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Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr 7 8

Haemoproteus minutus and Haemoproteus belopolskyi (Haemoproteidae): Complete sporogony in the biting midge Culicoides impunctatus (Ceratopogonidae), with implications on epidemiology of haemoproteosis

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 nas Rita Zˇiegyte˙ ⇑, Vaidas Palinauskas, Rasa Bernotiene˙, Tatjana A. Iezhova, Gediminas Valkiu Nature Research Centre, Akademijos 2, Vilnius 2100, LT-08412, Lithuania

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h i g h l i g h t s

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 Deadly Haemoproteus minutus is

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 Sporogony of H. belopolskyi completes

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 Sporogonic stages of H. minutus and

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g r a p h i c a l a b s t r a c t

transmitted by Culicoides impunctatus. in C. impunctatus. H. belopolskyi are described and illustrated.  Ookinetes and sporozoites of these parasites are readily distinguishable morphologically.  Culicoides impunctatus is an important vector of Haemoproteus parasites.

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a r t i c l e 3 7 3 4 34 35 36 37 38 39 40 41 42 43 44 45 46

i n f o

Article history: Received 31 March 2014 Received in revised form 21 July 2014 Accepted 25 July 2014 Available online xxxx Keywords: Haemoproteus Haemosporidian parasite Sporogony Culicoides Transmission Epidemiology

a b s t r a c t Species of Haemoproteus (Haemoproteidae) are cosmopolitan haemosporidian parasites, some of which cause severe diseases in birds. Numerous recent studies address molecular characterization, distribution and genetic diversity of haemoproteids. However, the information about their vectors is scarce. We investigated sporogonic development of two widespread species of Haemoproteus (Haemoproteus minutus and Haemoproteus belopolskyi) in the experimentally infected biting midge Culicoides impunctatus. Wildcaught flies were allowed to take blood meals on naturally infected common blackbirds Turdus merula and icterine warblers Hippolais icterina harboring mature gametocytes of H. minutus (lineage hTURDUS2) and H. belopolskyi (hHIICT1), respectively. The engorged flies were collected, transported to the laboratory, held at 15–18 °C, and dissected daily in order to obtain ookinetes, oocysts and sporozoites. Mature ookinetes of H. minutus developed blisteringly rapidly; they were numerous in the midgut content between 1 and 4 h post exposure. Ookinetes of H. belopolskyi developed slower and were reported 1 day post exposure (dpe). Oocysts of both parasites were seen in the midgut wall 3–4 dpe. Sporozoites of H. minutus and H. belopolskyi were first observed in the salivary glands preparations 7 dpe. The percentage of experimentally infected flies with sporozoites of H. minutus was 82.1% and 91.7% with H. belopolskyi. In accordance with microscopy data, polymerase chain reaction amplification and sequencing confirmed presence of the corresponding parasite lineages in experimentally infected biting midges. Sporogonic stages of these parasites were described and illustrated. This study indicates that C. impunctatus is involved in the transmission of deadly H. minutus, which kills captive parrots in Europe. This biting midge is an important vector of avian haemoproteids and worth more attention in epidemiology research of avian haemoproteosis. Ó 2014 Published by Elsevier Inc.

⇑ Corresponding author. Fax: +370 5 272 93 52. E-mail address: [email protected] (R. Zˇiegyte˙). http://dx.doi.org/10.1016/j.exppara.2014.07.014 0014-4894/Ó 2014 Published by Elsevier Inc.

Please cite this article in press as: Zˇiegyte˙, R., et al. Haemoproteus minutus and Haemoproteus belopolskyi (Haemoproteidae): Complete sporogony in the biting midge Culicoides impunctatus (Ceratopogonidae), with implications on epidemiology of haemoproteosis. Exp. Parasitol. (2014), http://dx.doi.org/ 10.1016/j.exppara.2014.07.014

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R. Zˇiegyte˙ et al. / Experimental Parasitology xxx (2014) xxx–xxx

