Feeding regimes affected the circadian rhythms of pancreatic digestive enzymes and somatic growth in flathead grey mullet (Mugil cephalus) fry.

The effect of different feeding regimes on the circadian rhythms of pancreatic digestive enzyme activities was evaluated in Mugil cephalus fry weighting 0.34 ± 0.01 g. Feeding regimes (feeding ration = 3% stocked biomass) differed on the number of meals offered per day: one, two and ten meals per day (R1, R2 and R3, respectively). The number of meals per day affected somatic growth; in particular, fry from the R3 group (0.80 ± 0.01 g) grew better than their congeners from R2 (0.70 ± 0.01 g) and R1 (0.63 ± 0.01 g) groups (P < 0.05). Feeding behaviour was modulated by the feeding regime, being the maximal gut fullness values found just after meal distribution in R1 and R2 groups, whereas this trend was not observed when feed was offered continuously during light hours (R3). Fry from R1 and R2 groups showed hyperphagia as they tended to store in their gut as much as possible feed particles to be later digested due to the limited daily meals. This strategy negatively affected feed digestion due to inappropriate enzyme to substrate ratio, changes in digestive enzyme activities and chyme transit times, which ultimately impaired growth performance. Enzyme activities were modulated by the number of meals, the more frequent the meals offered, the lower enzyme activities, supporting the hypothesis that digestive function is adapted to obtain a maximum benefit of the ingested nutrients. Present results showed that feeding grey mullet fry continuously during day light hours optimized feed digestion and promoted fry growth.

Perampanel in chronic insomnia.

OBJECTIVES: Insomnia is the most prevalent sleep disorder in the general population, and one of the most frequent reasons for consultation in the Sleep Units. Perampanel is an antiepileptic also effective on the structure of sleep, and in restless legs syndrome. We describe the first study that evaluates perampanel in patients with chronic insomnia. PATIENTS AND METHODS: Observational retrospective cohorts study of 66 patients with chronic resistant insomnia, 33 exposed to perampanel, other 33 as non-exposed group. All patients attended in Neurology or Psychiatry Consultation, from November 2017 to November 2018. Patients included had been treated with more than 4 different drugs in the previous 4 years. We reviewed age, sex, insomnia etiology, years of evolution, number of previously used drugs, and the results of perampanel for insomnia after 3 months of treatment in the exposed cohort, measured by the improvement of 3 or more points in the ISI and Pittsburgh scales, as well as the average of hours of sleep gained. Non-exposed patients were matched with this variables, but never treated with perampanel. RESULTS: We have included 66 patients. In the exposed cohort: we describe 33 patients with chronic resistant insomnia, 20 women (60 %), 13 men (40 %). Average age 53.48 years, average time of evolution: 11.25 years. Main etiology: depression 13 cases (40 %). After the combination of perampanel 2-4 mg (100 %) with antidepressants (17 cases, 51.5 %) or anxiolytics (12 cases, 36.36 %) along 3 months: the total number of hours of sleep improves in 2.5 h, the scale ISI improves by 6 points (± 2.1 SD, p = 0.02), and Pittsburgh scale improves in 4 points (± 1.7, p = 0.04). In non-exposed cohort, the improvement of the ISI scale was 2.2 points (±0.8, p = 0.06), on the Pittsburgh scale was 1.6 points (± 0.5, p = 0.01). The main adverse effect was irritability in 3 patients, without withdrawal perampanel. The treatment was abandoned by 4 patients (12.12%): 1 due to persistent irritability (3%), 2 due to lack of efficacy (6 %), 1 due to pregnancy wish (3 %). CONCLUSION: The combination of Perampanel with an antidepressant, or an anxiolytic, improves the quality of sleep measured by ISI and Pittsburgh scales (statistically significant), probably due to its antagonistic action on glutamate. A clinical trial compared with placebo would be necessary to corroborate these results.

Ontogeny of the digestive tract in stinging catfish, Heteropneustes fossilis (Bloch) larvae.

