The flying activity of biting midges (Ceratopogonidae: Culicoides) in Verkiai Regional Park, southeastern Lithuania.

Culicoides biting midges are small dipterous insects (Diptera: Ceratopogonidae) which are known to be vectors of arboviruses, bacteria, protozoan and helminth parasites that can cause disease and mortality in livestock and poultry globally. Detailed knowledge of the Culicoides species composition and biology is essential to assess the risk of the introduction and transmission of pathogens. The aim of this study was to obtain data on Culicoides species composition and flying activity in southeastern Lithuania and to determine the meteorological variables related to the abundance of Culicoides biting midges. Biting midges were collected in Verkiai Regional Park, southeastern Lithuania, using an Onderstepoort trap once a week from April to October 2016 and 2018, and from April to July 2019; 7332 Culicoides females belonging to 22 species were identified. Both morphology and DNA barcoding were used for identification. The number of specimens trapped was highest for the Obsoletus Group, followed by Culicoides kibunensis and Culicoides impunctatus. The highest relative abundance and diversity of biting midges were found in May and June. The number of trapped biting midges correlated positively with the mean air temperature. The first biting midges in spring were caught when the mean daily temperature rose higher than 10 °C. No Culicoides were detected when the air temperature dropped below 5 °C in autumn. Wind speed and air humidity had no statistically significant effect on Culicoides abundance.

De novo transcriptome assembly and preliminary analyses of two avian malaria parasites, Plasmodium delichoni and Plasmodium homocircumflexum.

Parasites of the genus Plasmodium infect a wide array of hosts, causing malaria in all major groups of terrestrial vertebrates including primates, reptiles, and birds. Molecular mechanisms explaining why some Plasmodium species are virulent, while other closely related malaria pathogens are relatively benign in the same hosts, remain unclear. Here, we present the RNA sequencing and subsequent transcriptome assembly of two avian Plasmodium parasites which can eventually be used to better understand the genetic mechanisms underlying Plasmodium species pathogenicity in an avian host. Plasmodium homocircumflexum, a cryptic, pathogenic species that often causes mortality and Plasmodium delichoni, a newly described, relatively benign malaria parasite that does not kill its hosts, were used to experimentally infect two Eurasian siskins (Carduelis spinus). RNA extractions were performed and RNA sequencing was completed using high throughput Illumina sequencing. Using established bioinformatics pipelines, the sequencing data from both species were used to generate transcriptomes using published Plasmodium species genomes as a scaffold. The finalized transcriptome of P. homocircumflexum contained 21,612 total contigs while that of P. delichoni contained 12,048 contigs. We were able to identify many genes implicated in erythrocyte invasion actively expressed in both P. homocircumflexum and P. delichoni, including the well described vaccine candidates Apical Membrane Antigen-1 (AMA-1) and Merozoite Surface Protein 1 (MSP1). This work acts as a stepping stone to increase available data on avian Plasmodium parasites, thus enabling future research into the evolution of pathogenicity in malaria.

Patterns of Plasmodium homocircumflexum virulence in experimentally infected passerine birds.

BACKGROUND: Avian malaria parasites (genus Plasmodium) are cosmopolitan and some species cause severe pathologies or even mortality in birds, yet their virulence remains fragmentally investigated. Understanding mechanisms and patterns of virulence during avian Plasmodium infections is crucial as these pathogens can severely affect bird populations in the wild and cause mortality in captive individuals. The goal of this study was to investigate the pathologies caused by the recently discovered malaria parasite Plasmodium homocircumflexum (lineage pCOLL4) in four species of European passeriform birds. METHODS: One cryopreserved P. homocircumflexum strain was multiplied and used for experimental infections. House sparrows (Passer domesticus), common chaffinches (Fringilla coelebs), common crossbills (Loxia curvirostra) and common starlings (Sturnus vulgaris) were exposed by subinoculation of infected blood. Experimental and control groups (8 individuals in each) were observed for over 1 month. Parasitaemia, haematocrit value and body mass were monitored. At the end of the experiment, samples of internal organs were collected and examined using histological and chromogenic in situ hybridization methods. RESULTS: All exposed birds were susceptible, with similar average prepatent period and maximum parasitaemia, yet virulence was different in different bird species. Mortality due to malaria was reported in chaffinches, house sparrows and crossbills (7, 5 and 3 individuals died respectively), but not in starlings. Exoerythrocytic meronts (phanerozoites) were observed in the brain of all dead experimental birds. Blockage of blood vessels in the brain led to cerebral ischaemia, invariably causing brain damage, which is likely the main reason of mortality. Phanerozoites were observed in parenchymal organs, heart and muscles of all infected individuals, except starlings. CONCLUSION: This study shows that P. homocircumflexum is generalist and the same lineage caused similar parasitaemia-related pathologies in different host species. Additionally, the mode of exo-erythrocytic development is different in different birds, resulting in different mortality rates. This should be taken into consideration in studies addressing pathology during avian malaria infections.

