been in the contact with the pathogen. All investigated parous females were identified using the main morphological characteristics for biting midge (wing coloration patterns and head features (palp and antenna)) [25] or blackfly (morphology of legs, head and terminalia) identification [26]. The insect head and wings were removed using flame-disinfected needles for Euparal-mounted preparations, and the rest of insect bodies were stored in 96% ethanol for further PCR-based analysis. Flame-disinfected needles were used to avoid contamination.

2.4. PCR-Based Screening and Statistics

Total DNA from blood of raptor bird nestlings and wild caught biting midges was extracted using ammonium-acetate DNA precipitation protocol [27]. After the extraction, DNA concentration was measured using Nanophotometer P330 (Implen, Germany). Samples which exceeded optimal DNA template concentration (20–50 ng/µL) for PCR were diluted using a 1x TE buffer. For trypanosomatid detection, a nested PCR protocol was used to amplify a 749 bp length DNA fragment encoding SSU 18S rRNA using two outer primers, Tryp763 (5′-CATATGCTTGTTCAAGGAC-3′) and Tryp 1016 (5′-CCCCATAATCTCCAATGGAC-3′), and two inner primers: Tryp99 (5′-TCAATCAGACGTAATCTGCC-3′) and Tryp957 (5′-CTGCTCCTTTGTTATCCCAT-3′) [28,29]. All PCRs were performed in 25 µL total volume: 2 µL total genomic DNA template, 12.5 µL DreamTaq Master Mix (Thermo Fisher Scientific, Lithuania, Vilnius), 8.5 µL nuclease-free water and 1 µL of each primer. Temperature profiles in all PCRs were the same as in the original protocol: first PCR started with denaturation at 95 °C (5 min), followed by 5 cycles of 95° (60 s), 45 °C (30 s), 65 °C (60 s) and 35 cycles of 95 °C (60 s), 50 °C (30 s), 72 °C (60 s), with a final extension at 65 °C (10 min). For the second PCR, temperatures were denaturation at 96 °C (3 min), followed by 25 cycles of 96 °C (30 s), 58 °C (60 s), 72 °C (30 s) and a final extension at 72 °C (7 min). All amplifications were evaluated using a multiSUB horizontal electrophoresis system (Cleaver Scientific Ltd., UK): 2 µL of the final PCR product was run on 2% agarose gel. One positive control (Trypanosoma sp. infection confirmed by microscopic examination and molecular methods) and one negative control (ultrapure water) were used during each amplification. All PCR positive samples were sequenced with corresponding primers, both strands, using a Big Dye Terminator V3.1 Cycle Sequencing kit and an ABI PRISMTM 3100 capillary sequencing robot (Applied Biosystems, Foster City, CA, USA). New-found haplotypes were submitted to the GenBank (OQ864985, OQ870298—OQ870302).
Species of biting midges which could not be reliably identified using morphological features (n = 118) were identified using primers LCO1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and HCO2198 (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′) in order to amplify a fragment of cytochrome c oxidase subunit 1 (COI) of insect mitochondrial DNA [30]. Insect species were identified using the ‘Basic Local Alignment Search Tool’ (NCBI BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 10 January 2023). The abundance of Culicoides at nests of different bird species was compared using t-test for independent samples. A p-value less than 0.05 was deemed to indicate statistical significance.

2.5. Phylogenetic Analysis

DNA sequences obtained in this study were aligned, a phylogenetic tree was constructed and genetic distances between sequences were calculated using Geneious Prime®2023.0.4 (Dotmatics, Auckland, New Zealand, https://www.geneious.com, accessed on 1 April 2023) software. Seven new sequences, five Crithidia spp., three Obscuromonas spp. and 17 Trypanosoma sequences closest to new-found haplotypes obtained from the GenBank and Cryptobia catostomi as an outgroup were used to construct a phylogenetic Bayesian tree. The best fit model (GTR+I+G) was suggested by the jModeltest-2.1.10 software [31,32]. The phylogenetic tree was run with the MrBayes plugin v3.2.6 [33]. The analysis was run for 6 million generations, with a sampling frequency of every 100th generation; 25% of the initial trees was discarded as a ‘burn in’ period before the construction of the consensus tree.