Heteropneustes fossilis (Bloch) is an important candidate species for diversification of freshwater aquaculture in India. However, high mortality rate during larval rearing is the most serious bottleneck in commercial production of this species. A proper understanding of the ontogenic development of digestive system provides the basis to understand the nutritional physiology of larvae and develop appropriate feeding strategies. In the present study, the ontogenical development of the digestive tract in H. fossilis larvae was studied from hatching until 30 day post-hatching (dph) at 29 °C. At hatching (2.8 ± 0.2 mm standard length, SL), the digestive tract was undifferentiated and attached dorsally to the yolk sac. At 1 dph (2.9 ± 0.2 mm SL), the mouth opened and oral valves were visible. At 2 dph (3.0 ± 0.3 mm SL), goblet cells were observed in the buccoparyngaeal cavity. At this age, exogenous feeding started and the intestine was differentiated into the anterior and posterior regions, and the rudimentary liver and pancreas were also seen. Small supranuclear vacuoles were observed in the enterocytes of the posterior intestine at 2 dph. Zymogen granules were observed in acinar cells of pancreas by 3 dph, and islets of Langerhans were visible at 4 dph (3.5 ± 0.1 mm SL). At the same age, most of the yolk sac reserves were consumed, whereas they were completely exhausted by 5 dph (3.9 ± 0.5 mm SL). Between 4 and 6 dph, the liver elongated in size and started to accumulate lipids in the hepatocytes. Gastric glands were detected at 4 dph, and the pyloric sphincter was completely differentiated at 9 dph (6.1 ± 0.4 mm SL) as an epithelial fold that separated stomach from the anterior intestine. By 13 dph (8.6 ± 0.2 mm SL), profuse gastric glands were visible inside longitudinal mucosal folds of the stomach. The formation of gastric glands and their development were noticed as the last events in the development of the digestive tract in H. fossilis. This indicated the end of the larval period and the commencement of the juvenile stage. Considering these observations, it is suggested that H. fossilis larvae have a morphologically complete digestive tract by 13 dph. The findings of the study on the development of the digestive system in H. fossilis may help in synchronising the larval stage of development and feeding strategies and would be helpful in improving larval rearing techniques for catfish species.

Lipase and Phospholipase Activity Methods for Marine Organisms.

Lipases are very important enzymes having a role in fat digestion and lipid metabolism in marine animals, plants, and microorganisms. The methods for measuring lipase and phospholipase activity have been applied in several studies; however, considering that lipases are water-soluble molecules and their substrates are generally water-insoluble molecules, several steps are required for measuring their digestion products. After a general review of the main type of methods used in marine lipase studies, and experimental procedures, a proposal of new or improved methods is described in order to facilitate the lipase activity measurements in marine organisms.

Skeletogenesis in the Persian sturgeon Acipenser persicus and its correlation with gene expression of vitamin K-dependent proteins during larval development.

The present study describes morphological development of the skeleton in the Persian sturgeon Acipenser persicus and discusses the hypothesis that expression of genes encoding vitamin K-dependent proteins (VKDP) might be correlated with the mineralization of skeletal tissues during early development in sturgeons. Results showed that development of cartilage started just after hatching (mean ± S.D., 10·9 ± 0·7 mm in total length, LT ) in the head and notochord, whereas the first signs of mineralization occurred in the dentary and in the dermopalatine and palatopterygoid elements of the upper jaw, coinciding with the onset of exogenous feeding (20·1 ± 1·5 mm LT ). All branchial arch elements developed between 19·3 and 22·3 mm LT , whereas mineralization was only observed in tooth plates associated with the hypobranchial 1 and gill rakers at 20·8 ± 1·5 mm LT and 48·4 ± 6·4 mm LT , respectively. Quantitative real-time PCR showed that transcripts of VKDP genes including bone Gla protein (bgp), matrix Gla protein (mgp) and Gla rich protein (grp) genes were significantly up-regulated during the transition to exogenous feeding, supporting hypotheses about relevance of the above-mentioned genes in chondrogenesis at early developmental stages. The strong mineralization of skeletal elements from 21·5 to 27·3 mm LT (20 days post hatch) was in accordance with the maximal levels of bgp, mgp and grp expression indicating a correlation between development of the skeleton and the expression of VKDP genes. These data are important for evaluating A. persicus larval quality, understanding the influence of rearing biotic and abiotic factors on skeletogenesis and recognizing the occurrence of skeletal deformities in this species.

