Combining histology, stable isotope analysis and ZooMS collagen fingerprinting to investigate the taphonomic history and dietary behaviour of extinct giant tortoises from the Mare aux Songes deposit on Mauritius

Taphonomic research of bones can provide additional insight into a site's formation and development, the burial environment and ongoing post-mortem processes. A total of 30 tortoise (Cylindraspis) femur bone samples from the Mare aux Songes site (Mauritius)were studied histologically, assessing parameters such as presence and type of microbial alteration, inclusions, staining/infiltrations, the degree of microcracking and birefringence. The absence of microbial attack in the 4200 year old Mare aux Songes bones suggests the animals rapidly entered the soil whole-bodied and were sealed anoxically, although they suffered frombiological and chemical degradation (i.e. pyrite formation/oxidation, mineral dissolution and staining) related to changes in the site's hydrology. Additionally, carbon and nitrogen stable isotopeswere analysed to obtain information on the animals' feeding behaviour. The results show narrowly distributed δ13C ratios, indicating a terrestrial C3 plant-based diet, combined with a wide range in δ15N ratios. This is most likely related to the tortoises' drought-adaptive ability to change their metabolic processes, which can affect the δ15N ratios. Furthermore, ZooMS collagen fingerprinting analysis successfully identified two tortoise species (C. triserrata and C. inepta) in the bone assemblage,which,when combined with stable isotope data, revealed significantly different δ15N ratios between the two tortoise species. As climatic changes around this period resulted in increased aridity in the Mascarene Islands, this could explain the extremely elevated δ15N ratio in our dataset. The endemic fauna was able to endure the climatic changes 4200 years ago, although human arrival in the 17th century changed the original habitat to such an extent that it resulted in the extinction of several species. Fortunately we are still able to study these extinct tortoises due to the beneficial conditions of their burial environment, resulting in excellent bone preservation.

Relative contribution of natural productivity and compound feed to tissue growth in blue shrimp (Litopenaeus stylirostris) reared in biofloc: Assessment by C and N stable isotope ratios and effect on key digestive enzymes

The aim of this study was to assess the relative contribution of natural productivity and compound food to the growth of the juvenile blue shrimp Litopenaeus stylirostris reared in a biofloc system. Two experiments were carried out based on the same protocol with three treatments: clear water with experimental diet (CW), biofloc with experimental diet (BF) and biofloc unfed (BU). Shrimp survival was significantly higher in biofloc rearing than in CW rearing. The contribution of the biofloc to shrimp diet was estimated through measurement of carbon and nitrogen stable isotope ratios in shrimp and food sources. Different isotopic compositions between feeds were obtained by feeding natural productivity with a mixture rich in fish meal and the shrimps with a pellet containing a high level of soy protein concentrate. Using a two source one-isotope mixing model, we found that the natural productivity of the biofloc system contributed to shrimp growth at a level of 39.8% and 36.9%, for C and N, respectively. The natural food consumed by the shrimps reared in the biofloc system resulted in higher gene expression (mRNA transcript abundance) and activities of two digestive enzymes in their digestive gland: α-amylase and trypsin. The growth of shrimp biomass reared in biofloc was, on average, 4.4 times that of those grown in clear water. Our results confirmed the best survival and promoted growth of shrimps using biofloc technology and highlighted the key role of the biofloc in the nutrition of rearing shrimps. Statement of relevance In this study, we have applied an original protocol to determine the respective contribution of natural productivity and artificial feeds on the alimentation of the juvenile blue shrimp L. stylirostris reared in biofloc system by using C and N natural stable isotope analysis. Moreover, we have compared, in shrimp digestive gland, the α-amylase and trypsin enzyme activities at biochemical and molecular levels for two different shrimp rearing systems, biofloc and clear water. In our knowledge, the use of molecular tool to study the influence of biofloc consumption on digest process of shrimp was never carried out. We think that our research is new and important to increase knowledge on biofloc topic.