1. Introduction Species of Haemoproteus (Haemosporida, Haemoproteidae) are widespread in birds in countries with temperate and tropical cli nas, 2005; Atkinson, 2008). Numerous recent studies mates (Valkiu addressed molecular characterization, distribution and genetic diversity of haemoproteids. However, few studies deal with vectors and transmission of avian Haemoproteus spp. (Atkinson, 1991;  nas et al., 2002; Martínez-de la Desser and Bennett, 1993; Valkiu Puente et al., 2011; Santiago-Alarcon et al., 2012; Levin et al., Q2 2012). Biting midges of Culicoides (Diptera, Ceratopogonidae) and louse flies (Hippoboscidae) transmit these parasites, but certain vector species remain unknown for the great majority of avian haemoproteids and their lineages (Atkinson, 2008; Clark et al., 2014). Avian Haemoproteus spp. have been traditionally considered relatively benign in their avian hosts (Bennett et al., 1993). However, numerous field studies (Nordling et al., 1998; Merino  nas, 2005; Atkinson, et al., 2000; Møller and Nielsen, 2007; Valkiu 2008) and limited experimental observations (Atkinson et al.,  nas et al., 2006) indicate negative 1988; Marzal et al., 2005; Valkiu influence of these parasites on bird fleetness. Several species of Haemoproteus have been reported to cause diseases, sometimes even lethal in non-adapted birds (Miltgen et al., 1981; Atkinson et al., 1988; Cardona et al., 2002; Ferrell et al., 2007; Donovan et al., 2008; Olias et al., 2011; Cannell et al., 2013). However, the true extent of pathology and mortality caused by Haemoproteus parasites remains unclear because the severe haemoproteosis and death of infected birds occur mainly during the tissue stage of parasite development, before the appearance of parasitemia (Cannell et al., 2013). Such diseases are difficult to diagnose both by microscopic and polymerase chain reaction (PCR)-based diagnostic methods. It is important to note that Haemoproteus infections are virulent in some vectors and even kills bird-biting  nas et al., 2014). dipteran insects (Levin and Parker, 2014; Valkiu These parasites are worthy of more attention in veterinary medicine, epidemiology and conservation biology studies. Recent PCR-based findings indicate that Haemoproteus minutus is responsible for some instances of mortality in captive parrots in Europe (Olias et al., 2011; Palinauskas et al., 2013). This parasite is widespread and relatively benign in common blackbirds Turdus merula in Europe, but it kills several species of captive parrots on the stage of megalomeronts, if these birds are exposed to the infection. Vectors of H. minutus parasite remain unknown. The sporogony of only few Haemoproteus spp. has been investi nas, 2005; gated in detail in biting midges (Linley, 1985; Valkiu Santiago-Alarcon et al., 2012); it is difficult to work with these insects due to their tiny size and difficulties to colonize the major nas, 2005; Atkinson, ity of their species (Miltgen et al., 1981; Valkiu 2008). It was shown that Culicoides impunctatus transmits several  nas, 2005). This biting species of haemoproteids in Europe (Valkiu midge was reported as a vector of Haemoproteus belopolskyi from  nas and Iezhova, 2004). Howblackcaps Sylvia atricapilla (Valkiu ever, recent molecular studies show that this blackcap parasite is  nas et al., 2007), Q3 actually Haemoproteus parabelopolskyi (Valkiu and the vector of H. belopolskyi, which parasitize icterine warblers Hippolais icterina needs to be identified. Because studies on vectors and transmission of avian Haemoproteus spp. are uncommon and vectors of H. minutus and H. belopolskyi are unknown, the aim of this study was to follow sporogony of these parasites in the biting midge C. impunctatus, which is widespread in Europe, willingly takes blood meal on birds and is susceptible to several haemoproteid infections (Glukhova, 1989;  nas, 2005). Blackwell, 1997; Valkiu

2. Materials and methods

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2.1. Study site, collection of blood samples and experimental birds

