This study presents data from a molecular survey of the species of the genus Culicoides from the region of Kalimok Field Station (NE Bulgaria) and haemosporidian parasites occurring in them in order to investigate the host-parasite specificity of haemosporidians to their dipteran vectors. The identification of Culicoides spp. was carried out by morphological and molecular-genetic methods. We collected and analysed 230 individuals of the genus Culicoides. Nine species were found. Eight species were identified morphologically; Culicoides obsoletus, C. riethi, C. newsteadi, C. circumscriptus, C. festivipennis, C. punctatus, C. pictipennis and C. puncticollis. The ninth species might be classified as either of C. nubeculosus or C. riethi and its identification needs additional investigations. The total prevalence of Haemoproteus in the examined biting midges was 2.17%. Three individuals of C. pictipennis were infected with the Haemoproteus lineage TURDUS2 (prevalence 16.67%), a common parasite of thrushes (Turdidae). Two individuals of C. circumscriptus contained Haemoproteus lineages (prevalence 2.78%); these were the lineage HAWF2 (previously reported from Coccothraustes coccothraustes) and a new lineage CULCIR1 not previously reported in the literature.

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.

DNA barcoding (molecular characterisation) is a useful tool for describing the taxonomy and systematics of organisms. Over 250 species of avian haemosporidian parasites have been described using morphological characters, yet molecular techniques based on polymerase chain reaction (PCR) suggest this diversity is underestimated. Moreover, molecular techniques are particularly useful for the detection of chronic infections and tissue stages of these parasites. Species delimitation is problematic among haemosporidians, and many questions about the mechanisms and patterns of speciation, host specificity and pathogenicity are still unresolved. Accumulation of additional genetic and morphological information is needed to approach these questions. Here, we combine microscopic examination with PCR-based methods to develop molecular characterisation of Haemoproteus (Parahaemoproteus) manwelli Bennett, 1978 and Haemoproteus (Parahaemoproteus) gavrilovi Valkiūnas & Iezhova, 1990, both of which parasitise the bee-eater Merops apiaster L. We also describe a new species, Haemoproteus (Parahaemoproteus) palloris n. sp., from the blood of the willow warbler Phylloscopus trochilus (L.). We performed phylogenetic analyses with a set of mitochondrial cytochrome b (cyt b) gene lineages, which have been linked to parasite morphospecies and are available in the MalAvi database. Our findings show that morphological characters, which have been traditionally used in the description of haemosporidians, exhibit phylogenetic congruence. This study contributes to a better understanding of avian haemosporidian diversity and provides new molecular markers (cyt b and apicoplast gene sequences) for the diagnostics of inadequately investigated haemosporidian infections.

The occurrence of haemosporidians in biting midges of the genus Culicoides is examined in North-East Bulgaria in order to reveal their potential role for parasite transmission. A PCR-based technique amplifying part of the mitochondrial cytochrome b gene of the parasite is applied on naturally infected biting midges. Totally, 640 parous individuals of four species and 95 blood-fed individuals of six species of Culicoides are examined for the presence of DNA of haemosporidians. Haemosporidian genetic lineages are identified in individuals of three insect species: Culicoides alazanicus (12 lineages, nine lineages of Haemoproteus and three lineages of Plasmodium), Culicoides festivipennis and Culicoides circumscriptus (with two and one lineages of Haemoproteus, respectively). Two genetic lineages of Haemoproteus are recorded in more than one vector species. These results demonstrate variations in the specificity of Haemoproteus genetic lineages to their potential vectors, since some lineages are recorded in a single vector species and others occur in two or more vector species.

The Aegean archipelago is among the largest on Earth with astonishing biodiversity within Europe. Its territory underwent a massive geotectonic transformation in Neogene that resulted in multitude of changes in land-sea configuration and disintegrated the formerly united Aegean land to a complicated mainland-archipelago system. Therefore, it represents an excellent laboratory for studying evolution of terrestrial fauna. In the present study, we use a model group of flightless bush crickets with annual reproduction cycle—Poecilimon jonicus species group—to trace correlation of lineage diversification with the known paleogeographic events in the Aegean area. The group belongs to the hyperdiverse genus Poecilimon and has a disjunct distribution along the Hellenic arc from southwestern Anatolia through Crete to the western Balkans and the Apennines. To test our hypothesis, we inferred phylogenetic relationships of the P. jonicus group sensu lato using a nuclear fragment covering two spacers of the ribosomal cistron (ITS1 + ITS2). To study intra-group phylogeny, we compared mitochondrial phylogenies based on two matrices—(1) a concatenated ND2 and COI dataset of 1656 bp and (2) a 16S rRNA + 12S rRNA dataset of 1835 bp. As a second step, we estimated divergence times applying Bayesian approach with BEAST and a relative rate framework with RelTime on the mitochondrial matrices. We compare trees calibrated based on evolutionary rates and tectonic events and discuss radiation scenarios in concordance with known paleogeographic events in the Aegean area. Our results revealed robust phylogeny of the Poecilimon jonicus group and confirmed a strong link between its evolution and the Aegean paleogeography. The phylogenetic relationships of the group supported reconsideration of its systematics.

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Immune response to pathogens such as avian malaria (Plasmodium spp.) and related haemosporidian parasites (Haemoproteus spp. and Leucocytozoon spp.) can lead to increases in energy demands of birds. Migrations are energetically costly for birds and require resources that could be otherwise invested in immune defence against pathogens. Here, we examined the prevalence and intensity of haemosporidian parasites (Apicomplexa, Haemosporida) in rosy starlings (Pastor roseus), an irruptive migrant on the Balkans, during their pre-migratory period. We further evaluated if these infections affected the stress levels of birds brought into overnight captivity, using leukocyte profiles (ratios of heterophils to lymphocytes, or H/L ratios). Haemosporidian parasites were detected based on microscopic examination, PCR and sequencing. We found high prevalence of haemosporidian parasites (83.9%) in rosy starlings (n=31), with predominant infections of Haemoproteus pastoris (cytochrome b lineage LAMPUR01) (80.1%). All birds had high heterophil/lymphocyte ratios (mean = 3.86), indicating the transition to captivity was stressful. However, there was no added stress from blood parasite infection during captivity, since infected birds did not have significantly higher heterophil/lymphocyte ratios than uninfected birds, despite all birds facing the same stress conditions. There was a negative correlation between intensity of H. pastoris and monocyte proportions, which may indicate an association with infection, or an artefact of the stress-induced shift in leukocyte profile following captivity. Our findings suggest that blood parasites have minimal additional impact on rosy starlings during an acute stress scenario (overnight captivity), arguing that they carry little cost to the energetically challenging migratory flight.