Medical and Veterinary Entomology (2015), doi: 10.1111/mve.12108
Host preferences of ornithophilic biting midges of the genus Culicoides in the Eastern Balkans A. B O B E V A 1 , P. Z E H T I N D J I E V 1 , M. I L I E V A 1,2 , D. D I M I T R O V 1,3 , A. M A T H I S 4 and S. B E N S C H 2 1
Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia, Bulgaria, 2 Department of Biology, Lund University, Lund, Sweden, 3 Institute of Ecology, Nature Research Centre, Vilnius, Lithuania and 4 Swiss National Centre for Vector Entomology, Institute of Parasitology, Vetsuisse Faculty, University of Zürich, Zürich, Switzerland
Abstract. Many biting midges of the genus Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) are competent vectors of a diverse number of pathogens. The identification of their feeding behaviour and of vector–host associations is essential for understanding their transmission capacity. By applying two different nested polymerase chain reaction (PCR) assays, of which one targeted the avian cyt b gene and the other targeted the COI gene of a wide range of vertebrates, we identified the blood hosts of six biting midge species including Culicoides circumscriptus, Culicoides festivipennis, Culicoides punctatus, Culicoides pictipennis, Culicoides alazanicus and Culicoides cf. griseidorsum, the latter two of which are reported in Bulgaria for the first time. Bird DNA was found in 50.6% of 95 investigated bloodmeals, whereas mammalian DNA was identified in 13.7%. Two Culicoides species were found to feed on both birds and mammals. There was remarkable diversity in the range of avian hosts: 23 species from four orders were identified in the abdomens of four Culicoides species. The most common bird species identified was the magpie, Pica pica (n = 7), which was registered in all four ornithophilic biting midge species. Six bloodmeals from the great tit, Parus major, were recorded only in C. alazanicus. None of the studied species of Culicoides appeared to be restricted to a single avian host. Key words. Culicoides, Haemoproteus, bloodmeal, host preferences, vector–host
associations.
Introduction Many biting midges of the genus Culicoides Latreille, 1809 are vectors of a diverse number of pathogens. A wide range of diseases of medical and veterinary importance, such as bluetongue virus (BTV) and African horse sickness (Mellor et al., 2000; Cêtre-Sossah et al., 2004), as well as avian trypanosomes (Miltgen & Landau, 1982) and haemosporidian infections (Garnham, 1966; Valki¯unas, 2005), are transmitted by these insect vectors. Avian haemosporidians (Haemosporida) use birds for asexual reproduction and development of gametocytes; they are transmitted exclusively by blood-sucking dipterans in which sexual reproduction and sporogony take place (Garnham, 1966; Valki¯unas, 2005). The majority of the avian
Haemoproteus (Haemosporida: Haemoproteidae) species are transmitted by biting midges of the genus Culicoides; however, hippoboscid flies (Diptera: Hippoboscidae) act as vectors of some of these parasites (Garnham, 1966; Valki¯unas, 2005). One species of the genus Leucocytozoon (Achromatorida: Leucocytozoidae), Leucocytozoon caulleryi, is also known to be transmitted by Culicoides (Valki¯unas, 2005). These insect vectors are widespread in almost all parts of the world and live in diverse habitats (Mellor et al., 2000). Prior to the present study, 36 species of biting midge had been reported from Bulgaria (Zilahi, 1934; Remm, 1988; Nedelchev, 2008). Most previous studies on avian haemosporidians have focused on their interactions with bird hosts, especially with respect to species diversity, prevalence and local transmission in different
Correspondence: Aneliya Bobeva, Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin Street, Sofia 1113, Bulgaria. Tel.: + 359 2 871 71 95/105; Fax: + 359 2 870 54 98; E-mail: [email protected] © 2015 The Royal Entomological Society
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2 A. Bobeva et al. geographical regions (Waldenström et al., 2002; Dimitrov et al., 2010). Few recent studies report potential associations between Haemoproteus species and vectors of the genus Culicoides (Martinez-de la Puente et al., 2011; Santiago-Alarcon et al., 2012, 2013; Bobeva et al., 2013, Synek et al., 2013). However, the roles of species of Culicoides in the transmission of certain haemoproteids, and in the geographic distribution of these parasites, remain poorly known. In order to understand the transmission capacity of Culicoides biting midges and their roles in the distribution of parasites, it is essential to identify the degree of specificity in the food preferences of insect vectors with reference to avian hosts. The host range of biting midge species remains largely undiscovered. Some recent studies indicate that most Culicoides spp. are mammaliophilic and ornithophilic, although some feed on reptiles and frogs (Borkent, 2005; Pettersson et al., 2012; Santiago-Alarcon et al., 2012). Because of their veterinary importance as vectors of BTV, trapping of biting midges has been conducted mainly within or close to farms containing livestock and therefore most attention has been focused on mammaliophilic species (Mellor et al., 2000; Borkent, 2005; Lassen et al., 2011, 2012). However, recent investigations have revealed some avian hosts of biting midges by examining bloodmeals found in the insects’ abdomens (Lassen et al., 2011, 2012; Pettersson et al., 2012; Santiago-Alarcon et al., 2012, 2013). Some Culicoides species feed preferentially on birds (Culicoides duddingstoni) or mammals (Culicoides deltus), whereas others seem to be host generalists (Culicoides circumscriptus, Culicoides festivipennis, Culicoides pictipennis) and feed on both birds and mammals (Alcaide et al., 2009; Cern´y et al., 2011, Pettersson et al., 2012; Santiago-Alarcon et al., 2012, 2013). Host generalist biting midges are of special interest because they are capable of feeding on different vertebrate groups (Santiago-Alarcon et al., 2013) and thus can facilitate the occurrence of emerging diseases. The prevalence and species diversity of haemosporidian parasites in birds, as well as the number of Haemoproteus species with local transmission, in the region of Kalimok Biological Station (northeast Bulgaria) have been intensely studied for the last 15 years (Valki¯unas et al., 1999, 2007, 2008; Shurulinkov & Golemansky, 2002, 2003; Zehtindjiev et al., 2008; Shurulinkov & Ilieva, 2009; Dimitrov et al., 2010). The present study focuses on the feeding preferences of ornithophilic biting midges in this region in order to reveal the potential vectorial capacity of these insects for the transmission of avian haemosporidians.
Materials and methods
Control (CDC) mini light traps with incandescent light, which use a 4-W CM-47 incandescent light bulb and down-draught suction motors and require a 6-V DC power supply or battery (BioQuip Products, Inc., Compton, CA, U.S.A.). Traps were placed on trees at heights of 1.5–4.0 m above the ground near two habitats, which were comprised of Phragmites australis, partly surrounded by dwarf elder (Sambucus ebulus), and deciduous forest of false acacia (Robinia pseudoacacia) and raywood ash (Fraxinus oxycarpa). The light traps were set to operate from approximately 1–2 h before sunset to 1–2 h after sunrise. Samples were subsequently processed in the laboratory under a stereomicroscope. Morphological identification of Culicoides spp. All Culicoides specimens were preserved in 70% ethanol. Species identification of representative (three to eight) specimens was conducted by microscopic analyses of wing patterns and by the observation of body parts (head, legs, wings and spermatheca of females) mounted on slides according to Delécolle (1985). The remains of the abdomen and thorax were stored in 2-mL round-bottomed Eppendorf tubes (Vaudaux-Eppendorf AG, Schönenbuch, Switzerland) at −20 ∘ C for DNA isolation. The other individuals included in the study were identified according to wing pattern only.