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This study was carried out at the Biological Station of the Zoological Institute of the Russian Academy of Sciences on the Curonian Spit in the Baltic Sea (55°090 N, 20°520 E) between 21 May and 2 July in 2013. The experiments described herein comply with the current laws of Lithuania and Russia. Experimental procedures of this study were approved by the International Research Co-operation Agreement between the Biological Station Rybachy of the Zoological Institute of the Russian Academy of Sciences and Institute of Ecology of Nature Research Centre (25-05-2010). All efforts were made to minimize handling time and potential suffering of birds. None of the experimental birds suffered apparent injury during experiments. Birds were captured with mist nets and identified. About 30 ll of blood was collected in heparinized microcapillaries by puncturing the brachial vein and stored in SET buffer (0.05 M Tris, 0.15 M NaCl, 0.5 M EDTA, pH 8.0) at ambient temperature while in the field, and then preserved at 20 °C in the laboratory. A drop of blood was taken from each bird to make two or three blood films. The smears were air-dried, fixed in absolute methanol and stained  nas et al. (2008). Blood films with Giemsa, as described by Valkiu were prepared and examined microscopically before each exposure of flies in order to check the level of parasitemia and to detect any other possible relapsed infections. Intensity of parasitemia was estimated as a percentage by counting the number of mature gametocytes per 1000 erythrocytes examined. The species of Hae nas (2005). Naturally moproteus were identified according to Valkiu infected birds with single infections were used as donors to infect biting midges. Blood samples from donor birds were examined for haemosporidian parasites by PCR amplification. Positive amplifications were sequenced and cytochrome b (cyt b) lineages of Haemoproteus parasites were determined in the laboratory (see description below). Two common blackbirds T. merula naturally infected with H. minutus (lineage hTURDUS2, parasitemia 0.1%) and two icterine warblers H. icterina infected with H. belopolskyi (lineage hHIICT1, parasitemia 0.2%) were used as donors of gametocytes to infect biting midges. One uninfected juvenile common crossbill Loxia curvirostra was used to feed a control group of flies. All birds were kept indoors in a vector-free room under controlled conditions [55–60% relative humidity (RH), 20 ± 1 °C, the natural light–dark photoperiod (L/D) 17:7 h]; they were fed standard diets for seed eating or insectivorous bird species. All birds survived to the end of this study and were released after experimental work.

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2.2. Wild-caught biting midges

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Experimental infection of biting midges C. impunctatus with two Haemoproteus species was performed near Lake Chaika, located close to the village of Rybachy, where density of the flies was high  nas, 1993; Liutkevicˇius, 2000; Valkiu  nas (Glukhova and Valkiu et al., 2002). To minimize the probability of natural infection of wild-caught midges with Haemoproteus, the first generation of naturally occurring flies was used in this study. All experimental infections were performed between 10 and 20 June when the first generation of C. impunctatus predominated (Liutkevicˇius, 2000). Unfed flies were collected by entomological net at this study site before experiments. Some were fixed in 70% ethanol and used for morphological species identification, and the remainder were fixed in 96% ethanol and used for PCR-based identification and

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determining natural prevalence of Haemoproteus infection (see description below). 2.3. Experimental design Wild-caught biting midges were infected as described by  nas (2005). Briefly, the crown feathers of birds were Valkiu removed from an area of about 1 cm2 to facilitate observation of feeding insects, which is helpful during work with such minute flies. Birds were held in hands covered by ruby gloves, and biting midges were allowed to feed naturally between 21:00 and 23:00 h on birds at a locality with a high density of flies. C. impunctatus willingly takes a blood meal on the feather-free region (Fig. 1). The bird’s head was inserted into an insect cage when several flies had started to feed. The cages (12  12  12 cm3) were made of fine-mesh bolting silk. A zip faster was sewn into one wall of the cage to permit entry of the bird’s head and removal of engorged midges, which flew off the bird’s head into the insect cage, which was then closed. The cages with engorged flies were transported to the laboratory and held at 15–18 °C, 70 ± 5% RH and L/D photoperiod of 17:7 h. Bowls with water were placed near each cage to maintain necessary relative humidity. The midges were supplied with 5–10% saccharose solution; pads of cotton wool moistened in this solution were placed on the top of each insect cage daily. 2.4. Dissection of biting midges and making preparations of ookinetes, oocysts and sporozoites Before dissection, biting midges were identified according to Gutsevich (1973). Engorged females were dissected daily in a drop of 0.85% saline as described by Kazlauskiene˙ et al. (2013). The flies were lightly anesthetized by putting them into a tube closed with a cotton pad wetted in 96% ethanol for several minutes. All biting midges were processed individually for microscopic and PCR-based detection of parasites. To eliminate contamination of samples, we used a new dissecting needle and a new microscope slide for each dissected biting midge. Midgut content was examined for ookinetes, the midgut wall for oocysts, and the salivary glands for sporozoites. Smears of midgut contents and salivary glands were dried in the air, fixed with methanol, stained with Giemsa, and examined in the same way as blood films. Permanent preparations of oocysts were made. The midgut was removed from the infected females, fixed in 10% formalin in normal saline, stained with Ehrlich’s hematoxylin and mounted in Canada balsam (for details  nas, 2005). The number of experimentally infected and see Valkiu dissected biting midges is given in Table 1. Seventeen females were

