First data on the genetic diversity of avian haemosporidians in China: cytochrome b lineages of the genera Plasmodium and Haemoproteus (Haemosporida) from Gansu Province

A total of 76 birds belonging to 23 species and 14 families was examined for the presence of Plasmodium spp. and Haemoproteus spp. Birds were trapped at four localities in Gansu Province, China, in June–July 2011. DNA was isolated from blood samples and parasite detection, and identification was based on PCR assays and sequences of 479 bp of cyt b gene. The total prevalence of haemosporidians was 21.0 %. Haemoproteus spp. were detected in 14 birds (prevalence 18.4 %). The lineage CYAPIC1 from Cyanopica cyanus, Parus major, Passer montanus and Pyrrhocorax pyrrhocorax was new; it is genetically distinct and probably represents a new species of the genus Haemoproteus. Three lineages represented known species: RBS4 (from Lanius tephronotus), a lineage of Haemoproteus lanii; COLL2 (from Turdus mupinensis), a lineage of Haemoproteus pallidus and TURDUS2 (from Turdus rubrocanus), a lineage of Haemoproteus minutus. The lineage RBS5 (from Lanius cristatus and L. tephronotus) differs by 1.4 % from RBS4 and probably represents an intraspecific entity of H. lanii. The lineages TUCHR1 (recorded from T. mupinensis), WW1 (recorded from Upupa epops) and YWT2 (recorded from Motacilla flava) have not been linked to any known species for the moment. Only one bird was positive for Plasmodium (prevalence 1.4 %), i.e. P. major infected with the lineage GRW4 of Plasmodium relictum. The latter lineage has been considered by previous studies as typical for migratory birds and having transmission in tropical areas only; its record in a sedentary bird in China suggests its transmission in temperate latitudes.     

Genetic diversity of avian blood parasites in SE Europe: Cytochrome <Emphasis Type="Italic">b</Emphasis> lineages of the genera <Emphasis Type="Italic">Plasmodium</Emphasis> and <Emphasis Type="Italic">Haemoproteus</Emphasis> (Haemosporida) from Bulgaria

We used a nested PCR protocol to examine the genetic diversity of cytochrome b (cyt b) lineages from blood parasites of the genera Plasmodium and Haemoproteus in birds in Bulgaria. In total, 460 birds of 43 species and 14 families (mostly passerines) were examined for the presence of infections. Of them, 267 were recognised as infected with haemosporidian parasites. Mixed infections were recorded in 24 individuals (9%). Besides the 24 individuals with mix infections, 114 (43%) were positive for Plasmodium spp. and 129 (48%) for Haemoproteus spp. We identified 52 genetic lineages of haemosporidian parasites: 38 of Haemoproteus and 14 of Plasmodium. Twelve new cyt b lineages of Haemoproteus were recorded; they occurred in the following hosts: grey-faced woodpecker (Picus canus), golden oriole (Oriolus oriolus), jay (Garrulus glandarius), barred warbler (Sylvia nisoria), song thrush (Turdus philomelos), spotted flycatcher (Muscicapa striata), spanish sparrow (Passer hispaniolensis), hawfinch (Coccothraustes coccothraustes), and cirl bunting (Emberiza cirlus). We also detected 22 new host records for previously known lineages. The most common lineage was SGS1 (Plasmodium relictum), which had a total prevalence of 14% and occurred in 8 host species belonging to 5 families. Three of the cyt b lineages of genus Haemoproteus (DURB1, DURB2 and SYNIS2) showed more than 5% divergence from all described morphologically lineages. These lineages probably represent at least 2 different morphospecies which remains to be identified.     

Evaluation of hematological values in indigenous chickens infected with <Emphasis Type="Italic">Plasmodium gallinaceum</Emphasis> and <Emphasis Type="Italic">Aegyptianella pullorum</Emphasis>

Plasmodium, Haemoproteus, and leukocytozoon are the most important hematozoa in birds, which have been reported in different areas of the world. The present study was undertaken to find which blood protozoans exist in indigenous chickens in Shiraz, southern Iran and to evaluate hematological parameters in birds infected with hematozoas. Plasmodium and Aegyptianella were the two parasites found in 740 blood samples examined from indigenous chickens of which 29 (3.91%) were positive for Aegyptianella pullorum, 106 (14.32%) for Plasmodium gallinaceum, and 12 (1.62%) for A. pullorum and Plasmodium gallinaceum together. There was no significant difference between hematological parameters of non-infected and naturally infected chickens with Plasmodium gallinaceum, A. pullorum, and both (P > 0.05). Low infection of indigenous chickens with A. pullorum, Plasmodium gallinaceum, and both had no significant effects on hematological parameters (P > 0.05), which is probably due to low parasitemia rate and immunity against these two parasites.     

