Gold-Conjugated Nanobodies for Targeted Imaging Using High-Resolution Secondary Ion Mass Spectrometry

Nanoscale imaging with the ability to identify cellular organelles and protein complexes has been a highly challenging subject in the secondary ion mass spectrometry (SIMS) of biological samples. This is because only a few isotopic tags can be used successfully to target specific proteins or organelles. To address this, we generated gold nanoprobes, in which gold nanoparticles are conjugated to nanobodies. The nanoprobes were well suited for specific molecular imaging using NanoSIMS at subcellular resolution. They were demonstrated to be highly selective to different proteins of interest and sufficiently sensitive for SIMS detection. The nanoprobes offer the possibility of correlating the investigation of cellular isotopic turnover to the positions of specific proteins and organelles, thereby enabling an understanding of functional and structural relations that are currently obscure.

Gold-conjugated Nanobodies for Targeted Imaging using High-resolution Secondary Ion Mass Spectrometry

Nanoscale imaging with the ability to identify cellular organelles and protein complexes has been a highly challenging subject in secondary ion mass spectrometry (SIMS) of biological samples. This is because only a few isotopic tags can be used successfully to target specific proteins or organelles. To address this, we have generated gold-nanoprobes, in which gold nanoparticles are conjugated to nanobodies. The nanoprobes were well suited for specific molecular imaging using NanoSIMS at subcellular resolution. They demonstrated to be highly selective to different proteins of interest, and sufficiently sensitive for SIMS detection. The nanoprobes offer the possibility of correlating the investigation of cellular isotopic turnover to the positions of specific proteins and organelles, thereby enabling an understanding of functional and structural relations that are currently obscure.

The Importance of Isotopic Turnover for Understanding Key Aspects of Animal Ecology and Nutrition

Stable isotope-based methods have proved to be immensely valuable for ecological studies ranging in focus from animal movements to species interactions and community structure. Nevertheless, the use of these methods is dependent on assumptions about the incorporation and turnover of isotopes within animal tissues, which are oftentimes not explicitly acknowledged and vetted. Thus, the purpose of this review is to provide an overview of the estimation of stable isotope turnover rates in animals, and to highlight the importance of these estimates for ecological studies in terrestrial, freshwater, and marine systems that may use a wide range of stable isotopes. Specifically, we discuss 1) the factors that contribute to variation in turnover among individuals and across species, which influences the use of stable isotopes for diet reconstructions, 2) the differences in turnover among tissues that underlie so-called ‘isotopic clocks’, which are used to estimate the timing of dietary shifts, and 3) the use of turnover rates to estimate nutritional requirements and reconstruct histories of nutritional stress from tissue isotope signatures. As we discuss these topics, we highlight recent works that have effectively used estimates of turnover to design and execute informative ecological studies. Our concluding remarks suggest several steps that will improve our understanding of isotopic turnover and support its integration into a wider range of ecological studies.

Incorporation of carbon and nitrogen isotopes in age-0 walleye (Sander vitreus) tissues following a laboratory diet switch experiment

Trophic dynamics are often described by following the exchange of naturally occurring isotopes through aquatic communities. However, without experimentally derived isotopic turnover rates and discrimination factors for each species, tissue, and life stage, these trophic models can be misleading. We conducted a laboratory diet shift experiment to describe isotopic turnover and discrimination in age-0 walleye (Sander vitreus) dorsal muscle and gutted carcass samples. Although turnover of dietary δ13C (half-life: 10–12 days) and δ15N (half-life: ∼13 days) signatures was relatively rapid, the diet change was undetected in both tissues during a short transitional period (up to 1.2 times shorter in muscle). Our discrimination estimates generally conform to those of other fishes (ΔCarbon= 0.91, ΔNitrogen= 1.6), but were 30%–50% higher in muscle tissues than in gutted carcass samples. The assumption that young walleye tissues are in equilibrium with their diet is untrue for weeks following a diet shift, and when incorporated, discrimination factors differ between tissues. We provide tissue-specific parameters that remove uncertainty associated with the analysis of field collected isotopic age-0 walleye data.

Landscape context alters cost of living in honeybee metabolism and feeding

Field metabolic rate (FMR) links the energy budget of an animal with the constraints of its ecosystem, but is particularly difficult to measure for small organisms. Landscape degradation exacerbates environmental adversity and reduces resource availability, imposing higher costs of living for many organisms. Here, we report a significant effect of landscape degradation on the FMR of free-flying Apis mellifera , estimated using 86 Rb radio-isotopic turnover. We validated the relationship between 86 Rb k b and metabolic rate for worker bees in the laboratory using flow-through respirometry. We then released radioisotopically enriched individuals into a natural woodland and a heavily degraded and deforested plantation. FMRs of worker bees in natural woodland vegetation were significantly higher than in a deforested landscape. Nectar consumption, estimated using 22 Na radio-isotopic turnover, also differed significantly between natural and degraded landscapes. In the deforested landscape, we infer that the costs of foraging exceeded energetic availability, and honeybees instead foraged less and depended more on stored resources in the hive. If this is generally the case with increasing landscape degradation, this will have important implications for the provision of pollination services and the effectiveness and resilience of ecological restoration practice.

Isotopic tissue turnover and discrimination factors following a laboratory diet switch in Colorado pikeminnow (Ptychocheilus lucius)

Stable isotope ecology has made great strides in quantifying energy transfer through food webs. However, trophic inferences gleaned from field-collected data can be limited when isotopic turnover and isotopic discrimination factors (Δ13C or Δ15N) are unknown. We quantified isotopic turnover and discrimination factors using an isotopic diet switch in the endangered Colorado pikeminnow (Ptychocheilus lucius). The estimated half-life for δ13C was 62 days or a 33% increase in mass and δ15N averaged 133 days or a 52% increase in mass. Growth and metabolic processes both contributed to rates of turnover, but metabolic processes had a stronger effect in δ13C than in δ15N. Lipid-corrected δ13C values resulted in discrimination factors of Δ13C between 0.67 and 0.82 and Δ15N between 2.31 and 2.93, values similar to other fishes. These results suggest sampling fin tissue may be a useful, nonlethal tool for isotopic studies. Fins also demonstrated enrichment in 13C that was not linked to the diet switch, highlighting the importance of controls in isotopic diet switch studies to verify species- and diet-specific estimates of isotopic turnover rates.