Fig. 1. Biting midges Culicoides impunctatus (arrow) taking blood meals on a feather-free region on head of the common blackbird Turdus merula.

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fed on uninfected common crossbill; they served as controls and were kept, dissected, and examined for ookinetes, oocysts, and sporozoites, as described above. Additionally, un-fed females of C. impunctatus were collected and tested by PCR amplification for detection of natural infection prevalence with haemoproteids, as described below.

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2.5. Microscopic examinations of preparations and parasite morphology

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An Olympus BX – 43 light microscope equipped with Olympus SZX2-FOF digital camera and imaging software QCapture Pro 6.0, Image Image – Pro Plius (Tokyo, Japan) was used to examine preparations, prepare illustrations and to take measurements. All preparations were first examined at low magnification (200, 600) and then at high magnification (1000). The morphometric fea nas (2005). tures studied (Table 2) were those defined by Valkiu The statistical analyses were carried out using the ‘Statistica 7’ package. Student’s t-test for independent samples was used to determine statistical significance between mean linear parameters of parasites. Percentages were compared by Fisher’s exact test. A P value of 0.05 or less was considered significant. 95% confidence limits of percentages are given in Table 1. Voucher specimens of ookinetes, oocysts and sporozoites of H. minutus (accession numbers 48810-48812 NS) and H. belopolskyi (48813-48815 NS) were deposited in the Institute of Ecology, Nature Research Centre, Vilnius, Lithuania.

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2.6. Polymerase chain reaction and sequencing

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Total DNA was extracted from all samples using ammonium acetate extraction method. For genetic analysis, we used a nested PCR protocol (Bensch et al., 2000; Hellgren et al., 2004). We amplified a segment of parasite mitochondrial cyt b gene using two pairs of initial primers, HaemNFI and HaemNR3 which amplify fragments of this gene of haemosporidians belonging to Haemoproteus, Plasmodium and Leucocytozoon. For the second PCR, we used primers HAEMF and HAEMR2, which are specific to Haemoproteus and Plasmodium spp. The amplification was evaluated by running 1.5 ll of the final PCR product on a 2% agarose gel. One negative control (nuclease-free water) and one positive control (one H. minutus microscopy positive blood sample, in the case of blood testing, and thoraxes of two flies experimentally infected with H. minutus, in the case of biting midge testing) were used per every 14 samples to control for false amplifications. No cases of false positive samples were found. Fragments of DNA from the positive samples were sequenced from the 50 end with the primer HAEMF. The ‘‘Basic Local Alignment Search Tool’’ (National Centre for Biotechnology Information website: http://www.ncbi.nlm.nih.gov/BLAST) was used to determine lineages of detected DNA sequences, which were deposited in GenBank (accessions KJ627800–KJ627802). DNA extracted from individual flies was used to confirm the identification of C. impunctatus used in our experiments (Table 1). For this purpose, the insect specific primers LCO149 and HCO2198 were applied to ampliphy a fragment of cytochrome oxydase subunit I of mitochondrial DNA (Folmer et al., 1994). All obtained sequences corresponded to the C. impunctatus DNA sequences, which are available in GenBank. Because we used wild-caught C. impunctatus in experiments, it was essential to determine prevalence of natural Haemoproteus infection in the biting midges. Unfed flies were collected at the same study site where we exposed donor birds to bites of C. impunctatus (see above). In all, 108 wild-caught females were tested by PCR amplification. DNA was extracted from 27 pools of biting midges, each containing 4 flies. Additionally, remains of all