DNA extraction and polymerase chain reaction protocol for identification of Culicoides spp. The molecular identification of representative biting midges (one to three individuals per species) by polymerase chain reaction (PCR) and sequencing was carried out as previously described by Wenk et al. (2012). Briefly, abdomens were ground in 180 𝜇L Tris–EDTA buffer (pH 8.4) using a mixer mill with a steel bead (3 mm in diameter) at 30 Hz for 1 min twice with an in-between chill-down step on ice. Total DNA was isolated using the QIAamp DNA Mini Kit (Qiagen Instruments AG, Hombrechtikon, Switzerland) according to the manufacturer’s instructions. Part (585 bp) of the mitochondrial cytochrome oxidase subunit I gene (mt COI) was amplified with the primers C1-J-1718 mod (5′ -GGWGGRTTTGGWAAYTGAYTAG-3′ ) and CW1 R (5′ -AGHWCCAAAAGTTTCYTTTTTCC-3′ ), and the sequences of the amplicons were determined after purification (MinElute PCR Purification Kit; Qiagen Instruments AG) by a private company (Synergene Biotech GmbH, Schlieren, Switzerland). Identification of Culicoides spp. with MALDI-TOF mass spectrometry
Insect collection and identification Midges of Culicoides were collected during May–October 2012 and May 2013 at Kalimok Biological Station (44∘ 00′ N, 26∘ 26′ E), Silistra District on 122 trapping nights. The insects were trapped by ultraviolet (UV) light/suction traps (OVI traps) manufactured by the Onderstepoort Veterinary Institute (Onderstepoort, South Africa) (Venter & Meiswinkel, 1994), which consist of an 8-W UV light bulb and a down-draught suction motor, powered by a car battery, and by Centers for Disease
The preparation of samples for MALDI-TOF mass spectrometry (MS) and implementation of the analysis were carried out as previously described (Kaufmann et al., 2011). Briefly, two C. festivipennis specimens without abdomens were individually triturated in 1.5-mL Eppendorf tubes in 10 μL of 25% formic acid using a manual homogenizer (Bio Vortexer; Fisher Scientific AG, Wohlen, Switzerland) with disposable pellet pestles. A
© 2015 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12108
Host preferences of ornithophilic midges 1-μL quantity of the homogenate was spotted in duplicate onto a steel target plate, and 1 μL of SA matrix (saturated solution of sinapic acid in 60% acetonitrile, 40% H2 O, 0.3% trifluoroacetic acid; all chemicals from Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) was added directly to the air-dried spots. Mass fingerprints were generated by a commercial company (Mabritec SA, Riehen, Switzerland) and subjected to automated identification against more than 4000 validated biomarker mass sets, including 15 Culicoides species-specific sets, as described by Kaufmann et al. (2012a). The diagnostic specificity of MALDI-TOF MS was evaluated in 1200 randomly chosen biting midges collected in the field, yielding the correct identification of 1173 (97.8%) specimens [as confirmed by PCR/sequencing of DNA extracted from the abdomens (Wenk et al., 2012) of 95 randomly selected specimens]; 14 specimens revealed novel mass spectra (and the species were eventually identified by PCR/sequencing), and 13 specimens yielded no identification as a result of poor-quality spectra (Kaufmann et al., 2012b).
DNA extraction and PCR protocol for identification of bloodmeals Female Culicoides individuals (whole insects) were homogenized into 1.5-mL tubes with 100 μL of lysis buffer (0.1 m Tris, 0.005 m EDTA, 0.2% SDS, 0.2 m NaCl, pH 8.5) (Laird et al., 1991) and 1.5 μL proteinase K (20 mg/mL) and incubated for 3 h at 56 ∘ C. After incubation, the samples were centrifuged for 10 min at 7378g. The supernatant was placed into new tubes and processed using standard ethanol precipitation. Pelleted DNA was dissolved in 25 μL ddH2 O and quantified using a NanoDrop 2000 microvolume spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, U.S.A.). The quantified DNA was diluted to a standard concentration of 25–50 ng/μL for PCR and used as a template for amplification of mitochondrial DNA from the vertebrate bloodmeal.
Primer design strategy and procedure for bloodmeal identification Avian and mammalian mitochondrial cytochrome b (cyt b) sequences were downloaded from GenBank and aligned using BioEdit software (Hall, 1999). These multiple alignments revealed regions that were highly conserved for bird species and contained diverse fragments for mammals. Based on sequence homology among aligned sequences of the avian cyt b gene, the following primers were devised to amplify 219-bp fragments (excluding primers): cytbPF1 5′ -CCTGAG GACAAATATCATTCTGAGG-3′ , and cytbBirdR 5′ -GGGTGG AATGGGATTTTATCGC-3′ . In order to improve the efficiency of detection, a second forward primer was designed for semi-nested PCR (cytbBirdF2 5′ -TCAGCAATCCCATACAT TGGCCAA-3′ ). One microlitre of the first PCR was used as a template for the second PCR including cytbBirdF2 and cytbBirdR primer pairs to amplify 169-bp fragments. Both PCRs were performed
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separately in 25-μL volumes with the same proportions of reagents [2.5 μL PCR 10× buffer, 0.125 mm of each nucleotide, 0.4 mm of each primer, 0.1 μL DreamTaq DNA polymerase (Thermo Fisher Scientific, Inc.)] and 1 μL of the DNA template. The PCR was conducted according to a thermal protocol of 94 ∘ C for 2 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. The final cycle was completed with a 10-min extension at 72 ∘ C. Negative controls were used with every PCR in order to detect possible contamination. Positive or negative samples were indicated by the presence or absence of bands on 2% agarose gels using 4.0 μL of the reaction. In order to reveal bloodmeals of non-avian origin, all negative samples (n = 51) were further processed with primers capable of amplifying DNA from the blood of a diverse number of vertebrate species. Two primer pairs were used for a nested PCR assay, as described in Alcaide et al. (2009). The initial PCR was conducted with M13BCV-FW (5′ -TGTAAAA CGACGGCCAGTH-AAYCAYAARGAYATYGG-3′ ) and BCV -RV1 (5′ -GCYCANACYATNCCYATRTA-3′ ) and the nested step was performed with M13 (5′ -GTAAAACGACGGCCAG TG-3′ ) and BCV-RV2 (5′ -ACYATNCCYATRTANCCRAANG G-3′ ) primer pairs. Both PCRs were performed separately in 25-𝜇L volumes using the proportions of reagents given above and with temperature profiles as described in Alcaide et al. (2009). Amplified fragments were sequenced with the primers cytbBirdF2 and BCV-RV2 at Macrogen Ltd (Amsterdam, the Netherlands). The sequences obtained were edited using BioEdit (Hall, 1999) and compared with available sequences in the GenBank DNA database.