3. Results

3.1. Species Composition of Culicoides Biting Midges

Overall, 1248 Culicoides and 3 Simuliidae females were caught and examined (909 insects were caught at the common buzzard nests and 342 insects at the lesser spotted eagle nests). Overall, eleven Culicoides species, belonging to five subgenera, and two Simulium species of two subgenera were identified using microscopy and PCR-based methods (Table 1). Culicoides species, belonging to subgenus Oecacta, were the most abundant in this study, and three species (C. festivipennis, C. kibunensis and C. pictipennis) together accounted for 94.8% of all caught biting midges. Only three biting midge species were collected in 2020 (n = 72), and C. kibunensis was the dominant among them (65.3%). The most abundant species were C. pictipennis (52.4%), C. festivipennis (31.8%) and C. kibunensis (9.6%) in 2021 (n = 1179), and individuals from the other species accounted for the small percentage of analyzed insects (6.2%). Differences in insect diversity were observed at the nests of different raptor bird species: C. pictipennis was the most abundant insect species at the common buzzard nests (61.9% of all collected Culicoides), and C. festivipennis was the most abundant at the lesser spotted eagle nests (60.3% from all caught and analyzed biting midges). The mean abundance of C. festivipennis caught at common buzzard (17.6 ± 6.3 (SE)) and at lesser spotted eagle nests (55.3 ± 29.0) per night per one trap differed statistically (t = 1.88, p = 0.04), while statistical differences between the abundance of other Culicoides species were not detected. Three Culicoides species were detected only in nests of the common buzzard and were not detected in nests of the lesser spotted eagle: C. impunctatus (n = 23), C. archayi (n = 1) and C. reconditus (n = 1). In general, the abundance of Culicoides midges collected per night per one trap was similar for nests of birds of both species (91.5 ± 32.5 for common buzzard and 85 ± 28 for lesser spotted eagle) and did not differ statistically. Simulium vernum was caught at common buzzard nests and S. angustipes was caught at lesser spotted eagle nests.

3.2. Biting Midge Infection with Trypanosomatids

Out of 1251 PCR-tested insect females (Ceratopogonidae and Simuliidae), 139 (11.1%) were found to be positive for the presence of trypanosomatids (Table 1). Trypanosomatid prevalence in insect females collected at the common buzzard nests was 6.2% (n = 56) and the prevalence in females collected at the lesser spotted eagle nests was 24.3% (n = 83). Two groups of trypanosomes (T. avium and Trypanosoma bennetti group) and two monoxenous trypanosomatid genera (Crithidia and Obscuromonas) were detected. More than half (61.2%) of all detected sequences showed mixed Trypanosoma infection in investigated insects, which was illustrated by the double peaks in the chromatograms. The vast majority (n = 43) of positive biting midges harbored DNA of trypanosomatids phylogenetically very similar to the Trypanosoma bennetti group (Figure 2). Six new haplotypes of the Trypanosoma bennetti group were found, and haplotype Hap-5 was the most common (n = 34) among them (Table 1). Trypanosoma avium was detected in Culicoides segnis, C. pictipennis females and in one female Simulium vernum. One Culicoides kibunensis was found to be positive for Obscuromonas sp., and five biting midges belonging to three different species (C. festivipennis, C. kibunensis and C. pictipennis) were positive for Crithidia brevicula or Critidia sp. (Table 1). Biting midges in which monoxenous trypanosomatids were detected were caught only at the common buzzard nests.

3.3. Raptor Nestling Blood Analysis for the Presence of Trypanosoma Parasites

During the investigation, 85 nestlings, belonging to three bird species—common buzzard (n = 15), lesser spotted eagle (n = 37) and white-tailed eagle (n = 33)—were tested for the presence of trypanosomatids in their blood. Only one nestling bird (common buzzard) sample was positive for Trypanosoma avium infection.

3.4. Phylogenetic Analysis

Phylogenetic relationships of detected haplotypes were determined (Figure 2). We detected three sequences in investigated insects which 100% matched with sequence of T. avium deposited in the GenBank (MT269500). New-found Trypanosoma haplotypes Hap-1, Hap-2, Hap-3, Ha-4, Ha-5 and Hap-6 clustered together with other parasites from the Trypanosoma bennetti group. Genetic distances between these new haplotypes varied between 0.8 and 2.9%. One new-found haplotype clustered together with Obscuromonas parasites. The genetic distance between it and Obscuromonas sp. (MW694350) was 3.6%. Five sequences detected in insect females 100% matched with Crithidia sp. or Crithidia brevicula deposited in the GenBank (Figure 2).