Isotope and fatty acid trends along continental shelf depth gradients: Inshore versus offshore hydrological influences on benthic trophic functioning

Anthropogenic activities and land-based inputs into the sea may influence the trophic structure and functioning of coastal and continental shelf ecosystems, despite the numerous opportunities and services the latter offer to humans and wildlife. In addition, hydrological structures and physical dynamics potentially influence the sources of organic matter (e.g., terrestrial versus marine, or fresh material versus detrital material) entering marine food webs. Understanding the significance of the processes that influence marine food webs and ecosystems (e.g., terrestrial inputs, physical dynamics) is crucially important because trophic dynamics are a vital part of ecosystem integrity. This can be achieved by identifying organic matter sources that enter food webs along inshore–offshore transects. We hypothesised that regional hydrological structures over wide continental shelves directly control the benthic trophic functioning across the shelf. We investigated this issue along two transects in the northern ecosystem of the Bay of Biscay (north-eastern Atlantic). Carbon and nitrogen stable isotope analysis (SIA) and fatty acid analysis (FAA) were conducted on different complementary ecosystem compartments that include suspended particulate organic matter (POM), sedimentary organic matter (SOM), and benthic consumers such as bivalves, large crustaceans and demersal fish. Samples were collected from inshore shallow waters (at ∼1 m in depth) to more than 200 m in depth on the offshore shelf break. Results indicated strong discrepancies in stable isotope (SI) and fatty acid (FA) compositions in the sampled compartments between inshore and offshore areas, although nitrogen SI (δ15N) and FA trends were similar along both transects. Offshore the influence of a permanently stratified area (described previously as a “cold pool”) was evident in both transects. The influence of this hydrological structure on benthic trophic functioning (i.e., on the food sources available for consumers) was especially apparent across the northern transect, due to unusual carbon isotope compositions (δ13C) in the compartments. At stations under the cold pool, SI and FA organism compositions indicated benthic trophic functioning based on a microbial food web, including a significant contribution of heterotrophic planktonic organisms and/or of SOM, notably in stations under the cold pool. On the contrary, inshore and shelf break areas were characterised by a microalgae-based food web (at least in part for the shelf break area, due to slope current and upwelling that can favour fresh primary production sinking on site). SIA and FAA were relevant and complementary tools, and consumers better medium- to long-term system integrators than POM samples, for depicting the trophic functioning and dynamics along inshore–offshore transects over continental shelves.

Incorporation of dietary nitrogen from fish meal and pea meal ( Pisum sativum ) in muscle tissue of Pacific white shrimp ( Litopenaeus vannamei ) fed low protein compound diets

Stable isotope analyses were applied to explore the relative dietary nitrogen contributions from fish meal and pea meal (Pisum sativum) to muscle tissue of Pacific white shrimp postlarvae (141 ± 31 mg) fed low protein diets having different proportions of both ingredients as the sole dietary protein sources. A negative control diet was formulated to contain 100% pea meal and six more isoproteic diets to have decreasing levels of pea meal-derived nitrogen: 95%, 85%, 70%, 55%, 40% and 0% of the initial level. Growth rates were negatively correlated to dietary pea protein inclusion due to progressive essential amino acid deficiencies (sulphur amino acids, threonine, lysine, histidine). The nitrogen turnover rate significantly increased in muscle tissue of shrimps fed diets having high levels of pea meal; however, contrary to observations from a previous study using soy protein, the relative contributions of dietary nitrogen from pea meal to shrimp muscle tissue were equal or higher than expected contributions established by the dietary formulations. Results highlight the effectiveness of stable isotope analysis in assessing the nutritional contributions of alternative ingredients for aquaculture feeds and the potential suitability of pea as a source of protein (provided the diets are nutritionally balanced)

Simultaneous estimation of the nutritional contribution of fish meal, soy protein isolate and corn gluten to the growth of Pacific white shrimp (Litopenaeus vannamei) using dual stable isotope analysis