Results A total of 4476 Culicoides specimens were identified as belonging to six species of Culicoides: C. circumscriptus Kieffer, 1918 (n = 373); C. festivipennis Kieffer, 1914 (n = 1354); Culicoides punctatus (Meigen, 1804) (n = 288); C. pictipennis (Staeger, 1839) (n = 146); Culicoides alazanicus Dzhafarov, 1961 (n = 2290), and Culicoides griseidorsum Kieffer, 1918 (n = 25). Ninety-five specimens were identified as blood-fed individuals. These included specimens of C. circumscriptus (n = 6), C. festivipennis (n = 23), C. punctatus (n = 15), C. pictipennis (n = 3), C. alazanicus (n = 40) and C. griseidorsum (n = 8). Culicoides alazanicus, identified by both morphological and molecular methods, has not been reported previously from Bulgaria. One species was identified as C. griseidorsum on the basis of morphological characteristics, but this was not confirmed by sequence analysis. blast analysis in GenBank showed a 99% match with C. duddingstoni for one individual and < 90% identity with certain Culicoides species for the others. Therefore, the identification of this species requires additional investigation. In the current study, the species will be referred to as C. griseidorsum with the abbreviation cf . (confer) thus: C. cf . griseidorsum. The application of the new primers designed for the purposes of the present study allowed us to obtain good-quality cyt b
© 2015 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12108
4 A. Bobeva et al. 27 13 5 6 2 2 55 1∗ — — — — 1 2 1 — 1 — — — 2 1∗ — — — — — 1 — 1 — — — — 1 2 1 3 1 — — 7 1 — — — — — 1
1 — — — — — 1
1 — — — — — 1
1 — — — — — 1
— — — 1∗ — — 1
1 — — — — — 1
— 2 — — — — 2
4 1 — — — — 5
6 — — — — — 6
— 1 — — — — 1
— 2 — — — — 2
— 1 — — — — 1
1 — — — — — 1
Discussion
Streptopelia decaocto Sylvia borin Passer montanus Phylloscopus trochilus Pica pica Passer domesticus Passer hispaniolensis Oriolus oriolus Parus major Muscicapa striata Nycticorax nycticorax Delichon urbica Ixobrychus minutus Luscinia luscinia Luscinia megarhynchos Coccothraustes coccothraustes Columba palumbus Acrocephalus palustris
— — — 1 — — 1 C. alazanicus (n = 40) C. festivipennis (n = 23) C. circumscriptus (n = 6) C. cf. griseidorsum (n = 8) C. pictipennis (n = 3) C. punctatus (n = 15) Total
∗Mixed bloodmeals.
Asio otus Anthus trivialis Ardea purpurea Culicoides spp.
1 1 — — — — 2
3 — — — — — 3
1 3 1 — — — 5
— — — 1 — — 1
Bos taurus Turdus merula Turdus philomelos
Avian species
1 — — — — — 1
— — — — 1 — 1
Cervus elaphus
Bloodmeals identified from vertebrate hosts, n
— — — 2∗ 1 1∗ 4
Homo sapiens
Mammalian species
Total Table 1. Number of bloodmeals of avian and mammalian origin identified to species level.
sequences from 44 bloodmeals. The ornithophilic biting midges were found to have fed on 23 avian species (Table 1). In two individuals, mixed bloodmeals were revealed by double peaks at several positions in the sequence chromatograms. Analyses of these two cases gave the best, but not perfect, blast hits to Luscinia megarhynchos (95% similarity) and Sylvia borin (89% similarity), respectively. Eleven good-quality COI sequences were obtained using the protocol described by Alcaide et al. (2009). DNA of three mammalian (Bos taurus, Cervus elaphus and Homo sapiens) and four avian (Columba palumbus, Ixobrychus minutus, Oriolus oriolus and Passer hispaniolensis) species were amplified. In three individuals, we identified mixed bloodmeals with the best, but not perfect, blast hits to H. sapiens (one with 95% similarity) and C. elaphus (two with 99 and 94% similarity, respectively) (Table 1).
The identification of the host preferences of haematophagous insects is crucial for elucidating their role in the transmission of various pathogens. The development of molecular approaches and their application to ecological and vector-borne disease studies have revealed a high diversity of vector–host associations (Pettersson et al., 2012; Santiago-Alarcon et al., 2012, 2013). However, opportunities to use host DNA markers for their identification are limited by the digestion of bloodmeals and the rapid degradation of DNA in the mid-guts of insects. As the stage of blood digestion increases, the likelihood of successfully identifying the bloodmeal source may decrease (Oshaghi et al., 2006; Martinez-de la Puente et al., 2013). Primers designed in the current study are capable of amplifying DNA of a remarkable diversity of bird species and give convincing results despite the relatively short length of the amplicons (169 bp). This increases the probability of the successful amplification of partly degraded DNA. To optimize the protocol, a nested PCR was conducted to enhance the specificity of the method and to obtain suitable concentrations of PCR products for sequencing. This approach allowed the precise identification of 21 species of birds bitten by biting midges of four species (Table 1). In comparison, similar studies have identified between three and 10 bird species (Lassen et al., 2012; Pettersson et al., 2012; Santiago-Alarcon et al., 2013). Four avian species (C. palumbus, I. minutus, O. oriolus and P. hispaniolensis) were identified using the primers from Alcaide et al. (2009). Two of them (C. palumbus and I. minutus) increase the diversity of avian hosts identified in the studied area. DNA from the other two avian species was found in two biting midges that had been found to be negative for bird DNA using avian-specific primers. The percentage of insects containing bird DNA in abdomens in our study is relatively high (50.6%) compared with those in previous studies. For example, Pettersson et al. (2012) found that 12.6% of bloodmeals originated from birds, and Santiago-Alarcon et al. (2013) and Lassen et al. (2012) reported rates of 14.5% and 5.8%, respectively. Contrary to what might have been expected based on earlier findings (Lassen et al., 2011, 2012; Pettersson et al., 2012), only seven of 51 (13.7%) individuals were found to have fed on
© 2015 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12108
Host preferences of ornithophilic midges
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Table 2. Bloodmeal hosts of the Culicoides spp. investigated in this study as identified in previous studies. Culicoides species
Previously known vertebrate host
References
C. alazanicus C. griseidorsum C. circumscriptus
N/A Mammalian: Bos taurus Mammalian: Homo sapiens
N/A Ayllón et al. (2014) Lassen et al. (2012) Pettersson et al. (2012)
C. festivipennis
Avian: Pica pica, Turdus merula, Phylloscopus trochilus, Corvus corone, Turdus philomelos, Columba palumbus Mammalian: Homo sapiens
C. pictipennis
Avian: Columba palumbus, Turdus philomelos, Pica pica Mammalian: Homo sapiens
C. punctatus
Avian: Pica pica, Turdus merula, Erithacus rubecula, Parus major Mammalian: Bos taurus, Capra hircus, Capreolus capreolus, Equus callabus, Alces alces, Bos taurus, Ovis aries Avian: Anas platyrhynchos, Columba palumbus, Luscinia svecica
Lassen et al. (2012) Santiago-Alarcon et al. (2012, 2013) Pettersson et al. (2012) Lassen et al. (2012) Santiago-Alarcon et al. (2012, 2013) Pettersson et al. (2012) Lassen et al. (2011, 2012) Pettersson et al. (2012)
N/A, no available information.