4. Discussion

Understanding the value of vectors in the transmission of pathogens and their ecological properties that allow us to understand the patterns of pathogen transmission are extremely important, but not sufficiently investigated. In this study, we aimed to obtain new knowledge on Culicoides spp. that were caught at about 15 m height from the ground at the nests of raptor birds, and their infection with flagellate parasites.
Biting midges have been suggested as potential trypanosome vectors since the middle of the last century [11,34]. Recent studies showed that avian trypanosomes of three species—T. avium, T. bennetti and T. everetti—can complete their development in C. nubeculosus, and T. everetti can also develop in one of the most abundant Culicoides species in North Europe—C. impunctatus [4,10]. Additionally, some studies [13] proved the transmission of T. avium by blackflies (Simulium spp.), and birds become infected due to the ingestion of the vectors or active penetration of the metacyclic trypomastigotes via the host conjunctiva. In this study, we found that C. festivipennis, C. pictipennis and C. kibunensis were the most abundant biting midges at the raptor nests at about 15 m from the ground. The biting midge species diversity determined was similar to that in the study which examined the spatial feeding preferences of ornithophilic insects, including Culicoides spp., in canopies in Czech Republic, where C. festivipennis and C. kibunensis accounted for 62.3% of all collected biting midges [35]. During a study of Culicoides attacking raptor nestlings in the same country, C. festivipennis and C. kibunensis accounted for 77.2% [21], with the trypanosomatid prevalence in insects (1.5%) being lower compared with our study (11.1%). We determined 11 Culicoides species near the nests of raptor birds, but some species were detected based on a single individual (C. reconditus, C. archayi), which means that midges of these species are rare in the study area, or they are opportunistic in their feeding preferences. Faunistic studies of nulliparous females would provide more information on this issue.
Trypanosoma avium was the first trypanosomatid described from avian blood and found to be infecting raptor birds [17]. Ornithophilic blackflies (Simulium (Eusimulium) spp.) were described as vectors of T. avium [21]. However, in this study, we found three parasites of the same haplotype in three different invertebrate hosts—in S. vernum, C. segnis and C. pictipennis. During our previous study [10], T. avium was also found in C. segnis females. These results indicate that some Culicoides might be considered potential vectors of this trypanosome, but the infection of Culicoides can be also the result of their feeding on infected bird blood; however, bird blood had been already digested in insect females in our study. Further research, dedicated to the possibility of females of certain Culicoides species transmitting avian trypanosomes, is needed.
During our research, the biggest proportion of all biting midges positive for trypanosomatid infection were found to be infected with trypanosomatids belonging to the T. bennetti group. Trypanosoma bennetti was described by Kirkpatrick et al. in 1986 from Falco sparverius, and later strains belonging to the T. bennetti group were found in European passerine birds as well as in raptors [3]; however, at that time vectors for this trypanosome species remained unknown [18]. Only thirty years later, based on high susceptibility to artificial infections and on findings of T. bennetti-infected wild-caught Culicoides studies, it was shown that biting midges are possible vectors of avian trypanosomes belonging to the T. bennetti group [4]. Trypanosoma bennetti was found to be very similar genetically to T. everetti (the genetic distance in partial 18S rRNA sequences is 0.4–1.3%) [10]. Originally, T. everetti was discovered and described from the black-rumped waxbill Estrilda troglodytes in Nigeria [36], and readily infects passerine birds [6,10]. Recent research [10] has shown that T. everetti readily developed and produced metacyclic stages in two different widespread Culicoides biting midge species, C. nubeculosus and C. impunctatus. The genetic distance within the T. bennetti group sequences analyzed in this study varied up to 2.9%, which is why it is not possible to determine trypanosome species based solely on partial 18S rRNA sequences, and further research is needed. Only molecular methods were used for this research, so the results of this paper cannot conclude that dixenous trypanosomatids develop in analyzed insects; however, biting midge species, individuals of which we found to be positive for Trypanosoma infection, can be considered as being ornithophilic and also likely to use raptor bird blood as their food source.