High susceptibility of the laboratory-reared biting midges Culicoides nubeculosus to Haemoproteus infections, with review on Culicoides species that transmit avian haemoproteids.

Haemosporidian parasites belonging to Haemoproteus cause avian diseases, however, vectors remain unidentified for the majority of described species. We used the laboratory-reared biting midges Culicoides nubeculosus to determine if the sporogonic development of three widespread Haemoproteus parasites completes in this insect. The midges were reared and fed on one common blackbird, white wagtail and thrush nightingale naturally infected with Haemoproteus minutus, Haemoproteus motacillae and Haemoproteus attenuatus, respectively. The engorged females were dissected in order to follow their sporogonic development. Microscopic examination was used to identify sporogonic stages. Bayesian phylogeny based on partial cytochrome b gene was constructed in order to determine phylogenetic relationships among Culicoides species-transmitted haemoproteids. All three parasites completed sporogony. Phylogenetic analysis placed Culicoides species transmitted haemoproteids in one well-supported clade, proving that such analysis readily indicates groups of dipteran insects transmitting avian haemoproteids. Available data show that 11 species of Culicoides have been proved to support complete sporogony of 18 species of avian haemoproteids. The majority of Culicoides species can act as vectors for many Haemoproteus parasites, indicating the low specificity of these parasites to biting midges, whose are globally distributed. This calls for control of haemoproteid infections during geographical translocation of infected birds.

Characterization of Plasmodium relictum, a cosmopolitan agent of avian malaria.

BACKGROUND: Microscopic research has shown that Plasmodium relictum is the most common agent of avian malaria. Recent molecular studies confirmed this conclusion and identified several mtDNA lineages, suggesting the existence of significant intra-species genetic variation or cryptic speciation. Most identified lineages have a broad range of hosts and geographical distribution. Here, a rare new lineage of P. relictum was reported and information about biological characters of different lineages of this pathogen was reviewed, suggesting issues for future research. METHODS: The new lineage pPHCOL01 was detected in Common chiffchaff Phylloscopus collybita, and the parasite was passaged in domestic canaries Serinus canaria. Organs of infected birds were examined using histology and chromogenic in situ hybridization methods. Culex quinquefasciatus mosquitoes, Zebra finch Taeniopygia guttata, Budgerigar Melopsittacus undulatus and European goldfinch Carduelis carduelis were exposed experimentally. Both Bayesian and Maximum Likelihood analyses identified the same phylogenetic relationships among different, closely-related lineages pSGS1, pGRW4, pGRW11, pLZFUS01, pPHCOL01 of P. relictum. Morphology of their blood stages was compared using fixed and stained blood smears, and biological properties of these parasites were reviewed. RESULTS: Common canary and European goldfinch were susceptible to the parasite pPHCOL01, and had markedly variable individual prepatent periods and light transient parasitaemia. Exo-erythrocytic and sporogonic stages were not seen. The Zebra finch and Budgerigar were resistant. Neither blood stages nor vector stages of all examined P. relictum lineages can be distinguished morphologically. CONCLUSION: Within the huge spectrum of vertebrate hosts, mosquito vectors, and ecological conditions, different lineages of P. relictum exhibit indistinguishable, markedly variable morphological forms. Parasites of same lineages often develop differently in different bird species. Even more, the variation of biological properties (parasitaemia dynamics, blood pathology, prepatent period) in different isolates of the same lineage might be greater than the variation in different lineages during development in the same species of birds, indicating negligible taxonomic value of such features. Available lineage information is excellent for parasite diagnostics, but is limited in predictions about relationships in certain host-parasite associations. A combination of experiments, field observations, microscopic and molecular diagnostics is essential for understanding the role of different P. relictum lineages in bird health.

Molecular characterization and distribution of Plasmodium matutinum, a common avian malaria parasite.

