Evaluation of the catalytic specificity,biochemical properties,and milk clotting abilities of an aspartic peptidase from <Emphasis Type="Italic">Rhizomucor miehei</Emphasis>

In this study, we detail the specificity of an aspartic peptidase from Rhizomucor miehei and evaluate the effects of this peptidase on clotting milk using the peptide sequence of k-casein (Abz-LSFMAIQ-EDDnp) and milk powder. Molecular mass of the peptidase was estimated at 37 kDa, and optimum activity was achieved at pH 5.5 and 55 °C. The peptidase was stable at pH values ranging from 3 to 5 and temperatures of up 45 °C for 60 min. Dramatic reductions in proteolytic activity were observed with exposure to sodium dodecyl sulfate, and aluminum and copper (II) chloride. Peptidase was inhibited by pepstatin A, and mass spectrometry analysis identified four peptide fragments (TWSISYGDGSSASGILAK, ASNGGGGEYIFGGYDSTK, GSLTTVPIDNSR, and GWWGITVDRA), similar to rhizopuspepsin. The analysis of catalytic specificity showed that the coagulant activity of the peptidase was higher than the proteolytic activity and that there was a preference for aromatic, basic, and nonpolar amino acids, particularly methionine, with specific cleavage of the peptide bond between phenylalanine and methionine. Thus, this peptidase may function as an important alternative enzyme in milk clotting during the preparation of cheese.     

Global genetic diversity of Aedes aegypti

Mosquitoes, especially Aedes aegypti, are becoming important models for studying invasion biology. We characterized genetic variation at 12 microsatellite loci in 79 populations of Ae. aegypti from 30 countries in six continents, and used them to infer historical and modern patterns of invasion. Our results support the two subspecies Ae. aegypti formosus and Ae. aegypti aegypti as genetically distinct units. Ae. aegypti aegypti populations outside Africa are derived from ancestral African populations and are monophyletic. The two subspecies co‐occur in both East Africa (Kenya) and West Africa (Senegal). In rural/forest settings (Rabai District of Kenya), the two subspecies remain genetically distinct, whereas in urban settings, they introgress freely. Populations outside Africa are highly genetically structured likely due to a combination of recent founder effects, discrete discontinuous habitats and low migration rates. Ancestral populations in sub‐Saharan Africa are less genetically structured, as are the populations in Asia. Introduction of Ae. aegypti to the New World coinciding with trans‐Atlantic shipping in the 16th to 18th centuries was followed by its introduction to Asia in the late 19th century from the New World or from now extinct populations in the Mediterranean Basin. Aedes mascarensis is a genetically distinct sister species to Ae. aegypti s.l. This study provides a reference database of genetic diversity that can be used to determine the likely origin of new introductions that occur regularly for this invasive species. The genetic uniqueness of many populations and regions has important implications for attempts to control Ae. aegypti, especially for the methods using genetic modification of populations.     

Robust estimation of survival and contribution of captive‐bred Mallards Anas platyrhynchos to a wild population in a large‐scale release programme

The survival of captive‐bred individuals from release into the wild to their first breeding season is crucial to assess the success of reintroduction or translocation programmes, and to assess their potential impact of wild populations. However, assessing the survival of captive‐bred individuals following their release is often complicated by immediate dispersal once in the wild. Here, we apply Lindberg's robust design model, a method that incorporates emigration from the study site, to obtain true estimates of survival of captive‐bred Mallards Anas platyrhynchos, a common duck species released on a large scale in Europe since the 1970s. Overall survival rate from release in July until the onset of the next breeding season in April was low (0.18 ± 0.07 se) and equivalent to half the first‐year survival of local wild Mallards. Higher overall detectability and temporary emigration during the hunting period revealed movements in response to hunting pressure. Such low survival of released Mallards during their first year may help prevent large‐scale genetic mixing with the wild population. Nevertheless, by combining our results with regional waterfowl counts, we estimated that a minimum of 34% of the Mallards in the region were of captive origin at the onset of the breeding season. Although most released birds quickly die, restocking for hunting may be of sufficient magnitude to affect the wild population through genetic homogenization or loss of local adaptation. Robust design protocols allow for the estimation of true survival estimates by controlling for permanent and temporary emigration and may require only a moderate increase in fieldwork effort.     