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Table 1 Percentages of experimentally infected Culicoides impunctatus with two species of Haemoproteus. Sporogonic stage

Number of engorged flies/number of infected flies

Ookinete Oocyst Sporozoite Total

Control

H. minutus

H. belopolskyi

5/0 (0, 0.0–18.7)a 6/0 (0, 0.0–15.8) 6/0 (0, 0.0–15.8) 17/0 (0, 0.0–5.8)

26/21 15/15 28/23 69/59

18/18 11/11 12/11 41/40

(80.7, 64.5–97.0) (100, 93.5–100) (82.1, 82.1, 67.0–97.3) (85.5, 88.0–94.0)

(100, 94.5–100) (100, 80.1–100) (91.7, 73.3–100) (97.5, 92.6–100)

a Percentages are given in parentheses, followed by 95% confidence limits of the percentage. All infections were determined by microscopic examination of preparations, except for control group, which was also tested by PCR-based methods.

Table 2 Morphometry of ookinetes, oocysts and sporozoites of two species of Haemoproteus in Culicoides impunctatus. Feature

Measurementsa H. minutus

H. belopolskyi

Ookinete Length Width Area Area of nucleus

5.7–9.1 (7.6 ± 0.8) 1.2–2.9 (2.1 ± 0.4) 7.9–19.5 (12.7 ± 3.3) 1.0–2.7 (1.7 ± 0.5)

11.5–20.8 (16.5 ± 2.5) 1.6–2.5 (2.0 ± 0.2) 17.5–34.2 (26.6 ± 4.5) 2.0–4.5 (3.3 ± 0.6)

Oocyst Minimum diameter Maximum diameter Area

3.7–6.8 (5.0 ± 0.9) 4.1–7.2 (5.6 ± 0.9) 14.1–36.8 (23.8 ± 7.8)

3.9–6.9 (4.8 ± 0.8) 4.4–7.6 (5.5 ± 1.0) 15.2–42.8 (23.0 ± 9.6)

Sporozoite Length Width Area Area of nucleus

10.9–14.5 (13.2 ± 1.1) 1.0–1.4 (1.1 ± 0.1) 9.3–15.7 (12.6 ± 1.7) 0.7–1.2 (0.9 ± 0.1)

7.6–10.6 (9.0 ± 0.7) 0.8–1.3 (1.0 ± 0.1) 5.6–10.4 (7.3 ± 1.1) 0.7–1.5 (1.1 ± 0.2)

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Measurements of ookinetes (n = 21, methanol-fixed preparations), oocysts (n = 12, formalin-fixed preparations) and sporozoites (n = 21, methanol-fixed preparations) are given in micrometers. Minimum and maximum values are provided, followed in parentheses by the arithmetic mean and standard deviation.

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flies were collected after dissection (Table 1); they were fixed in 96% ethanol and tested by PCR amplification in order to confirm the identity of parasite lineages in experimentally infected flies.

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3. Results

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Microscopic identification and PCR-based testing showed that all experimental flies belonged to C. impunctatus. According to PCR-based analysis, no natural infection was found in wild-caught biting midges. Parasites were also not detected in control flies (Table 1) both by PCR and microscopic methods. H. minutus and H. belopolskyi completed sporogony in C. impunctatus. In accordance to microscopic observation, the PCR and sequencing confirmed the presence of corresponding parasite lineages in experimentally infected biting midges (Table 1). Mature ookinetes of H. minutus were seen in the midgut contents of experimentally infected flies between 1 and 4 h post exposure, and they were not seen 6 h post exposure, indicating rapid development and movement of the parasites in the midgut. Ookinetes of H. belopolskyi developed more slowly; they were seen 1 dpe and also reported 3 dpe. No difference was discernable in the percentage of midges with ookinetes between two parasite species (P = 0.06, Table 1). Ookinetes of both species were elongate wormlike bodies with prominent, slightly off center located nuclei and visible vacuoles (Fig. 2e and f). Occasionally, a few pigment granules were discernable in the cytoplasm of ookinetes of both parasite species (Fig. 2e). There was a significant difference in the length and area of ookinetes of two parasites (Table 1). Ookinetes of H. belopolskyi were significantly longer and greater in area than those of H. minutus (P < 0.001, both for length and area).