non-avian vertebrates (Table 1). DNA from B. taurus, C. elaphus and H. sapiens was isolated from C. punctatus, C. pictipennis, C. cf. griseidorsum and C. alazanicus. The proportions of avian and mammalian bloodmeals obtained most probably reflect the richness of the habitat and the abundance of birds in the area of study. The use of two different nested PCR assays to identify the feeding sources of biting midges allowed us to identify more precise associations between haematophagous insects of the genus Culicoides and their vertebrate hosts in the area studied. Thus, the present study adds important information to existing knowledge of the feeding preferences of the Culicoides species investigated (Table 2). Recent studies have shown that most Culicoides species are opportunistic and readily feed on a variety of mammals and birds; these opportunistic species include C. circumscriptus, C. festivipennis, C. pictipennis and C. punctatus (Lassen et al., 2012; Pettersson et al., 2012; Santiago-Alarcon et al., 2013). According to our data, two Culicoides species (C. alazanicus and C. cf. griseidorsum) were found to feed from both birds and mammals. However, some species demonstrated predominant ornithophilic (C. pictipennis) or mammophilic (C. punctatus) feeding preferences (Lassen et al., 2012; Pettersson et al., 2012). Despite its predominantly ornithophilic preferences, C. pictipennis was found to be able to feed on cattle (B. taurus) and red deer (C. elaphus). Therefore, host availability plays an important role in the feeding behaviour of biting midges (Lassen et al., 2012). Culicoides cf. griseidorsum and C. alazanicus were recorded in Bulgaria for the first time in the present study. Moreover, C. alazanicus was identified as the most abundant species in our samples and therefore should be regarded as the most probable vector of haemoproteids in the region. However, information on the feeding preferences of both species is lacking in the literature. There is increasing interest in the vectorial capacity of Culicoides biting midges for BTV, as well as in the transmission biology of the highly diverse group of haemosporidian parasites.
Recent studies have revealed interesting associations between various Culicoides spp. (C. circumscriptus, C. pictipennis, C. festivipennis, Culicoides salinarius, C. duddingstoni, Culicoides kibunensis, C. punctatus, Culicoides simulator and Culicoides segnis) and Haemoproteus lineages, and show variations in the specificity of parasites to vectors (Martinez-de la Puente et al., 2011; Santiago-Alarcon et al., 2012; Bobeva et al., 2013; Synek et al., 2013). In addition, revealing the feeding preferences of biting midges sheds light on the transmission routes of avian haemosporidians and contributes to better understanding of the ecological and evolutionary traits of vector–host–parasite interactions. In the present study, C. circumscriptus, C. festivipennis, C. alazanicus and C. cf. griseidorsum were found to feed on a variety of bird species. As these species have rather opportunistic feeding patterns, they can be expected to represent important vectors of diverse haemoproteid parasites in the studied area. Acknowledgements This study represents Report No. 57 from Kalimok Field Station. We acknowledge Dr Bruno Mathieu (University of Strasbourg, Strasbourg, France) for his contribution to the morphological identification of Culicoides biting midges. We are grateful to Dr Boyko B. Georgiev, Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia, Bulgaria, for useful suggestions offered during the course of the present study and in the preparation of the present manuscript, and to Dr Ognyan Mikov, National Centre for Infectious and Parasitic Diseases, Sofia, Bulgaria, for valuable suggestions. The authors are grateful to the staff of Kalimok Field Station and to Christoffer Sjöholm, for their support with the collection of insects, and to Nikola Bankov, Master degree student in the Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia, Bulgaria, for his help with laboratory work. Facilities developed in
© 2015 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12108
6 A. Bobeva et al. the frames of the projects WETLANET (FP7 CAPACITIES grant 229802), CEBDER (National Science Fund, Ministry of Education, Youth and Science of the Republic of Bulgaria; grant DO 02-15/2009) and ‘Development of scientific potential in the field of faunistic diversity and environment protection’ (funded by the Ministry of Education, Youth and Science and the European Social Fund, Operational Programme ‘Human Resources Development’; grant BG 051 PO001-3.3.04/41) were used in the course of the present study.
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identification by MALDI-TOF mass spectrometry. Parasites & Vectors, 5, 246. Laird, P.W., Zijderveld, A., Linders, K., Rudnicki, M.A., Jaenisch, R. & Berns, A. (1991) Simplified mammalian DNA isolation procedure. Nucleic Acids Research, 19, 4293. Lassen, S.B., Nielsen, S.A., Skovgård, H. & Kristensen, M. (2011) Molecular identification of bloodmeals from biting midges (Diptera: Ceratopogonidae: Culicoides Latreille) in Denmark. Parasitology Research, 108, 823–829. Lassen, S.B., Nielsen, S.A. & Kristensen, M. (2012) Identity and diversity of blood meal hosts of biting midges (Diptera: Ceratopogonidae: Culicoides Latreille) in Denmark. Parasites & Vectors, 5, 143. Martinez-de la Puente, J., Martínez, J., Rivero-de Aguilar, J., Herrero, J. & Merino, S. (2011) On the specificity of avian blood parasites: revealing specific and generalist relationships between haemosporidians and biting midges. Molecular Ecology, 20, 3275–3287. Martinez-de la Puente, J., Ruiz, S., Soriguer, R. & Figuerola, J. (2013) Effect of blood meal digestion and DNA extraction protocol on the success of blood meal source determination in the malaria vector Anopheles atroparvus. Malaria Journal, 12, 109. Mellor, P.S., Boorman, J. & Baylis, M. (2000) Culicoides biting midges: their role as arbovirus vectors. Annual Review of Entomology, 45, 307–340. Miltgen, F. & Landau, I. (1982) Culicoides nubeculosus, vecteur experimental d’un nouveau trypanosome des Psittaciformes: Trypanosoma avium bakeri n. sp. Annales de Parasitologie Humaine et Comparée, 57, 423–428. Nedelchev, N. (2008) Prouchvane varkhu nasekomite ot rod Culicoides (Diptera: Ceratopogonidae) v Balgariya. Central Research Diagnostic Veterinary Institute, Sofia. Oshaghi, M.A., Chavshin, A.R., Vatandoost, H., Yaaghoobi, F., Mohtarami, F. & Noorjah, N. (2006) Effects of post-ingestion and physical conditions on PCR amplification of host blood meal DNA in mosquitoes. Experimental Parasitology, 112, 232–236. Pettersson, E., Bensch, S., Ander, M., Chirico, J., Sigvald, R. & Ignell, R. (2012) Molecular identification of bloodmeals and species composition in Culicoides biting midges. Medical and Veterinary Entomology, 27, 104–112. Remm, H. (1988) Ceratopogonidae. Catalogue of Palaearctic Diptera (ed. by A. Soos), pp. 11–110. Academiai Kiado, Budapest. Santiago-Alarcon, D., Havelka, P., Schaefer, H.M. & Segelbacher, G. (2012) Bloodmeal analysis reveals avian Plasmodium infections and broad host preferences of Culicoides (Diptera: Ceratopogonidae) vectors. PLoS One, 7, e31098. Santiago-Alarcon, D., Havelka, P., Pineda, E., Segelbacher, G. & Schaefer, H.M. (2013) Urban forests as hubs for novel zoonosis: blood meal analysis, seasonal variation in Culicoides (Diptera: Ceratopogonidae) vectors, and avian haemosporidians. Parasitology, 140, 1799–1810. Shurulinkov, P. & Golemansky, V. (2002) Haemoproteids (Haemosporida: Haemoproteidae) of wild birds in Bulgaria. Acta Protozoologica, 41, 359–374. Shurulinkov, P. & Golemansky, V. (2003) Plasmodium and Leucocytozoon (Sporozoa: Haemosporida) of wild birds in Bulgaria. Acta Protozoologica, 42, 205–214. Shurulinkov, P. & Ilieva, M. (2009) Spatial and temporal differences in the blood parasite fauna of passerine birds during the spring migration in Bulgaria. Parasitology Research, 104, 1453–1458. Synek, P., Munclinger, P., Albrecht, T. & Vot´ypka, J. (2013) Avian haemosporidians in haematophagous insects in the Czech Republic. Parasitology Research, 112, 839–845.