Data strategy for environmental assessment of agricultural regions via LCA: case study of a French catchment

Purpose

Various approaches have been carried out to extrapolate environmental assessments of farms to the regional level, some of them oversimplified and thus leading to high uncertainty. Key challenges include selection of a representative sample, construction of a farm/land use typology, the extrapolation strategy and dealing with data limitations. This work proposes a method for addressing these issues by means of statistically supported approaches.

Methods

We applied a novel approach combining a sampling strategy, estimation of farm-level environmental impacts via life cycle assessment (LCA), a farm typology based on principal component analysis, a statistical method for extending the farm sample given data constraints and finally linear extrapolation based on regional production and land use, taking into account the regional import–export balance. The approach was applied to a French case study, the Lieue de Grève catchment in the dairy-intensive Brittany region. A decision flowchart was developed to generalise the approach for similar applications dealing with farm and LCA data constraints. Additionally, innovative farm practices were modelled and their impacts propagated to the regional level.

Results and discussion

The typology developed identified “dairy”, “beef”, “dairy + beef” and “swine” farms as the dominant farm types in the region. While swine farms had the highest mean impacts per hectare, dairy and dairy + beef farms had impacts two to five times as high as those of beef and swine farms, when extrapolated to the entire catchment. Multiple linear regressions based on an extended farm and LCA dataset were used to predict environmental impacts of dairy farms lacking LCA results, thus increasing their sample size before extrapolation. The inclusion of farm and LCA data from a neighbouring region did not contribute to the accuracy of predicted impacts, as determined by comparing them to those of the farm closest to the dairy cluster’s centre, but rather produced significantly larger coefficients of variation. Results of tests of including two extra-regional farm and LCA datasets helped determine decision rules for the decision flowchart. Modelling of innovative agricultural practices yielded regional impacts consistent with previous estimates.

Conclusions

This approach provides a generalisable approach for farm typologies, data handling and regional extrapolation of farm-level LCAs, applicable to estimate environmental impacts of any agricultural area if requirements of a representative farm sample are met. We demonstrate the utility of the method for estimating effects of innovative agricultural practices on a region’s impacts by modelling practices on virtual farms and extrapolating their results.

     

A constitutive expression system for <Emphasis Type="Italic">Pichia pastoris</Emphasis> based on the <Emphasis Type="Italic">PGK1</Emphasis> promoter

Objectives

To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter.

Results

P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants.

Conclusions

A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.

     

The effects of different cycles of starvation and re‐feeding on growth and body composition in rainbow trout (Oncorhynchus mykiss,Walbaum, 1792)

This study was designed to investigate the effects of starvation and re‐feeding cycles on the growth performance and body chemical composition of Oncorhynchus mykiss juveniles. A total of 360 juveniles with initial mean weights (IW) of 8.46 ± 0.07 g (n = 360) were stocked into 400‐L tanks in triplicate for each group, with 30 juveniles per tanks. The control group received regular feed, as is the common practice. The three other groups were periodically starved: 1 day starvation followed by 6 days re‐feeding (S1), 2 days starvation followed by 5 days re‐feeding (S2) and 3 days starvation followed by 4 days re‐feeding (S3). The experiment lasted for 10 weeks, over the course of which the water flow rate was 4 L min^?1 and the water quality parameters determined as: temperature 14.4 ± 1.1°C, oxygen 8.2 ± 0.4 mg L^?1 and pH 7.5 ± 0.2. At the end of the study, S1 had the best growth performance (final weight, specific growth rate, average daily growth) of all test groups (P < 0.05). The lowest daily feed intake (DFI) and growth performance parameters were observed in S3 (P < 0.05), while protein efficiency ratio (PER), net protein utilization (NPU) and lipid efficiency ratio (LER) were higher in the S3 fish group than in the other groups (P < 0.05). Whole body protein and lipid contents were highest in S1 fish. The hepatosomatic index (HSI) and viscerosomatic index (VSI) were significantly different among groups (P < 0.05). Feed conversion ratio (FCR) was significantly lower in starvation groups S1, S2 and S3 than in the control (P < 0.05). Compensation coefficient (CC) values were higher than 1 in all starvation groups. The concluding indicate that rainbow trout exposed to 1 and 2 days of starvation in week cycles could achieve over compensation compared to the control. Additionally, partial growth compensation and improved feed utilization could be achieved in a starvation group within 3 days in a week, by beginning with the juvenile size over a 10‐week experimental period.