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Oocysts both of H. minutus and H. belopolskyi were first seen in the midgut wall 3 dpe, and they were reported in the midgut preparations until 6 dpe. No difference was discernable in the percentage of midges with oocysts between two parasite species (P = 1, Table 1). In formalin fixed preparations, oocysts appeared as small roundish bodies (Fig. 2g and h, Table 2). Pigment was visible in some oocysts. There was no significant difference in the diameter or area of oocysts between these parasite species 4 dpe (P > 0.8, both for diameter and area). Sporozoites of H. minutus and H. belopolskyi were seen in the salivary glands of the biting midges 7 dpe. They were reported in salivary gland preparations of the majority of dissected insects between 8 and 12 dpe (the period of observation). No difference was discernable in the percentage of midges with sporozoites between two parasite species (P = 0.41, Table 1). Sporozoites had fusiform bodies with slightly off center placed nuclei and approximately equally pointed ends (Fig. 2i and j). Sporozoites of H. belopolskyi were significantly shorter than those of H. minutus (P < 0.001, Table 2). It is worth mentioning that sporozoites were significantly longer than ookinetes in H. minutus (P < 0.001, Table 2, Fig. 2e and i) in contrast to H. belopolskyi, in which sporozoites were significantly shorter than ookinetes (P < 0.001, Table 2, Fig. 2f and j).

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4. Discussion

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The key result of this study is that two species of widespread Haemoproteus parasites, i.e. H. minutus (lineage hTURDUS2) and H. belopolskyi (hHIICT1) complete sporogony and produce sporozoites in C. impunctatus. This biting midge is common in Europe and is  nas, 1993; abundant at our study site (Glukhova and Valkiu  nas et al., 2002). Sporogony of six species Liutkevicˇius, 2000; Valkiu of Haemoproteus completes in C. impunctatus; these are H. balmorali, Q4 H. dolniki, H. fringillae, H. lanii, H. parabelopolskyi and H. tartakovskyi  nas et al., 2002; Valkiu  nas, 2005). The present study adds (Valkiu two species to the group of avian haemoproteids. Former experimental studies proved that infective sporozoites of H. fringillae  nas and and H. parabelopolskyi developed in C. impunctatus (Valkiu  nas, 2005), indicating that this fly is an imporIezhova, 2004; Valkiu tant vector of avian haemoproteids and worth more attention in epidemiological studies of avian haemoproteosis. The present study supports former conclusions about important role of biting midges of Culicoides in transmission of avian Haemoproteus spp. (Garnham, 1966; Atkinson, 2008). It worth mentioning that several molecular studies detected DNA of Haemoproteus spp. in mosquitoes and speculated about possible involvement of these insects in haemoproteid transmission (Ishtiaq et al., 2008; Kimura et al., 2010; Njabo et al., 2011; Kim and Tsuda, 2012; Ventim et al., 2012). Recent experimental research shows that this speculation likely is incorrect due to DNA amplification of Haemoproteus parasites, which abort development and persist in resistant insects,  nas et al., 2013). Because sometimes as long as two weeks (Valkiu haemosporidians have complicated life cycles and their abortive

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Fig. 2. Gametocytes (a–d) and sporogonic stages (e–j) of Haemoproteus minutus (a, c, e, g, i) and H. belopolskyi (b, d, f, h, j): mature macrogametocytes (a and b) and microgametocytes (c and d) in the peripheral blood of donor birds before experimental infection of biting midges Culicoides impunctatus, ookinetes (e and f), oocysts (g and h), sporozoites (i and j). Methanol-fixed and Giemsa-stained thin films (a–d, e, f, i, j). Formalin-fixed whole mounts stained with Erlich’s hematoxylin (g and h). Long simple arrows – nuclei of parasites, arrowheads – pigment granules, short simple arrows – oocysts. Scale bar = 10 lm.

