Recent in vitro experimental studies reported the complex patterns of haemosporidian (Haemosporid... more Recent in vitro experimental studies reported the complex patterns of haemosporidian (Haemosporida) between-lineage interactions, which prevent mixing of lineages during simultaneous sexual process. Numerous anomalous ookinetes have been observed; these are not involved in sporogony. Massive development of such ookinetes might influence parasite transmission but is insufficiently investigated. The simultaneous sexual process of several lineages is a common phenomenon in vectors due to high prevalence of haemosporidian co-infections in wildlife. It remains unclear if the number of anomalous ookinetes changes during dual-infection sporogony in comparison with the single-infection process. We calculated proportions of the anomalous and normal ookinetes, which developed during single-infection (control) and dual-infection experiments in vitro conditions. Three mitochondrial cytochrome b lineages belonging to three Haemoproteus spp. (Haemosporida, Haemoproteidae) were isolated from naturally infected passerine birds. Sexual process and ookinete development were initiated in vitro by mixing blood containing mature gametocytes of two different parasites; the following experiments were performed: (1) Haemoproteus tartakovskyi (lineage hSISKIN1) × Haemoproteus lanii (lineage hRBS4) and (2) Haemoproteus belopolskyi (hHIICT3) × H. lanii (hRBS4). Genetic difference between lineages was 5.0-5.9%. Normal and anomalous ookinetes developed in all control and dual-infection experiments. The number of anomalous ookinetes markedly decreased, and normal ookinetes increased in all dual-infection experiments in comparison with those in controls, except for H. belopolskyi, in which proportion of the anomalous and normal ookinetes did not change. This study shows that simultaneous sexual process of two genetically distant lineages of haemosporidian parasites might increase the efficiency of reproductive cells, resulting in the development of a greater number of normal ookinetes. The marked increase of the number of normal ookinetes, which is involved in sporogony, indicates the success of sporogony in dual infections. Some haemosporidian lineages might benefit from simultaneous sporogony. Widespread avian Haemoproteus spp. are convenient and laboratory-friendly organisms for in vitro experimental research addressing between-lineage interaction in parasites during the sexual process.
Mixed infections of different species and genetic lineages of haemosporidian parasites (Haemospor... more Mixed infections of different species and genetic lineages of haemosporidian parasites (Haemosporida) predominate in wildlife, and such infections are particularly virulent. However, currently used polymerase chain reaction (PCR)-based detection methods often do not read mixed infections. Sensitivity of different PCR assays in detection of mixed infections has been insufficiently tested, but this knowledge is essential in studies addressing parasite diversity in wildlife. Here, we applied five different PCR assays, which are broadly used in wildlife avian haemosporidian research, and compared their sensitivity in detection of experimentally designed mixed infections of Haemoproteus and Plasmodium parasites. Three of these PCR assays use primer sets that amplify fragments of cytochrome b gene (cyt b), one of cytochrome oxidase subunit I (COI) gene, and one target apicoplast genome. We collected blood from wild-caught birds and, using microscopic and PCR-based methods applied in parallel, identified single infections of ten haemosporidian species with similar parasitemia. Then, we prepared 15 experimental mixes of different haemosporidian parasites, which often are present simultaneously in wild birds. Similar concentration of total DNA was used in each parasite lineage during preparation of mixes. Positive amplifications were sequenced, and the presence of mixed infections was reported by visualising double-base calling in sequence electropherograms. This study shows that the use of each single PCR assay markedly underestimates biodiversity of haemosporidian parasites. The application of at least 3 PCR assays in parallel detected the majority, but still not all lineages present in mixed infections. We determined preferences of different primers in detection of parasites belonging to different genera of haemosporidians during mixed infections.
ABSTRACT Up to the year 2000 there have been known 118 species of biting midges in Lithuania. It ... more ABSTRACT Up to the year 2000 there have been known 118 species of biting midges in Lithuania. It should be pointed out, however, that the latest investigations of the fauna of biting midges in Lithuania were performed by H. Remm in 1957–1962. The article presents the data on biting midges collected and defined in 1997–2002. As a result of the studies performed there were 67 species of biting midges identified, 13 new for the Lithuanian fauna among them. Information on the locality and time of midge sampling is provided therein, too.