Species of Plasmodium (Plasmodiidae, Haemosporida) are widespread and cause malaria, which can be severe in avian hosts. Molecular markers are essential to detect and identify parasites, but still absent for many avian malaria and related haemosporidian species. Here, we provide first molecular characterization of Plasmodium matutinum, a common agent of avian malaria. This parasite was isolated from a naturally infected thrush nightingale Luscinia luscinia (Muscicapidae). Fragments of mitochondrial, apicoplast and nuclear genomes were obtained. Domestic canaries Serinus canaria were susceptible after inoculation of infected blood, and the long-lasting light parasitemia developed in two exposed birds. Clinical signs of illness were not reported. Illustrations of blood stages of P. matutinum (pLINN1) are given, and phylogenetic analysis identified the closely related avian Plasmodium species. The phylogeny based on partial cytochrome b (cyt b) sequences suggests that this parasite is most closely related to Plasmodium tejerai (cyt b lineage pSPMAG01), a common malaria parasite of American birds. Both these parasites belong to subgenus Haemamoeba, and their blood stages are similar morphologically, particularly due to marked vacuolization of the cytoplasm in growing erythrocytic meronts. Molecular data show that transmission of P. matutinum (pLINN1) occurs broadly in the Holarctic, and the parasite likely is of cosmopolitan distribution. Passeriform birds and Culex mosquitoes are common hosts. This study provides first molecular markers for detection of P. matutinum.

Mortality and pathology in birds due to Plasmodium (Giovannolaia) homocircumflexum infection, with emphasis on the exoerythrocytic development of avian malaria parasites.

BACKGROUND: Species of avian malaria parasites (Plasmodium) are widespread, but their virulence has been insufficiently investigated, particularly in wild birds. During avian malaria, several cycles of tissue merogony occur, and many Plasmodium spp. produce secondary exoerythrocytic meronts (phanerozoites), which are induced by merozoites developing in erythrocytic meronts. Phanerozoites markedly damage organs, but remain insufficiently investigated in the majority of described Plasmodium spp. Avian malaria parasite Plasmodium (Giovannolaia) homocircumflexum (lineage pCOLL4) is virulent and produces phanerozoites in domestic canaries Serinus canaria, but its pathogenicity in wild birds remains unknown. The aim of this study was to investigate the pathology caused by this infection in species of common European birds. METHODS: One individual of Eurasian siskin Carduelis spinus, common crossbill Loxia curvirostra and common starling Sturnus vulgaris were exposed to P. homocircumflexum infection by intramuscular sub-inoculation of infected blood. The birds were maintained in captivity and parasitaemia was monitored until their death due to malaria. Brain, heart, lungs, liver, spleen, kidney, and a piece of breast muscle were examined using histology and chromogenic in situ hybridization (ISH) methods. RESULTS: All exposed birds developed malaria infection, survived the peak of parasitaemia, but suddenly died between 30 and 38 days post exposure when parasitaemia markedly decreased. Numerous phanerozoites were visible in histological sections of all organs and were particularly easily visualized after ISH processing. Blockage of brain capillaries with phanerozoites may have led to cerebral ischaemia, causing cerebral paralysis and is most likely the main reason of sudden death of all infected individuals. Inflammatory response was not visible around the brain, heart and muscle phanerozoites, and it was mild in parenchymal organs. The endothelial damage likely causes dysfunction and failure of parenchymal organs. CONCLUSION: Plasmodium homocircumflexum caused death of experimental passerine birds due to marked damage of organs by phanerozoites. Patterns of phanerozoites development and pathology were similar in all exposed birds. Mortality was reported when parasitaemia decreased or even turned into chronic stage, indicating that the light parasitaemia is not always indication of improved health during avian malaria. Application of traditional histological and ISH methods in parallel simplifies investigation of exoerythrocytic development and is recommended in avian malaria research.

Mechanisms of mortality in Culicoides biting midges due to Haemoproteus infection.

We examined the effects of Haemoproteus infection on the survival and pathology caused in the biting midges. Forty-six females of Culicoides impunctatus were exposed experimentally by allowing them to feed on a naturally infected red-backed shrike infected with Haemoproteus lanii (lineage hRB1, gametocytaemia 5·2%). Seventeen females were fed on an uninfected bird (controls). Dead insects were collected, counted and used for dissection, histological examination and polymerase chain reaction-based testing. Parasites were present in all experimentally infected biting midges, but absent from control insects. Survivorship differed significantly between the control and infected groups. Twelve hours post-exposure (PE), 45 (98%) experimentally infected midges were dead, but all control midges remained alive, and many of them survived until 7 day PE. The migrating ookinetes of H. lanii overfilled midgut, markedly damaged the midgut wall, entered the haemocoel and overfilled the abdomen and thorax of exposed biting midges. Massive infection by migrating ookinetes led to damage of abdomen and thorax of biting midges. The parasites often present in large clumps in the haemocoel in abdomen and thorax, leading to the interruption of the haemolymph circulation. These are the main reasons for rapid death of biting midges after feeding on high-intensity infections of Haemoproteus parasites.