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infections are common, demonstration of sporozoites in bloodsucking insects is essential for definitively demonstrating the insects are vectors. The pattern of sporogony of H. balmorali, H. belopolskyi, H. dolniki, H. fringillae, H. lanii, H. minutus, H. parabelopolskyi and H. tartakovskyi in C. impunctatus is similar under the same conditions  nas, 2005; this study). Mainly, mature ookinetes first seen (Valkiu in midguts approximately 1 dpe and oocysts 3 dpe, and sporozoites

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reported in salivary glands 5–7 dpe at 15–18 °C. Rapid sporogony has been also reported in H. mansoni (Fallis and Bennett, 1960; Atkinson, 1991). An exception is H. minutus, whose mature ookinetes develop within several hours after exposure biting midges. Interestingly, ookinetes of this parasite develop more rapidly than any other species of haemosporidian parasites (Garnham, 1966;  nas, 2005). Biological meaning of such rapid Sinden, 1998; Valkiu development of ookinetes is unclear. Tiny size probably contributes to the rapid ookinete development of this parasite (Table 2). It is worth mentioning that the rate of maturation and the morphology of H. minutus ookinetes were the same during develop nas, 2005; this study). Tiny ment both in vivo and in vitro (Valkiu ookinetes, which are similar to H. minutus, have been described only in Haemoproteus pallidus, but vectors of the later parasite remain unknown. Ookinetes of other investigated Haemoproteus spp. are approximately 2-fold longer than those of H. minutus  nas et al., 2002; Valkiu  nas, 2005). Interestand H. pallidus (Valkiu ingly, in spite of marked size differences between ookinetes of H. minutus on the one hand, and ookinetes of H. belopolskyi and other investigated Haemoproteus spp. on the other hand, the size and the rate of maturation of oocysts of all investigated Haemoproteus species are similar in C. impunctatus. Further studies are needed for better understanding this phenomenon. Both ends of sporozoites are approximately equally pointed in H. minutus and H. belopolskyi (Fig. 2i and j); this is a diagnostic feature of haemoproteid species belonging to subgenus Parahaemoproteus (Fallis and Bennett, 1960; Garnham, 1966; Atkinson, 1991). This feature is readily distinguishable in salivary gland preparations in all investigated species of Parahaemoproteus  nas, 2005) and can be used in determining subgeneric iden(Valkiu tity of parasites based on morphology of sporozoites. Species of subgenus Haemoproteus are transmitted by louse flies; these parasites produce large (>20 lm in diameter) oocysts, in which develop sporozoites with one end more pointed than the other end (Garnham, 1966; Baker, 1966; Atkinson, 2008). H. minutus is widespread, prevalent and relatively benign in common blackbirds in Europe, but it killed several species of captive parrots at the stage of tissue megalomeronts (Olias et al., 2011; Palinauskas et al., 2013). Our study showed that C. impunctatus transmits H. minutus and seven other haemoproteid species. This biting midge is important vector of avian Haemoproteus parasites and worth attention during development of prevention measures against avian haemoproteosis.

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Acknowledgments

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We would like to thank the staff of the Biological Station ‘‘Rybachy’’, for assistance in the field. The director of the Biological Station ‘‘Rybachy’’, Casimir V. Bolshakov, is acknowledged for generously providing facilities for the experimental research and  te˙, Dovile˙ Bukauskaite˙ and Asta KrizˇanauskZˇivile˙ Prontkelevicˇiu iene˙, for participation in fieldwork. The experiments described herein comply with the current laws of Lithuania and Russia. This study was funded by the European Social Fund under the Global Grant measure (VPI-3.1.-ŠMM-07-K-01-047).

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