Ontogeny changes and weaning effects in gene expression patterns of digestive enzymes and regulatory digestive factors in spotted rose snapper (Lutjanus guttatus) larvae.

The study of digestive physiology is an important issue in species that have been introduced in aquaculture like the spotted rose snapper (Lutjanus guttatus). The aims of this study were to describe the expression of digestive enzymes (trypsinogen, chymotrypsinogen, α-amylase, lipoprotein lipase, phospholipase A and pepsinogen) and their relation with orexigenic (neuropeptide Y, NPY) and anorexigenic (cholecystokinin, CCK) factors during the larval development and to evaluate the effect of weaning in their expression. The results showed that the transcripts of all the assayed digestive enzymes, with the exception of pepsinogen, and NPY and CCK were already present in L. guttatus from the hatching stage. The expression of all the enzymes was low during the yolk-sac stage (0-2 days after hatching, DAH), whereas after the onset of exogenous feeding at 2 DAH, their expression increased and fluctuated throughout larval development, which followed a similar pattern as in other marine fish species and reflected changes in different types of food items and the progressive maturation of the digestive system. On the other hand, weaning of L. guttatus larvae from live prey onto a microdiet between 25 and 35 DAH significantly affected the relative expression of most pancreatic digestive enzymes during the first weaning days, whereas chymotrypsinogen 2 and lipoprotein lipase remained stable during this period. At the end of co-feeding, larvae showed similar levels of gene expression regardless of the diet (live prey vs. microdiet), which indicated that larvae of L. guttatus were able to adapt their digestive capacities to the microdiet. In contrast, feeding L. guttatus larvae with live feed or microdiet did not affect the expression of CCK and NPY. The relevance of these findings with regard to current larval rearing procedures of L. guttatus is discussed.

Development of digestive tract and enzyme activities during the early ontogeny of the tropical gar Atractosteus tropicus.

Changes in digestive enzyme activity and histology were studied in Atractosteus tropicus embryos, larvae and juvenile periods. Alkaline protease, chymotrypsin, carboxypeptidase A, lipase and α-amylase were detected in all periods and gradually increased until reaching the maximum peak in juveniles; meanwhile, acid protease was first detected at 5 days after hatching (dah) when first feeding started and trypsin and leucine aminopeptidase activities were detected from 19 dah, their values being increased gradually until reaching a maximum value at 31 dah. Acid and alkaline phosphatase activities increased from yolk-sac absorption (3 dah) until day 31 after hatching. Zymogram for acid protease showed two bands in active forms (0.4 and 0.5 Rfs) from day 5 after hatching and a third protease form (0.3 Rf) that appears at 31 dah. Two active forms (26.3 and 24.9 kDa) were detected using SDS-PAGE alkaline proteases zymogram at 5 dah, and an additional active form (44.1 kDa) was detected at 7 dah. Regarding the histological development of the digestive system, the exocrine pancreas containing zymogen granules was already visible at 3 dah, whereas at 5 dah first gastric glands were already detected in the stomach. Between 7 and 9 dah, the digestive tract of A. tropicus resembled that of a juvenile specimen with a well-developed and short oesophagus, stomach divided into a glandular and non-glandular (pyloric) stomach, folded intestine with pyloric caeca and a well-developed spiral valve (posterior intestine). Considering this, larvae of A. tropicus are capable of digesting several foods from yolk absorption (3 dah), maximizing its activities at 15 dah, age at which the organisms maximize its capability to absorb nutrients from diets provided.

Spray-dried plasma promotes growth, modulates the activity of antioxidant defenses, and enhances the immune status of gilthead sea bream (Sparus aurata) fingerlings.