Description of Haemoproteus ciconiae sp. nov. (Haemoproteidae, Haemosporida) from the white stork Ciconia ciconia, with remarks on insensitivity of established polymerase chain reaction assays to detect this infection.

Haemoproteus ciconiae sp. nov. (Haemosporida, Haemoproteidae) was found in the white stork Ciconia ciconia (Ciconiiformes, Ciconiidae) after spring migration in Lithuania. This organism is illustrated and described based on the morphology of its blood stages. The new species can be readily distinguished from all other haemoproteids parasitizing ciconiiform birds due to the presence of large number (approximately 20 on average) small (<1 µm) pigment granules in its mature gametocytes. Growing and mature gametocytes of H. ciconiae were readily visible in all blood films (parasitemia of 0.001 %). However, experienced researchers were unable to detect sequences of its mitochondrial cytochrome b (cyt b) or apicoplast genes from the microscopically positive sample by using five established assays for polymerase chain reaction (PCR)-based detection of avian haemosporidian parasites. The white stork cyt b sequence was readily detectable, indicating the well-optimised PCR protocols and the good quality of total DNA in the sample containing the new species. The failure to amplify this parasite DNA indicates insufficient sensitivity of the currently used PCR-based assays in diagnostics of avian haemosporidian infections. We suggest possible explanations of this observation. To minimize number of the false negative PCR reports, we call for the continued use of optical microscopy in parallel with molecular diagnostics in studies of haemosporidian parasites, particularly in wildlife.

Plasmodium delichoni n. sp.: description, molecular characterisation and remarks on the exoerythrocytic merogony, persistence, vectors and transmission.

Malaria parasite Plasmodium (Novyella) delichoni n. sp. (Haemosporida, Plasmodiidae) was found in a widespread Eurasian songbird, the common house martin Delichon urbicum (Hirundinidae). It is described based on the morphology of its blood stages and segments of the mitochondrial cytochrome b and apicoplast genes, which can be used for molecular identification of this species. Erythrocytic meronts and gametocytes are strictly nucleophilic, and mature gametocytes possess pigment granules of markedly variable size, including large ones (1 µm in length). Due to these features, P. delichoni can be readily distinguished from all described species of avian malaria parasites belonging to subgenus Novyella. Additionally, mature erythrocytic merozoites contain a dense clump of chromatin, a rare character in avian malaria parasites. Erythrocytic merogony is asynchronous. Illustrations of blood stages of the new species are given, and phylogenetic analysis identifies DNA lineages closely related to this parasite. Domestic canary Serinus canaria and Eurasian siskin Carduelis spinus were infected after subinoculation of infected blood obtained from the house martin. Parasitemia was long lasting in both these hosts, but it was high (up to 70 %) in Eurasian siskins and low (up to 1 %) in canaries. Mortality was not observed, and histological examination and chromogenic in situ hybridisation did not reveal secondary exoerythrocytic meronts (phanerozoites) in the exposed birds. It is likely that persistence of this infection occurs due to long-lasting parasitemia in avian hosts. Sporogony was abortive in mosquitoes Culex pipiens pipiens form molestus, Culex quinquefasciatus and Aedes aegypti at gametogenesis or ookinete stages. The new species is absent from juvenile birds at breeding sites in Europe, indicating that transmission occurs at African wintering grounds.

Plasmodium spp.: an experimental study on vertebrate host susceptibility to avian malaria.

