Prediction of apparent protein digestibility of ingredients and diets by in vitro pH-stat degree of protein hydrolysis with species-specific enzymes for juvenile Pacific white shrimp Litopenaeus vannamei

Aquafeed production faces global issues related to availability of feed ingredients. Feed manufacturers require greater flexibility in order to develop nutritional and cost-effective formulations that take into account nutrient content and availability of ingredients. The search for appropriate ingredients requires detailed screening of their potential nutritional value and variability at the industrial level. In vitro digestion of feedstuffs by enzymes extracted from the target species has been correlated with apparent protein digestibility (APD) in fish and shrimp species. The present study verified the relationship between APD and in vitro degree of protein hydrolysis (DH) with Litopenaeus vannamei hepatopancreas enzymes in several different ingredients (n = 26): blood meals, casein, corn gluten meal, crab meal, distiller`s dried grains with solubles, feather meal, fish meals, gelatin, krill meals, poultry by-product meal, soybean meals, squid meals and wheat gluten. The relationship between APD and DH was further verified in diets formulated with these ingredients at 30% inclusion into a reference diet. APD was determined in vivo (30.1 +/- 0.5 degrees C, 32.2 +/- 0.4%.) with juvenile L vannamei (9 to 12 g) after placement of test ingredients into a reference diet (35 g kg(-1) CP: 8.03 g kg(-1) lipid; 2.01 kcal g(-1)) with chromic oxide as the inert marker. In vitro DH was assessed in ingredients and diets with standardized hepatopancreas enzymes extracted from pond-reared shrimp. The DH of ingredients was determined under different assay conditions to check for the most suitable in vitro protocol for APD prediction: different batches of enzyme extracts (HPf5 or HPf6), temperatures (25 or 30 degrees C) and enzyme activity (azocasein): crude protein ratios (4 U: 80 mg CP or 4 U: 40 mg CP). DH was not affected by ingredient proximate composition. APD was significantly correlated to DH in regressions considering either ingredients or diets. The relationships between APD and DH of the ingredients could be suitably adjusted to a Rational Function (y = (a + bx)/(1 + cx + dx2), n = 26. Best in vitro APD predictions were obtained at 25 degrees C, 4 U: 80 mg CP both for ingredients (R(2) = 0.86: P = 0.001) and test diets (R(2) = 0.96; P = 0.007). The regression model including all 26 ingredients generated higher prediction residuals (i.e., predicted APD - determined APD) for corn gluten meal, feather meal. poultry by-product meal and krill flour. The remaining test ingredients presented mean prediction residuals of 3.5 points. A model including only ingredients with APD>80% showed higher prediction precision (R(2) = 0.98: P = 0.000004; n = 20) with average residual of 1.8 points. Predictive models including only ingredients from the same origin (e.g., marine-based, R(2) = 0.98; P = 0.033) also displayed low residuals. Since in vitro techniques have been usually validated through regressions against in vivo APD, the DH predictive capacity may depend on the consistency of the in vivo methodology. Regressions between APD and DH suggested a close relationship between peptide bond breakage by hepatopancreas digestive proteases and the apparent nitrogen assimilation in shrimp, and this may be a useful tool to provide rapid nutritional information. (C) 2009 Elsevier B.V. All rights reserved.

Relative abundance and population biology of the non-indigenous crab Charybdis hellerii (Crustacea: Brachyura: Portunidae) in a southwestern Atlantic estuary-bay complex

Invasive species are potential threats to biodiversity, especially if they become established and outnumber native species. In this study, a population of the non-indigenous crab Charybdis hellerii was analyzed in an estuary-bay complex on the southeastern Brazilian coast, with respect to its abundance relative to sympatric native brachyuran species, as well as the size structure, sexual maturity, sex ratio, frequency of mutilation, reproductive period, and development of the reproductive system. Crabs were sampled monthly both in the intertidal zone of rocky shores and on sublittoral soft-bottom. Nine species were recorded on the rocky shores, where C. hellerii was the second most abundant species; only three individuals of C. hellerii were collected in the sublittoral samples. This population of C. hellerii showed a unimodal size structure composed mainly of mature individuals; males were larger than females, and the sex ratio was skewed toward males (3.1:1). About 46.9% of the individuals (75 of 160 crabs) had mutilated or regenerating appendages, more frequent in males (56.8%) than in females (28.2%), which may reflect both inter- and intraspecific agonistic interactions. A continuous reproductive pattern is suggested for this population, although ovigerous females occurred unevenly during the year, with 58.82% of them being collected in winter. There was evidence of multiple spawning, since the ovigerous females with an initial egg mass showed mature ovaries as well as seminal receptacles filled with sperm. C. hellerii is well established in the estuary-bay complex, but is concentrated in intertidal and shallow subtidal rocky shores, where it may compete with and replace other species such as the portunid Cronius ruber. This study also highlights the importance of systematic monitoring studies to evaluate the effects of the introduction of non-indigenous species on ecologically similar natives.

