Rapid quantification of biological nitrogen fixation using optical spectroscopy.

Biological nitrogen fixation (BNF) provides a globally important input of nitrogen (N); its quantification is critical but technically challenging. Leaf reflectance spectroscopy offers a more rapid approach than traditional techniques to measure plant N concentration ([N]) and isotopes (δ15N). Here we present a novel method for rapidly and inexpensively quantifying BNF using optical spectroscopy. We measured plant [N], δ15N, and the amount of N derived from atmospheric fixation (Ndfa) following the standard traditional methodology using isotope ratio mass spectrometry (IRMS) from tissues grown under controlled conditions and taken from field experiments. Using the same tissues, we predicted the same three parameters using optical spectroscopy. By comparing the optical spectroscopy-derived results with traditional measurements (i.e. IRMS), the amount of Ndfa predicted by optical spectroscopy was highly comparable to IRMS-based quantification, with R2 being 0.90 (slope=0.90) and 0.94 (slope=1.02) (root mean square error for predicting legume δ15N was 0.38 and 0.43) for legumes grown in glasshouse and field, respectively. This novel application of optical spectroscopy facilitates BNF studies because it is rapid, scalable, low cost, and complementary to existing technologies. Moreover, the proposed method successfully captures the dynamic response of BNF to climate changes such as warming and drought.

To Eat or Not to Eat: Novel Stable Isotope Models Reveal a Shift in Carnivory with Nutrient Availability for Aquatic Utricularia spp.

BACKGROUND AND AIMS: Freshwater nitrogen inputs are increasing globally, altering the structure and function of wetland ecosystems adapted to low nutrient conditions. Carnivorous wetland plants, Utricularia spp., are hypothesised to reduce their reliance on carnivory and increase their assimilation of environmental nutrients when the supply of ambient nutrients increases. Despite success in using stable isotope approaches to quantify carnivory of terrestrial carnivorous plants, quantifying carnivory of aquatic Utricularia requires improvement. METHODS: We developed stable isotope mixing models to quantify aquatic plant carnivory and used these models to measure dietary changes of three Utricularia species: Utricularia australis, U. gibba, and U. uliginosa in 11 wetlands across a 794 km gradient in eastern Australia. Diet was assessed using multiple models that compared variations in the natural abundance nitrogen isotope composition (δ15N) of Utricularia spp. with that of non-carnivorous plants, and environmental and carnivorous nitrogen sources. KEY RESULTS: Carnivory supplied 40 - 100 % of plant nitrogen. The lowest carnivory rates coincided with the highest availability of ammonium and dissolved organic carbon. CONCLUSIONS: Our findings suggest that Utricularia populations may adapt to high nutrient environments by shifting away from energetically costly carnivory. This has implications for species conservation as anthropogenic impacts continue to affect global wetland ecosystems.

Mandibular bone mass density in a medieval population and its relationship with stable isotopes δ13C and δ15N.

The aim of this study was to compare the level of bone mass in digital orthopantomograms in two populations (medieval and current) using two radiomorphometric indexes, and to correlate the mandibular bone mass value, in the medieval mandible population, with stable isotope data δ13C and δ15N. An observational, cross-sectional, and analytical study on mandibles from two diachronic groups, 15 mandibles from the medieval settlement of La Torrecilla (Granada, Spain) and 15 mandibles from current patients at the Faculty of Dentistry of the University of Granada (Spain), matched by age and sex was conducted. The bone mass density was determined using the Mandibular Cortical Width Index (MCW) and the Mandibular Panoramic Index (PMI) in digital panoramic radiographs. In the medieval group, the values of bone mass density were correlated with those of two stable isotopes (δ13C and δ15N). The mean value of MCW in mm in the medieval group was 3.96 ± 0.60 (mean ± standard deviation) and in the current group was 4.02 ± 1.01. The PMI was 0.33 ± 0.06 and 0.35 ± 0.08 in the medieval and current groups respectively, with similar results in both groups (p = 0.820 and p = 0.575). A negative correlation was found between both morphometric indices and the δ15N isotope (rs = 0.56, p = 0.030 and rs = 0.61, p = 0.016, respectively). The bone mass density in mandibles belonging to the two compared populations, determined by two quantitative radiomorphometric indices, is similar. Within the medieval population, there is an inverse correlation between the δ15N value and bone mass density.

