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.

High dietary arachidonic acid levels affect the process of eye migration and head shape in pseudoalbino Senegalese sole Solea senegalensis early juveniles.

The effect of high dietary levels of arachidonic acid (ARA) on the eye migration and cranial bone remodelling processes in Senegalese sole Solea senegalensis early juveniles (age: 50 days post hatch) was evaluated by means of geometric morphometric analysis and alizarin red staining of cranial skeletal elements. The incidence of normally pigmented fish fed the control diet was 99·1 ± 0·3% (mean ± s.e.), whereas it was only 18·7 ± 7·5% for those fed high levels of ARA (ARA-H). The frequency of cranial deformities was significantly higher in fish fed ARA-H (95·1 ± 1·5%) than in those fed the control diet (1·9 ± 1·9%). Cranial deformities were significantly and negatively correlated with the incidence of normally pigmented animals (r² = -0·88, P < 0·001, n = 16). Thus, fish displaying pigmentary disorders differed in the position of their eyes with regard to the vertebral column and mouth axes, and by the interocular distance and head height, which were shorter than in fish not displaying pigmentary disorders. In addition to changes in the positioning of both eyes, pseudoalbino fish showed some ARA-induced osteological differences for some of the skeletal elements from the splanchnocranium (e.g. right premaxillary, dentary, angular, lacrimal, ceratohyal and branchiostegal rays) and neurocranium (e.g. sphenotic, left lateral ethmoid and left frontal) by comparison to normally pigmented specimens. Pseudoalbino fish also had teeth in both lower and upper jaws. This is the first study in Pleuronectiformes that describes impaired metamorphic relocation of the ocular side eye, the right eye in the case of S. senegalensis, whereas the left eye migrated into the ocular side almost normally.

Gene expression patterns during the larval development of European sea bass (dicentrarchus labrax) by microarray analysis.

During the larval period, marine teleosts undergo very fast growth and dramatic changes in morphology, metabolism, and behavior to accomplish their metamorphosis into juvenile fish. Regulation of gene expression is widely thought to be a key mechanism underlying the management of the biological processes required for harmonious development over this phase of life. To provide an overall analysis of gene expression in the whole body during sea bass larval development, we monitored the expression of 6,626 distinct genes at 10 different points in time between 7 and 43 days post-hatching (dph) by using heterologous hybridization of a rainbow trout cDNA microarray. The differentially expressed genes (n = 485) could be grouped into two categories: genes that were generally up-expressed early, between 7 and 23 dph, and genes up-expressed between 25 and 43 dph. Interestingly, among the genes regulated during the larval period, those related to organogenesis, energy pathways, biosynthesis, and digestion were over-represented compared with total set of analyzed genes. We discuss the quantitative regulation of whole-body contents of these specific transcripts with regard to the ontogenesis and maturation of essential functions that take place over larval development. Our study is the first utilization of a transcriptomic approach in sea bass and reveals dynamic changes in gene expression patterns in relation to marine finfish larval development.

Characterization of a partial alpha-amylase clone from red porgy (Pagrus pagrus): expression during larval development.

A partial alpha-amylase cDNA was isolated from red porgy (Pagrus pagrus, Teleostei: Sparidae) and its tissue specific expression during larval development was examined. The cDNA was 949 bp long and showed 90% identity with other fish amylases. A 545 bp fragment was used to study amylase expression using in situ hybridization and RT-PCR techniques. Both methods showed a similar pattern: high and relatively constant expression for the first 30 days after hatching (dah), subsequently decreasing until the end of the experiment at 60 dah. The goal of this work was to extend the existing knowledge of the functionality of larval fish digestive systems and to provide new information about alpha-amylase gene expression.

Organogenesis of the digestive tract in the white seabream, Diplodus sargus. Histological and histochemical approaches.

The ontogeny of the digestive tract of the white seabream, Diplodus sargus during the larval development up to day 45 post-hatching (dph) has been studied using histological and histochemical techniques. The oesophageal goblet cells appeared around 6 dph and contained neutral and acid mucosubstances (PAS/diastase-PAS and Alcian Blue pH 2.5 positive reactions). An incipient stomach can be distinguished from 2 dph but the first sign of gastric gland development was detected around 13-15 dph, increasing in number and size by 22-23 dph. Gastric glands were concentrated in the cardiac stomach region and they had a high content of protein rich in tyrosine, arginine and tryptophan. Acidophilic supranuclear inclusions related to pynocitosis of proteins, were already observed in the intestinal cells of the posterior intestine around 4-6 dph (exogenous feeding) and they were present until 25 dph. The intestinal mucous cells appeared between 15-18 dph and contained a mixture of neutral and acid mucosubstances/glycoconjugates, carboxylated ones being more abundant than the sulphated ones. The stomach and gastric glands were fully developed by the first month of life marking the beginning of digestive features characteristic of the juvenile stage. Around 4-6 dph, glycogen, proteins and neutral lipids were observed in the granular cytoplasm of hepatocytes. Strongly acidophilic zymogen granules were also present, at this time, in the basophilic cytoplasm of the exocrine pancreatic acinar cells and contained abundant proteins, especially rich in arginine, tyrosine and tryptophan.