Only 0.48% of all analyzed insects were infected with monoxenous parasites. One C. kibunensis biting midge was found to be infected with Obscuromonas sp. This genus was originally described by Votýpka and Lukeš in 2021, from heteropteran bugs [37]. Together with other monoxenous insect parasites (Crithidia spp.), Obscuromonas was previously found in the guts of tsetse flies Glossina (Austenia) tabaniformis and Glossina (Nemorhina) fuscipes fuscipes [38]. Obscuromonas has never been found in biting midges before this study; this is the first case in which Obscuromonas has been found in Culicoides biting midges. Genetic distances between sequences found during this study were 12.9% with O. modryi (MW177946), 14.5% with O. eliasi (MW177947), and 3.6% with Obscuromonas sp. (MW694350) [37,38].
Flagellate belonging to genus Crithidia infects a variety of different insect hosts [16,39]. In this study, two different Crithidia haplotypes were detected in individuals belonging to C. pictipennis, C. kibunensis and C. festivipennis. Crithidia brevicula was originally known to infect heteropteran bugs [40]; however, different studies have found these parasites in mosquitoes [41,42]. Two Crithidia species had already been found in Culicoides peregrinus in the study carried out in Thailand, which showed high similarity with sequences of Crithidia found in Glossina (Nemorhina) fuscipes fuscipes [43]. A previous study from Lithuania [10] also detected Crithidia sp. infections with a 0.4% prevalence in C. pictipennis biting midges. The comparison of sequences showed 100% similarity with sequences deposited in the GenBank (Figure 2): MG255956, MT232055, MT232056, MT232057, KJ443354 and KJ443355 [40,41,44]. This study supplemented information on the presence of Crithidia parasites in Culicoides biting midges, but further research is needed to confirm trypanosomatids development in these bloodsucking insects.
In previous studies [13,21], potential vectors of avian trypanosomes were noted attacking nestlings in raptor nests. Moreover, insects found near the raptors could have previously been feeding on other birds or mammals and subsequently seeking raptor birds for their next bloodmeal, and could possibly transmit parasites to different host species. In this study, we found T. avium in one juvenile common buzzard, with the prevalence of infection being 1.2%. Trypanosoma avium is a known raptor parasite and has previously been found in ornithophilic blackflies attacking buzzard nestlings [13]. It was determined that the prevalence of all parasites increased with the age of nestlings, because prolonged exposure to vectors is thus positively correlated with prevalence [20]. This could explain the low Trypanosoma prevalence in nestlings, as the blood of 4–7 weeks old nestlings was examined using PCR. We also detected differences in the Culicoides species composition collected at the common buzzard and lesser spotted eagle nests. These differences, however, can be related not to the host of the nest, but to the different collection times because of differences in breeding phenology of these two raptor species [45]. The common buzzard breeds early in the season, and nestlings in the nests grow usually during May–June (eight out of nine collections were made in June), while lesser spotted eagle nestlings grow during June–July (all four collections were made in July), so biting midges were collected at different times at nests of birds of different species. The results of this research show that nestlings of different bird species can be attacked by different species of blood-sucking insects, although this may be related to the differences in breeding time.
The results of this study improved the understanding of the vertical distribution of biting midges (as usually these insects are investigated at ground level [10]), and revealed the most abundant Culicoides species near raptor nests. We also obtained new data on the prevalence of trypanosomatids, both avian trypanosomes and monoxenous insect parasites, in wild-caught biting midges and blackflies collected in the canopy.