Analysis of δ13C and δ15N values in Croatian honey by EA-IRMS and possibility of their application in botanical origin verification.

The aim of this work was to give characteristic stable carbon and nitrogen isotope ratio (δ13Choney, δ13Cprotein and δ15N) ranges and examine their relation with botanical origin of honey. Despite that δ13C parameter has primary purpose to detect honey adulteration, stable isotopes generally have become important parameter for detection its botanical and geographical origin. The data about stable isotopes are scarce in comparison to other well-known parameters in honey, and in Croatia there is no data about stable isotopes in unifloral honey. This research includes six characteristic honey types (black locust, chestnut, lime, rape, winter savory, and sage honey) from Croatia. Large number of differences between honey types were found in the analyzed IRMS parameters. PCA analysis has successfully separated winter savory from all other honey types, except sage honey, whose samples differed from black locust samples. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05888-9.

A distinctive latitudinal trend of nitrogen isotope signature across urban forests in eastern China.

Rapid urbanization has greatly altered nitrogen (N) cycling from regional to global scales. Compared to natural forests, urban forests receive much more external N inputs with distinctive abundances of stable N isotope (δ15 N). However, the large-scale pattern of soil δ15 N and its imprint on plant δ15 N remain less well understood in urban forests. By collecting topsoil (0-20 cm) and leaf samples from urban forest patches in nine large cities across a north-south transect in eastern China, we analyzed the latitudinal trends of topsoil C:N ratio and δ15 N as well as the correlations between tree leaf δ15 N and topsoil δ15 N. We further explored the spatial variation of topsoil δ15 N explained by corresponding climatic, edaphic, vegetation-associated, and anthropogenic drivers. Our results showed a significant increase of topsoil C:N ratio towards higher latitudes, suggesting lower N availability at higher latitudes. Topsoil δ15 N also increased significantly at higher latitudes, being opposite to the latitudinal trend of soil N availability. The latitudinal trend of topsoil δ15 N was mainly explained by mean annual temperature, mean annual precipitation, and atmospheric deposition of both ammonium and nitrate. Consequently, tree leaf δ15 N showed significant positive correlations with topsoil δ15 N across all sampled plant species and functional types. Our findings reveal a distinctive latitudinal trend of δ15 N in urban forests and highlight an important role of anthropogenic N sources in shaping the large-scale pattern of urban forest 15 N signature.

Isotopic evidence for increased carbon and nitrogen exchanges between peatland plants and their symbiotic microbes with rising atmospheric CO2 concentrations since 15,000 cal. year BP.

Whether nitrogen (N) availability will limit plant growth and removal of atmospheric CO2 by the terrestrial biosphere this century is controversial. Studies have suggested that N could progressively limit plant growth, as trees and soils accumulate N in slowly cycling biomass pools in response to increases in carbon sequestration. However, a question remains over whether longer-term (decadal to century) feedbacks between climate, CO2 and plant N uptake could emerge to reduce ecosystem-level N limitations. The symbioses between plants and microbes can help plants to acquire N from the soil or from the atmosphere via biological N2 fixation-the pathway through which N can be rapidly brought into ecosystems and thereby partially or completely alleviate N limitation on plant productivity. Here we present measurements of plant N isotope composition (δ15 N) in a peat core that dates to 15,000 cal. year BP to ascertain ecosystem-level N cycling responses to rising atmospheric CO2 concentrations. We find that pre-industrial increases in global atmospheric CO2 concentrations corresponded with a decrease in the δ15 N of both Sphagnum moss and Ericaceae when constrained for climatic factors. A modern experiment demonstrates that the δ15 N of Sphagnum decreases with increasing N2 -fixation rates. These findings suggest that plant-microbe symbioses that facilitate N acquisition are, over the long term, enhanced under rising atmospheric CO2 concentrations, highlighting an ecosystem-level feedback mechanism whereby N constraints on terrestrial carbon storage can be overcome.

Enhanced foliar 15 N enrichment with increasing nitrogen addition rates: Role of plant species and nitrogen compounds.