The interest in experimental studies on avian malaria caused by Plasmodium species has increased recently due to the need of direct information about host-parasite interactions. Numerous important issues (host susceptibility, development of infection, the resistance and tolerance to avian malaria) can be answered using experimental infections. However, specificity of genetically different lineages of malaria parasites and their isolates is largely unknown. This study reviews recent experimental studies and offers additional data about susceptibility of birds to several widespread cytochrome b (cyt b) lineages of Plasmodium species belonging to four subgenera. We exposed two domesticated avian hosts (canaries Serinus canaria and ducklings Anas platyrhynchos) and also 16 species of common wild European birds to malaria infections by intramuscular injection of infected blood and then tested them by microscopic examination and PCR-based methods. Our study confirms former field and experimental observations about low specificity and wide host-range of Plasmodium relictum (lineages SGS1 and GRW11) and P. circumflexum (lineage TURDUS1) belonging to the subgenera Haemamoeba and Giovannolaia, respectively. However, the specificity of different lineages and isolates of the same parasite lineage differed between species of exposed hosts. Several tested Novyella lineages were species specific, with a few cases of successful development in experimentally exposed birds. The majority of reported cases of mortality and high parasitaemia were observed during parasite co-infections. Canaries were susceptible mainly for the species of Haemamoeba and Giovannolaia, but were refractory to the majority of Novyella isolates. Ducklings were susceptible to three malaria infections (SGS1, TURDUS1 and COLL4), but parasitaemia was light (<0.01%) and transient in all exposed birds. This study provides novel information about susceptibility of avian hosts to a wide array of malaria parasite lineages, outlining directions for future experimental research on various aspects of biology and epidemiology of avian malaria.

Complete sporogony of Plasmodium relictum (lineage pGRW4) in mosquitoes Culex pipiens pipiens, with implications on avian malaria epidemiology.

Plasmodium relictum (lineage pGRW4) causes malaria in birds and is actively transmitted in countries with warm climates and also temperate regions of the New World. In Europe, the lineage pGRW4 has been frequently reported in many species of Afrotropical migrants after their arrival from wintering grounds, but is rare in European resident birds. Obstacles for transmission of this parasite in Europe have not been identified. Culex quinquefasciatus is an effective vector of pGRW4 malaria, but this mosquito is absent from temperate regions of Eurasia. It remains unclear if the lineage pGRW4 completes sporogony in European species of mosquitoes. Here we compare the sporogonic development of P. relictum (pGRW4) in experimentally infected mosquitoes Culex pipiens pipiens form molestus, C. quinquefasciatus, and Ochlerotatus cantans. The pGRW4 parasite was isolated from a garden warbler Sylvia borin, multiplied, and used to infect laboratory-reared Culex spp. and wild-caught Ochlerotatus mosquitoes by allowing them to take blood meals on infected birds. The exposed females were maintained at a mean laboratory temperature of 19 °C, which ranged between 14 °C at night and 24 °C during daytime. They were dissected on intervals to study the development of sporogonic stages. Only ookinetes developed in O. cantans; sporogonic development was abortive. The parasite completed sporogony in both Culex species, with similar patterns of development, and sporozoites were reported in the salivary glands 16 days after infection. The presence of sporogonic stages of the lineage pGRW4 in mosquitoes was confirmed by PCR-based testing of (1) the sporozoites present in salivary glands and (2) the single oocysts, which were obtained by laser microdissection from infected mosquito midguts. This study shows that P. relictum (pGRW4) completes sporogony in C. p. pipiens at relatively low temperatures. We conclude that there are no restrictions for spreading this bird infection in Europe from the point of view of vector availability and temperature necessary for sporogony. Other factors should be considered and were discussed for the explanation of rare reports of this malaria parasite in Europe.

Haemoproteus infections (Haemosporida, Haemoproteidae) kill bird-biting mosquitoes.

Haemoproteus parasites (Haemosporida, Haemoproteidae) are widespread; some species cause severe diseases in avian hosts. Heavy Haemoproteus infections are often lethal for biting midges (Ceratopogonidae), which transmit avian haemoproteids, but there is no information regarding detrimental effect on other blood-sucking insects. We examined effects of Haemoproteus tartakovskyi (lineage hSISKIN1), Haemoproteus lanii (lineages hRB1and hRBS2) and Haemoproteus balmorali (lineage hCOLL3) on the survival of Ochlerotatus cantans, a widespread Eurasian mosquito. Wild-caught females were infected by allowing them to feed on naturally infected birds with light (0.01%) and high (3.0-9.6%) parasitaemia. Mosquitoes fed on uninfected birds were used as controls. Both experimental and control groups were maintained under the same laboratory conditions until 20 days post-exposure (dpe). Dead insects were counted daily and used for parasitological examination and PCR-based testing. No difference was discernible in the survival rate of control mosquitoes and those fed on meal with light parasitaemia. There was a highly significant difference in the survival rate between the control group and all groups fed on meals with high parasitaemia, with the greatest mortality reported 1-3 dpe. For 4 dpe, the percentage of survived control mosquitoes (88%) was 2.2-, 3.6- and 4-fold greater than that of groups fed on meals with high parasitaemia of H. balmorali, H. tartakovskyi and H. lanii, respectively. Numerous ookinetes were observed in the gut area and adjacent tissues located in the head, thorax and abdomen of infected insects 0.5-1 dpe. The migrating parasites damage organs throughout the entire body of mosquitoes; that is the main reason of mortality. To the end of this study, 46% of mosquitoes survived in control group, but the survival rates of experimental mosquitoes fed on meals with high parasitaemia were between 2.6- and 5.8-fold lower. This study indicates that widespread Haemoproteus infections are markedly virulent for bird-biting mosquitoes, which rapidly die after feeding on heavily infected blood meals.