Species Distribution Modeling for Conservation Purposes

Species distribution models (SDMs) can be useful for different conservation purposes. We discuss the importance of fitting spatial scale and using current records and relevant predictors aiming conservation. We choose jaguar (Panthera onca) as a target species and Brazil and Atlantic Forest biome as study areas. We tested two different extents (continent and biome) and resolutions (similar to 4 Km and similar to 1 Km) in Maxent with 186 records and 11 predictors (bioclimatic, elevation, land-use and landscape structure). All models presented satisfactory AUC values (>0.70) and low omission errors (<23%). SDMs were scale-sensitive as the use of reduced extent implied in significant gains to model performance generating more constrained and real predictive distribution maps. Continental-scale models performed poorly in predicting potential current jaguar distribution, but they reached the historic distribution. Specificity increased significantly from coarse to finer-scale models due to the reduction of overprediction. The variability of environmental space (E-space) differed for most of climatic variables between continental and biome-scale and the representation of the E-space by predictors differed significantly (t = 2.42, g.I. = 9, P < 0.05). Refining spatial scale, incorporating landscape variables and improving the quality of biological data are essential for improving model prediction for conservation purposes.

Species identification, distributioin and reproductive interactions of common smoot-hound and blackspotted smooth-hound shark (Genus mustelus) in the Adriatic Sea

Longstanding taxonomic ambiguity and uncertainty exist in the identification of the common (M. mustelus) and blackspotted (M. punctulatus) smooth-hound in the Adriatic Sea. The lack of a clear and accurate method of morphological identification, leading to frequent misidentification, prevents the collation of species-specific landings and survey data for these fishes and hampers the delineation of the distribution ranges and stock boundaries of the species. In this context, adequate species-specific conservation and management strategies can not be applied without risks of population declining and local extinction. In this thesis work I investigated the molecular ecology of the two smooth-hound sharks which are abundant in the demersal trawl surveys carried out in the NC Adriatic Sea to monitor and assess the fishery resources. Ecological and evolutionary relationships were assessed by two molecular tests: a DNA barcoding analysis to improve species identification (and consequently the knowledge of their spatial ecology and taxonomy) and a hybridization assay based on the nuclear codominant marker ITS2 to evaluate reproductive interactions (hybridization or gene introgression). The smooth-hound sharks (N=208) were collected during the MEDITS 2008 and 2010 campaigns along the Italian and Croatian coasts of the Adriatic Sea, in the Sicilian Channel and in the Algerian fisheries. Since the identification based on morphological characters is not strongly reliable, I performed a molecular identification of the specimens producing for each one the cytochrome oxidase subunit 1 (COI) gene sequence (ca. 640 bp long) and compared them with reference sequences from different databases (GenBank and BOLD). From these molecular ID data I inferred the distribution of the two target species in the NC Adriatic Sea. In almost the totality of the MEDITS hauls I found no evidence of species sympatry. The data collected during the MEDITS survey showed an almost different distribution of M. mustelus (confined along the Italian coasts) and M. punctulatus (confined along the Croatian coasts); just one sample (Gulf of Venice, where probably the ranges of the species overlap) was found to have catches of both the species. Despite these data results suggested no interaction occurred between my two target species at least during the summertime (the period in which MEDITS survey is carried out), I still wanted to know if there were inter-species reproductive interactions so I developed a simple molecular genetic method to detect hybridization. This method is based on DNA sequence polymorphism among species in the nuclear ribosomal Internal Transcribed Spacer 2 locus (ITS2). Its application to the 208 specimens collected raised important questions regarding the ecology of this two species in the Adriatic Sea. In fact results showed signs of hybridization and/or gene introgression in two sharks collected during the trawl survey of 2008 and one collected during the 2010 one along the Italian and Croatian coasts. In the case that it will be confirmed the hybrid nature of these individuals, a spatiotemporal overlapping of the mating behaviour and ecology must occur. At the spatial level, the northern part of the Adriatic Sea (an area where the two species occur with high frequency of immature individuals) could likely play the role of a common nursery area for both species.

