Background: Accurate quantification of infection intensity is essential to estimate infection patterns of avian haemosporidian parasites in order to understand the evolution of host-parasite associations. Traditional microscopy is cost-effective but requires high-quality blood smears and considerable experience, while the widely used semi-quantitative qPCR methods are mostly employed with ideal, laboratory-based golden samples and standard curves, which may limit the comparison of parasitemia from different laboratories. Methods: Here we present a digital droplet PCR (ddPCR) protocol for absolute quantification of avian haemosporidians in raptors. Novel primers were designed that target a conserved fragment of a rRNA region of the mitochondrial genome of the parasites. Sensitivity and repeatability were evaluated compared to qPCR and other assays. Results: This ddPCR assay enables accurate quantification of haemosporidian parasites belonging to Plasmodium, Haemoproteus and Leucocytozoon with minimum infection quantities of 10⁻⁵ (i.e. one parasite copy in 10⁵ host genomes) without the use of standard curves. Quantities assessed by ddPCR were more accurate than qPCR using the same primers through reduction of non-specific amplification in low-intensity samples. The ddPCR technique was more consistent among technical duplicates and reactions, especially when infection intensities were low, and this technique demonstrated equal sensitivity with high correspondence (R² = 0.97) compared to the widely used qPCR assay. Both ddPCR and qPCR identified more positive samples than the standard nested PCR protocol for the cytb gene used for barcoding avian haemosporidians. Conclusions: We developed a novel ddPCR assay enabling accurate quantification of avian haemosporidians without golden samples or standard curves. This assay can be used as a robust method for investigating infection patterns in different host-parasite assemblages and can facilitate the comparison of results from different laboratories.

Cabot's tragopan Tragopan caboti, a pheasant that inhabits subtropical montane forests in south-east China, is categorized as Vulnerable on the IUCN Red List. Nesting in trees, it routinely makes use of natural platforms and the old nests of other species, both of which may sometimes be in short supply. This study was designed to test how much use would be made of artificial nest platforms, and to identify factors influencing their occupation. Basketry platforms made of bamboo were erected in parts of Wuyanling National Nature Reserve, Zhejiang Province, China. The tragopans used 16 platforms (8%) in 2002 and 12 (5%) in the following year. A census of the population in spring suggested that a high proportion of the females (41% in 2002, 36% in 2003) in the study area made use of the platforms. There were significant preferences for platforms in mixed conifer/broadleaf forest, as opposed to pure stands, as well as for sites on the upper part of hill slopes. Principal component scores were used to represent variation in 11 variables measured at each platform site. Binary logistic regressions employing these scores as predictors for each year separately did not reveal a consistent model for distinguishing used from unused platforms, although short distances to the forest edge were associated with use in both years. These nest platforms are cheap and easy to erect, and may have the potential to halt or reverse a perceived downward population trend for this species across the whole of its fragmented range. Further trials are therefore advocated.

Brood parasitic cuckoos and their hosts serve as model systems for studying host–parasite coevolution. Egg-rejection behavior constitutes an effective defense against brood parasitism, but some host species show phenotypic plasticity in egg-rejection behavior. Direct exposure to a cuckoo near the nest can increase egg-rejection likelihood, and long-term studies have shown that increased the egg-rejection rates generally correlate with higher parasite prevalence. However, it remains unclear whether such increases result from interactions between parasites and hosts, as these can be surprisingly common, or whether the mere presence of cuckoos in the breeding area is sufficient. Daurian redstarts Phoenicurus auroreus are a common host of the common cuckoo Cuculus canorus that defend against cuckoo parasitism mainly by ejecting the parasitic egg from the nest. This species is unique, as its first breeding attempt of the year takes place before the arrival of cuckoos, excluding the possibility for direct interactions. We simulated the ambient presence of cuckoos or hoopoes Upupa epops (control) in sub-populations of redstarts during their first egg-laying period by presenting taxidermic models and playing back vocalizations. Redstarts in cuckoo-treated plots showed significantly higher egg-ejection rates than individuals in control plots, even though females in both groups were equally likely to recognize the parasitic egg. Among females that did recognize the parasitic egg, those exposed to the cuckoo treatment were more likely to eject it than those exposed to the control treatment. Our results demonstrate unequivocally that the mere presence of cuckoos in the environment is sufficient to provoke egg-ejection behavior.

The arms race between brood parasites and their hosts provides a classic model to study coevolution. Hosts often reject the parasitic egg, and brood parasites should therefore select host nests in which the colour of the eggs best matches that of their own. Although this hypothesis has received some support, direct experimental evidence is still lacking. Here, we report on a study of Daurian redstarts, which show a distinct egg-colour dimorphism, with females laying either blue or pink eggs. Redstarts are often parasitized by common cuckoos, which lay light blue eggs. First, we showed that cuckoo eggs were more similar in spectral reflectance to the blue than to the pink redstart egg morph. Second, we report that the natural parasitism rate was higher in blue than in pink host clutches. Third, we performed a field experiment in which we presented a dummy clutch of each colour morph adjacent to active redstart nests. In this set-up, cuckoos almost always chose to parasitize a blue clutch. Our results demonstrate that cuckoos actively choose redstart nests in which the egg colour matches the colour of their own eggs. Our study thus provides direct experimental evidence in support of the egg matching hypothesis.

In species that are subject to brood parasitism, individuals often vary in their responses to parasitic eggs, with some rejecting the eggs while others do not. While some factors, such as host age (breeding experience), the degree of egg matching and the level of perceived risk of brood parasitism have been shown to influence host decisions, much of the variation remains unexplained. The host personality hypothesis suggests that personality traits of the host influence its response to parasitic eggs, but few studies have tested this. We investigated the relationship between two personality traits (exploration and neophobia) and a physiological trait (breathing rate) of the host, and egg-rejection behaviour in a population of Daurian redstartsPhoenicurus auroreusin northeast China. We first show that exploratory behaviour and the response to a novel object are repeatable for individual females and strongly covary, indicating distinct personality types. We then show that fast-exploring and less neophobic hosts were more likely to reject parasitic eggs than slow-exploring and more neophobic hosts. Variation in breathing rate—a measure of the stress-response—did not affect rejection behaviour. Our results demonstrate that host personality, along the bold-shy continuum, predicts the responses to parasitic eggs in Daurian redstarts, with bold hosts being more likely to reject parasitic eggs.