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.
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.
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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