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Host preferences of ornithophilic midges Valki¯unas, G. (2005) Avian Malaria Parasites and other Haemosporidia. CRC Press, Boca Raton, FL. Valki¯unas, G., Iezhova, T., Golemansky, V., Pilarska, D. & Zehtindjiev, P. (1999) Blood protozoan parasites (Protozoa: Kinetoplastida and Haemosporida) in wild birds from Bulgaria. Acta Zoologica Bulgarica, 51, 127–129. Valki¯unas, G., Zehtindjiev, P., Hellgren, O., Ilieva, M., Iezhova, T.A. & Bensch, S. (2007) Linkage between mitochondrial cytochrome b lineages and morphospecies of two avian malaria parasites, with a description of Plasmodium (Novyella) ashfordi sp. nov. Parasitology Research, 100, 1311–1322. Valki¯unas, G., Zehtindjiev, P., Dimitrov, D., Križanauskien˙e, A., Iezhova, T.A. & Bensch, S. (2008) Polymerase chain reaction-based identification of Plasmodium (Huffia) elongatum, with remarks on species identity of haemosporidian lineages deposited in GenBank. Parasitology Research, 102, 1185–1193. Venter, G.J. & Meiswinkel, R. (1994) The virtual absence of Culicoides imicola (Diptera: Ceratopogonidae) in a light-trap survey of the colder, high-lying area of the eastern Orange Free State, South Africa,
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and implications for the transmission of arboviruses. Onderstepoort Journal of Veterinary Research, 61, 327–340. Waldenström, J., Bensch, S., Kiboi, S., Hasselquist, D. & Ottosson, U. (2002) Cross-species infection of blood parasites between resident and migratory songbirds in Africa. Molecular Ecology, 11, 1545–1554. Wenk, C.E., Kaufmann, C., Schaffner, F. & Mathis, A. (2012) Molecular characterization of Swiss Ceratopogonidae (Diptera) and evaluation of real-time PCR assays for the identification of Culicoides biting midges. Veterinary Parasitology, 184, 258–266. Zehtindjiev, P., Ilieva, M., Westerdahl, H., Hansson, B., Valki¯unas, G. & Bensch, S. (2008) Dynamics of parasitemia of malaria parasites in a naturally and experimentally infected migratory songbird, the great read warbler Acrocephalus arundinaceus. Experimental Parasitology, 119, 99–110. Zilahi, G. (1934) Beiträge zur fliegenfauna Bulgariens. I. Chironomiden. Bulletin de la Société Entomologique de Bulgarie, 8, 152–158. Accepted 7 July 2014
© 2015 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12108
Host preferences of ornithophilic biting midges of the genus Culicoides in the Eastern Balkans A. B O B E V A 1 , P. Z E H T I N D J I E V 1 , M. I L I E V A 1,2 , D. D I M I T R O V 1,3 , A. M A T H I S 4 and S. B E N S C H 2 1
Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia, Bulgaria, 2 Department of Biology, Lund University, Lund, Sweden, 3 Institute of Ecology, Nature Research Centre, Vilnius, Lithuania and 4 Swiss National Centre for Vector Entomology, Institute of Parasitology, Vetsuisse Faculty, University of Zürich, Zürich, Switzerland
Abstract. Many biting midges of the genus Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) are competent vectors of a diverse number of pathogens. The identification of their feeding behaviour and of vector–host associations is essential for understanding their transmission capacity. By applying two different nested polymerase chain reaction (PCR) assays, of which one targeted the avian cyt b gene and the other targeted the COI gene of a wide range of vertebrates, we identified the blood hosts of six biting midge species including Culicoides circumscriptus, Culicoides festivipennis, Culicoides punctatus, Culicoides pictipennis, Culicoides alazanicus and Culicoides cf. griseidorsum, the latter two of which are reported in Bulgaria for the first time. Bird DNA was found in 50.6% of 95 investigated bloodmeals, whereas mammalian DNA was identified in 13.7%. Two Culicoides species were found to feed on both birds and mammals. There was remarkable diversity in the range of avian hosts: 23 species from four orders were identified in the abdomens of four Culicoides species. The most common bird species identified was the magpie, Pica pica (n = 7), which was registered in all four ornithophilic biting midge species. Six bloodmeals from the great tit, Parus major, were recorded only in C. alazanicus. None of the studied species of Culicoides appeared to be restricted to a single avian host. Key words. Culicoides, Haemoproteus, bloodmeal, host preferences, vector–host
associations.
