Parasitology ResearchFounded as Zeitschrift für Parasitenkunde

The simultaneous occurrence of male and female gametocytes inside a single host blood cell has been suggested to enhance apicomplexan transmission [ double gametocyte infection (DGI) hypothesis ]. We did a bibliographic search and a direct screen of blood smears from wild birds and reptiles to answer, for the first time, how common are these infections in the wild. Taking these two approaches together, we report here cases of DGIs in Plasmodium, Haemoproteus, Leucocytozoon and Hepatozoon, and cases of male–female DGIs in Haemoproteus of birds and reptiles and in Leucocytozoon of birds. Thus, we suggest that DGIs and male–female DGIs are more widespread than previously thought, opening a new research avenue on apicomplexan transmission. In Plasmodium and related apicomplexan parasites, the occurrence of two or more gametocytes inside a single host blood cell recently attracted the attention of researchers for their potential to enhance parasite transmission between vertebrate hosts (Jovani 2002). In these microparasites, transmission occurs when the gametocytes contained in the host blood cells are ingested within the bloodmeal of a suitable vector. Once in the vector, male and female gametes differentiate from male and female gametocytes, respectively, and at least one of the mobile male gametes must search for and fertilize one of the immobile female gametes contained in the same bloodmeal. Gametocytes are normally alone within single host blood cells (e.g. erythrocytes), but they are also found in couples (double gametocyte infections, DGIs) or in trios (TGIs) within a given blood cell. Female gametes are very small compared with the bloodmeal volume ingested by the vector; and male gametes do not seem to have any searching mechanism to find the female gamete (Gaillarg et al. 2003). Thus, it seems logical that some mechanism operating while in the vertebrate host could have evolved to increase the proximity of gametocytes and thus gametes when on the bloodmeal; and two non-mutually exclusive hypotheses have been proposed. Jovani (2002) proposed that male–female DGIs (i.e. one blood cell infected by one male and one female gametocyte) might reduce the time needed for male gametes to encounter female gametes, facilitating parasite transmission between vertebrate hosts. Gaillard et al. (2003) recently proposed that erythrocytes infected by gametocytes could adhere among them in peripheral host capillaries, enhancing the probability of gamete encounter once in the vector bloodmeal. The hypothesis proposed by Gaillard et al. (2003) agrees with the discovered pattern of aggregated distribution of erythrocytes infected by malaria gametocytes, both in human blood (Gaillard et al. 2003) and in the vector bloodmeal (Pichon et al. 2000). The DGI hypothesis relies on the assumption that DGIs, and especially male–female DGIs, are widespread in nature, but this is currently unconfirmed (Jovani 2002). The goal of this note is to show that DGIs are widespread among in vivo apicomplexan parasites of wild populations of birds and reptiles. Moreover, we also report the occurrence of other multiple gametocyte infections (MGIs, i.e. more than one gametocyte inside a single erythrocyte), because they inform us that the needed process to produce a DGI does occur in a given host–parasite system, i.e. the entrance and growth into a given erythrocyte of more than one gametocyte. We first systematically searched in the parasite literature for cases of MGIs. Information regarding this kind of infection is little likely to appear in the title, abstract, or keywords of a publication. Consequently, we were forced to search our files for papers on apicomplexan infections; and then we accurately revised each paper for any mention of MGIs. Our survey found any reference on the possible relevance of MGIs for any aspect of the biology of the parasite. Indeed, some papers even contained photographs of DGIs, but without any mention in the text (e.g. Mushi et al. 1999). Despite this evident lack of attention of researchers for DGIs and MGIs as a whole, we found eight works with information on DGIs and TGIs (Table 1). Table 1 Some examples of published data on the occurrence of multiple gametocyte infections in apicomplexan parasites. DGI Double gametocyte infection Host Parasite Observation Reference Haemoproteus columbae DGIs in 17 of 30 host individuals infected, with 14–97 DGIs in each individual host Graczyk et al. (1994) H. columbae A photograph of a male–female DGI Bennett and Peirce (1990) H. columbae A photograph of a male–female DGI, a photograph of a TGI Mushi et al. (1999) Columba livia Haemoproteus spp A photograph of a female–female DGI Tsai et al. (1986) Geochelone denticulate H. geochelonis Drawings and photographs of