Outbreaks of African Swine Fever (ASF) in domestic pig farms in Lithuania typically begin in June... more Outbreaks of African Swine Fever (ASF) in domestic pig farms in Lithuania typically begin in June and are detected through October, suggesting that insects might be involved in the transmission of the virus. Entomological collecting was performed to obtain two data sets: from farms with ASF outbreaks, and from farms without ASF outbreaks but in an ASF‐infected area. Target insects from the families Muscidae, Calliphoridae and Tabanidae were analysed for the presence of ASF Virus (ASFV) DNA. Musca domestica L., Calliphoridae flies and Stomoxys calcitrans (L.) collected by entomological net during ASF outbreaks were confirmed to be ASFV positive. Viral DNA detected in insects collected by Nzi traps from farms with no ASFV outbreaks indicate that Culex, Lucilia, M. domestica and S. calcitrans are likely to play a role in spreading the ASFV mechanically. This finding could suggest contamination from outside of the farms: from infected wild boar or their carcasses. The role of Stomoxys f...
Numerous recent studies have addressed the molecular characterization, distribution and genetic d... more Numerous recent studies have addressed the molecular characterization, distribution and genetic diversity of Haemoproteus spp. (Haemoproteidae). Some species of these blood parasites cause severe disease in birds, and heavy infections are often lethal in biting midges (Ceratopogonidae) and other blood-sucking insects. However, information about the vectors of haemoproteids is scarce. This presents an obstacle for better understanding the mechanisms of host-parasite interactions and the epidemiology of haemoproteosis. Here we investigated the sporogonic development of Haemoproteus tartakovskyi, a widespread bird parasite, in experimentally infected biting midges, Culicoides nubeculosus. These biting midges are widespread in the Europe. The insects were cultivated under laboratory conditions. Unfed females were allowed to take blood meals on wild caught siskins Carduelis spinus naturally infected with H. tartakovskyi (lineage hSISKIN1). Engorged females were maintained at 22-23 °C, di...
Haemoproteus parasites are widespread, and several species cause diseases both in birds and blood... more Haemoproteus parasites are widespread, and several species cause diseases both in birds and blood-sucking insects. These pathogens are transmitted by dipterans belonging to the Ceratopogonidae and Hippoboscidae, however certain vector species remain unknown for the majority of Haemoproteus spp. Owls are often infected by Haemoproteus parasites, but experimental studies on vectors of these infections are lacking. The aim of this study was to investigate sporogonic development of two widespread Haemoproteus parasites of owls, H. noctuae and H. syrnii in experimentally infected biting midges Culicoides impunctatus and Culicoides nubeculosus. We also followed in vitro sporogonic development of these infections and determined their phylogenetic relationships with Haemoproteus spp., for which vectors have been identified. Wild-caught C. impunctatus and laboratory reared C. nubeculosus were infected experimentally by allowing them to take blood meals on one individual long-eared owl (Asio ...
BackgroundCulicoides biting midges (Diptera, Ceratopogonidae) are known to be vectors of avian Ha... more BackgroundCulicoides biting midges (Diptera, Ceratopogonidae) are known to be vectors of avian Haemoproteus parasites. These parasites cause disease and pathology in birds. The diversity of biting midges in Europe is great, but only four Culicoides species are known to be vectors of avian Haemoproteus parasites. In general, our knowledge about the role of the particular Culicoides species in transmission of Haemoproteus parasites remains insufficient. Information gaps hinder a better understanding of parasite biology and the epizootiology of parasite-caused diseases. The aim of this study was to determine new ornithophilic Culicoides species potentially involved in local transmission of Haemoproteus parasites. To do this we collected biting midges in bird nest boxes, identified their species and prevalence of haemoproteids in insects as well as in juvenile birds during breeding season.Methods Biting midges were collected from bird nest boxes, identified and parous females were teste...