An integrated closed system for fish-plankton aquaculture in Amazonian fresh water.

A prototype of an integrated closed system for fish-plankton aquaculture was developed in Iquitos (Peruvian Amazonia) in order to cultivate the Tiger Catfish, Pseudoplatystoma punctifer (Castelnau, 1855). This freshwater recirculating system consisted of two linked sewage tanks with an intensive rearing unit (a cage) for P. punctifer placed in the first, and with a fish-plankton trophic chain replacing the filters commonly used in clear water closed systems. Detritivorous and zooplanktivorous fishes (Loricariidae and Cichlidae), maintained without external feeding in the sewage volume, mineralized organic matter and permitted the stabilization of the phytoplankton biomass. Water exchange and organic waste discharge were not necessary. In this paper we describe the processes undertaken to equilibrate this ecosystem: first the elimination of an un-adapted spiny alga, Golenkinia sp., whose proliferation was favored by the presence of a small rotifer, Trichocerca sp., and second the control of this rotifer proliferation via the introduction of two cichlid species, Acaronia nassa Heckel, 1840 and Satanoperca jurupari Heckel, 1840, in the sewage part. This favored some development of the green algae Nannochloris sp. and Chlorella sp. At that time we took the opportunity to begin a 3-month rearing test of P. punctifer. The mean specific growth rate and feed conversion ratio (FCR) of P. punctifer were 1.43 and 1.27, respectively, and the global FCR, including fish in the sewage part, was 1.08. This system has proven to be suitable for growing P. punctifer juveniles out to adult, and provides several practical advantages compared with traditional recirculating clear water systems, which use a combination of mechanical and biological filters and require periodic waste removal, leading to water and organic matter losses.

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.

Dietary vitamin mix levels influence the ossification process in European sea bass (Dicentrarchus labrax) larvae.

The influence of dietary vitamins on growth, survival, and morphogenesis was evaluated until day 38 of posthatching life in European sea bass larvae (Dicentrarchus labrax). A standard vitamin mix (VM), at double the concentration of the U.S. National Research Council's recommendations, was incorporated into larval feeds at 0.5%, 1.5%, 2.5%, 4.0%, and 8.0% to give treatments VM 0.5, VM 1.5, VM 2.5, VM 4.0, and VM 8.0, respectively. The group fed the VM 0.5 diet all died before day 30. At day 38, the larvae group fed VM 1.5 had 33% survival, while the other groups, with higher vitamin levels, showed at least 50% survival. The higher the percentage VM in the diet, the lower the percentage of column deformities. High dietary vitamin levels positively influenced the formation of mineralized bone in larvae: the higher the dietary vitamin level, the higher the ossification status. In the larvae group fed at the highest vitamin levels, we observed a temporal sequence of coordinated growth factor expression, in which the expression of bone morphometric protein (BMP-4) preceded the expression of IGF-1, which stimulated the maturation of osteoblasts (revealed by high osteocalcin expression levels). In groups fed lower proportions of vitamins, elevated proliferator peroxisome-activated receptors (PPAR-gamma) expression coincided with low BMP-4 expression. Our results suggest that high levels of PPAR-gamma transcripts in larvae-fed diets with a low VM content converted some osteoblasts into adipocytes during the first two weeks of life. This loss of osteoblasts is likely to have caused skeletal deformities.

Larval organogenesis of Pagrus pagrus L., 1758 with special attention to the digestive system development.