Pharmaceutical residues in aquatic systems: mode of action and effects on mussel physiology

Pharmaceutical residues contaminate aquatic ecosystems as a result of their widespread human and veterinary usage. Since continuously released and not efficiently removed, certain pharmaceuticals exhibit pseudo-persistence thus generating concerns for the health of aquatic wildlife. This work aimed at assessing on mussels Mytilus galloprovincialis, under laboratory conditions, the effects of three pharmaceuticals, carbamazepine (antiepileptic), propranolol (β-blocker) and oxytetracycline (antibiotic), to evaluate if the human-based mode of action of these molecules is conserved in invertebrates. Furthermore, in the framework of the European MEECE Programme, mussels were exposed to oxytetracycline and copper at increasing temperatures, simulating variations due to climate changes. The effects of these compounds were assessed evaluating a battery of biomarkers, the expression of HSP70 proteins and changes in cAMP-related parameters. A decrease in lysosomal membrane stability, induction of oxidative stress, alterations of cAMP-dependent pathway and the induction of defense mechanisms were observed indicating the development of a stress syndrome, and a worsening in mussels health status. Data obtained in MEECE Programme confirmed that the toxicity of substances can be enhanced following changes in temperature. The alterations observed were obtained after exposure to pharmaceuticals at concentrations sometimes lower than those detected in the aquatic environment. Hence, further research is advisable regarding subtle effects of pharmaceuticals on non-target organisms. Furthermore, results obtained during a research stay in the laboratories of Cádiz University (Spain) are presented. The project aimed at measuring possible effects of polluted sediments in Algeciras Bay (Spain) and in Cádiz Bay, by assessing different physiological parameters in caged crabs Carcinus maenas and clams Ruditapes decussatus exposed in situ for 28 days. The neutral red retention assay was adapted to these species and proved to be a sensitive screening tool for the assessment of sediment quality.

Phytoplankton Response to Environmental Variables and Organic Pollutants. Laboratory Cultures and Numerical Simulations Experiments

Herbicides are becoming emergent contaminants in Italian surface, coastal and ground waters, due to their intensive use in agriculture. In marine environments herbicides have adverse effects on non-target organisms, as primary producers, resulting in oxygen depletion and decreased primary productivity. Alterations of species composition in algal communities can also occur due to the different sensitivity among the species. In the present thesis the effects of herbicides, widely used in the Northern Adriatic Sea, on different algal species were studied. The main goal of this work was to study the influence of temperature on algal growth in the presence of the triazinic herbicide terbuthylazine (TBA), and the cellular responses adopted to counteract the toxic effects of the pollutant (Chapter 1 and 2). The development of simulation models to be applied in environmental management are needed to organize and track information in a way that would not be possible otherwise and simulate an ecological prospective. The data collected from laboratory experiments were used to simulate algal responses to the TBA exposure at increasing temperature conditions (Chapter 3). Part of the thesis was conducted in foreign countries. The work presented in Chapter 4 was focused on the effect of high light on growth, toxicity and mixotrophy of the ichtyotoxic species Prymnesium parvum. In addition, a mesocosm experiment was conducted in order to study the synergic effect of the pollutant emamectin benzoate with other anthropogenic stressors, such as oil pollution and induced phytoplankton blooms (Chapter 5).