Molecular characterization of five widespread avian haemosporidian parasites (Haemosporida), with perspectives on the PCR-based detection of haemosporidians in wildlife.

Haemosporidians (Haemosporida) are cosmopolitan in birds. Over 250 species of these blood parasites have been described and named; however, molecular markers remain unidentified for the great majority of them. This is unfortunate because linkage between DNA sequences and identifications based on morphological species can provide important information about patterns of transmission, virulence, and evolutionary biology of these organisms. There is an urgent need to remedy this because few experts possess the knowledge to identify haemosporidian species and few laboratories are involved in training these taxonomic skills. Here, we describe new mitochondrial cytochrome b markers for the polymerase chain reaction (PCR)-based detection of four widespread species of avian Haemoproteus (Haemoproteus hirundinis, Haemoproteus parabelopolskyi, Haemoproteus pastoris, Haemoproteus syrnii) and 1 species of Plasmodium (Plasmodium circumflexum). Illustrations of blood stages of the reported species are given, and morphological and phylogenetic analyses identify the DNA lineages that are associated with these parasites. This study indicates that morphological characters, which have been traditionally used in taxonomy of avian haemosporidian parasites, have a phylogenetic value. Perspectives on haemosporidian diagnostics using microscopic and PCR-based methods are discussed, particularly the difficulties in detection of light parasitemia, coinfections, and abortive parasite development. We emphasize that sensitive PCR amplifies more infections than can be transmitted; it should be used carefully in epidemiology studies, particularly in wildlife parasitology research. Because molecular studies are describing remarkably more parasite diversity than previously expected, the need for traditional taxonomy and traditional biological knowledge is becoming all the more crucial. The linkage of molecular and morphological approaches is worth more of the attention of researchers because this approach provides new knowledge for better understanding insufficiently investigated lethal diseases caused by haemosporidian infections, particularly on the exoerythrocytic (tissue) and vector stages. That requires close collaboration between researchers from different fields with a common interest.

Differences in haemosporidian parasite prevalence and diversity in migratory and resident birds of prey species revealed by a non-invasive sampling method.

Avian haemosporidian parasites are widespread globally and infect numerous wild bird species. However, they have primarily been studied in passerine birds. Accordingly, the prevalence and diversity of these parasites in birds of prey remain understudied. This lack of research is primarily due to the international protection status of many of these species, their sparse distribution across ecosystems and difficulty to capture in the wild. The aim of this study was to examine the prevalence and diversity of haemosporidian parasites in two species of birds of prey, namely white-tailed eagle and lesser spotted eagle. To achieve this, a non-invasive approach was employed, involving the extraction of DNA from blood spots present in moulted feathers. Freshly moulted feathers were collected from the ground under nests or within the nests of these birds during the breeding season. A visible blood spot located at the junction of the calamus and rachis was removed and fixed in SET buffer for molecular analysis. The identification of haemosporidian parasites (Plasmodium, Haemoproteus and Leucocytozoon) was conducted using PCR-based methods. Overall, parasite DNA was successfully detected in shed feathers that were kept in their original form at least decade prior to analysis. Among the studied individuals, resident white-tailed eagles showed significantly lower infection rates of haemosporidian parasites compared to migratory lesser spotted eagles. A total of nine genetic lineages of haemosporidian parasites were detected, with seven of them being new to science based on partial sequences of the cytb gene. Additionally, the phylogenetic relationships among these new lineages and previously described ones were established. These findings highlight the suitability of non-invasive sampling for investigating the prevalence and diversity of haemosporidian parasites in wild birds of prey populations. Moreover, this approach holds promise for studying other challenging-to-reach and protected bird species. According to our research, there is a greater chance of finding haemosporidian parasites in freshly gathered feathers.