Determining the abundance of N isotope (δ15 N) in natural environments is a simple but powerful method for providing integrated information on the N cycling dynamics and status in an ecosystem under exogenous N inputs. However, whether the input of different N compounds could differently impact plant growth and their 15 N signatures remains unclear. Here, the response of 15 N signatures and growth of three dominant plants (Leymus chinensis, Carex duriuscula, and Thermopsis lanceolata) to the addition of three N compounds (NH4 HCO3 , urea, and NH4 NO3 ) at multiple N addition rates were assessed in a meadow steppe in Inner Mongolia. The three plants showed different initial foliar δ15 N values because of differences in their N acquisition strategies. Particularly, T. lanceolata (N2 -fixing species) showed significantly lower 15 N signatures than L. chinensis (associated with arbuscular mycorrhizal fungi [AMF]) and C. duriuscula (associated with AMF). Moreover, the foliar δ15 N of all three species increased with increasing N addition rates, with a sharp increase above an N addition rate of ~10 g N m-2  year-1 . Foliar δ15 N values were significantly higher when NH4 HCO3 and urea were added than when NH4 NO3 was added, suggesting that adding weakly acidifying N compounds could result in a more open N cycle. Overall, our results imply that assessing the N transformation processes in the context of increasing global N deposition necessitates the consideration of N deposition rates, forms of the deposited N compounds, and N utilization strategies of the co-existing plant species in the ecosystem.

Using 15N to determine the metabolic fate of dietary nitrogen in North Pacific spiny dogfish (Squalus acanthias suckleyi).

Marine elasmobranchs are ureosmotic, retaining large concentrations of urea to balance their internal osmotic pressure with that of the external marine environment. The synthesis of urea requires the intake of exogenous nitrogen to maintain whole-body nitrogen balance and satisfy obligatory osmoregulatory and somatic processes. We hypothesized that dietary nitrogen may be directed toward the synthesis of specific nitrogenous molecules in post-fed animals; specifically, we predicted the preferential accumulation and retention of labelled nitrogen would be directed towards the synthesis of urea necessary for osmoregulatory purposes. North Pacific spiny dogfish (Squalus acanthias suckleyi) were fed a single meal of 7 mmol l-1 15NH4Cl in a 2% ration by body mass of herring slurry via gavage. Dietary labelled nitrogen was tracked from ingestion to tissue incorporation and the subsequent synthesis of nitrogenous compounds (urea, glutamine, bulk amino acids, protein) in the intestinal spiral valve, plasma, liver and muscle. Within 20 h post-feeding, we found labelled nitrogen was incorporated into all tissues examined. The highest δ15N values were seen in the anterior region of the spiral valve at 20 h post-feeding, suggesting this region was particularly important in assimilating the dietary labelled nitrogen. In all tissues examined, enrichment of the nitrogenous compounds was sustained throughout the 168 h experimental period, highlighting the ability of these animals to retain and use dietary nitrogen for both osmoregulatory and somatic processes.

Stable isotope ratios of nitrogen and carbon as biomarkers of a vegan diet.

PURPOSE: Dietary biomarkers can potentially overcome the limitations of self-reported dietary data. While in ecology and archaeology, stable isotope ratios of carbon and nitrogen are widely used as biomarkers, this is not the case in nutrition research. Since the abundance of the 13C and the 15N isotope differ in food sources from plant and animal origin, stable isotope ratios of carbon and nitrogen (δ13C and δ15N) may differ in human biological material. Here, we investigated the stable isotope ratios of nitrogen and carbon in serum and urine from vegans and omnivores. METHOD: Measurement of δ15N and δ13C in serum and 24 h urine was performed by Elemental Analyzer-Isotope Ratio Mass Spectrometer in the cross-sectional study "Risks and Benefits of a Vegan Diet". The study included 36 vegans and 36 omnivores with a median age of 37.5 years (matched for age and sex), who adhered to their diet for at least 1 year. RESULTS: Both δ15N and δ13C were significantly lower in both the serum and 24 h urine of vegans compared to omnivores. δ15N either in serum or urine had 100% specificity and sensitivity to discriminate between vegans and omnivores. Specificity of δ13C was also > 90%, while sensitivity was 93% in serum and 77% in urine. CONCLUSION: δ15N both in serum and urine was able to accurately identify vegans and thus appears to be a promising marker for dietary habits.

Nitrogen retention in stream biofilms - A potential contribution to the self-cleaning capacity.