The nutritional contribution of the dietary nitrogen, carbon and total dry matter supplied by fish meal (FM), soy protein isolate (SP) and corn gluten (CG) to the growth of Pacific white shrimp Litopenaeus vannamei was assessed by means of isotopic analyses. As SP and CG are ingredients derived from plants having different photosynthetic pathways which imprint specific carbon isotope values to plant tissues, their isotopic values were contrasting. FM is isotopically different to these plant meals with regards to both, carbon and nitrogen. Such natural isotopic differences were used to design experimental diets having contrasting isotopic signatures. Seven isoproteic (36% crude protein), isoenergetic (4.7 kcal g−1) diets were formulated; three diets consisted in isotopic controls manufactured with only one main ingredient supplying dietary nitrogen and carbon: 100% FM (diet 100F), 100% SP (diet 100S) and 100% CG (diet 100G). Four more diets were formulated with varying mixtures of these three ingredients, one included 33% of each ingredient on a dietary nitrogen basis (diet 33FSG) and the other three included a proportion 50:25:25 for each of the three ingredients (diets 50FSG, 50SGF and 50GFS). At the end of the bioassay there were no significant differences in growth rate in shrimps fed on the four mixed diets and diet 100F (k=0.215–0.224). Growth rates were significantly lower (k=0.163–0.201) in shrimps grown on diets containing only plant meals. Carbon and nitrogen stable isotope values (δ13C and δ15N) were measured in experimental diets and shrimp muscle tissue and results were incorporated into a three-source, two-isotope mixing model. The relative contributions of dietary nitrogen, carbon and total dry matter from FM, SP and CG to growth were statistically similar to the proportions established in most of the diets after correcting for the apparent digestibility coefficients of the ingredients. Dietary nitrogen available in diet 33FSG was incorporated in muscle tissue at proportions representing 24, 35 and 41% of the respective ingredients. Diet 50GSF contributed significantly higher amounts of dietary nitrogen from CG than from FM. When the level of dietary nitrogen derived from FM was increased in diet 50FSG, nutrient contributions were more comparable to the available dietary proportions as there was an incorporation of 44, 29 and 27% from FM, SP and CG, respectively. Nutritional contributions from SP were very consistent to the dietary proportions established in the experimental diets.

Application of stable isotope analysis to differentiate shrimp extracted by industrial fishing or produced through aquaculture practices.

Carbon and nitrogen stable isotope values were determined in Pacific white shrimp (Litopenaeus vannamei) with the objective of discriminating animals produced through aquaculture practices from those extracted from the wild. Farmed animals were collected at semi-intensive shrimp farms in Mexico and Ecuador. Fisheries-derived shrimps were caught in different fishing areas representing two estuarine systems and four open sea locations in Mexico and Ecuador. Carbon and nitrogen stable isotope values (13CVPDB and 15NAIR) allowed clear differentiation of wild from farmed animals. 13CVPDB and 15NAIR values in shrimps collected in the open sea were isotopically enriched (−16.99‰ and 11.57‰), indicating that these organisms belong to higher trophic levels than farmed animals. 13CVPDB and 15NAIR values of farmed animals (−19.72‰ and 7.85‰, respectively) partially overlapped with values measured in animals collected in estuaries (−18.46‰ and 5.38‰, respectively). Canonical discriminant analysis showed that when used separately and in conjunction, 13CVPDB and I5NAIR values were powerful discriminatory variables and demonstrate the viability of isotopic evaluations to distinguish wild-caught shrimps from aquaculture shrimps. Methodological improvements will define a verification tool to support shrimp traceability protocols.

Assessing land–ocean connectivity via submarine groundwater discharge (SGD) in the Ria Formosa Lagoon (Portugal): combining radon measurements and stable isotope hydrology