Urine proteome in animals of veterinary interest: species comparison and new biomarkers of nephropathy

Urine is considered an ideal source of biomarkers, however in veterinary medicine a complete study on the urine proteome is still lacking. The present work aimed to apply proteomic techniques to the separation of the urine proteome in dogs, cats, horses, cows and some non-conventional species. High resolution electrophoresis (HRE) was also validated for the quantification of albuminuria in dogs and cats. In healthy cats, applying SDS-PAGE and 2DE coupled to mass spectrometry (MS), was produced a reference map of the urine proteome. Moreover, 13 differentially represented urine proteins were linked with CKD, suggesting uromodulin, cauxin, CFAD, Apo-H, RBP and CYSM as candidate biomarkers to be investigated further. In dogs, applying SDS-PAGE coupled to MS, was highlighted a specific pattern in healthy animals showing important differences in patients affected by leishmaniasis. In particular, uromodulin could be a putative biomarker of tubular damage while arginine esterase and low MW proteins needs to be investigated further. In cows, applying SDS-PAGE, were highlighted different patterns between heifers and cows showing some interesting changes during pregnancy. In particular, putative alpha-fetoprotein and b-PAP needs to be further investigated. In horses, applying SDS-PAGE, was produced a reference profile characterized by 13±4 protein bands and the most represented one was the putative uromodulin. Proteinuric horses showed the decrease of the putative uromodulin band and the appearance of 2 to 4 protein bands at higher MW and a greater variability in the range of MW between 49 and 17 kDa. In felids and giraffes was quantified proteinuria reporting the first data for UTP and UPC. Moreover, by means of SDS-PAGE, were highlighted species-specific electrophoretic patterns in big felids and giraffes.

INTEGRATED MANAGEMENT OF THE INVASIVE SPECIES CIRSIUM ARVENSE, CANADA THISTLE, AND PHRAGMITES AUSTRALIS, COMMON REED: USING ECOLOGICALLY BASED CONTROL MEASURES AT EACH STAGE OF THE INVASION PROCESS

Invasive plant species threaten natural areas by reducing biodiversity and altering ecosystem functions. They also impact agriculture by reducing crop and livestock productivity. Millions of dollars are spent on invasive species control each year, and traditionally, herbicides are used to manage invasive species. Herbicides have human and environmental health risks associated with them; therefore, it is essential that land managers and stakeholders attempt to reduce these risks by utilizing the principles of integrated weed management. Integrated weed management is a practice that incorporates a variety of measures and focuses on the ecology of the invasive plant to manage it. Roadways are high risk areas that have high incidence of invasive species. Roadways act as conduits for invasive species spread and are ideal harborages for population growth; therefore, roadways should be a primary target for invasive species control. There are four stages in the invasion process which an invasive species must overcome: transport, establishment, spread, and impact. The aim of this dissertation was to focus on these four stages and examine the mechanisms underlying the progression from one stage to the next, while also developing integrated weed management strategies. The target species were Phragmites australis, common reed, and Cisrium arvense, Canada thistle. The transport and establishment risks of P. australis can be reduced by removing rhizome fragments from soil when roadside maintenance is performed. The establishment and spread of C. arvense can be reduced by planting particular resistant species, e.g. Heterotheca villosa, especially those that can reduce light transmittance to the soil. Finally, the spread and impact of C. arvense can be mitigated on roadsides through the use of the herbicide aminopyralid. The risks associated with herbicide drift produced by application equipment can be reduced by using the Wet-Blade herbicide application system.

Species mobility and frequency dynamics in a Swiss limestone grassland

Community dynamics in a calcareous grassland (Mesobrometum) in Egerkingen (Jura mountains, Switzerland) were investigated for 53 non-woody species in 25 1-m2 plots over 6 years. 50 0.0 1-m2 subplots per plot were recorded. The derived variables were spatial frequency, temporal frequency, frequency fluctuation, turnover, and cumulative frequency (each species), and cumulative species richness (all species). Spectra for 53 species of all variables were different for the two investigated spatial scales (0.0 1 m2, 1 m2). The comparison with other investigations of similar grass lands showed that the behaviour of some species is specific for this type of vegetation in general (e.g. Achillea millefolium, Arrhenatherum elatius, Bromus erectus ), but most species behaved in a stand-specific way, i.e. they may play another (similar or completely different) role in another grassland stand. Six spatio-temporal patterns were defined across species. To understand community dynamics, not only the dynamics of mobility but also of frequency fluctuations and spatial distribution of the species are fundamental. In addition, the understanding of temporal behaviour of all species present should be included. Averages always hide important information of vegetation dynamics, as was shown by the present investigation.

