Further Observations on In Vitro Hybridization of Hemosporidian Parasites: Patterns of Ookinete Development inHaemoproteusSpp.

Abstract

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.

Hybridization and pre-zygotic reproductive barriers inPlasmodium

“…We demonstrate that asymmetric reproductive interference occurs in Plasmodium, in which the self-fertilization success of P. berghei, but not P. yoelii , is reduced in the presence of heterospecifics ( figure 3 ). Similar results have been obtained by Paul et al [ 42 ] and Valkiunas et al [ 24 ] for avian Plasmodium and Haemoproteus , respectively. There are several mechanisms that could underpin this phenomenon, including (i) host immune factors produced in P. yoelii infections that act in the blood meal (e.g.…”

“…Therefore, our approach has identified proteins important for reproductive isolation, which is of particular relevance for taxa where hybridization is suspected (e.g. Haemoproteus [ 23 , 24 ]). We then examined the ecology of mixed-species mating groups and show that asymmetric reproductive interference occurs.…”

The buffy coat method: a tool for detection of blood parasites without staining procedures

“…Thirdly, BCM is not recommended to be used for diagnostics of Plasmodium and Leucocytozoon infections. BCM fails to detect Plasmodium infections mainly because malaria parasites do not exflagellate when the blood is simply exposed to air, as is the case in all tested Haemoproteus species [40,41]. Haemoproteus parasites develop readily visible moving stages (exflagellating microgametocytes and microgametes) in preparations prepared using BCM (Fig.…”

“…3i). The feature of Haemoproteus parasites to extensively exflagellate quickly (within 10 minutes after infected blood is exposed to air) has been used in haemosporidian genomic research [40,80] as well as gametogenesis and ookinete development studies [2,81,82], hybridization experiments [41] and vector studies [62,83]. Sixthly, BCM not only leads to a concentration of parasites in one layer but it also uses a larger volume of blood (around 10 times more) than ME, which makes it more sensitive to detect a low parasitaemia [59].…”

“…Quick detection of infected animals in the wild is essential for collection of parasite donor-hosts, which are often needed for helminthology [39], experimental parasitology [40,41], parasite identification [31,35,42] and host pathology [43,44] research. This is a particularly sensitive issue aimed at minimizing the harm to wildlife, when only a few parasitized individuals could be caged or euthanized if essentially necessary for collection of pathogens, and all other sampled animals can be quickly released according to permit requirements at study sites.…”