Terrestrial animal byproduct meals, including nonruminant blood meal and blood products, represent the largest and largely untapped safe source of animal protein available within the international market for the aquafeed industry. Spray-dried blood and spray-dried plasma (SDP) proteins have long been recognized as high-quality feed ingredients for farmed animals. In this study, we evaluated the inclusion of SDP from porcine blood (SDPP) in growing diets for gilthead sea bream. Three isonitrogenous (CP = 51.2%) and isolipidic (fat = 12.4%) diets manufactured by cold extrusion (0.8 to 1.5 mm pellet size) were prepared by substituting high-quality fish meal with 0, 3, and 6% SDPP. The diets were tested for a period of 60 d at 22°C with 4 replicates each (400-L cylindroconical tanks, 150 fish per tank, and initial density = 0.5 kg/m(3)). The SDPP inclusion in diets for gilthead sea bream fingerlings were evaluated in terms of growth performance, feed utilization, histological organization of the intestinal mucosa, activity of oxidative stress enzymes (catalase, glutathione S-transferase, glutathione peroxidase, and glutathione reductase) in the intestine, and nonspecific serum immune parameters (lysozyme and bactericidal activity). Results from this study indicated that dietary SDPP promoted fish growth in terms of BW and length; fish fed 3% SDPP were 10.5% heavier (P < 0.05) than those fed the control diet. Spray-dried plasma from porcine blood modulated the activity of the antioxidative defenses in the intestine (P < 0.05) and increased the density of goblet cells in the intestine (P < 0.05) and benefited the host by providing an effective immune barrier against gut pathogenic microbiota. The nonspecific serum immune response in fish fed diets with SDPP was greater (P < 0.05) than in fish fed the control diet. These results indicated that the inclusion of SDPP in gilthead sea bream feed could be beneficial for the fish by enhancing intestinal and serum innate immune function and the activity of antioxidative stress enzymes of the intestine and promoting growth performance.

Diplectanum sciaenae (Van Beneden & Hesse, 1863) (Monogenea) infecting meagre, Argyrosomus regius (Asso, 1801) broodstock in Catalonia, Spain. A case report.

Recurrent infection of captive meagre, Argyrosomus regius, by a monogenean parasite has been observed in the broodstock facilities of Institut de Recerca i Tecnologia Agroalimentàries (IRTA) in Catalonia, Spain, between 2008 and 2015. Following handling procedures related with hormonal treatment for spawning induction, one fish died. Post-mortem examination and detailed microscopical examination using light microscopy, SEM, and histology revealed intense infection by Diplectanum sciaenae (Monogenea, Diplectanidae) which caused noteworthy gill pathology. In the present study, we provide detailed description of the parasite and the pathology caused to its host. This is the first report associating this parasite with disease and mortality in farmed meagre.

Feeding habits and ontogenic changes in digestive enzyme patterns in five freshwater teleosts.

Feeding habits and the activity of digestive enzymes (total alkaline proteases, α-amylase and lipase) from dace Leuciscus leuciscus, roach Rutilus rutilus, Prussian carp Carassius auratus gibelio, perch Perca fluviatilis and pikeperch Sander lucioperca fry were studied in the Malye Chany Lake-Kargat Estuary (western Siberia, Russia). The diet of fry from all studied species was mainly composed of chironomid larvae and zooplanktonic organisms (i.e. cladocera and copepoda), whereas carnivorous species such as P. fluviatilis and S. lucioperca also preyed on fry from other fishes while detritus and microalgae were also important in the diet of ommivorous species. When comparing diet similarity (Sørensen-Dice index, Q(S)) among fry at different stages of development, both omnivorous and carnivorous species showed a high level of similarity (0.67 < Q(S) < 0.89 and 0.73 < Q(S)< 0.89, respectively). Diet similarity values were in agreement with the overall digestive activity profile analysed by cluster analysis. Diet similarity suggested potential trophic competition when zooplanktonic and benthic prey began to decline towards autumn. The analysis of pancreatic digestive enzymes revealed a correlation among their activities and fry feeding habits with α-amylase:total proteases (A:P) values higher than 1 in omnivorous species and lower (A:P ≤ 1) in carnivorous species.

