The mechanisms of parasite persistence in host populations are still poorly understood. In the present study, we studied blood parasites belonging to three genera in a breeding sparrowhawk population that has been followed in a long-term survey. Repeated sampling of adult individuals allowed us to follow persistence, incidence and age-related patterns of the infections.
The detected lineages of the L. toddi complex were previously reported from sparrowhawks; however, to our knowledge, we are the first to find lineages belonging to the H. elani complex in the sparrowhawk (for review see [8]). Phylogenetic analysis revealed that the detected Haemoproteus, together with lineages found previously and assigned morphologically by G. Valkiunas to the morphospecies H. elani de Mello, 1937 [32], might represent a distinct genus. Parasites belonging to this clade have previously been found in other accipitrid raptors. Krone et al. [15] assigned it to the genus Plasmodium, but its position was unsupported. Outlaw and Ricklefs [34] designated it as an “unknown genus,ˮ potentially being Plasmodium. The taxonomic position of parasites of the H. elani complex needs to be elucidated.
There are only few records of molecularly characterized trypanosomes infecting the genus Accipiter. In addition to strains originating from the sparrowhawk population studied here, T. avium s.s. was found in the Goshawk (Accipiter gentilis) and Japanese sparrowhawk (Accipiter gularis) [5, 19]. Barcoding of trypanosome isolates obtained in our study revealed that the vast majority of the birds harbored trypanosomes belonging to T. avium s.s. In buzzard populations sampled in Czechia, the spectrum of trypanosome lineages was similar, with only three out of 83 barcoded isolates belonging to T. corvi, while 96% belonged to T. avium s.s. (Svobodová and Kassahun, unpublished). We hypothesize that striking differences in the prevalence of different trypanosome lineages are not caused by raptor resistence to these lineages but rather by different exposure to vectors or by different transmission modes of the respective trypanosome species. Vectors differ in their height preferences, with black flies being found almost exclusively in the canopy level [35], thus facilitating transmission of T. avium to birds that perch or build their nests in the canopy. Moreover, all avian trypanosomes with life-cycles that have been elucidated to date are transmitted by vector ingestion; some of them may also use transconjuctival transmission via prediuresis of infectious stages (T. avium s.s., Trypanosoma thomasbancrofti [36, 37]). These species do not depend exclusively on vector ingestion and thus may enter potential hosts that are not willing to eat the infected vector.
The prevalence of trypanosomes based on their detection on blood smears is usually low in birds, including members of order Accipitriformes. Munoz et al. [38] did not find any infection in 22 sparrowhawks screened; however, Hanel et al. [39] found trypanosomes in nine out of 15 sampled goshawks. In our sample, we found trypomastigotes on 14 slides out of the 254 screened (5.5%), while using the culture method, 74% of adults were positive for trypanosomes [20]. Thus, cultivation was more sensitive than microscopy for trypanosome detection by an order of magnitude.
PCR diagnosis of raptor haemosporidians is also limited by a number of pitfalls. The most popular protocol used to detect haemosporidia has been developed for passerines, and there is evidence that this protocol is not optimal for the detection of raptor parasites [8, 24, 39]. In the present study, we used the DW primers designed by Perkins and Schall [23] for the detection of Leucocytozoon infections, but we developed a specific PCR protocol, including newly designed degenerate primers, for the detection of Haemoproteus infections.
Our previous study, based on blood culturing methods and blood smears, revealed a prevalence of 74% for Trypanosoma, 88% for Leucocytozoon and 30% for Haemoproteus [20]. The present study shows that sex of the sampled birds has a significant influence on the respective prevalence; the largest difference between males and females was the 31% pooled prevalence of Haemoproteus. This difference is substantial, and host sex should be taken into account in comparative studies in addition to host species and age.
In both females and males, the prevalence of trypanosomes and of Haemoproteus increased with age, although in females the increase was greater; Leucocytozoon prevalence remained high and stable in both sexes. A high prevalence of haemosporidians in the sparrowhawk has been found previously in Scotland, with 92% of adult females and 93% of adult males testing positive for Leucocytozoon; in that study the prevalence of Haemoproteus was lower and, similarly to our study, differed between females and males (32% vs 17%) [12]. Similar factors may drive prevalence patterns across different populations of the same species. On the contrary, no sex differences in the prevalence of Haemoproteus and Leucocytozoon infections were found in the Black Sparrowhawk (A. melanoleucus) in South Africa, but the exact age of the adults was not assessed in that study [40].
The higher prevalence of infections in females might result from increased exposure to parasite vectors at the nest and/or, in the case of the sparrowhawk, to a larger size, with females being larger than males, from a higher production of kairomones that attract vectors. The lower infection prevalence in males of the same age might also result from differences in ontogenetic development early in life. Males mature faster, become feathered earlier and leave nests 3–4 days earlier than their female counterparts (sisters) [41]. If a substantial part of the infections is acquired at the nest, these factors could also partly explain the higher prevalence of infections in females.
Ashford et al. [42] speculated that transmission of haemosporidia in a sparrowhawk population occurs almost exclusively during breeding, based on the observations that prevalence in adults is not higher than in nestlings and that besides breeding, the possibility of an infectious bite by an individual vector infected with a specific parasite is very low. This vector-mediated parent-to-offspring transmission was later confirmed in another common accipitrid species, the buzzard, and its Leucocytozoon parasite [43]. Parent-to-nestling transmission probably occurs in other avian apicomplexan parasites as well [44].
