Telomere length measurement by qPCR in birds is affected by storage method of blood samples.

Given the potential role of telomeres as biomarkers of individual health and ageing, there is an increasing interest in studying telomere dynamics in a wider range of taxa in the fields of ecology and evolutionary biology. Measuring telomere length across the lifespan in wild animal systems is essential for testing these hypotheses, and may be aided by archived blood samples collected as part of longitudinal field studies. However, sample collection, storage, and DNA extraction methods may influence telomere length measurement, and it may, therefore, be difficult to balance consistency in sampling protocol with making the most of available samples. We used two complementary approaches to examine the impacts of sample storage method on measurements of relative telomere length (RTL) by qPCR, particularly focusing on FTA (Flinders Technology Associates) cards as a long-term storage solution. We used blood samples from wandering albatrosses collected over 14 years and stored in three different ways (n = 179), and also blood samples from captive zebra finches (n = 30) that were each stored using three different methods. Sample storage method influenced RTL in both studies, and samples on FTA cards had significantly shorter RTL measurements. There was no significant correlation between RTL measured in zebra finch blood on FTA cards and the same samples stored either as frozen whole blood or as extracted DNA. These results highlight the importance of consistency of sampling protocol, particularly in the context of long-term field studies, and suggest that FTA cards should not be used as a long-term storage solution to measure RTL without validation.

Consequences of measurement error in qPCR telomere data: A simulation study.

The qPCR method provides an inexpensive, rapid method for estimating relative telomere length across a set of biological samples. Like all laboratory methods, it involves some degree of measurement error. The estimation of relative telomere length is done subjecting the actual measurements made (the Cq values for telomere and a control gene) to non-linear transformations and combining them into a ratio (the TS ratio). Here, we use computer simulations, supported by mathematical analysis, to explore how errors in measurement affect qPCR estimates of relative telomere length, both in cross-sectional and longitudinal data. We show that errors introduced at the level of Cq values are magnified when the TS ratio is calculated. If the errors at the Cq level are normally distributed and independent of true telomere length, those in the TS ratio are positively skewed and proportional to true telomere length. The repeatability of the TS ratio declines sharply with increasing error in measurement of the Cq values for telomere and/or control gene. In simulated longitudinal data, measurement error alone can produce a pattern of low correlation between successive measures of relative telomere length, coupled with a strong negative dependency of the rate of change on initial relative telomere length. Our results illustrate the importance of reducing measurement error: a small increase in error in Cq values can have large consequences for the power and interpretability of qPCR estimates of relative telomere length. The findings also illustrate the importance of characterising the measurement error in each dataset-coefficients of variation are generally unhelpful, and researchers should report standard deviations of Cq values and/or repeatabilities of TS ratios-and allowing for the known effects of measurement error when interpreting patterns of TS ratio change over time.

Gastrointestinal nematode species diversity in Soay sheep kept in a natural environment without active parasite control.

Molecular methods based on ITS2 sequence analysis were used to identify strongylid parasites and describe their diversity in a management intervention and anthelmintic drug treatment-free sheep flock. Fourteen different nematode parasite species were identified in the flock and the results showed a greater level of nematode species diversity than is normally reported in managed farmed flocks, with the presence of parasites such as Bunostomum trigonocephalum, Ostertagia leptospicularis, Spiculopteragia houdemeri and Trichostrongylus retortaeformis that are considered to be absent or rare in sheep kept in comparable localities. The implied prevalences of Haemonchus contortus in lambs, and of Trichostrongylus axei in lambs, ewes and rams, were higher than those in farmed sheep kept in similar regions, while those of Teladorsagia circumcincta and Trichostrongylus vitrinus were lower. Comparison of the patterns of nematode parasite infection between the summer and autumn sampling periods showed differences from the scenarios that are commonplace in comparable managed flocks; with T. vitrinus burdens of the lambs being higher in the summer than in the winter, and Oesophagostomum venulosum being the predominant nematode species in the adult sheep during the summer, while more-or-less absent from these groups during the winter. Rams played an important role in the epidemiology of certain parasitic nematode species. The relatively non-pathogenic O. venulosum was the only parasitic nematode species to predominate in any group during the study. This preliminary characterisation of the nematode parasite burdens of sheep extensively grazed on diverse unimproved pastures will aid in the understanding of the parasitological consequences of intensive grazing management and of the manner in which modern agriculture upsets the equilibrium between parasites and their hosts. These factors must be accounted for when defining the concept of sustainable parasite control and informing sustainability with reference to commercially efficient sheep farming.

Contrasting patterns of phenotypic plasticity in reproductive traits in two great tit (Parus major) populations.

Phenotypic plasticity is an important mechanism via which populations can respond to changing environmental conditions, but we know very little about how natural populations vary with respect to plasticity. Here we use random-regression animal models to understand the multivariate phenotypic and genetic patterns of plasticity variation in two key life-history traits, laying date and clutch size, using data from long-term studies of great tits in The Netherlands (Hoge Veluwe [HV]) and UK (Wytham Woods [WW]). We show that, while population-level responses of laying date and clutch size to temperature were similar in the two populations, between-individual variation in plasticity differed markedly. Both populations showed significant variation in phenotypic plasticity (IxE) for laying date, but IxE was significantly higher in HV than in WW. There were no significant genotype-by-environment interactions (GxE) for laying date, yet differences in GxE were marginally nonsignificant between HV and WW. For clutch size, we only found significant IxE and GxE in WW but no significant difference between populations. From a multivariate perspective, plasticity in laying date was not correlated with plasticity in clutch size in either population. Our results suggest that generalizations about the form and cause of any response to changing environmental conditions across populations may be difficult.