Introduction Many biting midges of the genus Culicoides Latreille, 1809 are vectors of a diverse number of pathogens. A wide range of diseases of medical and veterinary importance, such as bluetongue virus (BTV) and African horse sickness (Mellor et al., 2000; Cêtre-Sossah et al., 2004), as well as avian trypanosomes (Miltgen & Landau, 1982) and haemosporidian infections (Garnham, 1966; Valki¯unas, 2005), are transmitted by these insect vectors. Avian haemosporidians (Haemosporida) use birds for asexual reproduction and development of gametocytes; they are transmitted exclusively by blood-sucking dipterans in which sexual reproduction and sporogony take place (Garnham, 1966; Valki¯unas, 2005). The majority of the avian
Haemoproteus (Haemosporida: Haemoproteidae) species are transmitted by biting midges of the genus Culicoides; however, hippoboscid flies (Diptera: Hippoboscidae) act as vectors of some of these parasites (Garnham, 1966; Valki¯unas, 2005). One species of the genus Leucocytozoon (Achromatorida: Leucocytozoidae), Leucocytozoon caulleryi, is also known to be transmitted by Culicoides (Valki¯unas, 2005). These insect vectors are widespread in almost all parts of the world and live in diverse habitats (Mellor et al., 2000). Prior to the present study, 36 species of biting midge had been reported from Bulgaria (Zilahi, 1934; Remm, 1988; Nedelchev, 2008). Most previous studies on avian haemosporidians have focused on their interactions with bird hosts, especially with respect to species diversity, prevalence and local transmission in different
Correspondence: Aneliya Bobeva, Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin Street, Sofia 1113, Bulgaria. Tel.: + 359 2 871 71 95/105; Fax: + 359 2 870 54 98; E-mail: [email protected] © 2015 The Royal Entomological Society
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2 A. Bobeva et al. geographical regions (Waldenström et al., 2002; Dimitrov et al., 2010). Few recent studies report potential associations between Haemoproteus species and vectors of the genus Culicoides (Martinez-de la Puente et al., 2011; Santiago-Alarcon et al., 2012, 2013; Bobeva et al., 2013, Synek et al., 2013). However, the roles of species of Culicoides in the transmission of certain haemoproteids, and in the geographic distribution of these parasites, remain poorly known. In order to understand the transmission capacity of Culicoides biting midges and their roles in the distribution of parasites, it is essential to identify the degree of specificity in the food preferences of insect vectors with reference to avian hosts. The host range of biting midge species remains largely undiscovered. Some recent studies indicate that most Culicoides spp. are mammaliophilic and ornithophilic, although some feed on reptiles and frogs (Borkent, 2005; Pettersson et al., 2012; Santiago-Alarcon et al., 2012). Because of their veterinary importance as vectors of BTV, trapping of biting midges has been conducted mainly within or close to farms containing livestock and therefore most attention has been focused on mammaliophilic species (Mellor et al., 2000; Borkent, 2005; Lassen et al., 2011, 2012). However, recent investigations have revealed some avian hosts of biting midges by examining bloodmeals found in the insects’ abdomens (Lassen et al., 2011, 2012; Pettersson et al., 2012; Santiago-Alarcon et al., 2012, 2013). Some Culicoides species feed preferentially on birds (Culicoides duddingstoni) or mammals (Culicoides deltus), whereas others seem to be host generalists (Culicoides circumscriptus, Culicoides festivipennis, Culicoides pictipennis) and feed on both birds and mammals (Alcaide et al., 2009; Cern´y et al., 2011, Pettersson et al., 2012; Santiago-Alarcon et al., 2012, 2013). Host generalist biting midges are of special interest because they are capable of feeding on different vertebrate groups (Santiago-Alarcon et al., 2013) and thus can facilitate the occurrence of emerging diseases. The prevalence and species diversity of haemosporidian parasites in birds, as well as the number of Haemoproteus species with local transmission, in the region of Kalimok Biological Station (northeast Bulgaria) have been intensely studied for the last 15 years (Valki¯unas et al., 1999, 2007, 2008; Shurulinkov & Golemansky, 2002, 2003; Zehtindjiev et al., 2008; Shurulinkov & Ilieva, 2009; Dimitrov et al., 2010). The present study focuses on the feeding preferences of ornithophilic biting midges in this region in order to reveal the potential vectorial capacity of these insects for the transmission of avian haemosporidians.
Materials and methods
Control (CDC) mini light traps with incandescent light, which use a 4-W CM-47 incandescent light bulb and down-draught suction motors and require a 6-V DC power supply or battery (BioQuip Products, Inc., Compton, CA, U.S.A.). Traps were placed on trees at heights of 1.5–4.0 m above the ground near two habitats, which were comprised of Phragmites australis, partly surrounded by dwarf elder (Sambucus ebulus), and deciduous forest of false acacia (Robinia pseudoacacia) and raywood ash (Fraxinus oxycarpa). The light traps were set to operate from approximately 1–2 h before sunset to 1–2 h after sunrise. Samples were subsequently processed in the laboratory under a stereomicroscope. Morphological identification of Culicoides spp. All Culicoides specimens were preserved in 70% ethanol. Species identification of representative (three to eight) specimens was conducted by microscopic analyses of wing patterns and by the observation of body parts (head, legs, wings and spermatheca of females) mounted on slides according to Delécolle (1985). The remains of the abdomen and thorax were stored in 2-mL round-bottomed Eppendorf tubes (Vaudaux-Eppendorf AG, Schönenbuch, Switzerland) at −20 ∘ C for DNA isolation. The other individuals included in the study were identified according to wing pattern only.
DNA extraction and polymerase chain reaction protocol for identification of Culicoides spp. The molecular identification of representative biting midges (one to three individuals per species) by polymerase chain reaction (PCR) and sequencing was carried out as previously described by Wenk et al. (2012). Briefly, abdomens were ground in 180 𝜇L Tris–EDTA buffer (pH 8.4) using a mixer mill with a steel bead (3 mm in diameter) at 30 Hz for 1 min twice with an in-between chill-down step on ice. Total DNA was isolated using the QIAamp DNA Mini Kit (Qiagen Instruments AG, Hombrechtikon, Switzerland) according to the manufacturer’s instructions. Part (585 bp) of the mitochondrial cytochrome oxidase subunit I gene (mt COI) was amplified with the primers C1-J-1718 mod (5′ -GGWGGRTTTGGWAAYTGAYTAG-3′ ) and CW1 R (5′ -AGHWCCAAAAGTTTCYTTTTTCC-3′ ), and the sequences of the amplicons were determined after purification (MinElute PCR Purification Kit; Qiagen Instruments AG) by a private company (Synergene Biotech GmbH, Schlieren, Switzerland). Identification of Culicoides spp. with MALDI-TOF mass spectrometry
Insect collection and identification Midges of Culicoides were collected during May–October 2012 and May 2013 at Kalimok Biological Station (44∘ 00′ N, 26∘ 26′ E), Silistra District on 122 trapping nights. The insects were trapped by ultraviolet (UV) light/suction traps (OVI traps) manufactured by the Onderstepoort Veterinary Institute (Onderstepoort, South Africa) (Venter & Meiswinkel, 1994), which consist of an 8-W UV light bulb and a down-draught suction motor, powered by a car battery, and by Centers for Disease
The preparation of samples for MALDI-TOF mass spectrometry (MS) and implementation of the analysis were carried out as previously described (Kaufmann et al., 2011). Briefly, two C. festivipennis specimens without abdomens were individually triturated in 1.5-mL Eppendorf tubes in 10 μL of 25% formic acid using a manual homogenizer (Bio Vortexer; Fisher Scientific AG, Wohlen, Switzerland) with disposable pellet pestles. A
© 2015 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12108
Host preferences of ornithophilic midges 1-μL quantity of the homogenate was spotted in duplicate onto a steel target plate, and 1 μL of SA matrix (saturated solution of sinapic acid in 60% acetonitrile, 40% H2 O, 0.3% trifluoroacetic acid; all chemicals from Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) was added directly to the air-dried spots. Mass fingerprints were generated by a commercial company (Mabritec SA, Riehen, Switzerland) and subjected to automated identification against more than 4000 validated biomarker mass sets, including 15 Culicoides species-specific sets, as described by Kaufmann et al. (2012a). The diagnostic specificity of MALDI-TOF MS was evaluated in 1200 randomly chosen biting midges collected in the field, yielding the correct identification of 1173 (97.8%) specimens [as confirmed by PCR/sequencing of DNA extracted from the abdomens (Wenk et al., 2012) of 95 randomly selected specimens]; 14 specimens revealed novel mass spectra (and the species were eventually identified by PCR/sequencing), and 13 specimens yielded no identification as a result of poor-quality spectra (Kaufmann et al., 2012b).
DNA extraction and PCR protocol for identification of bloodmeals Female Culicoides individuals (whole insects) were homogenized into 1.5-mL tubes with 100 μL of lysis buffer (0.1 m Tris, 0.005 m EDTA, 0.2% SDS, 0.2 m NaCl, pH 8.5) (Laird et al., 1991) and 1.5 μL proteinase K (20 mg/mL) and incubated for 3 h at 56 ∘ C. After incubation, the samples were centrifuged for 10 min at 7378g. The supernatant was placed into new tubes and processed using standard ethanol precipitation. Pelleted DNA was dissolved in 25 μL ddH2 O and quantified using a NanoDrop 2000 microvolume spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, U.S.A.). The quantified DNA was diluted to a standard concentration of 25–50 ng/μL for PCR and used as a template for amplification of mitochondrial DNA from the vertebrate bloodmeal.