Avian haemosporidians in haematophagous insects in the Czech Republic

The degree to which avian haemosporidian parasites can exploit different vectors as a definitive host has ecological implications for their transmission and biogeography. Studies targeting haemosporidian parasites using precise molecular detection methods are almost lacking in Central Europe, however. Here, we utilized PCR-based molecular methods to detect avian haemosporidians in insect vectors in the Czech Republic. Nine lineages of parasites belonging to three genera, Haemoproteus, Plasmodium, and Leucocytozoon, were detected in pooled samples of insect individuals, of which three lineages had not yet been discovered in previous studies. All three Leucocytozoon lineages were found exclusively in black flies, while five Haemoproteus lineages were found in biting midges. The most abundant insect species Culicoides kibunensis harbored three Haemoproteus lineages, and the second-most numerous species Culicoides segnis even four. The positive mosquitoes of Culex pipiens complex hosted two parasite lineages, one Plasmodium and one Haemoproteus, the latter of which, however, could suggest the aberrant development of this parasite in an unusual invertebrate host. The co-occurrence of Haemoproteus ROFI1 and TURDUS2 lineages in both insects and birds at the same study plot suggests a transmission of these lineages during breeding season of birds.     

Description and molecular characterization of Haemoproteus macrovacuolatus n. sp. (Haemosporida,Haemoproteidae), a morphologically unique blood parasite of black-bellied whistling duck (Dendrocygna autumnalis) from South America

During a surveillance programme on avian influenza in wild birds in the east of Colombia, 42 % of examined wild black-bellied whistling ducks (Dendrocygna autumnalis) were infected with undescribed Haemoproteus sp., which macrogametocytes possess one or several huge (2.5 μm in largest diameter) conspicuous roundish vacuoles, a unique character of avian haemoproteids. This parasite is named Haemoproteus (Parahaemoproteus) macrovacuolatus and described here using data on the morphology of its gametocytes, host cells and sequences of the complete mitochondrial genome and cytochrome b fragments. Illustrations of blood stages of the new species and DNA sequence information are provided. The phylogenetic analysis identified a closely related lineage C033, reported in South Asian ducks belonging to Dendrocygna. We also found that all Haemoproteus lineages from Passeriformes conformed a monophyletic group. Whereas we cannot exclude that this pattern could be an artefact of the limited taxonomic sampling in non-passeriform birds, thus this finding is worthy of attention. This study adds to our knowledge of the phylogenetic relationships among species of avian haemoproteids and describes a new haemoparasite in a non-passerine host.     

Haemosporidian parasites of a European passerine wintering in South Asia: diversity, mixed infections and effect on host condition

We studied haemosporidian parasites in the scarlet rosefinch Carpodacus erythrinus in a small isolated semicolony during an eight-year period using molecular methods of parasite detection. The scarlet rosefinch is an interesting model of parasite host species. It winters in South Asia which represents a rare exception among European passerines. Males express yellow to red carotenoid-based plumage ornament which is a good predictor of male reproductive success. In 240 blood samples originating from 199 adult individuals, the total parasite prevalence reached 60 %. Prevalence varied among years from 36 to 81 % in Haemoproteus, 8 to 22 % in Plasmodium, and 0 to 14 % in Leucocytozoon. Twenty parasite lineages were detected (Haemoproteus: 5 lineages, Plasmodium: 10 lineages, and Leucocytozoon: 5 lineages). Among them, the Haemoproteus ROFI2 lineage, which is a host-specific parasite lineage of the scarlet rosefinch, was the most frequently found. Parasite lineages showed varying degree of lineage specificity. While Haemoproteus lineages detected in the scarlet rosefinch have relatively narrow host breadth restricted mainly to Fringillidae family, Leucocytozoon and Plasmodium lineages generally showed wider host range. The presence of some parasite lineages hitherto detected in sedentary European passerines (SISKIN1, CCF3, BT2) or in Culicoides biting midges at the same locality (ROFI1) suggest local transmission. On the contrary, lineages LK05 and FANTAIL1 that were previously reported exclusively from Asian hosts imply parasite transmission at the scarlet rosefinch wintering sites in South Asia. Mixed infections were found in 17 % of infected samples and comprised mainly the most frequent lineages. The pattern of concomitant infections seemed to be rather random and matched expected levels based on lineage frequencies. Between-year comparisons revealed that in a majority of the repeatedly captured individual hosts the infection status remained unchanged (individuals stayed uninfected or possessed the same parasite lineages). However, 16 gains and 8 losses of lineages were also reported. We have not found any effect of haemosporidians on male carotenoid ornament expression or host body mass.     