There is some evidence that blood parasites (Haemoproteus) cause selective avian mortality, leading to a lower prevalence in the older age classes [45]. In our case, the decrease in prevalence was only statistically significant for Haemoproteus, but only after including the two oldest individuals (two 9-year-old females) to the analysis. The maximal life span of a female sparrowhawk is around 10 years, and there is evidence for lower survival rates in the older age classes (7–10 years) [46]. It is possible that parasites influence survival in concordance with senescence, which leads to decreased immunocompetence [47, 48].
In our previous study, modeled Leucocytozoon prevalence at fledging was around 30% [20]. Since the prevalence in year-after-hatching adults exceeds 80%, most individuals must become infected with Leucocytozoon during their first year of life. This prepatent period (first nestling found positive for Leucocytozoon at the age of 17 days [20]) implies that the majority of the individuals are infected before postfledging dispersal. The predicted prevalence of trypanosomes at fledging was similar in both studies (present study and previous study [20]), but its increase was slower in our previous study, which again corresponds to lower trypanosome prevalence (56%) in the year-after-hatching birds in the present study.
We suggested previously that the unexpected lack of association between Leucocytozoon and trypanosome infections in adult sparrowhawks might be due to the occurrence of trypanosomes other than T. avium, which are not transmitted by black flies [20]. Another trypanosome species infecting raptors, T. bennetti, has recently been shown to be transmitted by biting midges [49]; however, barcoding of trypanosomes occurring in the studied sparrowhawk population revealed that the vast majority of the isolates (96%) belong to T. avium s.s., which is transmitted by black flies [36]. It should be noted that, based on indirect evidence, Ashford et al. [13] suggested that Leucocytozoon is transmitted to sparrowhawks by biting midges. Recent studies of avian blood parasite life-cycles that include transmission by vectors are scarce; for example, the wide range of avian trypanosome vectors has been revealed only recently (see [37]). In this context, to simply suppose that the distinct raptorial “Haemoproteusˮ lineage is transmitted by biting midges is perhaps not appropriate. The use of similar vectors might explain the positive association of Haemoproteus and Leucocytozoon infections. Nevertheless, if we follow the conservative presumption that the sparrowhawk Leucocytozoon is transmitted by black flies, then the lack of a significant association between Leucocytozoon and trypanosomes seems surprising. The proximate mechanism of transmission might influence the apparent discrepancy: Leucocytozoon is transmitted by a vector’s bite, inoculating sporozoites with saliva, while Trypanosoma is transmitted by ingestion of the vector (not probable in raptors) or via the conjuctiva through prediuresis, a process during which infective stages are expelled with prediuretic liquid while the vector feeds, as was recently demonstrated for Trypanosoma avium sensu lato [37]. Consumption of infected prey has been suggested as a mode of transmission for avian trypanosomes in sparrowhawks [50] and since the species is a specialist that feeds almost exclusively on small birds, this additional mode of trypanosome transmission should be considered as well.
At the generic level, Leucocytozoon had the highest incidence and persistence of infection. Most individuals acquire their Leucocytozoon infection early in life (see preceding text); thus, there is no effect of adult age on prevalence. The incidence of Trypanosoma was lower but its persistence was high. Since the incidence does not increase with age, increasing prevalence of trypanosomes with age is probably caused by the accumulation of chronic trypanosome infections. In one study, trypanosome infections caused by the same species (T. avium s.s.) were mostly lost in passerines (Geothlypis trichas) that were repeatedly sampled [29]. The detection of T. avium by culture methods is about twofold more sensitive than by PCR ([37] and Svobodová et al., unpublished), probably due to very low blood parasitemia. Consequently, chronic infections with lower parasitemia might remain undetected, leading to an underestimation of prevalence. Moreover, only seven parasite lineages out of 54 were barcoded in the passerine study; thus, the diversity of the parasites might remain undetected. Haemoproteus had the lowest incidence and persistence; nevertheless, Haemoproteus prevalence increased with age as well.
The persistence of infection detected at the parasite genus level may in fact hide more or less intensive lineage turnover due to reinfections, since lineages may change while the apparent infection status remains the same. This applies mostly for those parasites with the highest (almost saturated) prevalence (Leucocytozoon). Indeed, Leucocytozoon sp. lineage changed in one-third of the samples, and species status changed in two-thirds of samples. This result is in concordance with Leucocytozoon in the Great Tits (Parus major) where lineage turnover was also high; in that study, as many as 17 haplotypes were found in a single population [51]. On the other hand, we found that sparrowhawk individuals that were repeatedly sampled retained their Haemoproteus lineages (but the sample size is small).
A high turnover of Leucocytozoon lineage further supports the need for parasite barcoding to improve detailed monitoring of intraindividual lineage turnover. On the other hand, the vast majority of trypanosomes found in sparrowhawks belonged to a single T. avium lineage, probably not due to host specificity of trypanosomes that belong to other trypanosome lineages but instead due to constraints given by different vectors and transmission modes of those lineages (see preceding text).