Primer design strategy and procedure for bloodmeal identification Avian and mammalian mitochondrial cytochrome b (cyt b) sequences were downloaded from GenBank and aligned using BioEdit software (Hall, 1999). These multiple alignments revealed regions that were highly conserved for bird species and contained diverse fragments for mammals. Based on sequence homology among aligned sequences of the avian cyt b gene, the following primers were devised to amplify 219-bp fragments (excluding primers): cytbPF1 5′ -CCTGAG GACAAATATCATTCTGAGG-3′ , and cytbBirdR 5′ -GGGTGG AATGGGATTTTATCGC-3′ . In order to improve the efficiency of detection, a second forward primer was designed for semi-nested PCR (cytbBirdF2 5′ -TCAGCAATCCCATACAT TGGCCAA-3′ ). One microlitre of the first PCR was used as a template for the second PCR including cytbBirdF2 and cytbBirdR primer pairs to amplify 169-bp fragments. Both PCRs were performed
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separately in 25-μL volumes with the same proportions of reagents [2.5 μL PCR 10× buffer, 0.125 mm of each nucleotide, 0.4 mm of each primer, 0.1 μL DreamTaq DNA polymerase (Thermo Fisher Scientific, Inc.)] and 1 μL of the DNA template. The PCR was conducted according to a thermal protocol of 94 ∘ C for 2 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. The final cycle was completed with a 10-min extension at 72 ∘ C. Negative controls were used with every PCR in order to detect possible contamination. Positive or negative samples were indicated by the presence or absence of bands on 2% agarose gels using 4.0 μL of the reaction. In order to reveal bloodmeals of non-avian origin, all negative samples (n = 51) were further processed with primers capable of amplifying DNA from the blood of a diverse number of vertebrate species. Two primer pairs were used for a nested PCR assay, as described in Alcaide et al. (2009). The initial PCR was conducted with M13BCV-FW (5′ -TGTAAAA CGACGGCCAGTH-AAYCAYAARGAYATYGG-3′ ) and BCV -RV1 (5′ -GCYCANACYATNCCYATRTA-3′ ) and the nested step was performed with M13 (5′ -GTAAAACGACGGCCAG TG-3′ ) and BCV-RV2 (5′ -ACYATNCCYATRTANCCRAANG G-3′ ) primer pairs. Both PCRs were performed separately in 25-𝜇L volumes using the proportions of reagents given above and with temperature profiles as described in Alcaide et al. (2009). Amplified fragments were sequenced with the primers cytbBirdF2 and BCV-RV2 at Macrogen Ltd (Amsterdam, the Netherlands). The sequences obtained were edited using BioEdit (Hall, 1999) and compared with available sequences in the GenBank DNA database.
Results A total of 4476 Culicoides specimens were identified as belonging to six species of Culicoides: C. circumscriptus Kieffer, 1918 (n = 373); C. festivipennis Kieffer, 1914 (n = 1354); Culicoides punctatus (Meigen, 1804) (n = 288); C. pictipennis (Staeger, 1839) (n = 146); Culicoides alazanicus Dzhafarov, 1961 (n = 2290), and Culicoides griseidorsum Kieffer, 1918 (n = 25). Ninety-five specimens were identified as blood-fed individuals. These included specimens of C. circumscriptus (n = 6), C. festivipennis (n = 23), C. punctatus (n = 15), C. pictipennis (n = 3), C. alazanicus (n = 40) and C. griseidorsum (n = 8). Culicoides alazanicus, identified by both morphological and molecular methods, has not been reported previously from Bulgaria. One species was identified as C. griseidorsum on the basis of morphological characteristics, but this was not confirmed by sequence analysis. blast analysis in GenBank showed a 99% match with C. duddingstoni for one individual and < 90% identity with certain Culicoides species for the others. Therefore, the identification of this species requires additional investigation. In the current study, the species will be referred to as C. griseidorsum with the abbreviation cf . (confer) thus: C. cf . griseidorsum. The application of the new primers designed for the purposes of the present study allowed us to obtain good-quality cyt b
© 2015 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12108
4 A. Bobeva et al. 27 13 5 6 2 2 55 1∗ — — — — 1 2 1 — 1 — — — 2 1∗ — — — — — 1 — 1 — — — — 1 2 1 3 1 — — 7 1 — — — — — 1
1 — — — — — 1
1 — — — — — 1
1 — — — — — 1
— — — 1∗ — — 1
1 — — — — — 1
— 2 — — — — 2
4 1 — — — — 5
6 — — — — — 6
— 1 — — — — 1
— 2 — — — — 2
— 1 — — — — 1
1 — — — — — 1
Discussion
Streptopelia decaocto Sylvia borin Passer montanus Phylloscopus trochilus Pica pica Passer domesticus Passer hispaniolensis Oriolus oriolus Parus major Muscicapa striata Nycticorax nycticorax Delichon urbica Ixobrychus minutus Luscinia luscinia Luscinia megarhynchos Coccothraustes coccothraustes Columba palumbus Acrocephalus palustris
— — — 1 — — 1 C. alazanicus (n = 40) C. festivipennis (n = 23) C. circumscriptus (n = 6) C. cf. griseidorsum (n = 8) C. pictipennis (n = 3) C. punctatus (n = 15) Total
∗Mixed bloodmeals.
Asio otus Anthus trivialis Ardea purpurea Culicoides spp.
1 1 — — — — 2
3 — — — — — 3
1 3 1 — — — 5
— — — 1 — — 1
Bos taurus Turdus merula Turdus philomelos
Avian species
1 — — — — — 1
— — — — 1 — 1
Cervus elaphus
Bloodmeals identified from vertebrate hosts, n
— — — 2∗ 1 1∗ 4
Homo sapiens
Mammalian species
Total Table 1. Number of bloodmeals of avian and mammalian origin identified to species level.
sequences from 44 bloodmeals. The ornithophilic biting midges were found to have fed on 23 avian species (Table 1). In two individuals, mixed bloodmeals were revealed by double peaks at several positions in the sequence chromatograms. Analyses of these two cases gave the best, but not perfect, blast hits to Luscinia megarhynchos (95% similarity) and Sylvia borin (89% similarity), respectively. Eleven good-quality COI sequences were obtained using the protocol described by Alcaide et al. (2009). DNA of three mammalian (Bos taurus, Cervus elaphus and Homo sapiens) and four avian (Columba palumbus, Ixobrychus minutus, Oriolus oriolus and Passer hispaniolensis) species were amplified. In three individuals, we identified mixed bloodmeals with the best, but not perfect, blast hits to H. sapiens (one with 95% similarity) and C. elaphus (two with 99 and 94% similarity, respectively) (Table 1).