Occurrence of haemosporidian parasites in the paddyfield warbler, <Emphasis Type="Italic">Acrocephalus agricola</Emphasis> (Passeriformes,Sylviidae)

The blood parasite diversity was studied in paddyfield warblers (Acrocephalus agricola) breeding in NE Bulgaria, SW Russia and S. Kazakhstan. Nine cytochrome b gene lineages were recorded, 4 belonging to Haemoproteus spp. and 5 to Plasmodium spp. The overall prevalence of haemosporidians was 33.3%. The composition of parasites varied geographically, with six lineages recorded in Russia, five lineages in Bulgaria and two lineages in Kazakhstan. Two lineages are described for the first time, i.e. ACAGR1 (belonging to Plasmodium sp. and recorded from a single bird in Russia) and ACAGR2 (belonging to Haemoproteus sp., recorded from Bulgaria and Russia). The latter lineage is the most widespread parasite in the Bulgarian population, scarce in Russia and absent in Kazakhstan. It is supposed that ACAGR2 has originated from the widespread lineage ACDUM1 differing from it by a single nucleotide. One lineage only (ACDUM2) occurs in all the three populations studied and is a nonspecific parasite known from various passerines. Six of the registered lineages have been found in a single population of A. agricola and also represent non-specific parasites occurring in a wide range of passerine birds. Their records in A. agricola may indicate the high transmission rate of these parasites in the habitats where this host co-occurs with other passerines. The variation of the composition of the haemosporidian parasite communities through the breeding range of A. agricola makes up heterogeneous selection pressures that may drive intraspecific variation in important life-history traits.     

Genetic characterization of avian malaria (Protozoa) in the endangered lesser kestrel, <Emphasis Type="Italic">Falco naumanni</Emphasis>

We genetically analyzed avian malaria (Protozoa) isolated from lesser kestrels (Falco naumanni) breeding in La Mancha, Central Spain. A total of 586 adult individuals were screened for blood parasites using a very efficient polymerase chain reaction approach that amplifies a partial segment (498 bp) of the cytochrome b gene of avian malaria of the genera Haemoproteus and Plasmodium. The prevalence of Plasmodium was 8.2%, and the prevalence of Haemoproteus was 4.1%. Sequence analyses revealed six unique lineages of avian malaria, three Plasmodium (LK5, LK6, RTSR1) and three Haemoproteus (LK2, LK3, LK4). According to sequence divergence, these lineages seem to correspond to at least three different species, although all recovered lineages could be independent evolutionary units. The third most common lineage (RTSR1) has been previously retrieved from two other avian host species, including a resident African bird species and a trans-Saharan migrant passerine, suggesting that lesser kestrels could acquire this Plasmodium lineage at their winter quarters in Africa.     

Hidden haemosporidian infections in Ruffs (Philomachus pugnax) staging in Northwest Europe en route from Africa to Arctic Europe

In their African freshwater wintering habitats, shorebirds show a high prevalence of blood parasites, whereas no parasites are detected elsewhere along the migration route. We looked at two genera of haemosporidian parasites, Haemoproteus and Plasmodium, in the long-distance migrating Ruff (Philomachus pugnax) along a geographical/seasonal gradient to verify the infection pattern and examine possible hidden organ infections at European staging areas. We amplified parasite DNA from blood of 53 healthy birds wintering in Mali, 53 samples of seven organ tissues (spleen, liver, kidneys, heart, lungs, brain, and pectoral muscle) from healthy individuals caught during spring migration, and 18 weak birds found sick in summer in The Netherlands. We confirm that Ruffs wintering in Africa carried blood infections and that some infections developed into hidden organ infections during spring migration. Moreover, sick birds either had new infections (in one juvenile) or relapses (in an adult harboring an African lineage). Our results suggest that some parasites develop latency. This strategy may be beneficial for the parasite as it may take control over reappearance in the blood to help further transmission.     

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.     

