Lipid-based transfection as a method for gene delivery in zebrafish (Danio rerio) embryos

A major challenge to the widespread production of transgenic, knockout and knockdown zebrafish has been the absence of a simple and effective procedure for introducing macromolecules into the fertilized egg. None of the existing techniques for gene transfer in fish embryos has proven to be a major advance over cytoplasm microinjection, which is a technically demanding and time‐consuming procedure. This report addresses this need, considering that the development of protocols for lipid‐based transfection with fish embryos would considerably simplify gene transfer in this complex biological model. In this study, lipid‐based transfection with two different reporter vectors was carried out in zebrafish embryos at different developmental stages. The parameters tested included different plasmid/transfection reagent ratios as well as the influence of an added transfection enhancer reagent. When embryos were transfected in the blastula stage with a pEGFP‐N1 vector, more than 35% successfully incorporated the plasmid and expressed the fluorescent protein 24 h after transfection. The transfection enhancer did not show any significant effect in our experiments. This work presents an approach to implement this technique as a faster, cheaper and more practical alternative than microinjection.     

Effect of industrial dehydration on the soluble carbohydrates and dietary fiber fractions in legumes

The effects of soaking, cooking, and industrial dehydration treatments on soluble carbohydrates, including raffinose family oligosaccharides (RFOs), and also on total dietary fiber (TDF), insoluble dietary fiber (IDF), and soluble (SDF) dietary fiber fractions were studied in legumes (lentil and chickpea). Ciceritol and stachyose were the main alpha-galactosides for chickpea and lentil, respectively. The processing involved a drastic reduction of soluble carbohydrates of these legumes, 85% in the case of lentil and 57% in the case of chickpea. The processed legume flours presented low residual levels of alpha-galactosides, which are advisable for people with digestive problems. Processing of legumes involved changes in dietary fiber fractions. A general increase of IDF (27-36%) due to the increase of glucose and Klason lignin was observed. However, a different behavior of SDF was exhibited during thermal dehydration, this fraction increasing in the case of chickpea (32%) and decreasing in the case of lentil (27%). This is probably caused by the different structures and compositions of the cell wall networks of the legumes.     

Intensive rearing of juvenile crayfish (Pacifastacus leniusculus,Astacidae) during the first 6 months: effects of size grading

Recent advances in crayfish feeding have enabled the development of size grading studies from the start of first‐feeding. A 180‐day experiment aimed at intensive rearing of Pacifastacus leniusculus was carried out under controlled conditions, evaluating the effects of size grading at two different periods from the onset of exogenous feeding. Stage 2 juveniles were stocked in fibreglass tanks at a density of 100 m², and fed a dry diet for salmonids combined with restricted amounts of Artemia cysts. Five groups were tested: no grading, grading at 60 days (large and small size) and grading at 100 days (large and small size). After 6 months, no significant differences were found in the survival among groups (mean: 73.06%). The highest final growth (pooled results from upper and lower classes: 17.39 mm carapace length, 1.43 g weight) was achieved by the crayfish sorted at 60 days, showing significant differences from the ungraded group. Smaller crayfish graded at 60 days grew significantly faster than smaller crayfish graded at 100 days. The food conversion ratio was lower in the graded groups (mean: 2.64), showing significant differences from the ungraded group (3.23). This study shows that size grading allows a better performance and an improved feeding efficiency.     

Nursery areas of Micromesistius poutassou and Sardina pilchardus unveil their reproductive strategies in the northwestern Mediterranean Sea

Winter conditions in the NW Mediterranean cause instability of the water column and non-geostrophic dynamics, such as vertical mixing and convection are significant. These events involve nutrient supply to the photic zone that can sustain high productivity. In this study, we aim to investigate the role of winter hydrodynamics on the spawning strategies of Sardina pilchardus and Micromesistius poutassou. Data were obtained on two oceanographic cruises (February 2017 and 2018) off the Catalan coast. The occurrence of S. pilchardus eggs very close to the coast indicated a clear preference of the species for spawning in coastal areas. Preflexion and postflexion larvae exhibited a slightly wider distribution showing a clear association with the cold, less saline and more productive coastal waters. Preflexion larvae of M. poutassou were found on the upper slope and over the shelf, being offshore limited by the shelf/slope front present all along the slope. The front would act as a barrier preventing their dispersion towards the open sea. M. poutassou larvae in advanced developmental stages were located close to the coast in the productive shelf waters, with instabilities of the front contributing to larval transport from offshore waters to the coast. The vertical distribution of both species showed high variability, not only related with the daily cycle or developmental stage, but also with the vertical structure of the water column. Overall, the results provide some clues on how the spawning strategies of both species may evolve under future scenarios of higher winter-stratification, because of the global warming.     