Molecular detection of Babesia vesperuginis in bats from Lithuania.

Babesia vesperuginis is an intraerythrocytic protozoan parasite that circulates among bats and ticks in many countries worldwide. However, the distribution of B. vesperuginis in the Baltic region has not been studied. A total of 86 dead bats from eight different species were collected and screened for Babesia spp. using real-time PCR. Overall, 52.3% (45/86) of the bats were found positive for Babesia spp. The prevalence of Babesia spp. in different organs varied, with the highest prevalence observed in heart tissues (37.0%) and the lowest in liver tissues (22.2%). However, the observed differences in prevalence among organs were not statistically significant. Blood samples from 125 bats of nine different species were also analyzed for Babesia spp. prevalence using real-time PCR and nested PCR. The results showed a prevalence of 35.2% and 22.4%, respectively. Moreover, 28.3% (17/60) of the examined blood samples were confirmed positive for Babesia spp. through blood smear analysis. The total of 32 partial sequences of the 18S rRNA gene derived in this study were 100% identical to B. vesperuginis sequences from GenBank. In eight species of bats, Pipistrellus nathusii, Pipistrellus pipistrellus, Pipistrellus pygmaeus, Vespertilio murinus, Eptesicus nilssonii, Eptesicus serotinus, Myotis daubentonii and Nyctalus noctula, Babesia parasites were identified. In E. nilssonii, Babesia spp. was identified for the first time.

High Abundance of Haemoproteus Parasites in Culicoides (Diptera, Ceratopogonidae), with a Confirmation of Culicoides reconditus as a New Vector of These Avian Blood Parasites.

Haemoproteus parasites are the most diverse among Haemosporida. However, their natural vectors (Culicoides) are still poorly investigated and were identified for only a few parasite species and lineages. The application of an integrative approach (insect dissection, microscopic analysis, and molecular-based methods) is necessary in these studies, which have been carried out by a few research groups, mainly in Europe. The aim of this study was (i) to determine the Culicoides species that are naturally infected by Haemoproteus parasites, and which can support its complete sporogonic development, and (ii) to investigate the prevalence of Culicoides species and Haemoproteus parasite lineages in different study sites. In total, 1953 parous Culicoides females, from 11 species, were collected in four different localities in Lithuania and were dissected and analyzed using an integrative approach. The most abundant was C. pictipennis (30.3%). Parasite DNA was found in 7.9% of all investigated Culicoides, of which ~30% had sporozoites in their salivary glands, confirming their vector competence for these parasites. The Botanical Garden presented the highest number of Culicoides parous females, Culicoides species, and parasite lineages, as well as the highest positivity for sporozoites. Culicoides reconditus was confirmed as a natural vector of Haemoproteus parasites, sporozoites of six Haemoproteus lineages were reported for the first time, and 12 new interactions between Haemoproteus parasite lineages and Culicoides species were identified. Haemoproteus parasites seem to be transmitted by a high number of Culicoides species, with C. kibunensis, C. pictipennis, and C. segnis being the most important vectors.

Unexpected absence of exo-erythrocytic merogony during high gametocytaemia in two species of Haemoproteus (Haemosporida: Haemoproteidae), including description of Haemoproteus angustus n. sp. (lineage hCWT7) and a report of previously unknown residual bodies during in vitro gametogenesis.

Neglected avian blood parasites of the genus Haemoproteus (Haemoproteidae) have recently attracted attention due to the application of molecular diagnostic tools, which unravelled remarkable diversity of their exo-erythrocytic (or tissue) stages both regarding morphology and organ tropism levels. The development of haemoproteids might result in pathologies of internal organs, however the exo-erythrocytic development (EED) of most Haemoproteus species remains unknown. Seven individual birds - Curruca communis (1) and Phylloscopus trochilus (6) - with high gametocytaemia (between 1% and 24%) of Haemoproteus angustus n. sp. (hCWT7) and Haemoproteus palloris (lineage hWW1) were sampled in Lithuania, and their internal organs were examined extensively by parallel application of histology and chromogenic in situ hybridization methods. Tissue stages were apparently absent, suggesting that the parasitaemia was not accompanied by detectable tissue merogony. Haemoproteus angustus n. sp. was described and characterized morphologically and molecularly. Sexual process and ookinete development of the new species readily occurred in vitro, and a unique character for Haemoproteus parasites was discovered - the obligatory development of several tiny residual bodies, which were associated with intracellular transformation of both macrogametocytes and microgametocytes before their escape from the host cells and formation of gametes. A DNA haplotype network was constructed with lineages that cluster in one clade with the lineage hCWT7. This clade consists of lineages mostly found in Curruca birds, indicating specificity for birds of this genus. The lineage hCWT7 is mainly a parasite of C. communis. Most reports of this lineage came from Turkey, with only a few records in Europe, mostly in birds wintering in Africa where transmission probably occurs. This study highlights unexpected difficulties in the research of EED even when using sensitive molecular diagnostic tools and extends information about transformation in early stages of gametogenesis in haemosporidian parasites.