Abortive long-lasting sporogony of two Haemoproteus species (Haemosporida, Haemoproteidae) in the mosquito Ochlerotatus cantans, with perspectives on haemosporidian vector research

Haemoproteus spp. are cosmopolitan vector-born haemosporidian parasites, some species of which cause diseases in non-adapted birds. Recent polymerase chain reaction (PCR)-based studies have detected mitochondrial cytochrome b gene lineages of these Haemoproteus parasites in blood-sucking mosquitoes and speculated about possible involvement of these insects in transmission of avian haemoproteids. However, development of Haemoproteus lineages has not been documented in mosquitoes. We infected 304 individuals of Ochlerotatus cantans, a widespread Eurasian mosquito, with Haemoproteus tartakovskyi (lineage hSISKIN1) and Haemoproteus balmorali (lineage hROBIN1). Mosquitoes were allowed to take non-infected and infected blood meals and maintained in the laboratory until 17 days post-infection (dpi). They were tested for presence of sporogonic stages by microscopic and PCR-based methods. Microscopic examination revealed partial development of both parasites in the infected insects. Numerous ookinetes were seen in the gut area and adjacent tissues located in the head, thorax and abdomen of mosquitoes between 1 and 5 dpi. Numerous oocysts were seen in the midgut wall between 4 and 15 dpi; they were also present in the head and thorax of infected mosquitoes testifying to the active movement of ookinetes throughout the body. Oocysts degenerated between 11 and 17 dpi. Sporozoites were not seen in oocysts or mosquito salivary glands, indicating abortive sporogonic development at the oocyst stage. In accordance with microscopy data, PCR and sequencing revealed presence of the lineages hSISKIN1 and hROBIN1 in experimental mosquitoes as long as 15 and 17 dpi, respectively, demonstrating relatively long survival of Haemoproteus parasites in the resistant insects without DNA degeneration. The present study shows that PCR-based diagnostics should be carefully used in vector studies of haemosporidians because it detects parasites in insects for several weeks after initial infection, but does not distinguish abortive parasite development. Demonstration of infective sporozoites in insects is essential for definitively demonstrating the insects are vectors.     

Molecular characterization of avian malaria parasites in three Mediterranean blue tit (Cyanistes caeruleus) populations

We genetically analysed malaria parasites (Protozoa) in three Mediterranean blue tit (Cyanistes caeruleus) populations from central Spain. A total of 853 breeding individuals were screened for parasites of the genera Plasmodium and Haemoproteus using a very efficient polymerase chain reaction approach that amplifies a partial segment of the mitochondrial cytochrome b gene of these parasites. We have found six lineages of Plasmodium (SGS1, GRW11, COLL1, DELURB4, GRW04 and BLUTI10) parasitizing the studied populations but we did not detect any infection by Haemoproteus. One of the detected lineages (BLUTI10) has not been previously described in any bird species and this is the first study recording lineages DELURB4 and GRW04 in blue tits. SGS1 (belonging to the morphospecies Plasmodium relictum) was the most frequent lineage (overall prevalence, 24?%), whereas the other lineages showed a much lower prevalence (<4?%). Only a small proportion (12.2?%) of positive amplifications of the most common lineage (SGS1) was detected in blood smears using light microscopy and infection intensities were very low (mean?±?SE, 2.0?±?1.4 parasites/2,000 erythrocytes). We have also found strong inter-population variability in prevalence patterns (12–41?% for lineage SGS1), suggesting important differences in parasite transmission rates among the geographically close studied localities.     

Low prevalence of haemosporidian parasites in the introduced house sparrow (<Emphasis Type="Italic">Passer domesticus</Emphasis>) in Brazil

Species that are introduced to novel environments can lose their native pathogens and parasites during the process of introduction. The escape from the negative effects associated with these natural enemies is commonly employed as an explanation for the success and expansion of invasive species, which is termed the enemy release hypothesis (ERH). In this study, nested PCR techniques and microscopy were used to determine the prevalence and intensity (respectively) of Plasmodium spp. and Haemoproteus spp. in introduced house sparrows and native urban birds of central Brazil. Generalized linear mixed models were fitted by Laplace approximation considering a binomial error distribution and logit link function. Location and species were considered as random effects and species categorization (native or non-indigenous) as fixed effects. We found that native birds from Brazil presented significantly higher parasite prevalence in accordance with the ERH. We also compared our data with the literature, and found that house sparrows native to Europe exhibited significantly higher parasite prevalence than introduced house sparrows from Brazil, which also supports the ERH. Therefore, it is possible that house sparrows from Brazil might have experienced a parasitic release during the process of introduction, which might also be related to a demographic release (e.g. release from the negative effects of parasites on host population dynamics).     