Black flies (Diptera: Simuliidae) are among the most bothersome blood-sucking dipterans causing s... more Black flies (Diptera: Simuliidae) are among the most bothersome blood-sucking dipterans causing severe irritation and distress to poultry, wild birds, animals, and humans globally. These insects are vectors of viruses, bacteria, parasitic protozoa and nematodes of humans and animals. Parasitic protozoa belonging to Haemosporida (Apicomplexa) are distributed worldwide and black flies are the principal vectors of avian haemosporidian parasites of the genus Leucocytozoon, a common parasite of birds. Based on the detection of parasite DNA in insects, 13 black fly species were reported to be potential vectors of Leucocytozoon in Europe. Information about which species of Simulium can play a role in the transmission of Leucocytozoon parasites is insufficient and needs to be developed. The aim of our study was to determine which black fly species are involved in the transmission of Leucocytozoon parasites in the Eastern Europe. The black fly females were collected in Lithuania using entomological net. They were morphologically identified, dissected to prepare salivary glands preparations, and then screened for the presence of Leucocytozoon parasites using microscopy and PCR-based methods. In all, we collected 437 black fly females belonging to eight species. The DNA of Leucocytozoon (genetic lineage lCOCO18) was detected in one of analysed females identified as Simulium maculatum. All salivary gland preparations were negative for the presence of Leucocytozoon sporozoites. Our results included S. maculatum as a potential vector of Leucocytozoon parasites. Increasing the knowledge on vector ecology, behaviour and improving collection methods may be the key to understand the evolution and diversity of these parasites.
Increasingly frequent outbreaks of zoonotic infections call for studies of wildlife parasites to ... more Increasingly frequent outbreaks of zoonotic infections call for studies of wildlife parasites to reach a better understanding of the mechanisms of host switch, leading to the evolution of new diseases. However, speciation processes have been insufficiently addressed in experimental parasitology studies, primarily due to difficulties in determining and measuring mate-recognition signals in parasites. We investigated patterns of sexual process and ookinete development in avian Haemoproteus (Parahaemoproteus) spp. (Haemosporida, Haemoproteidae) using in vitro experiments on between-lineage hybridization. Eleven mitochondrial cytochrome b (cyt b) lineages belonging to 9 species of hemoproteid were isolated from naturally infected passerine birds. The parasites were identified to species on the basis of morphology of their gametocytes and polymerase chain reaction amplification of segments of the cyt b gene. Sexual process and ookinete development were initiated in vitro by mixing blood containing mature gametocytes with a 3.7% solution of sodium citrate and exposing the mixture to air. Ookinetes of all lineages except Haemoproteus payevskyi (lineage hRW1) and Haemoproteus nucleocondensus (hGRW1) developed; the 2 latter species did not exflagellate. Between-lineage hybridization was initiated by mixing blood containing mature gametocytes of 2 different parasites; the following experiments were performed: (1) Haemoproteus pallidus (lineage hPFC1) × Haemoproteus minutus (lineage hTURDUS2); (2) H. pallidus (hPFC1) × Haemoproteus tartakovskyi (hSISKIN1); (3) Haemoproteus belopolskyi (hHIICT3) × Haemoproteus lanii (hRB1); (4) Haemoproteus balmorali (hSFC1) × H. pallidus (hPFC1); (5) H. belopolskyi (hHIICT1) × Haemoproteus parabelopolskyi (hSYBOR1); (6) H. tartakovskyi (hHAWF1) × H. tartakovskyi (hSISKIN1); (7) H. pallidus (hPFC1) × H. lanii (hRB1); (8) H. tartakovskyi (hHAWF1) × H. parabelopolskyi (hSYBOR1). We report 4 patterns of between-lineage interactions that seem to be common and might prevent mixing lineages during simultaneous sexual process in wildlife: (1) the blockage of ookinete development of both parasites; (2) the development of ookinetes of 1 parasite and blockage of ookinete development of the other; (3) selective within-lineage mating resulting in ookinete development of both parent species and absence of hybrid organisms; (4) absence of selective within-lineage mating resulting in presence of ookinetes of both parents and also development of hybrid organisms with unclear potential for further sporogony. The present study indicates directions for collection of source material in the investigation of mechanisms of reproductive isolation leading to speciation in these parasites. The next steps in these studies should be the development of nuclear markers for distinguishing hemosporidian hybrid organisms and the experimental observation of further development of hybrid ookinetes in vectors.