Changes in species composition in alpine snowbeds with climate change inferred from small-scale spatial patterns

Alpine snowbeds are characterised by a very short growing season. However, the length of the snow-free period is increasingly prolonged due to climate change, so that snowbeds become susceptible to invasions from neighbouring alpine meadow communities. We hypothesised that spatial distribution of species generated by plant interactions may indicate whether snowbed species will coexist with or will be out-competed by invading alpine species – spatial aggregation or segregation will point to coexistence or competitive exclusion, respectively. We tested this hypothesis in snowbeds of the Swiss Alps using the variance ratio statistics. We focused on the relationships between dominant snowbed species, subordinate snowbed species, and potentially invading alpine grassland species. Subordinate snowbed species were generally spatially aggregated with each other, but were segregated from alpine grassland species. Competition between alpine grassland and subordinate snowbed species may have caused this segregation. Segregation between these species groups increased with earlier snowmelt, suggesting an increasing importance of competition with climate change. Further, a dominant snowbed species (Alchemilla pentaphyllea) was spatially aggregated with subordinate snowbed species, while two other dominants (Gnaphalium supinum and Salix herbacea) showed aggregated patterns with alpine grassland species. These dominant species are known to show distinct microhabitat preferences suggesting the existence of hidden microhabitats with different susceptibility to invaders. These results allow us to suggest that alpine snowbed areas are likely to be reduced as a consequence of climate change and that invading species from nearby alpine grasslands could outcompete subordinate snowbed species. On the other hand, microhabitats dominated by Gnaphalium or Salix seem to be particularly prone to invasions by non-snowbed species.

Assessing the effectiveness of a long-standing rocky intertidal protected area and its contribution to the regional conservation of species, habitats and assemblages

1. The acceptance of reserves as a useful management strategy relies on evidence of their effectiveness in preserving stocks of harvested species and conserving biodiversity. A history of ad hoc decisions in terrestrial and marine protected area planning has meant that many of these areas are contributing inefficiently to conservation goals. The conservation value of existing protected areas should be assessed when planning the placement of additional areas in a reserve network. 2. This study tested (1) the effectiveness of protection for intertidal molluscs of a marine reserve (Bouddi Marine Extension, NSW, Australia) established in 1971, and (2) the contribution of the protected area to the conservation of regional species, assemblages, and habitats. 3. The shell length and population density of one harvested (Cellana tramoserica), and three non-harvested species (Bembicium nanum, Morula marginalba, Nerita atramentosa) of intertidal molluscs were examined in the protected area and two reference locations over two seasons. 4. The heavily collected limpet C. tramoserica was significantly larger in the protected area and was the only species to exhibit a significant difference. No species significantly differed in population density between the protected area and reference locations. 5. Temporally replicated surveys of macro-molluscs at 21 locations over 75km of coastline identified that the existing protected area included 50% of species, two of five assemblage types and 19 of 20 intertidal rocky shore habitats surveyed in the study region. Reservation of a further three rocky reefs would protect a large proportion of species (71%), a representative of each assemblage and all habitat types. 6. Despite originally being selected in the absence of information on regional biodiversity, the protected area is today an effective starting point for expansion to a regional network of intertidal protected areas.

Little evidence for release from herbivores as a driver of plant invasiveness from a multi-species herbivore-removal experiment

Enemy release is frequently posed as a main driver of invasiveness of alien species. However, an experimental multi-species test examining performance and herbivory of invasive alien, non-invasive alien and native plant species in the presence and absence of natural enemies is lacking. In a common garden experiment in Switzerland, we manipulated exposure of seven alien invasive, eight alien non-invasive and fourteen native species from six taxonomic groups to natural enemies (invertebrate herbivores), by applying a pesticide treatment under two different nutrient levels. We assessed biomass production, herbivore damage and the major herbivore taxa on plants. Across all species, plants gained significantly greater biomass under pesticide treatment. However, invasive, non-invasive and native species did not differ in their biomass response to pesticide treatment at either nutrient level. The proportion of leaves damaged on invasive species was significantly lower compared to native species, but not when compared to non-invasive species. However, the difference was lost when plant size was accounted for. There were no differences between invasive, non-invasive and native species in herbivore abundance. Our study offers little support for invertebrate herbivore release as a driver of plant invasiveness, but suggests that future enemy release studies should account for differences in plant size among species.