Molecular characterisation of Cryptosporidium isolates from pet reptiles

In this study, we investigated the presence of Cryptosporidium in 171 faecal samples from reptiles commonly used as pet animals. These include lizards belonging to the genera Eublepharis, Pogona, Chlamydosaurus, Hemiteconyx, Teratoscincus, Tiliqua, Iguana, and Chamaeleo, snakes of the genera Lampropeltis, Elaphe, Python, Boa and Corallus, and tortoises belonging to the genera Testudo and Kinixys. Cryptosporidium oocysts were detected by immunofluorescence using a commercially available kit and cryptosporidial DNA by amplification of a polymorphic fragment of the 18S rDNA and the HSP70 locus.Cryptosporidium was detected in 38.6% and 25.1% of the samples analysed by immunofluorescence and PCR, respectively. Molecular characterisation of the isolates confirmed that C. serpentis and C. varanii (syn. C. saurophilum) are the main species involved in infection in pet reptiles but also showed the presence of C. parvum and C. muris, as well as other species or genotypes of this parasite including the Cryptosporidium mouse genotype and Cryptosporidium tortoise genotype previously described in reptiles. In addition, a Cryptosporidium sp. was isolated from a chameleon and a python.     

Fatal toxoplasmosis and concurrent Calodium hepaticum infection in Korean squirrels (Tanias sibericus)

Four Korean squirrels (Tanias siberius) imported in Spain from People's Republic of China died 2 days after their arrival at a pet shop. They had neurological signs associated with generalized toxoplasmosis involving brain, lungs, liver, and the heart. Toxoplasma gondii-like tachyzoites and tissue cysts were found in organs of all four squirrels. The protozoa stained positively with T. gondii polyclonal antibodies and were ultrastructurally similar to T. gondii. Calodium (Capillaria) hepaticum infection was found in the liver of one squirrel.     

Decapsulated Artemia Cysts: A Suitable Dietary Supplement for Juvenile Tench (Tinca tinca L.)

There are no specifically formulated dry foods for tench (Tinca tinca L.), which forces farmers to use diets formulated for other fish species. This has major drawbacks, such as high mortality, slow growth, and body deformities. A 120 day experiment was performed with five-month-old juvenile tench (initial mean weight: 0.388 g; total length: 31.78 mm) to evaluate decapsulated Artemia cysts as a supplement to a dry diet for other fish species. Three treatments, differing in the daily supplement, were tested: 1,800 freshly hatched nauplii, 1,800 cysts, and 300 cysts per g of tench biomass. Final survival ranged between 95.3% and 97.9%. Juvenile tench that received the supplement of 1,800 decapsulated Artemia cysts had a specific growth rate (1.28), weight (1.83 g), and total length (52.30 mm) significantly higher than those with the same amount of nauplii. The lowest supplement (300 cysts/g of fish biomass) allowed significantly lower growth and higher condition coefficient (1.40) than the rest. Animals with body deformities (1.06%) were only recorded in the groups that received the lowest cyst supplement. Results showed Artemia cysts are a suitable dietary supplement for juvenile tench, being an advantageous alternative to live nauplii.     

Effects of different bronopol treatments on final survival rates in the artificial incubation of crayfish eggs (Pacifastacus leniusculus,Astacidae)

Two experiments were conducted on the effects of bronopol in the artificial incubation of crayfish eggs (Pacifastacus leniusculus) with the aim to search an alternative to formaldehyde. In the first experiment, 50, 250, 500 and 1000 ppm bronopol and 3000 ppm formaldehyde (control) in periodical administrations were tested on a density of 6.6 eggs cm^?2. After 44 days of incubation, the highest survival was obtained with 1000 ppm bronopol (81.9% to stage 2 juvenile, with no significant difference from formaldehyde), whereas lower bronopol concentrations resulted in significantly lower survival. In the second experiment, 1000, 3000 and 5000 ppm bronopol and 3000 ppm formaldehyde (control) administered for 15 min every second day were tested on eggs at a density of 20 eggs cm^?2. After 78 days of incubation, bronopol at 3000 ppm allowed for a stage 2 juvenile survival rate of 65.0% (with no significant difference from formaldehyde), whereas significantly lower survival was obtained with 1000 ppm or 5000 ppm. This study shows that bronopol may constitute an alternative to formaldehyde in the artificial incubation of crayfish eggs. A concentration of 3000 ppm administered for 15 min every second day may be adequate even on long incubations at high densities (at least 20 eggs cm^?2, one complete layer).     

One-Step Extraction Procedure as an Easy Tool for Monitoring a Full-Scale Composting Plant

ABSTRACT. Composting is an efficient technique for managing the increasing production of organic wastes since it transforms them into a valuable product that can be applied to soil to improve it. However, it is necessary to ensure the adequate properties of this product for a proper agricultural or environmental application. Various parameters have been proposed as estimators of the evolution of the composting process, nevertheless there is a lack of easy techniques with a wide application range for monitoring it. This study evaluated some parameters that can be used to test compost evolution in samples of composted sewage sludge under full-scale conditions. Total extractable carbon (TEC) by alkaline solution of sodium pyrophosphate, humic acids (HA), fulvic acids (FA), and the ratio of them (HA/FA) were obtained by a one-step extraction procedure. Results were compared with those obtained after an official extraction method for the same parameters. Total organic matter was calculated by calcination (TOM%). The objective was to assess the usefulness of the one-step extraction as an easy and cheap tool for monitoring the composting process. The results indicate that both extraction procedures show a statistically significant correlation between them and with TOM%. Therefore, TEC obtained by the one-step extraction procedure can provide useful information about the evolution of the composting process of compost samples with the easiest methodology.