Factors affecting the relapse of Haemoproteus belopolskyi infections and the parasitaemia of Trypanosoma spp. in a naturally infected European songbird, the blackcap, Sylvia atricapilla

The effects of photoperiod and stress on the relapse of Haemoproteus belopolskyi infections and the parasitaemia of Trypanosoma spp. were studied in naturally infected blackcaps, Sylvia atricapilla. Twenty-one birds (12 infected with H. belopolskyi, 5 with Trypanosoma spp.) were kept indoors and investigated by microscopic examination of stained blood films. All infections were latent before the experiments started on 7 February 2002. The relapse of H. belopolskyi and the parasitaemia of Trypanosoma spp. after the latent stage of infection were induced by increased day length and stress due to a change of room. At 44 days after the beginning of the experiment, all latent infections were patent in the experimental birds, while no parasites were recorded in the control birds. The relapse of avian Haemoproteus spp. infection and of the parasitaemia of avian Trypanosoma spp. may be related to gonadal hormones, corticosterone and melatonin.     

The ecto-, gastro-intestinal and haemo-parasites of live pigeons (Columba livia) in Kampala,Uganda

In a study from October, 1996 to March, 1997, 34 pigeons in three different locations were studied for parasites. The majority of birds in Nakulabye appeared clinically sick, while those within Makerere University and Wandegeya were apparently healthy. Biometric data were taken for each bird and, during the process, ectoparasites were collected. Faecal and blood samples were obtained. The study of ectoparasites revealed that Pseudolynchia canariensis (the 'pigeon fly') was the most prevalent parasite (100%). The louse Columbicola columbae was next in prevalence (94.1%). It is postulated that the pigeon fly transports this parasite. Three lice of economic importance were found: Menopon gallinae, Menacanthus stramineus and Chelopistes meleagridis. Cestodes were the only helminths found, occurring in 23.5% of the birds. Identification of the cestodes was not possible. Haemoparasites were mainly of two genera, Haemoproteus and Plasmodium. Haemaproteus was the most prevalent (76.5%). This was possibly due to the high abundance of its vector, P. canariensis.     

New malaria parasites of the subgenus <Emphasis Type="Italic">Novyella</Emphasis> in African rainforest birds,with remarks on their high prevalence,classification and diagnostics

Blood samples from 655 passerine birds were collected in rainforests of Ghana and Cameroon and examined both by microscopy and polymerase chain reaction (PCR)-based techniques. The overall prevalence of Plasmodium spp. was 46.6%, as determined by combining the results of both these diagnostic methods. In comparison to PCR-based diagnostics, microscopic examination of blood films was more sensitive in determining simultaneous infection of Plasmodium spp., but both detection methods showed similar trends of prevalence of malaria parasites in the same study sites. Plasmodium (Novyella) lucens n. sp., Plasmodium (Novyella) multivacuolaris n. sp. and Plasmodium (Novyella) parahexamerium n. sp. were found in the olive sunbird Cyanomitra olivacea (Nectariniidae), yellow-whiskered greenbul Andropadus latirostris (Picnonotidae), and white-tailed alethe Alethe diademata (Turdidae), respectively. These parasites are described based on the morphology of their blood stages and a segment of the mitochondrial cytochrome b (cyt b) gene, which can be used for molecular identification and diagnosis of these species. Illustrations of blood stages of new species are given, and phylogenetic analysis identifies DNA lineages closely related to these parasites. Malaria parasites of the subgenus Novyella with small erythrocytic meronts clearly predominate in African passerines. It is probable that the development of such meronts is a characteristic feature of evolution of Plasmodium spp. in African rainforest birds. Subgeneric taxonomy of avian Plasmodium spp. is discussed based on the recent molecular phylogenies of these parasites. It is concluded that a multi-genome phylogeny is needed before revising the current subgeneric classification of Plasmodium. We supported a hypothesis by Hellgren, Križanauskienė, Valkiūnas, Bensch (J Parasitol 93:889–896, 2007), according to which, haemosporidian species with a genetic differentiation of over 5% in mitochondrial cyt b gene are expected to be morphologically differentiated. This study emphasises the importance of employing both PCR-based and microscopic methods in taxonomic, ecological and evolutionary investigations of avian haemosporidian parasites.     

In vitro development of Haemoproteus parasites: the efficiency of reproductive cells increase during simultaneous sexual process of different lineages

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.