Surface waters are under increasing pressure due to human activities, such as nutrient emissions from wastewater treatment plants (WWTPs). Using the retention of nitrogen (N) released from WWTPs as a proxy, we assessed the contribution of biofilms grown on inorganic and organic substrates to the self-cleaning capacity of second-order streams within the biosphere reserve Vosges du Nord/Palatinate forest (France/Germany). The uptake of N from anthropogenic sources, which is enriched with the heavy isotope 15N, into biofilms was assessed up- and downstream of WWTPs after five weeks of substrate deployment. Biofilms at downstream sites showed a significant positive linear relationship between δ15N and the relative contribution of wastewater to the streams' discharge. Furthermore, δ15N substantially increased in areas affected by WWTP effluent (∼8.5‰ and ∼7‰ for inorganic and organic substrate-associated biofilms, respectively) and afterwards declined with increasing distance to the WWTP effluent, approaching levels of upstream sections. The present study highlights that biofilms contribute to nutrient retention and likely the self-cleaning capacity of streams. This function seems, however, to be limited by the fact that biofilms are restricted in their capacity to process excessive N loads with large differences between individual reaches (e.g., δ15N: -3.25 to 12.81‰), influenced by surrounding conditions (e.g., land use) and modulated through climatic factors and thus impacted by climate change. Consequently, the impact of WWTPs located close to the source of a stream are dampened by the biofilms' capacity to retain N only to a minor share and suggest substantial N loads being transported downstream.

Stable C and N isotopes of PM2.5 and size-segregated particles emitted from incense stick and cigarette burning.

This work measured the δ13C and δ15N signatures in PM2.5 and size-segregated particles emitted from incense stick and cigarette burning in different brands or nicotine contents for pollution source identification indoors. Three popular brands of incense stick and cigarette were selected for experiments. A personal environmental monitoring sampler and a Sioutas cascade impactor were used to collect PM2.5 and size-segregated particles, respectively, for isotopic signatures analyses. Our data showed that both δ13C and δ15N values were heavier from incense stick burning (δ13C: 27.3 ± 0.5; δ15N: 8.63 ± 1.35) than cigarette (δ13C: 28.5 ± 0.2; δ15N: 4.15 ± 0.69). The scatter plots of δ13C and TC/PM2.5 and of δ15N and TN/PM2.5 can be applied to distinguish particle pollution sources and assess the influence of cigarette burning to PM2.5 according to different nicotine contents. The δ13C values in size-segregated particles were similar to incense stick or cigarette burning; the δ13C values in PM2.5 were significantly higher than those in size-segregated particles. However, the nitrogen amount was too low in most of the size-segregated particles to analyze δ15N from incense stick and cigarette burning. These results suggest that the δ13C signatures on PM2.5 cannot represent the isotopic characteristics of size-segregated particles and δ15N has limitation for pollution source identification of different particle sizes.

Assessment of the temporal retention of mercury and nutrient records within the mangrove sediments of a highly impacted estuary.

Mangrove ecosystems are dynamic and biodiverse environments with the capacity to sequester more organic carbon per unit area, per time, than terrestrial forests, yet are among one of the most heavily degraded ecosystems on Earth. Here, we quantify trace metal, nutrient and carbon accumulation rates in a tropical mangrove environment in northeast Brazil, a region that has been rapidly developed over the past seven decades. Carbon accumulation rate results show modest or no increase since the 1950's, when major development occurred in the region. Organic carbon isotope (δ13C) and C:N molar ratios indicate that the OM is primarily derived from autochthonous C3 plant sources. However, the most recent sediments revealed changes from terrestrial to alga-derived source of OM, which is consistent with the increase of total nitrogen, δ15N and total phosphorous content in the last seven decades, suggesting anthropogenic impact. Furthermore, the Hg enrichment factor (EF) in mangrove sediments is shown to have increased 13-fold since the 1960's, highlighting the ability of tropical mangrove systems in trap filtering pollutants from proximal urban development.

Comparative trophic ecology of microhabitat-associated guilds of reef fishes in the Adriatic Sea.