The identification of the host preferences of haematophagous insects is crucial for elucidating their role in the transmission of various pathogens. The development of molecular approaches and their application to ecological and vector-borne disease studies have revealed a high diversity of vector–host associations (Pettersson et al., 2012; Santiago-Alarcon et al., 2012, 2013). However, opportunities to use host DNA markers for their identification are limited by the digestion of bloodmeals and the rapid degradation of DNA in the mid-guts of insects. As the stage of blood digestion increases, the likelihood of successfully identifying the bloodmeal source may decrease (Oshaghi et al., 2006; Martinez-de la Puente et al., 2013). Primers designed in the current study are capable of amplifying DNA of a remarkable diversity of bird species and give convincing results despite the relatively short length of the amplicons (169 bp). This increases the probability of the successful amplification of partly degraded DNA. To optimize the protocol, a nested PCR was conducted to enhance the specificity of the method and to obtain suitable concentrations of PCR products for sequencing. This approach allowed the precise identification of 21 species of birds bitten by biting midges of four species (Table 1). In comparison, similar studies have identified between three and 10 bird species (Lassen et al., 2012; Pettersson et al., 2012; Santiago-Alarcon et al., 2013). Four avian species (C. palumbus, I. minutus, O. oriolus and P. hispaniolensis) were identified using the primers from Alcaide et al. (2009). Two of them (C. palumbus and I. minutus) increase the diversity of avian hosts identified in the studied area. DNA from the other two avian species was found in two biting midges that had been found to be negative for bird DNA using avian-specific primers. The percentage of insects containing bird DNA in abdomens in our study is relatively high (50.6%) compared with those in previous studies. For example, Pettersson et al. (2012) found that 12.6% of bloodmeals originated from birds, and Santiago-Alarcon et al. (2013) and Lassen et al. (2012) reported rates of 14.5% and 5.8%, respectively. Contrary to what might have been expected based on earlier findings (Lassen et al., 2011, 2012; Pettersson et al., 2012), only seven of 51 (13.7%) individuals were found to have fed on
© 2015 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12108
Host preferences of ornithophilic midges
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Table 2. Bloodmeal hosts of the Culicoides spp. investigated in this study as identified in previous studies. Culicoides species
Previously known vertebrate host
References
C. alazanicus C. griseidorsum C. circumscriptus
N/A Mammalian: Bos taurus Mammalian: Homo sapiens
N/A Ayllón et al. (2014) Lassen et al. (2012) Pettersson et al. (2012)
C. festivipennis
Avian: Pica pica, Turdus merula, Phylloscopus trochilus, Corvus corone, Turdus philomelos, Columba palumbus Mammalian: Homo sapiens
C. pictipennis
Avian: Columba palumbus, Turdus philomelos, Pica pica Mammalian: Homo sapiens
C. punctatus
Avian: Pica pica, Turdus merula, Erithacus rubecula, Parus major Mammalian: Bos taurus, Capra hircus, Capreolus capreolus, Equus callabus, Alces alces, Bos taurus, Ovis aries Avian: Anas platyrhynchos, Columba palumbus, Luscinia svecica
Lassen et al. (2012) Santiago-Alarcon et al. (2012, 2013) Pettersson et al. (2012) Lassen et al. (2012) Santiago-Alarcon et al. (2012, 2013) Pettersson et al. (2012) Lassen et al. (2011, 2012) Pettersson et al. (2012)
N/A, no available information.
non-avian vertebrates (Table 1). DNA from B. taurus, C. elaphus and H. sapiens was isolated from C. punctatus, C. pictipennis, C. cf. griseidorsum and C. alazanicus. The proportions of avian and mammalian bloodmeals obtained most probably reflect the richness of the habitat and the abundance of birds in the area of study. The use of two different nested PCR assays to identify the feeding sources of biting midges allowed us to identify more precise associations between haematophagous insects of the genus Culicoides and their vertebrate hosts in the area studied. Thus, the present study adds important information to existing knowledge of the feeding preferences of the Culicoides species investigated (Table 2). Recent studies have shown that most Culicoides species are opportunistic and readily feed on a variety of mammals and birds; these opportunistic species include C. circumscriptus, C. festivipennis, C. pictipennis and C. punctatus (Lassen et al., 2012; Pettersson et al., 2012; Santiago-Alarcon et al., 2013). According to our data, two Culicoides species (C. alazanicus and C. cf. griseidorsum) were found to feed from both birds and mammals. However, some species demonstrated predominant ornithophilic (C. pictipennis) or mammophilic (C. punctatus) feeding preferences (Lassen et al., 2012; Pettersson et al., 2012). Despite its predominantly ornithophilic preferences, C. pictipennis was found to be able to feed on cattle (B. taurus) and red deer (C. elaphus). Therefore, host availability plays an important role in the feeding behaviour of biting midges (Lassen et al., 2012). Culicoides cf. griseidorsum and C. alazanicus were recorded in Bulgaria for the first time in the present study. Moreover, C. alazanicus was identified as the most abundant species in our samples and therefore should be regarded as the most probable vector of haemoproteids in the region. However, information on the feeding preferences of both species is lacking in the literature. There is increasing interest in the vectorial capacity of Culicoides biting midges for BTV, as well as in the transmission biology of the highly diverse group of haemosporidian parasites.
Recent studies have revealed interesting associations between various Culicoides spp. (C. circumscriptus, C. pictipennis, C. festivipennis, Culicoides salinarius, C. duddingstoni, Culicoides kibunensis, C. punctatus, Culicoides simulator and Culicoides segnis) and Haemoproteus lineages, and show variations in the specificity of parasites to vectors (Martinez-de la Puente et al., 2011; Santiago-Alarcon et al., 2012; Bobeva et al., 2013; Synek et al., 2013). In addition, revealing the feeding preferences of biting midges sheds light on the transmission routes of avian haemosporidians and contributes to better understanding of the ecological and evolutionary traits of vector–host–parasite interactions. In the present study, C. circumscriptus, C. festivipennis, C. alazanicus and C. cf. griseidorsum were found to feed on a variety of bird species. As these species have rather opportunistic feeding patterns, they can be expected to represent important vectors of diverse haemoproteid parasites in the studied area. Acknowledgements This study represents Report No. 57 from Kalimok Field Station. We acknowledge Dr Bruno Mathieu (University of Strasbourg, Strasbourg, France) for his contribution to the morphological identification of Culicoides biting midges. We are grateful to Dr Boyko B. Georgiev, Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia, Bulgaria, for useful suggestions offered during the course of the present study and in the preparation of the present manuscript, and to Dr Ognyan Mikov, National Centre for Infectious and Parasitic Diseases, Sofia, Bulgaria, for valuable suggestions. The authors are grateful to the staff of Kalimok Field Station and to Christoffer Sjöholm, for their support with the collection of insects, and to Nikola Bankov, Master degree student in the Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia, Bulgaria, for his help with laboratory work. Facilities developed in
© 2015 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12108
6 A. Bobeva et al. the frames of the projects WETLANET (FP7 CAPACITIES grant 229802), CEBDER (National Science Fund, Ministry of Education, Youth and Science of the Republic of Bulgaria; grant DO 02-15/2009) and ‘Development of scientific potential in the field of faunistic diversity and environment protection’ (funded by the Ministry of Education, Youth and Science and the European Social Fund, Operational Programme ‘Human Resources Development’; grant BG 051 PO001-3.3.04/41) were used in the course of the present study.
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