Recent in vitro experimental studies reported the complex patterns of haemosporidian (Haemosporid... more Recent in vitro experimental studies reported the complex patterns of haemosporidian (Haemosporida) between-lineage interactions, which prevent mixing of lineages during simultaneous sexual process. Numerous anomalous ookinetes have been observed; these are not involved in sporogony. Massive development of such ookinetes might influence parasite transmission but is insufficiently investigated. The simultaneous sexual process of several lineages is a common phenomenon in vectors due to high prevalence of haemosporidian co-infections in wildlife. It remains unclear if the number of anomalous ookinetes changes during dual-infection sporogony in comparison with the single-infection process. We calculated proportions of the anomalous and normal ookinetes, which developed during single-infection (control) and dual-infection experiments in vitro conditions. Three mitochondrial cytochrome b lineages belonging to three Haemoproteus spp. (Haemosporida, Haemoproteidae) were isolated from naturally infected passerine birds. Sexual process and ookinete development were initiated in vitro by mixing blood containing mature gametocytes of two different parasites; the following experiments were performed: (1) Haemoproteus tartakovskyi (lineage hSISKIN1) × Haemoproteus lanii (lineage hRBS4) and (2) Haemoproteus belopolskyi (hHIICT3) × H. lanii (hRBS4). Genetic difference between lineages was 5.0-5.9%. Normal and anomalous ookinetes developed in all control and dual-infection experiments. The number of anomalous ookinetes markedly decreased, and normal ookinetes increased in all dual-infection experiments in comparison with those in controls, except for H. belopolskyi, in which proportion of the anomalous and normal ookinetes did not change. This study shows that simultaneous sexual process of two genetically distant lineages of haemosporidian parasites might increase the efficiency of reproductive cells, resulting in the development of a greater number of normal ookinetes. The marked increase of the number of normal ookinetes, which is involved in sporogony, indicates the success of sporogony in dual infections. Some haemosporidian lineages might benefit from simultaneous sporogony. Widespread avian Haemoproteus spp. are convenient and laboratory-friendly organisms for in vitro experimental research addressing between-lineage interaction in parasites during the sexual process.
Mixed infections of different species and genetic lineages of haemosporidian parasites (Haemospor... more Mixed infections of different species and genetic lineages of haemosporidian parasites (Haemosporida) predominate in wildlife, and such infections are particularly virulent. However, currently used polymerase chain reaction (PCR)-based detection methods often do not read mixed infections. Sensitivity of different PCR assays in detection of mixed infections has been insufficiently tested, but this knowledge is essential in studies addressing parasite diversity in wildlife. Here, we applied five different PCR assays, which are broadly used in wildlife avian haemosporidian research, and compared their sensitivity in detection of experimentally designed mixed infections of Haemoproteus and Plasmodium parasites. Three of these PCR assays use primer sets that amplify fragments of cytochrome b gene (cyt b), one of cytochrome oxidase subunit I (COI) gene, and one target apicoplast genome. We collected blood from wild-caught birds and, using microscopic and PCR-based methods applied in parallel, identified single infections of ten haemosporidian species with similar parasitemia. Then, we prepared 15 experimental mixes of different haemosporidian parasites, which often are present simultaneously in wild birds. Similar concentration of total DNA was used in each parasite lineage during preparation of mixes. Positive amplifications were sequenced, and the presence of mixed infections was reported by visualising double-base calling in sequence electropherograms. This study shows that the use of each single PCR assay markedly underestimates biodiversity of haemosporidian parasites. The application of at least 3 PCR assays in parallel detected the majority, but still not all lineages present in mixed infections. We determined preferences of different primers in detection of parasites belonging to different genera of haemosporidians during mixed infections.
ABSTRACT Up to the year 2000 there have been known 118 species of biting midges in Lithuania. It ... more ABSTRACT Up to the year 2000 there have been known 118 species of biting midges in Lithuania. It should be pointed out, however, that the latest investigations of the fauna of biting midges in Lithuania were performed by H. Remm in 1957–1962. The article presents the data on biting midges collected and defined in 1997–2002. As a result of the studies performed there were 67 species of biting midges identified, 13 new for the Lithuanian fauna among them. Information on the locality and time of midge sampling is provided therein, too.