Reef ecosystems are characterized by highly heterogenous habitats and functionally diverse fish communities. Few studies have examined how functional diversity differs among habitats within these communities, i.e., species associated with a specific habitat may have similar trophic ecologies meaning that the functional diversity within the community is driven by habitat diversity or, conversely, high functional diversity within each habitat would indicate that resource segregation also occurs at the habitat level. We used stable isotope ratios of carbon and nitrogen to estimate trophic position, resource use and ontogenetic niche shifts of 15 reef fishes associated with four distinct habitat types (cryptobenthic, epibenthic sand, epibenthic rock and hyperbenthic) on the Croatian coast of the Adriatic Sea. Trophic ecology was quite similar across fish assemblages, but there was strong evidence of niche segregation among fish species within each assemblage showing high functional diversity within each microhabitat. The sampled fish community contained benthic and pelagic resource users, along with multiple intermediate generalists. Consumer stable isotope ratios revealed considerable interspecific variation in resource use among fishes within each habitat type. The cryptobenthic fishes were a notable exception to this trend with the narrow range of resource use values, indicating reliance of these species on a single resource. The greatest diversity of trophic positions within a guild was observed in cryptobenthic and rock-associated epibenthic fishes. The majority of observed ontogenetic variation in studied fish species reflected an increase in benthic resource use and trophic position. However, the degree of ontogenetic variation in trophic ecology of studied species, if present, was generally low, showing no dramatic change in the ecology of any species. The size structuring among guilds was considerable, with cryptobenthic fishes the smallest on average and hyperbenthic fishes the largest, despite guilds having similar ranges of trophic positions.

Natural abundance analysis of the role played by 15 N as indicator for the certification of organic-system deriving food.

BACKGROUND: The natural abundance of stable isotope 15 N (δ15 N) in production systems has emerged as an alternative to distinguish organic products from conventional ones. This study evaluated the use of δ15 N values recorded for nitrogen fertilizers, soil and plant tissue in order to set the differences between organic and conventional agricultural production systems applied to rice, potatoes, apple and banana crops. RESULTS: Values of δ15 N recorded for N sources ranged from +5.58‰ to +18.27‰ and from -3.55‰ to +3.19‰ in organic and synthetic fertilizers, respectively. Values recorded for δ15 N in food from organic rice, potatoes and banana farms were higher than values recorded for δ15 N in conventional farms; the same was observed for values recorded for δ15 N in leaves from the four crops. CONCLUSION: Results have allowed for differentiation between production systems due to values of δ15 N recorded in leaves of all crops and food, for rice, potatoes and banana trees. © 2021 Society of Chemical Industry.

Drought stress mitigation by nitrogen in boreal forests inferred from stable isotopes.

Forest growth in most parts of the boreal zone is originally limited by low temperatures and low nitrogen availability. Due to the rapid climate warming at high latitudes, an increasing forest area is switching to drought limitation, especially in continental and southern parts of the boreal forest. Studies addressing this issue were mostly dendrochronological and remote-sensing analyses focusing on climatic effects, but not answering the question whether drought is effective alone or in combination with nitrogen shortage at limiting the forests' productivity and vitality. Here we show in a case study from larch forests of Mongolia with a combination of stable isotope analyses, tree-ring analysis and bioindication of the local variability of livestock densities using epiphytic lichens that, in the studied highly drought-prone forests at the southern fringe of the boreal forest in Inner Asia, the trees' vulnerability to drought is modified by nitrogen fertilization from livestock kept in the vicinity and the edge of the forests. The most likely mechanism behind this drought-nitrogen interaction is the reduction of stomatal conductance, which is known to be induced by low nitrogen levels in plants. Nitrogen fertilization by the livestock could, thus, shorten the times of stomatal closure and thereby increase tree growth, which we measured as radial stem increment. Even though the underlying mechanisms, which were so far examined in angiosperms, should be experimentally tested for conifers, our results indicate that focusing on water alone is not enough to understand the climate change response of drought-limited boreal forests.

Landscape-scale patterns of nutrient enrichment in a coral reef ecosystem: implications for coral to algae phase shifts.