Organogenesis of the red porgy (Pagrus pagrus L., 1758) was examined from hatching until 63 days post-hatching (dph) using histological and histochemical techniques. At hatching, the heart appeared as a tubular structure which progressively developed into four differentiated regions at 2 dph: bulbus arteriosus, atrium, ventricle and sinus venosus. First ventricle and atrium trabeculae were appreciated at 6 and 26 dph, respectively. Primordial gill arches were evident at 2 dph. Primordial filaments and first lamellae were observed at 6 and 15 dph, respectively. At mouth opening (3dph), larvae exhausted their yolk-sac reserves. The pancreatic zymogen granules appeared at 6 dph. Glycogen granules, proteins and neutral lipids (vacuoles in paraffin sections) were detected in the cytoplasm of the hepatocytes from 4-6 dph. Hepatic sinusoids could be observed from 9 dph. Pharyngeal and buccal teeth were observed at 9 and 15 dph, respectively. Oesophageal goblet cells appeared around 6 dph, containing neutral and acid mucosubstances. An incipient stomach could be distinguished at 2 dph. The first signs of gastric gland development were detected at 26 dph, increasing in number and size by 35-40 dph. Gastric glands were concentrated in the cardiac stomach region and presented a high content of protein rich in tyrosine, arginine and tryptophan. The intestinal mucous cells appeared at 15 dph and contained neutral and acid glycoconjugates, the carboxylated mucins being more abundant than the sulphated ones. Acidophilic supranuclear inclusions in the intestinal cells of the posterior intestine, related to pynocitosis of proteins, were observed at 4-6 dph.

Ventilación dirigida contrastada y entrenamiento muscular aeróbico-anaeróbico mejoran un paciente con EPOC severo por enfisema pulmonar / Comprehensive directed breathing and aerobic-anaerobic muscle training strongly improve a patient with severe COPD by pulmonary emphysema

ObjetivoAnalizar los efectos de la asociación de ventilación dirigida contrastada con entrenamiento Square Wave Endurance Exercise Test (SWEET), 4-minutos aeróbicos y 1-minuto anaeróbico, repetido 9 veces en 45min, con oxigenoterapia al ejercicio, en un paciente (73 años) con EPOC severo, hiperinflación e hipoxemia al ejercicio.MetodologíaExamen clínico y funcional, y 4 pruebas de ejercicio: 6 min marcha, «escabel», ejercicio incremental y SWEET controlando la disnea (Borg), la SpO2 y la FC. La gasometría arterial (PaO2, PaCO2, pH, ácido láctico), el metabolismo (O2, CO2), el patrón ventilatorio (E, FR, VT) y la frecuencia cardiaca (FC) se comparan antes y después de 11 sesiones de 120min «personalizadas», vigiladas por el fisioterapeuta y rehabilitador especializados, y 6 meses después de autotratamiento a domicilio.ResultadosSe corrigen la asincronía tóraco abdominal, la ventilación paradójica y se mejora la espirometría discretamente, pero los flujos y las presiones respiratorias máximas aumentan mucho más (+132 y +119%), así como la capacidad aeróbica (+111%), la deambulación (+32%) y escabel (+119%). La eficacia ventilatoria aumenta, y el metabolismo disminuye en reposo. La disnea de esfuerzo (DE) se reduce en un 35%, y el volumen residual en un 33%.ConclusiónEl protocolo de ventilación dirigida contrastada más el entrenamiento Square Wave Endurance Exercise Test realizado ha sido efectivo para reducir la disnea de esfuerzo, aumentar la capacidad de ejercicio máximo, los flujos y las presiones respiratorias máximas en este paciente con enfisema pulmonar y EPOC severo (AU)

ObjectiveTo analyze the effects of the combination of Comprehensive Directed Breathing (CDB) with training Square Wave Endurance Exercise Test (SWEET), 4-min aerobic and 1-min anaerobic repeated nine times in 45min, with oxygen therapy during exercise, in a patient (73 years) with severe COPD, hyperinflation and hypoxemia during exercises.MethodologyClinical examination, functional testing and 4 exercise testing: 6-min walking test (6MWT), two step stool, incremental exercise and the SWEET, with control of exertional Dyspnea (Borg’ scale), SpO2 and heart rate (HR). Arterial blood gases (PaO2, PaCO2, pH, lactic acid), metabolism (O2, CO2), ventilatory pattern (E, FR, VT) and heart rate (HR) were compared before and immediately after 11 tailored sessions of 120-min supervised by the respiratory physiotherapist and the physiatrist specialists. Then, he continued at home applying himself the full learning protocol and a fast-control was made six months later.ResultsThe thoraco-abdominal asynchrony and paradoxical ventilation were corrected and the spirometry improved discreetly, but the maximal flows and respiratory pressures increase strongly (+132 and +119%), as well as aerobic capacity (+111%), ambulation (32%) and the two step stool (+119%). The ventilatory efficiency increases and the resting metabolism decreases. Exertional dyspnea is reduced by 35% as the residual volume by 33%.ConclusionThe protocol CDB plus SWEET has been made effective in reducing exertional Dyspnea, increased MVV, maximum exercise capacity and maximal respiratory flows and pressures in this patient with severe emphysema and COPD (AU)