ABSTRACT

Despite a commitment by the European Union to protect its migratory bat populations, conservation efforts are hindered by a poor understanding of bat migratory strategies and connectivity between breeding and wintering grounds. Traditional methods like mark-recapture are ineffective to study broad-scale bat migratory patterns. Stable hydrogen isotopes (δD) have been proven useful in establishing spatial migratory connectivity of animal populations. Before applying this tool, the method was calibrated using bat samples of known origin. Here we established the potential of δD as a robust geographical tracer of breeding origins of European bats by measuring δD in hair of five sedentary bat species from 45 locations throughout Europe. The δD of bat hair strongly correlated with well-established spatial isotopic patterns in mean annual precipitation in Europe, and therefore was highly correlated with latitude. We calculated a linear mixed-effects model, with species as random effect, linking δD of bat hair to precipitation δD of the areas of hair growth. This model can be used to predict breeding origins of European migrating bats. We used δ13C and δ15N to discriminate among potential origins of bats, and found that these isotopes can be used as variables to further refine origin predictions. A triple-isotope approach could thereby pinpoint populations or subpopulations that have distinct origins. Our results further corroborated stable isotope analysis as a powerful method to delineate animal migrations in Europe.

Starch and soluble sugars are the major photosynthetic products, and their carbon isotope signatures reflect external versus internal limitations of CO(2) fixation. There has been recent renewed interest in the isotope composition of carbohydrates, mainly for use in CO(2) flux partitioning studies at the ecosystem level. The major obstacle to the use of carbohydrates in such studies has been the lack of an acknowledged method to isolate starch and soluble sugars for isotopic measurements. We here report on the comparison and evaluation of existing methods (acid and enzymatic hydrolysis for starch; ion-exchange purification and compound-specific analysis for sugars). The selectivity and reproducibility of the methods were tested using three approaches: (i) an artificial leaf composed of a mixture of isotopically defined compounds, (ii) a C(4) leaf spiked with C(3) starch, and (iii) two natural plant samples (root, leaf). Starch preparation methods based on enzymatic or acid hydrolysis did not yield similar results and exhibited contaminations by non-starch compounds. The specificity of the acidic hydrolysis method was especially low, and we therefore suggest terming these preparations as HCl-hydrolysable carbon, rather than starch. Despite being more specific, enzyme-based methods to isolate starch also need to be further optimized to increase specificity. The analysis of sugars by ion-exchange methods (bulk preparations) was fast but produced more variable isotope compositions than compound-specific methods. Compound-specific approaches did not in all cases correctly reproduce the target values, mainly due to unsatisfactory separation of sugars and background contamination. Our study demonstrates that, despite their wide application, methods for the preparation of starch and soluble sugars for the analysis of carbon isotope composition are not (yet) reliable enough to be routinely applied and further research is urgently needed to resolve the identified problems.

In contrast to birds, bats are possibly limited in their capacity to use body fat as an energy source for long migrations. Here, we studied the fuel choice of migratory Pipistrellus nathusii (approximate weight: 8 g) by analysing the stable carbon isotope ratio (d C V-PDB of insect prey and adipocyte triacylglycerols, suggesting a mixed-fuel use of P. nathusii during autumn migration. To clarify the origin of oxidized fatty acids, we performed feeding experiments with captive P. nathusii. After an insect diet, bat breath was enriched in 13 C relative to the bulk and fat portion of insects, but not deviating from the non-fat portion of insects, suggesting that bats oxidized exogenous proteins and carbohydrates, but not exogenous fatty acids. A feeding experiment with 13 C-labelled substrates confirmed these findings. In conclusion, migratory P. nathusii oxidized dietary proteins directly from insects captured en route in combination with endogenous fatty acids from adipocytes, and replenished their body reserves by routing dietary fatty acids to their body reserves.

Flying vertebrates, such as bats, face exceptionally high energy costs during active flapping flight. Once airborne, energy turnover may exceed basal metabolic rate by a factor of up to 15. Here, we asked whether fuel that powers flight originates from exogenous (dietary nutrients), endogenous sources (mostly body lipids or glycogen), or a combination of both. Since most insectivorous bats fly continuously over relatively long time periods during foraging, we assumed that slowly mobilized glycogen, although suitable for supporting brief sallying flights, is inadequate to power aerial insect-hunting of bats. We hypothesized that the insect-feeding Noctilio albiventris rapidly mobilizes and combusts nutrients from insects it has just eaten instead of utilizing endogenous lipids. We used the stable carbon isotope ratio in the bats' exhaled breath (delta13C(brth)) to assess the origin of metabolized substrates of resting and flying N. albiventris in two nutritional conditions: fasted and recently fed. The breath of fasted resting bats was depleted in 13C in relation to their insect diet (delta13C(diet)), indicating the combustion of 13C depleted body lipids. In contrast to this, delta13C(brth) of bats that had recently fed closely matched delta13C(diet) in both resting and flying bats, suggesting a quick mobilization of ingested nutrients for metabolism. In contrast to most non-volant mammals, bats have evolved the ability to fuel their high energy expenditure rates through the rapid combustion of exogenous nutrients, enabling them to conquer the nocturnal niche of aerial insectivory.