Histological development of the digestive system of the Amazonian pimelodid catfish Pseudoplatystoma punctifer.

The organogenesis of the digestive system was described in the Amazonian pimelodid catfish species Pseudoplatystoma punctifer from hatching (3.5 mm total length, TL) to 41 days post-fertilization (dpf) (58.1 mm TL) reared at 28°C. Newly hatched larvae showed a simple digestive tract, which appeared as a straight undifferentiated and unfolded tube lined by a single layer of columnar epithelial cells (future enterocytes). During the endogenous feeding period, comprised between 20 and 96 h post-fertilization (3.5 to 6.1 mm TL), the larval digestive system experienced a fast transformation with the almost complete development and differentiation of most of digestive organs (buccopahrynx, oesophagus, intestine, liver and exocrine pancreas). Yolk reserves were not completely depleted at the onset of exogenous feeding (4 dpf, 6.1 mm TL), and a period of mixed nutrition was observed up to 6 to 7 dpf (6.8 to 7.3 mm TL) when yolk was definitively exhausted. The stomach was the organ that latest achieved its complete differentiation, characterized by the development of abundant gastric glands in the fundic stomach between 10 and 15 dpf (10.9 to 15.8 mm TL) and the formation of the pyloric sphincter at the junction of the pyloric stomach and the anterior intestine at 15 dpf (15.8 mm TL). The above-mentioned morphological and histological features observed suggested the achievement of a digestive system characteristic of P. punctifer juveniles and adults. The ontogeny of the digestive system in P. punctifer followed the same general pattern as in most Siluriform species so far, although some species-specific differences in the timing of differentiation of several digestive structures were noted, which might be related to different reproductive guilds, egg and larval size or even different larval rearing practices. According to present findings on the histological development of the digestive system in P. punctifer, some recommendations regarding the rearing practices of this species are also provided in order to improve the actual larval rearing techniques of this fast-growing Neotropical catfish species.

Partial characterisation of digestive proteases of the Mayan cichlid Cichlasoma urophthalmus.

The characterisation of digestive proteases in native freshwater fish such as the Mayan cichlid Cichlasoma urophthalmus provides scientific elements that may be used to design balanced feed that matches with the digestive capacity of the fish. The purpose of this study was to characterise the digestive proteases, including the effect of the pH and the temperature on enzyme activity and stability, as well as the effect of inhibitors using multienzymatic extracts of the stomach and intestine of C. urophthalmus juveniles. Results showed that the optimum activities of the acid and alkaline proteases occurred at pH values of 3 and 9, respectively, whereas their optimum temperatures were 55 and 65 °C, respectively. The acid proteases were most stable at pH values of 2­3 and at temperatures of 35­45 °C, whereas the alkaline proteases were most stable at pH values of 6­9 and at 25­55 °C. The inhibition assays recorded a residual activity of 4% with pepstatin A for the acid proteases. The inhibition of the alkaline proteases was greater than 80% with TPCK, TLCK, EDTA and ovalbumin, and of 60 and 43.8% with PMSF and SBT1, respectively. The results obtained in this study make it possible to state that C. urophthalmus has a sufficiently complete digestive enzyme machinery to degrade food items characteristic of an omnivorous fish species, although specimens showed a tendency to carnivory.

Comparative analysis of a teleost skeleton transcriptome provides insight into its regulation.