male– female DGIs; text: quite common occurrence of DGIs Lainson and Naiff (1998) Birds Haemoproteus, Plasmodium, Leucocytozoon spp Drawings of: immature DGIs from 20 Haemoproteus spp, one immature TGI in H. kairullaevi, one male–female DGI in H. bennetti, one male–male DGI in H. ortalidum, two immature DGIs in P. octamerium and P. columbae, one immature DGI and one male–female DGI in L. caulleryi Valkiunas (1997) Caiman c. crocodiles Hepatozoon caimani Photographs of DGIs and TGIs, sex unknown Lainson et al. (2003) Homalopsis buccata Hepatozoon spp Photograph of a DGI, sex unknown Salakij et al. (2002) Moreover, from our own blood smear collection, we selected those blood samples of 11 bird and four reptile species infected by some apicomplexan parasites. These blood smears were collected from wild hosts in Germany and Spain during the period 1996–2002 (see Table 1). On each smear, we actively searched for MGIs on a portion equivalent to ca. 2,000 erythrocytes for Haemoproteus and Hepatozoon infections and ca. 25,000 erythrocytes for Leucocytozoon infections, recording when possible the sex for mature gametocytes involved in a MGI. MGIs were found in most of the parasite–host associations we examined (Table 2). Table 2 Occurrence of DGIs, male–female (m-f)DGIs, and triple gametocyte infections (TGIs) in blood smears inspected for this study. N Number of infected individual hosts inspected, or number of individual hosts with at least one D/TGI. Numbers in parentheses indicate the number of D/TGIs in each infected host. – Sex of parasite could not be determined Host Country N N DGIs N m-f DGIs N TGIs Haemoproteus parasites Buteo buteo Germany 8 3 (1, 1, 2) 0 0 Falco tinnunculus Germany 3 0 – 0 F. subbuteo Germany 7 0 – 0 Tyto alba Spain 4 1 (3) 0 0 Bubo bubo Spain 21 3 (1, 2, 2) 0 0 Strix aluco Germany, Spain 21 10 (1, 2, 2, 2, 3, 4, 10, 11, 13, 27) 1 (1) 4 (1, 1, 3, 15) Parus caeruleus Spain 46 10 (1, 1, 1, 1, 1, 2, 2, 3, 4, 6) 0 0 Delichon urbica Spain 57 7 (1, 1, 1, 1, 1, 1, 2) 1 (1) 0 Lanius collurio Spain 11 0 – 1 (1) Passer domesticus Spain 39 11 (1, 1, 1, 1, 2, 2, 2, 2, 2, 5, 6) 4 (1, 1, 1, 1) 0 Leucocytozoon parasites B. bubo Spain 19 0 – 0 Athene noctua Spain 2 0 – 0 Strix aluco Spain 5 0 – 0 Parus caeruleus Spain 15 2 (1, 2) – 0 Hepatozoon parasites P. caeruleus Spain 9 0 – 0 Lacerta lepida Spain 38 0 – 0 L. monticola Spain 105 5 (1, 1, 1, 2, 3) – 0 Podarcis muralis Spain 28 0 – 0 P. hispanica Spain 19 0 – 0 Moreover, from a parasite survey conducted by Shurulinkov and Golemansky (2002, 2003), P.S. revised their own notes taken during blood smear inspection in a search for records of MGIs. Although not comparable with the other samples because it was not a systematic search for MGIs, it is of qualitative importance and also denotes the low reporting of these infections in publications, although present in the researchers notes. A total of 15 DGIs were recorded from 11 birds infected by Haemoproteus of five bird–parasite assemblages (bird–parasite [number of birds with DGIs, total number of DGIs]: Sylvia borin–H. belopolskyi [1, 3], Lanius minor–H. lanii [2, 2], Lanius collurio–H. lanii [2, 2], Acrocephalus arundinaceus–H. payevskyi [2, 3], Muscicapa striata–H. pallidus [4, 5]). Most of these DGIs were constituted by female gametocytes. However, H. payevskyi infecting A. arundinaceus showed a case of a male–female DGI. Moreover, two cases of DGIs were recorded in Plasmodium polare infecting one individual of Hirundo rustica. See Shurulinkov and Golemansky (2002, 2003) for the total numbers of species and individuals examined. Our results support the view that DGIs are widespread in a variety of host– parasite associations, including Plasmodium, Haemoproteus, and Leucocytozoon infections of birds and Haemoproteus and Hepatozoon parasites of reptiles. Moreover, we found male–female DGIs in Haemoproteus parasites of birds and reptiles and in Leucocytozoon of birds, validating the major assumption of the DGI hypothesis . The widespread occurrence of DGIs, and particularly of malefemale DGIs, opens a promising direction for future research on the transmission strategies of apicomplexan parasites. Acknowledgements The authors thank Juan A. Fargallo, Jesús Martínez, Marilo Romero, Alberto Lizarraga, Francisco Campos, Santiago Merino, Juan Moreno, Judith Morales for their fieldwork and Javier Caldera for allowing the collection of blood smears at the Wildlife Recuperation Centre of Los Hornos . Grants and funds are as follows: R.J. (Telefónica Móbiles S.A.), L.A. (C.S.I.C.–Ventorrillo, BOS 2002–00598, BOS 2002–00547), E.A. (C.S.I.C.– Ventorrillo, 07 M/0137/2000), A.M. (Junta de Extremadura, FIC01A043), G.T. (Residencia de Estudiantes; FPI from Comunidad de Madrid, BOS2000–1125, BOS2001–0587). This study has been done according to the current laws of each country.