Description of Haemoproteus asymmetricus n. sp. (Haemoproteidae), with remarks on predictability of the DNA haplotype networks in haemosporidian parasite taxonomy research.

Haemoproteus species (Haemosporida, Haemoproteidae) are cosmopolitan blood parasites, which have been neglected for over 100-years, but attracted attention recently due to reports of severe and even lethal haemoproteosis in birds and vectors. Approximately 150 species of avian Haemoproteus have been described and named, but molecular data suggest that hundreds of independently evolving molecular lineages might occur, indicating the existence of a remarkable undescribed species diversity. It is timely to develop a methodology, which allow the application of available genetic data in taxonomy of haemosporidians on species levels. This study aimed to test a hypothesis suggesting that DNA haplotype networks might aid in targeting genetically distinct, but still undescribed parasites, and might be used to direct taxonomic studies on haemosporidian species levels. Mainly, we tested a prediction that the lineage hTUPHI01, a common Haemoproteus parasite of Turdus philomelos, might be a new species, which is morphologically similar and genetically closely related to the parasites of Haemoproteus minutus group. Blood samples of T. philomelos naturally infected with this parasite lineage were collected and studied using microscopic examination of blood films and PCR-based methods. Haemoproteus asymmetricus n. sp. was found in this bird, described and characterised molecularly using partial cytochrome b (cytb) sequences. The new species shared some features with parasites of the H. minutus group, as was predicted by the DNA haplotype network. Due to the visualisation of closely related lineages as well as the evaluation of their host and geographic distributions, DNA haplotype networks can be recommended as the helpful methodology, able to direct and speed practical work on parasite species taxonomy and pathogen biodiversity. The combined molecular phylogenetic and morphological approaches showed that the well-supported clades in Bayesian phylogenetic trees based on the partial cytb gene sequences contain morphologically remarkably different Haemoproteus parasite species, which however, share some basic biological features. Phylogenetic analysis can be used for prediction of these basic features in still undescribed parasites. This study calls for further fusion of advanced molecular and microscopy approaches for better understanding haemosporidian parasite biology.

Lankesterella (Apicomplexa, Lankesterellidae) Blood Parasites of Passeriform Birds: Prevalence, Molecular and Morphological Characterization, with Notes on Sporozoite Persistence In Vivo and Development In Vitro.

Recent studies confirmed that some Hepatozoon-like blood parasites (Apicomplexa) of birds are closely related to the amphibian parasite Lankesterella minima. Little is known about the biology of these pathogens in birds, including their distribution, life cycles, specificity, vectors, and molecular characterization. Using blood samples of 641 birds from 16 species, we (i) determined the prevalence and molecular diversity of Lankesterella parasites in naturally infected birds; (ii) investigated the development of Lankesterella kabeeni in laboratory-reared mosquitoes, Culex pipiens forma molestus and Aedes aegypti; and (iii) tested experimentally the susceptibility of domestic canaries, Serinus canaria, to this parasite. This study combined molecular and morphological diagnostic methods and determined 11% prevalence of Lankesterella parasites in Acrocephalidae birds; 16 Lankesterella lineages with a certain degree of host specificity and two new species (Lankesterella vacuolata n. sp. and Lankesterella macrovacuolata n. sp.) were found and characterized. Lankesterella kabeeni (formerly Hepatozoon kabeeni) was re-described. Serinus canaria were resistant after various experimental exposures. Lankesterella sporozoites rapidly escaped from host cells in vitro. Sporozoites persisted for a long time in infected mosquitoes (up to 42 days post exposure). Our study demonstrated a high diversity of Lankesterella parasites in birds, and showed that several avian Hepatozoon-like parasites, in fact, belong to Lankesterella genus.