Nutrient pollution is altering coastal ecosystems worldwide. On coral reefs, excess nutrients can favor the production of algae at the expense of reef-building corals, yet the role of nutrients in driving community changes such as shifts from coral to macroalgae is not well understood. Here we investigate the potential role of anthropogenic nutrient loading in driving recent coral-to-macroalgae phase shifts on reefs in the lagoons surrounding the Pacific island of Moorea, French Polynesia. We use nitrogen (N) tissue content and stable isotopes (δ15 N) in an abundant macroalga (Turbinaria ornata) together with empirical models of nutrient discharge to describe spatial and temporal patterns of nutrient enrichment in the lagoons. We then employ time series data to test whether recent increases in macroalgae are associated with nutrients. Our results revealed that patterns of N enrichment were linked to several factors, including rainfall, wave-driven circulation, and distance from anthropogenic nutrient sources, especially human sewage. Reefs near large watersheds, where inputs of N from sewage and agriculture are high, have been consistently enriched in N for at least the last decade. In many of these areas, corals have decreased and macroalgae have increased, while reefs with lower levels of N input have maintained high cover of coral and low cover of macroalgae. Importantly, these patchy phase shifts to macroalgae have occurred despite substantial island-wide increases in the density and biomass of herbivorous fishes over the time period. Together, these results indicate that nutrient loading may be an important driver of coral-to-macroalgae phase shifts in the lagoons of Moorea even though the reefs harbor an abundant and diverse herbivore assemblage. These results emphasize the important role that bottom-up factors can play in driving coral-to-macroalgae phase shifts and underscore the critical importance of watershed management for reducing inputs of nutrients and other land-based pollutants to coral reef ecosystems.

Plant and soil nitrogen in oligotrophic boreal forest habitats with varying moss depths: does exclusion of large grazers matter?

The boreal forest consists of drier sunlit and moister-shaded habitats with varying moss abundance. Mosses control vascular plant-soil interactions, yet they all can also be altered by grazers. We determined how 2 decades of reindeer (Rangifer tarandus) exclusion affect feather moss (Pleurozium schreberi) depth, and the accompanying soil N dynamics (total and dissolvable inorganic N, δ15N), plant foliar N, and stable isotopes (δ15N, δ13C) in two contrasting habitats of an oligotrophic Scots pine forest. The study species were pine seedling (Pinus sylvestris L.), bilberry (Vaccinium myrtillus L.), lingonberry (V. vitis-idaea L.), and feather moss. Moss carpet was deeper in shaded than sunlit habitats and increased with grazer exclusion. Humus N content increased in the shade as did humus δ15N, which also increased due to exclusion in the sunlit habitats. Exclusion increased inorganic N concentration in the mineral soil. These soil responses were correlated with moss depth. Foliar chemistry varied due to habitat depending on species identity. Pine seedlings showed higher foliar N content and lower foliar δ15N in the shaded than in the sunlit habitats, while bilberry had both higher foliar N and δ15N in the shade. Thus, foliar δ15N values of co-existing species diverged in the shade indicating enhanced N partitioning. We conclude that despite strong grazing-induced shifts in mosses and subtler shifts in soil N, the N dynamics of vascular vegetation remain unchanged. These indicate that plant-soil interactions are resistant to shifts in grazing intensity, a pattern that appears to be common across boreal oligotrophic forests.

Body condition of returning Atlantic salmon Salmo salar L. correlates with scale δ13 C and δ15 N content deposited at the last marine foraging location.

Patterns of feeding and growth of Atlantic salmon Salmo salar L. in the marine environment are critical to understanding how observed declines in recruitment may reflect warming or other oceanic drivers. The isotopic composition of scales can provide insight into differences in marine feeding location and possibly temperature regime. The authors used archived scale samples to measure δ13 C and δ15 N deposited in the scales of one sea-winter (1SW) salmon during their last season of growth at sea before they returned to five Irish rivers. δ13 C values were related statistically to observed salmon body condition (Fulton's K), and fish with higher δ13 C values tended to show significantly better condition. In contrast, δ15 N values were negatively related to body condition. There was no important effect on condition of length at smolt migration, and the effect of duration of marine residence varied among rivers. It is likely that δ13 C values partly reflected ambient ocean temperature and recent marine feeding environment before return migration, such that the observed relationship between higher δ13 C values and increased body condition may express an advantage for adult fish feeding in warmer, potentially closer, waters. If greater body condition influences fitness, then a changing temperature regime in the Northeast Atlantic may drive shifts in salmon survival and reproduction. This study provides evidence that there is spatial and trophic variation at sea between salmon from rivers of origin that are located relatively close to each other, with potential consequences for body condition and, consequently, fitness and life history; this suggests that salmon populations from geographically proximate rivers within regions may exhibit differential responses to ocean-scale climatic changes across the Northeast Atlantic.