Human and ecological risk assessment of a crop protection chemical: a case study with the azole fungicide epoxiconazole.

Conventional risk assessments for crop protection chemicals compare the potential for causing toxicity (hazard identification) to anticipated exposure. New regulatory approaches have been proposed that would exclude exposure assessment and just focus on hazard identification based on endocrine disruption. This review comprises a critical analysis of hazard, focusing on the relative sensitivity of endocrine and non-endocrine endpoints, using a class of crop protection chemicals, the azole fungicides. These were selected because they are widely used on important crops (e.g. grains) and thereby can contact target and non-target plants and enter the food chain of humans and wildlife. Inhibition of lanosterol 14α-demethylase (CYP51) mediates the antifungal effect. Inhibition of other CYPs, such as aromatase (CYP19), can lead to numerous toxicological effects, which are also evident from high dose human exposures to therapeutic azoles. Because of its widespread use and substantial database, epoxiconazole was selected as a representative azole fungicide. Our critical analysis concluded that anticipated human exposure to epoxiconazole would yield a margin of safety of at least three orders of magnitude for reproductive effects observed in laboratory rodent studies that are postulated to be endocrine-driven (i.e. fetal resorptions). The most sensitive ecological species is the aquatic plant Lemna (duckweed), for which the margin of safety is less protective than for human health. For humans and wildlife, endocrine disruption is not the most sensitive endpoint. It is concluded that conventional risk assessment, considering anticipated exposure levels, will be protective of both human and ecological health. Although the toxic mechanisms of other azole compounds may be similar, large differences in potency will require a case-by-case risk assessment.

Herbaceous plant species invading natural areas tend to have stronger adaptive root foraging than other naturalized species

Although plastic root-foraging responses are thought to be adaptive, as they may optimize nutrient capture of plants, this has rarely been tested. We investigated whether nutrient-foraging responses are adaptive, and whether they pre-adapt alien species to become natural-area invaders. We grew 12 pairs of congeneric species (i.e., 24 species) native to Europe in heterogeneous and homogeneous nutrient environments, and compared their foraging responses and performance. One species in each pair is a USA natural-area invader, and the other one is not. Within species, individuals with strong foraging responses, measured as plasticity in root diameter and specific root length, had a higher biomass. Among species, the ones with strong foraging responses, measured as plasticity in root length and root biomass, had a higher biomass. Our results therefore suggest that root foraging is an adaptive trait. Invasive species showed significantly stronger root-foraging responses than non-invasive species when measured as root diameter. Biomass accumulation was decreased in the heterogeneous vs. the homogeneous environment. In aboveground, but not belowground and total biomass, this decrease was smaller in invasive than in non-invasive species. Our results show that strong plastic root-foraging responses are adaptive, and suggest that it might aid in pre-adapting species to becoming natural-area invaders.

A missing link between facilitation and plant species coexistence: nurses benefit generally rare species more than common ones

1. Positive interactions among plants can increase species richness by relaxing environmental filters and providing more heterogeneous environments. However, it is not known if facilitation could affect coexistence through other mechanisms. Most studies on plant coexistence focus on negative frequency-dependent mechanisms (decreasing the abundance of common species); here, we test if facilitation can enhance coexistence by giving species an advantage when rare. 2. To test our hypothesis, we used a global data set from drylands and alpine environments and measured the intensity of facilitation (based on co-occurrences with nurse plants) for 48 species present in at least 4 different sites and with a range of abundances in the field. We compared these results with the degree of facilitation experienced by species which are globally rare or common (according to the IUCN Red List), and with a larger data base including over 1200 co-occurrences of target species with their nurses. 3. Facilitation was stronger for rare species (i.e. those having lower local abundances or considered endangered by the IUCN) than for common species, and strongly decreased with the abundance of the facilitated species. These results hold after accounting for the distance of each species from its ecological optimum (i.e. the degree of functional stress it experiences). 4. Synthesis. Our results highlight that nurse plants not only increase the number of species able to colonize a given site, but may also promote species coexistence by preventing the local extinction of rare species. Our findings illustrate the role that nurse plants play in conserving endangered species and link the relationship between facilitation and diversity with coexistence theory. As such, they provide further mechanistic understanding on how facilitation maintains plant diversity.