An articulated endoskeleton that is calcified is a unifying innovation of the vertebrates, however the molecular basis of the structural divergence between terrestrial and aquatic vertebrates, such as teleost fish, has not been determined. In the present study long-read next generation sequencing (NGS, Roche 454 platform) was used to characterize acellular perichondral bone (vertebrae) and chondroid bone (gill arch) in the gilthead sea bream (Sparus auratus). A total of 15.97 and 14.53Mb were produced, respectively from vertebrae and gill arch cDNA libraries and yielded 32,374 and 28,371 contigs (consensus sequences) respectively. 10,455 contigs from vertebrae and 10,625 contigs from gill arches were annotated with gene ontology terms. Comparative analysis of the global transcriptome revealed 4249 unique transcripts in vertebrae, 4201 unique transcripts in the gill arches and 3700 common transcripts. Several core gene networks were conserved between the gilthead sea bream and mammalian skeleton. Transcripts for putative endocrine factors were identified in acellular gilthead sea bream bone suggesting that in common with mammalian bone it can act as an endocrine tissue. The acellular bone of the vertebra, in contrast to current opinion based on histological analysis, was responsive to a short fast and significant (p<0.05) down-regulation of several transcripts identified by NGS, osteonectin, osteocalcin, cathepsin K and IGFI occurred. In gill arches fasting caused a significant (p<0.05) down-regulation of osteocalcin and up-regulation of MMP9.

Bacillus cereus var. toyoi promotes growth, affects the histological organization and microbiota of the intestinal mucosa in rainbow trout fingerlings.

In this preliminary study, we evaluated the effects of a gram-positive soil bacteria Bacillus cereus var. toyoi on the growth performance, digestive enzyme activities, intestinal morphology, and microbiota in rainbow trout Oncorhynchus mykiss fingerlings. Trout were maintained in a recirculation system and fed 2 diets: 1) a commercial trout feed deprived of the probiotic and 2) the same diet but with the spores of the probiotic bacteria dissolved in fish oil during the manufacturing of the feed (final concentration = 2 × 10(4) cfu/g). Each diet was tested in three 400-L cylindroconical tanks (125 fish per tank; initial density = 1.3 kg/m(3); 13.2°C) for a period of 93 d. The probiotic-supplemented diet promoted growth, and the final mean BW and standard length in fish fed the probiotic were 3.4% and 2.1%, respectively, which was greater than the control group (P < 0.05). Fish fed the probiotic showed a more homogeneous distribution in the final BW, with a greater frequency of individuals around the modal of the normal distribution of the population. This result is of practical importance because homogenous production lots can improve rearing practices, reducing hierarchical dominance situations arising from individuals of larger sizes. In addition, the probiotic-supplemented diet increased the level of leukocyte infiltration in the lamina propria of the intestinal mucosa, the number of goblet cells (P < 0.010), and villi height (P < 0.001) but did not affect villi width. The administration of the probiotic changed the intestinal microbiota as indicated by 16S rDNA PCR-restriction fragment length polymorphism. In this sense, fish fed the probiotic formed a well-defined cluster composed of 1 super clade, whereas compared control fish had a greater degree of diversity in their gut microbiota. These changes in gut microbiota did not affect the specific activity of selected pancreatic and intestinal digestive enzymes. These results indicate that the inclusion of the probiotic bacteria in trout feeds could be beneficial for the host by enhancing its intestinal innate immune function and promoting growth.

Co-infection with Pseudomonas anguilliseptica and Delftia acidovorans in the European eel, Anguilla anguilla (L.): a case history of an illegally trafficked protected species.

Inspections by customs agents at Barcelona airport discovered 420 kg of contraband glass eels prepared for shipment to Hong Kong. After confiscation of these animals by police, they were transported to holding facilities to be maintained until after a judicial hearing. Upon arrival, they were separated into two groups and held under ambient flow-through conditions in fresh water. During their captivity period, several peaks in mortality occurred and multiple bacterial strains were isolated from moribund animals. Sequencing of 16S rDNA was used to determine specific identity of the isolates. An initial isolation of Pseudomonas anguilliseptica was treated with oxytetracycline. A subsequent isolation of Delftia acidovorans proved resistant to oxytetracycline and was treated with gentamicin in combination with sulphadiazine-trimethoprim. Once the health condition of the animals was stabilized, they were partitioned into groups and subsequently released as part of a restocking effort for the species following the guidelines of Regulation (EC) 1100/2007 (Anon 2007). This represents the first record for both bacterial species in the host Anguilla anguilla in the Spanish Mediterranean.