Natural radioactive tracer-based assessments of basin-scale submarine groundwater discharge (SGD) are well developed. However, SGD takes place in different modes and the flow and discharge mechanisms involved occur over a wide range of spatial and temporal scales. Quantifying SGD while discriminating its source functions therefore remains a major challenge. However, correctly identifying both the fluid source and composition is critical. When multiple sources of the tracer of interest are present, failure to adequately discriminate between them leads to inaccurate attribution and the resulting uncertainties will affect the reliability of SGD solute loading estimates. This lack of reliability then extends to the closure of local biogeochemical budgets, confusing measures aiming to mitigate pollution. Here, we report a multi-tracer study to identify the sources of SGD, distinguish its component parts and elucidate the mechanisms of their dispersion throughout the Ria Formosa – a seasonally hypersaline lagoon in Portugal. We combine radon budgets that determine the total SGD (meteoric + recirculated seawater) in the system with stable isotopes in water (δ2H, δ18O), to specifically identify SGD source functions and characterize active hydrological pathways in the catchment. Using this approach, SGD in the Ria Formosa could be separated into two modes, a net meteoric water input and another involving no net water transfer, i.e., originating in lagoon water re-circulated through permeable sediments. The former SGD mode is present occasionally on a multi-annual timescale, while the latter is a dominant feature of the system. In the absence of meteoric SGD inputs, seawater recirculation through beach sediments occurs at a rate of  ∼  1.4  ×  106 m3 day−1. This implies that the entire tidal-averaged volume of the lagoon is filtered through local sandy sediments within 100 days ( ∼  3.5 times a year), driving an estimated nitrogen (N) load of  ∼  350 Ton N yr−1 into the system as NO3−. Land-borne SGD could add a further  ∼  61 Ton N yr−1 to the lagoon. The former source is autochthonous, continuous and responsible for a large fraction (59 %) of the estimated total N inputs into the system via non-point sources, while the latter is an occasional allochthonous source capable of driving new production in the system.

Linking nitrogen sources utilised by seagrass in a temperate marine embayment to patterns of seagrass change during drought

Reductions in the extent of seagrass Zostera nigricaulis coverage in Port Phillip Bay (PPB), Australia, between 2000 and 2011 coincided with a prolonged period of drought (1997 to 2009) characterized by decreases in freshwater and nutrient inputs. This led us to hypothesize that patterns of seagrass expansion and decline in PPB may be linked to nutrient availability. Seagrasses in PPB can make use of a range of different nitrogen (N) sources depending on their relative availability. Accordingly, there is a need to identify the origin of the N utilised by seagrasses in order to understand how changes in the availability of nutrients from various sources may influence seagrass growth. This study used stable isotope analysis to estimate the contribution of different sources of N to seagrass growth in different parts of PPB. Source modelling indicated that regional patterns of N source utilisation matched changes in seagrass extent from 2000 to 2011. Regions in which seagrass declined contained a similar array of sources, including significant contributions from the catchment area, whereas regions where seagrass areas remained unchanged were largely dependent on a single N source (either fixation/recycled or sewage-derived). We propose that reductions in N from the catchment during the drought may have contributed to the decline of seagrasses in regions where N from the catchment is an important source. This finding is likely to have implications for the growth, distribution and resilience of Z. nigricaulis seagrass in PPB as well as in other parts of its range in southern Australia.

Stable isotope and multi-analytical investigation of Monte da Cegonha: A Late Antiquity population in southern Portugal

This study presents for the first time the diet of a Late Antiquity population in southern Portugal (Civitas of Pax Julia), from the Roman villa of Monte da Cegonha (predominantly 7th century CE). Stable isotope analysis (δ13C, δ15N, δ18O, 87Sr/86Sr) of human and faunal bone collagen and apatite was conducted in order to understand the influence of Roman subsistence strategies on the way of life of rural inhabitants of the area of Pax Julia and to explore their diet (types of ingested plants, amount of animal resources, terrestrial versus marine resources). X-ray diffraction (XRD) and Fourier transform infra-red spectroscopy (FTIR) analyses were used to determine the degree of bone diagenesis and assess the reliability of the bone stable isotopic composition for palaeodietary reconstruction. Anthropological analysis revealed a cariogenic diet, rich in starchy food and carbohydrates, in at least in two individuals based on the frequency of dental caries. Collagen and apatite carbon isotopic analysis suggested that C3 plants were the basis of the population's diet, complemented with some terrestrial meat and its by-products as reflected by the observed bone collagen nitrogen isotopic composition. Moreover, whilst the fairly low apatite-collagen spacing recorded in some skeletons (at around 4‰) may have been due to freshwater organisms intake, the relatively low nitrogen values observed indicate that this consumption did not occur very often, unless in the form of fresh fish of low trophic level or fish sauces. There were no significant differences in isotopic values depending on gender or burial type. Strontium and oxygen isotopic composition of bone apatite revealed a sedentary community, with the exception of a male individual who probably did not spend his childhood in Monte da Cegonha.