Outbreaks of African Swine Fever (ASF) in domestic pig farms in Lithuania typically begin in June... more Outbreaks of African Swine Fever (ASF) in domestic pig farms in Lithuania typically begin in June and are detected through October, suggesting that insects might be involved in the transmission of the virus. Entomological collecting was performed to obtain two data sets: from farms with ASF outbreaks, and from farms without ASF outbreaks but in an ASF‐infected area. Target insects from the families Muscidae, Calliphoridae and Tabanidae were analysed for the presence of ASF Virus (ASFV) DNA. Musca domestica L., Calliphoridae flies and Stomoxys calcitrans (L.) collected by entomological net during ASF outbreaks were confirmed to be ASFV positive. Viral DNA detected in insects collected by Nzi traps from farms with no ASFV outbreaks indicate that Culex, Lucilia, M. domestica and S. calcitrans are likely to play a role in spreading the ASFV mechanically. This finding could suggest contamination from outside of the farms: from infected wild boar or their carcasses. The role of Stomoxys f...
Numerous recent studies have addressed the molecular characterization, distribution and genetic d... more Numerous recent studies have addressed the molecular characterization, distribution and genetic diversity of Haemoproteus spp. (Haemoproteidae). Some species of these blood parasites cause severe disease in birds, and heavy infections are often lethal in biting midges (Ceratopogonidae) and other blood-sucking insects. However, information about the vectors of haemoproteids is scarce. This presents an obstacle for better understanding the mechanisms of host-parasite interactions and the epidemiology of haemoproteosis. Here we investigated the sporogonic development of Haemoproteus tartakovskyi, a widespread bird parasite, in experimentally infected biting midges, Culicoides nubeculosus. These biting midges are widespread in the Europe. The insects were cultivated under laboratory conditions. Unfed females were allowed to take blood meals on wild caught siskins Carduelis spinus naturally infected with H. tartakovskyi (lineage hSISKIN1). Engorged females were maintained at 22-23 °C, di...
Haemoproteus parasites are widespread, and several species cause diseases both in birds and blood... more Haemoproteus parasites are widespread, and several species cause diseases both in birds and blood-sucking insects. These pathogens are transmitted by dipterans belonging to the Ceratopogonidae and Hippoboscidae, however certain vector species remain unknown for the majority of Haemoproteus spp. Owls are often infected by Haemoproteus parasites, but experimental studies on vectors of these infections are lacking. The aim of this study was to investigate sporogonic development of two widespread Haemoproteus parasites of owls, H. noctuae and H. syrnii in experimentally infected biting midges Culicoides impunctatus and Culicoides nubeculosus. We also followed in vitro sporogonic development of these infections and determined their phylogenetic relationships with Haemoproteus spp., for which vectors have been identified. Wild-caught C. impunctatus and laboratory reared C. nubeculosus were infected experimentally by allowing them to take blood meals on one individual long-eared owl (Asio ...
BackgroundCulicoides biting midges (Diptera, Ceratopogonidae) are known to be vectors of avian Ha... more BackgroundCulicoides biting midges (Diptera, Ceratopogonidae) are known to be vectors of avian Haemoproteus parasites. These parasites cause disease and pathology in birds. The diversity of biting midges in Europe is great, but only four Culicoides species are known to be vectors of avian Haemoproteus parasites. In general, our knowledge about the role of the particular Culicoides species in transmission of Haemoproteus parasites remains insufficient. Information gaps hinder a better understanding of parasite biology and the epizootiology of parasite-caused diseases. The aim of this study was to determine new ornithophilic Culicoides species potentially involved in local transmission of Haemoproteus parasites. To do this we collected biting midges in bird nest boxes, identified their species and prevalence of haemoproteids in insects as well as in juvenile birds during breeding season.Methods Biting midges were collected from bird nest boxes, identified and parous females were teste...
