Parasite detection and quantification in avian blood is dependent on storage medium and duration.

Studies of parasites in wild animal populations often rely on molecular methods to both detect and quantify infections. However, method accuracy is likely to be influenced by the sampling approach taken prior to nucleic acid extraction. Avian Haemosporidia are studied primarily through the screening of host blood, and a range of storage mediums are available for the short- to long-term preservation of samples. Previous research has suggested that storage medium choice may impact the accuracy of PCR-based parasite detection, however, this relationship has never been explicitly tested and may be exacerbated by the duration of sample storage. These considerations could also be especially critical for sensitive molecular methods used to quantify infection (qPCR). To test the effect of storage medium and duration on Plasmodium detection and quantification, we split blood samples collected from wild birds across three medium types (filter paper, Queen's lysis buffer, and 96% ethanol) and carried out DNA extractions at five time points (1, 6, 12, 24, and 36 months post-sampling). First, we found variation in DNA yield obtained from blood samples dependent on their storage medium which had subsequent negative impacts on both detection and estimates of Plasmodium copy number. Second, we found that detection accuracy (incidence of true positives) was highest for filter-paper-stored samples (97%), while accuracy for ethanol and Queen's lysis buffer-stored samples was influenced by either storage duration or extraction yield, respectively. Lastly, longer storage durations were associated with decreased copy number estimates across all storage mediums; equating to a 58% reduction between the first- and third-year post-sampling for lysis-stored samples. These results raise questions regarding the utility of standardizing samples by dilution, while also illustrating the critical importance of considering storage approaches in studies of Haemosporidia comparing samples subjected to different storage regimes and/or stored for varying lengths of time.

Sex identification in embryos and adults of Darwin's finches.

Darwin's finches are an iconic example of adaptive radiation and evolution under natural selection. Comparative genetic studies using embryos of Darwin's finches have shed light on the possible evolutionary processes underlying the speciation of this clade. Molecular identification of the sex of embryonic samples is important for such studies, where this information often cannot be inferred otherwise. We tested a fast and simple chicken embryo protocol to extract DNA from Darwin's finch embryos. In addition, we applied minor modifications to two of the previously reported PCR primer sets for CHD1, a gene used for sexing adult passerine birds. The sex of all 29 tested embryos of six species of Darwin's finches was determined successfully by PCR, using both primer sets. Next to embryos, hatchlings and fledglings are also impossible to distinguish visually. This extends to juveniles of sexually dimorphic species which are yet to moult in adult-like plumage and beak colouration. Furthermore, four species of Darwin's finches are monomorphic, males and females looking alike. Therefore, sex assessment in the field can be a source of error, especially with respect to juveniles and mature monomorphic birds outside of the mating season. We caught 567 juveniles and adults belonging to six species of Darwin's finches and only 44% had unambiguous sex-specific morphology. We sexed 363 birds by PCR: individuals sexed based on marginal sex specific morphological traits; and birds which were impossible to classify in the field. PCR revealed that for birds with marginal sex specific traits, sexing in the field produced a 13% error rate. This demonstrates that PCR based sexing can improve field studies on Darwin's finches, especially when individuals with unclear sex-related morphology are involved. The protocols used here provide an easy and reliable way to sex Darwin's finches throughout ontogeny, from embryos to adults.

Avian disease surveillance on the island of San Cristóbal, Galápagos.

Endemic island species face unprecedented threats, with many populations in decline or at risk of extinction. One important threat is the introduction of novel and potentially devastating diseases, made more pressing due to accelerating global connectivity, urban development, and climatic changes. In the Galápagos archipelago two important wildlife diseases: avian pox (Avipoxvirus spp.) and avian malaria (Plasmodium spp. and related Haemosporidia) challenge endemic species. San Cristóbal island has seen a paucity of disease surveillance in avian populations, despite the island's connectedness to the continent and the wider archipelago. To survey prevalence and better understand the dynamics of these two diseases on San Cristóbal, we captured 1205 birds of 11 species on the island between 2016 and 2020. Study sites included urban and rural lowland localities as well as rural highland sites in 2019. Of 995 blood samples screened for avian haemosporidia, none tested positive for infection. In contrast, evidence of past and active pox infection was observed in 97 birds and identified as strains Gal1 and Gal2. Active pox prevalence differed significantly with contemporary climatic conditions, being highest during El Niño events (~11% in 2016 and in 2019 versus <1% in the La Niña year of 2018). Pox prevalence was also higher at urban sites than rural (11% to 4%, in 2019) and prevalence varied between host species, ranging from 12% in medium ground finches (Geospiza fortis) to 4% in Yellow Warblers (Setophaga petechial aureola). In the most common infected species (Small Ground Finch: Geospiza fuliginosa), birds recovered from pox had significantly longer wings, which may suggest a selective cost to infection. These results illustrate the threat future climate changes and urbanization may present in influencing disease dynamics in the Galápagos, while also highlighting unknowns regarding species-specific susceptibilities to avian pox and the transmission dynamics facilitating outbreaks within these iconic species.

Contrasting the seasonal and elevational prevalence of generalist avian haemosporidia in co-occurring host species.

Understanding the ecology and evolution of parasites is contingent on identifying the selection pressures they face across their infection landscape. Such a task is made challenging by the fact that these pressures will likely vary across time and space, as a result of seasonal and geographical differences in host susceptibility or transmission opportunities. Avian haemosporidian blood parasites are capable of infecting multiple co-occurring hosts within their ranges, yet whether their distribution across time and space varies similarly in their different host species remains unclear. Here, we applied a new PCR method to detect avian haemosporidia (genera Haemoproteus, Leucocytozoon, and Plasmodium) and to determine parasite prevalence in two closely related and co-occurring host species, blue tits (Cyanistes caeruleus, N = 529) and great tits (Parus major, N = 443). Our samples were collected between autumn and spring, along an elevational gradient in the French Pyrenees and over a three-year period. Most parasites were found to infect both host species, and while these generalist parasites displayed similar elevational patterns of prevalence in the two host species, this was not always the case for seasonal prevalence patterns. For example, Leucocytozoon group A parasites showed inverse seasonal prevalence when comparing between the two host species, being highest in winter and spring in blue tits but higher in autumn in great tits. While Plasmodium relictum prevalence was overall lower in spring relative to winter or autumn in both species, spring prevalence was also lower in blue tits than in great tits. Together, these results reveal how generalist parasites can exhibit host-specific epidemiology, which is likely to complicate predictions of host-parasite co-evolution.