In search of a hidden long-term isolated sub-chondritic 142Nd/144Nd reservoir in the deep mantle : implications for the Nd isotope systematics of the Earth

Here we search for evidence of the existence of a sub-chondritic 142Nd/144Nd reservoir that balances the Nd isotope chemistry of the Earth relative to chondrites. If present, it may reside in the source region of deeply sourced mantle plume material. We suggest that lavas from Hawai’i with coupled elevations in 186Os/188Os and 187Os/188Os, from Iceland that represent mixing of upper mantle and lower mantle components, and from Gough with sub-chondritic 143Nd/144Nd and high 207Pb/206Pb, are favorable samples that could reflect mantle sources that have interacted with an Early-Enriched Reservoir (EER) with sub-chondritic 142Nd/144Nd. High-precision Nd isotope analyses of basalts from Hawai’i, Iceland and Gough demonstrate no discernable 142Nd/144Nd deviation from terrestrial standards. These data are consistent with previous high-precision Nd isotope analysis of recent mantle-derived samples and demonstrate that no mantle-derived material to date provides evidence for the existence of an EER in the mantle. We then evaluate mass balance in the Earth with respect to both 142Nd/144Nd and 143Nd/144Nd. The Nd isotope systematics of EERs are modeled for different sizes and timing of formation relative to ε143Nd estimates of the reservoirs in the μ142Nd = 0 Earth, where μ142Nd is ((measured 142Nd/144Nd/terrestrial standard 142Nd/144Nd)−1 * 10−6) and the μ142Nd = 0 Earth is the proportion of the silicate Earth with 142Nd/144Nd indistinguishable from the terrestrial standard. The models indicate that it is not possible to balance the Earth with respect to both 142Nd/144Nd and 143Nd/144Nd unless the μ142Nd = 0 Earth has a ε143Nd within error of the present-day Depleted Mid-ocean ridge basalt Mantle source (DMM). The 4567 Myr age 142Nd–143Nd isochron for the Earth intersects μ142Nd = 0 at ε143Nd of +8 ± 2 providing a minimum ε143Nd for the μ142Nd = 0 Earth. The high ε143Nd of the μ142Nd = 0 Earth is confirmed by the Nd isotope systematics of Archean mantle-derived rocks that consistently have positive ε143Nd. If the EER formed early after solar system formation (0–70 Ma) continental crust and DMM can be complementary reservoirs with respect to Nd isotopes, with no requirement for significant additional reservoirs. If the EER formed after 70 Ma then the μ142Nd = 0 Earth must have a bulk ε143Nd more radiogenic than DMM and additional high ε143Nd material is required to balance the Nd isotope systematics of the Earth.

Porous PLGA microspheres effectively loaded with BSA protein by electrospraying combined with phase separation in liquid nitrogen

Polymer microspheres loaded with bioactive particles, biomolecules, proteins, and/or growth factors play important roles in tissue engineering, drug delivery, and cell therapy. The conventional double emulsion method and a new method of electrospraying into liquid nitrogen were used to prepare bovine serum albumin (BAS)-loaded poly(lactic-co-glycolic acid) (PLGA) porous microspheres. The particle size, the surface morphology and the internal porous structure of the microspheres were observed using scanning electron microscopy (SEM). The loading efficiency, the encapsulation efficiency, and the release profile of the BSA-loaded PLGA microspheres were measured and studied. It was shown that the microspheres from double emulsion had smaller particle sizes (3-50 m), a less porous structure, a poor loading efficiency (5.2 %), and a poor encapsulation efficiency (43.5%). However, the microspheres from the electrospraying into liquid nitrogen had larger particle sizes (400-600 m), a highly porous structure, a high loading efficiency (12.2%), and a high encapsulation efficiency (93.8%). Thus the combination of electrospraying with freezing in liquid nitrogen and subsequent freeze drying represented a suitable way to produce polymer microspheres for effective loading and sustained release of proteins.