Author Contributions

Conceptualization, R.B., R.T. and D.B.; methodology, M.K., R.B., R.T. and D.B.; software, D.B.; formal analysis, M.K., R.B., R.T. and D.B.; investigation, M.K., R.B., R.T. and D.B.; resources, R.B. and D.B., writing—original draft preparation, M.K.; writing—review and editing, R.B., D.B. and R.T.; visualization, D.B.; funding, D.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Research Council of Lithuania, grant number S-MIP-20-57.

Institutional Review Board Statement

The experiments described herein comply with the current laws of Lithuania and were performed by licensed researchers according to procedures approved by the Environmental Protection Agency, Vilnius, Lithuania (29 May 2020, no. 31; 11 June 2021 no. (26)-SR-104; 2022-06-03 no. SR-168). None of the birds suffered apparent injury during this study, and they were released after sampling.

Data Availability Statement

The data supporting reported results are available upon email inquiry.

Acknowledgments

We are very thankful to Carolina Romeiro Fernandes Chagas and Mélanie Yvonne Ludivine Duc for their help with material collection and sample preparation for the investigation.

Conflicts of Interest

The authors declare no conflict of interest.

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Diversity 15 00692 g001 550
Figure 1. Map of the study area showing the raptor bird’s nests locations with UV light traps. Square figures designate Buteo buteo nests, and triangle figures Clanga pomarina nests.
Figure 1. Map of the study area showing the raptor bird’s nests locations with UV light traps. Square figures designate Buteo buteo nests, and triangle figures Clanga pomarina nests.
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Figure 2. Bayesian phylogenetic tree of Trypanosoma sp., Obscuromonas sp. and Crithidia sp. using 18S rRNA. The tree was rooted using a Cryptobia catostomi. Names of parasites in bold font represent sequences detected in wild biting midges during this investigation, of which those marked with a dot represents new haplotypes. Hosts in which parasites have been detected are provided, and in case of multiple hosts detected during this investigation are marked as: 1—parasites have been also detected in Culicoides kibunensis, C. segnis, 2—in C. kibunensis, 3—C. festivipennis, C. kibunensis, C. pictipennis, C. segnis, 4—C. pictipennis, C. segnis, Simulium vernum, 5—C. festivipennis, C. kibunensis, C. pictipennis, 6—C. festivipennis.
Figure 2. Bayesian phylogenetic tree of Trypanosoma sp., Obscuromonas sp. and Crithidia sp. using 18S rRNA. The tree was rooted using a Cryptobia catostomi. Names of parasites in bold font represent sequences detected in wild biting midges during this investigation, of which those marked with a dot represents new haplotypes. Hosts in which parasites have been detected are provided, and in case of multiple hosts detected during this investigation are marked as: 1—parasites have been also detected in Culicoides kibunensis, C. segnis, 2—in C. kibunensis, 3—C. festivipennis, C. kibunensis, C. pictipennis, C. segnis, 4—C. pictipennis, C. segnis, Simulium vernum, 5—C. festivipennis, C. kibunensis, C. pictipennis, 6—C. festivipennis.
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Table
Table 1. Investigated Culicoides and Simulium females and their infection with trypanosomatids in temperate forests at nests of raptor birds of two species in 2020–2021.
Table 1. Investigated Culicoides and Simulium females and their infection with trypanosomatids in temperate forests at nests of raptor birds of two species in 2020–2021.
Insect SpeciesBird NestNo of IndividualsNo of Positive InsectsDetected Kinetoplastids
Culicoides (Oecacta) festivipennisButeo buteo1781Crithidia brevicula
1Crithidia sp.
5Trypanosoma mixed infection
4T. bennetti group Hap 5
Clanga pomarina22138Trypanosoma mixed infection
1T. bennetti group Hap 2
21T. bennetti group Hap 5
C. (O.) kibunensisB. buteo9310Trypanosoma mixed infection
1C. brevicula
1Obscuromonas sp.
C. pomarina677Trypanosoma mixed infection
2Trypanosoma sp.
2T. bennetti group Hap 1
1T. bennetti group Hap 3
3T. bennetti group Hap 5
1T. bennetti group Hap 6
C. (O.) pictipennisB. buteo59922Trypanosoma mixed infection
2C. brevicula
1Trypanosoma avium
4T. bennetti group Hap 4
3T. bennetti group Hap 5
C. pomarina211Trypanosoma mixed infection
2T. bennetti group Hap 5
C. (Avaritia) obsoletusB. buteo30
C. pomarina40
C. (Culicoides) impunctatusB. buteo230
C. (C.) newsteadiB. buteo20
C. (C.) punctatusB. buteo30
C. pomarina91Trypanosoma mixed infection
C. (Silvaticulicoides) archayiB. buteo10
C. (S.) pallidicornisB. buteo10
C. pomarina20
C. (Wirthomyia) reconditusB. buteo10
C. (W.) segnisB. buteo50
C. pomarina151T. avium
1T. bennetti group Hap 1
1T. bennetti group Hap 5
Simulium (Eusimulium) angustipesC. pomarina20
S. (Nevermannia) vernumB. buteo11T. avium
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Kazak, M.; Bernotienė, R.; Treinys, R.; Bukauskaitė, D. Trypanosomatids in Bloodsucking Diptera Insects (Ceratopogonidae and Simuliidae) Wild-Caught at Raptor Bird Nests in Temperate Forests. Diversity 2023, 15, 692. https://doi.org/10.3390/d15050692

AMA Style

Kazak M, Bernotienė R, Treinys R, Bukauskaitė D. Trypanosomatids in Bloodsucking Diptera Insects (Ceratopogonidae and Simuliidae) Wild-Caught at Raptor Bird Nests in Temperate Forests. Diversity. 2023; 15(5):692. https://doi.org/10.3390/d15050692

Chicago/Turabian Style

Kazak, Margarita, Rasa Bernotienė, Rimgaudas Treinys, and Dovilė Bukauskaitė. 2023. "Trypanosomatids in Bloodsucking Diptera Insects (Ceratopogonidae and Simuliidae) Wild-Caught at Raptor Bird Nests in Temperate Forests" Diversity 15, no. 5: 692. https://doi.org/10.3390/d15050692

APA Style

Kazak, M., Bernotienė, R., Treinys, R., & Bukauskaitė, D. (2023). Trypanosomatids in Bloodsucking Diptera Insects (Ceratopogonidae and Simuliidae) Wild-Caught at Raptor Bird Nests in Temperate Forests. Diversity, 15(5), 692. https://doi.org/10.3390/d15050692

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