Unorthodox approaches can yield unexpected findings about host-vector-parasite interactions. In this case, we aimed to discover whether closely related raptor species attract different simuliid species which may transmit distinct parasite lineages. In the process, we used natural hosts as bait, combined with manual netting. This approach managed to collect new knowledge about the diet of some of the most common European blackfly species. While most studies typically consider only engorged blackflies, we managed to derive much information by analysing every available specimen for Leucocytozoon-lineages and combined it with existent knowledge on typical host groups.
Blackfly species and host preference
One of the main findings of our study was the determination of certain blackfly species foraging at great heights in the canopy and attracted to avian hosts. All but two out of the 154 identified individuals belonged to the subgenera Nevermannia and Eusimulium, and nearly 30% of the individuals of both subgenera had fed on birds, as revealed by their Leucocytozoon load. This finding indicates Nevermannia and Eusimulium as the dominant ornithophilic subgenera of the genus Simulium and as the most probable vectors of Leucocytozoon-lineages in Central Europe (c.f. ). This corresponds to the findings that other European species of Nevermannia attack thrushes and warblers, while blackflies of the S. (N.) vernum and S. (E.) aureum-groups are less distributed under 10 m in spruce and pine forests [8, 12, 26]. So far, feeding preferences of Nevermannia and Eusimulium species were known only from S. (N.) silvestre, S (N.) curvans, S. (E.) angustipes and S. (E.) aureum, the latter being one of the best examined ornithophilic blackflies and vectors of Leucocytozoon [10, 13, 14, 26, 27]. This species, however, appears to be rather rare in the upper canopy.
The composition of Leucocytozoon lineages found in this study shows that Nevermannia and Eusimulium species attack most avian host groups represented in this canopy layer, which are sufficiently large and/or abundant, such as thrushes, corvids, pigeons, raptors, owls and tits. A similar size-and-abundance pattern of prey was found among ornithophilic and mammalophilic blackflies close to ground level in Scandinavia . In contrast to the pattern found in Scandinavia, we did not find a strong association between blackfly species or haplotype and Leucocytozoon lineages, or their corresponding vertebrate host group . In the study by Hellgren et al. , a limited number of engorged S. (N.) silvestre suggested a preference for thrushes. However, a much larger sample of S. (N.) silvestre from North America harboured Leucocytozoon lineages across the avian phylogeny, and thus parasitizes a great variety of host groups . This pattern corresponds much better to our findings from Central Europe and supports the notion that species of Nevermannia and Eusimulium have habitat preferences but are otherwise indiscriminately ornithophilic (Fig. 1, Table 1). Our results deliver no information in which vector species the respective Leucocytozoon lineage can complete their development and life cycle. However, experimental evidence suggests that most parasites of the genus Leucocytozoon are rather restricted by the ecology of the vector than by its physiology .
The absence of non-Leucocytozoon parasites also grants insight into the behaviour of blackflies. Plasmodium and Haemoproteus are not transmitted and cannot fulfil their development in blackflies, but our protocol was apt to detect them, and their prevalence in the putative hosts is relatively high [14, 28]. Therefore, their complete absence indicates that blackflies swiftly leave the foraging habitat after feeding and remain distant and inactive until the blood meal and potential abortive stages of Plasmodium and Haemoproteus are digested . Individual blackflies possibly return after oviposition, as the period between two feedings has been measured to take 5-7 days in S. (Byssodon) rugglesi .
To our knowledge, this is the first study of haemosporidian parasites being present in individual non-engorged blackflies. We found that nearly 30% of the blackfly individuals active in the upper canopy layers of central Europe are Leucocytozoon-carriers. Blackflies are likely to return to a large stationary food source such as a raptor brood after oviposition [29, 30]. Nonetheless, 30% infected vectors is likely representative of the blackfly population in this habitat, since raptor lineages potentially belonging to our “bait” accounted for only 15 % of all infected blackflies. Previous studies have either analysed pools of non-engorged blackflies or individual visibly engorged blackflies [10, 12, 13, 27]. Engorged blackflies are very rare in forests (0.1-0.3% of all blackfly individuals), but can account for up to 23.6% in an alpine habitat [26, 31]. The frequency of Leucocytozoon-carriers among freshly engorged blackflies in Scandinavia was 62%, being more representative of the Leucocytozoon prevalence in avian hosts, which is expected to be higher there than in Central Europe [12, 14]. On the other hand, close to 50% of pools of non-engorged blackflies seem to contain Leucocytozoon lineages, providing an informative upper limit for the prevalence of Leucocytozoon in blackfly populations [10, 13, 27]. Thus, we complement previous studies of Leucocytozoon-carrying blackflies with a lower individual-based estimate, which may be more precise but is specific to the upper canopy habitat of Central Europe.
Vector and host habitat choice
Finally, we found a substantially different composition of blackfly species around the nests of three closely related avian hosts. Simulium (E.) velutinum were overrepresented around nests of red kites, and S. (N.) vernum* were the only blackflies present around goshawk nests. At the same time, all species and the greatest diversity were represented around nests of common buzzards. This pattern could be due to host preference. Red kites incorporate a great share of carrion and garbage in their nests and food, which lead to a distinct smell of the whole brood. Buzzards, on the other hand, commonly have dead and decaying voles deposited around the nest. Goshawks feed mainly on birds and do not keep unconsumed prey remains at the nest. Therefore, the three raptor species would be identifiable by odour, which is a primary sense for prey recognition outside of the visible range of blackflies [4, 32]. However, it seems unlikely that the involved blackfly species would discriminate against any of the raptor species, given the patterns outlined by the distribution of Leucocytozoon lineages (see above).
Alternatively, the choice of breeding habitat by the three raptor species may predispose them to accumulation of different blackfly species around their nests. Red kites have a preference for open, dispersed deciduous and mixed forests, while goshawks prefer the core of bigger forests with a higher proportion of coniferous trees, and buzzards cover the whole continuum from single trees to the core of big forests. These preferences may co-vary with the microhabitat foraging preferences of the different blackfly species, which are very poorly known . Such a difference in the blackfly community, however, may have catalysed an ecological speciation of parasites, leading to the cryptic Leucocytozoon species infecting currently sympatric raptor species [15, 16, 33].
Vectors are currently the least explored members of host-vector-parasite assemblages . Knowledge of vector ecology and behaviour may be key to understanding the evolution, diversity, prevalence and health impact of parasite populations. Unfortunately, revealing the behaviour of minute arthropods remains extremely challenging and beyond the capacity even of the current bio-logging revolution. Our study approached this aim by relying on the vast knowledge of associations between avian hosts and molecular lineages of blood parasites, which has accumulated in the last decades . It thereby reaffirms the role of parasites as biological markers which can be extremely useful to unveil details of vector biology .