Recombinant Bovine Growth Hormone-Induced Metabolic Remodelling Enhances Growth of Gilthead Sea-Bream (Sparus aurata): Insights from Stable Isotopes Composition and Proteomics.

Growth hormone and insulin-like growth factors (GH/IGF axis) regulate somatic growth in mammals and fish, although their action on metabolism is not fully understood in the latter. An intraperitoneal injection of extended-release recombinant bovine growth hormone (rbGH, Posilac®) was used in gilthead sea bream fingerlings and juveniles to analyse the metabolic response of liver and red and white muscles by enzymatic, isotopic and proteomic analyses. GH-induced lipolysis and glycogenolysis were reflected in liver composition, and metabolic and redox enzymes reported higher lipid use and lower protein oxidation. In white and red muscle reserves, rBGH increased glycogen while reducing lipid. The isotopic analysis of muscles showed a decrease in the recycling of proteins and a greater recycling of lipids and glycogen in the rBGH groups, which favoured a protein sparing effect. The protein synthesis capacity (RNA/protein) of white muscle increased, while cytochrome-c-oxidase (COX) protein expression decreased in rBGH group. Proteomic analysis of white muscle revealed only downregulation of 8 proteins, related to carbohydrate metabolic processes. The global results corroborated that GH acted by saving dietary proteins for muscle growth mainly by promoting the use of lipids as energy in the muscles of the gilthead sea bream. There was a fuel switch from carbohydrates to lipids with compensatory changes in antioxidant pathways that overall resulted in enhanced somatic growth.

Determining the appropriate pretreatment procedures and the utility of liver tissue for bulk stable isotope (δ13 C and δ15 N) studies in sharks.

Stable-isotope analysis (SIA) provides a valuable tool to address complex questions pertaining to elasmobranch ecology. Liver, a metabolically active, high turnover tissue (~166 days for 95% turnover), has the potential to reveal novel insights into recent feeding/movement behaviours of this diverse group. To date, limited work has used this tissue, but ecological application of SIA in liver requires consideration of tissue preparation techniques given the potential for high concentrations of urea and lipid that could bias δ13 C and δ15 N values (i.e., result in artificially lower δ13 C and δ15 N values). Here we investigated the effectiveness of (a) deionized water washing (WW) for urea removal from liver tissue and (b) chloroform-methanol for extraction of lipids from this lipid rich tissue. We then (a) established C:N thresholds for deriving ecologically relevant liver isotopic values given complications of removing all lipid and (b) undertook a preliminary comparison of δ13 C values between tissue pairs (muscle and liver) to test if observed isotopic differences correlated with known movement behaviour. Tests were conducted on four large shark species: the dusky (DUS, Carcharhinus obscurus), sand tiger (RAG, Carcharias taurus), scalloped hammerhead (SCA, Sphyrna lewini) and white shark (GRE, Carcharodon carcharias). There was no significant difference in δ15 N values between lipid-extracted (LE) liver and lipid-extracted/water washed (WW) treatments, however, WW resulted in significant increases in %N, δ13 C and %C. Following lipid extraction (repeated three times), some samples were still biased by lipids. Our species-specific "C:N thresholds" provide a method to derive ecologically viable isotope data given the complexities of this lipid rich tissue (C:N thresholds of 4.0, 3.6, 4.7 and 3.9 for DUS, RAG, SCA and GRE liverLEWW tissue, respectively). The preliminary comparison of C:N threshold corrected liver and muscle δ13 C values corresponded with movement/habitat behaviours for each shark; minor differences in δ13 C values were observed for known regional movements of DUS and RAG (δ13 CDiffs = 0.24 ± 0.99‰ and 0.57 ± 0.38‰, respectively), while SCA and GRE showed greater differences (1.24 ± 0.63‰ and 1.08 ± 0.71‰, respectively) correlated to large-scale movements between temperate/tropical and pelagic/coastal environments. These data provide an approach for the successful application of liver δ13 C and δ15 N values to examine elasmobranch ecology.