Black flies (Diptera: Simuliidae) are among the most bothersome blood-sucking dipterans causing s... more Black flies (Diptera: Simuliidae) are among the most bothersome blood-sucking dipterans causing severe irritation and distress to poultry, wild birds, animals, and humans globally. These insects are vectors of viruses, bacteria, parasitic protozoa and nematodes of humans and animals. Parasitic protozoa belonging to Haemosporida (Apicomplexa) are distributed worldwide and black flies are the principal vectors of avian haemosporidian parasites of the genus Leucocytozoon, a common parasite of birds. Based on the detection of parasite DNA in insects, 13 black fly species were reported to be potential vectors of Leucocytozoon in Europe. Information about which species of Simulium can play a role in the transmission of Leucocytozoon parasites is insufficient and needs to be developed. The aim of our study was to determine which black fly species are involved in the transmission of Leucocytozoon parasites in the Eastern Europe. The black fly females were collected in Lithuania using entomological net. They were morphologically identified, dissected to prepare salivary glands preparations, and then screened for the presence of Leucocytozoon parasites using microscopy and PCR-based methods. In all, we collected 437 black fly females belonging to eight species. The DNA of Leucocytozoon (genetic lineage lCOCO18) was detected in one of analysed females identified as Simulium maculatum. All salivary gland preparations were negative for the presence of Leucocytozoon sporozoites. Our results included S. maculatum as a potential vector of Leucocytozoon parasites. Increasing the knowledge on vector ecology, behaviour and improving collection methods may be the key to understand the evolution and diversity of these parasites.
Increasingly frequent outbreaks of zoonotic infections call for studies of wildlife parasites to ... more Increasingly frequent outbreaks of zoonotic infections call for studies of wildlife parasites to reach a better understanding of the mechanisms of host switch, leading to the evolution of new diseases. However, speciation processes have been insufficiently addressed in experimental parasitology studies, primarily due to difficulties in determining and measuring mate-recognition signals in parasites. We investigated patterns of sexual process and ookinete development in avian Haemoproteus (Parahaemoproteus) spp. (Haemosporida, Haemoproteidae) using in vitro experiments on between-lineage hybridization. Eleven mitochondrial cytochrome b (cyt b) lineages belonging to 9 species of hemoproteid were isolated from naturally infected passerine birds. The parasites were identified to species on the basis of morphology of their gametocytes and polymerase chain reaction amplification of segments of the cyt b gene. Sexual process and ookinete development were initiated in vitro by mixing blood containing mature gametocytes with a 3.7% solution of sodium citrate and exposing the mixture to air. Ookinetes of all lineages except Haemoproteus payevskyi (lineage hRW1) and Haemoproteus nucleocondensus (hGRW1) developed; the 2 latter species did not exflagellate. Between-lineage hybridization was initiated by mixing blood containing mature gametocytes of 2 different parasites; the following experiments were performed: (1) Haemoproteus pallidus (lineage hPFC1) × Haemoproteus minutus (lineage hTURDUS2); (2) H. pallidus (hPFC1) × Haemoproteus tartakovskyi (hSISKIN1); (3) Haemoproteus belopolskyi (hHIICT3) × Haemoproteus lanii (hRB1); (4) Haemoproteus balmorali (hSFC1) × H. pallidus (hPFC1); (5) H. belopolskyi (hHIICT1) × Haemoproteus parabelopolskyi (hSYBOR1); (6) H. tartakovskyi (hHAWF1) × H. tartakovskyi (hSISKIN1); (7) H. pallidus (hPFC1) × H. lanii (hRB1); (8) H. tartakovskyi (hHAWF1) × H. parabelopolskyi (hSYBOR1). We report 4 patterns of between-lineage interactions that seem to be common and might prevent mixing lineages during simultaneous sexual process in wildlife: (1) the blockage of ookinete development of both parasites; (2) the development of ookinetes of 1 parasite and blockage of ookinete development of the other; (3) selective within-lineage mating resulting in ookinete development of both parent species and absence of hybrid organisms; (4) absence of selective within-lineage mating resulting in presence of ookinetes of both parents and also development of hybrid organisms with unclear potential for further sporogony. The present study indicates directions for collection of source material in the investigation of mechanisms of reproductive isolation leading to speciation in these parasites. The next steps in these studies should be the development of nuclear markers for distinguishing hemosporidian hybrid organisms and the experimental observation of further development of hybrid ookinetes in vectors.
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