Osmoregulatory responses to abrupt salinity changes in the euryhaline gilthead sea bream (Sparus aurata L.)

• 1.1. Gilthead sea breams (Sparus aurata L.) adapted to sea water (SW, 39‰ salinity) and brackish water (BW, 7‰) were submitted to abrupt osmotic stress by transferring the specimens to 7‰ and 39‰, respectively.
• 2.2. Plasma osmolality, Na,⁺ Cl, ⁻ K, ⁺ Ca, ²⁺ cortisol and glucose were measured before and after the transfers.
• 3.3. The transfer from SW to BW led to transitory hypomineralization and hyperglycemia. In long-term adapted fish cortisol level increased, and osmolality slightly decreased.
• 4.4. Conversely, the transfer from BW to SW provoked transitory hypermineralization. In adapted fish, cortisol levels strongly decreased, and osmolality slightly increased.

     

Characterization of digestive carbohydrase activity in the gilthead seabream (Sparus aurata)

Six species of Phallodrilinae are reported from Hainan Island in southern China. The mesopsammic Aktedrilus yiboi sp. nov. is new to science. It is characterized by a combination of (1) small, straight, conical, strongly cuticularized penis sheaths, (2) posterior prostate glands being associated with the most ectal parts of the atria, and (3) small spermathecal ampulla. Bathydrilus ampliductus Erséus, 1997 is recorded for the first time since it was originally described from Australia's Northern Territory.     

Juvenile Sparus aurata L. on the south coast of Ireland

Records of gilthead sea bream Sparus aurata L. in Irish and British waters since 1967 are provided together with the first observations on the ecology of 0 year group gilthead sea bream in these islands.     

Effects of four anaesthetics on the innate immune response of gilthead seabream (Sparus aurata L.)

Anaesthesia may depress the immune system in mammals, but there is no available information on this topic in fish. In the present work, four anaesthetics that are used in aquaculture, MS222 (0 19 mM), benzocaine (0.21 mM), 2-phenoxyethanol (16 mM) and quinaldine sulphate (0.083 mM), were tested in order to observe their effects on the gilthead seabream (Sparus aurata L.) innate immune system. The results showed that the four anaesthetics produced increased blood glucose levels after an hour. In addition, benzocaine and 2-phenoxyethanol depressed complement activity and phagocytosis, while MS222 and quinaldine sulphate did not. Some anaesthesia is a common practice in aquaculture, the data obtained should be taken into account to avoid possible immunodepression in farmed fish.     

Effects of short-term crowding stress on the gilthead seabream (Sparus aurata L) innate immune response

Gilthead seabream specimens were subjected to an intense short-term crowding stress of 100 kg m(-3) for 2 h. After 0, 1, 2, 3 and 4 days, blood glucose and serum cortisol levels, serum complement activity, phagocytic and respiratory burst activities of head-kidney leucocytes, and the percentage of monocyte/ macrophages and granulocytes in head-kidney and circulating blood were determined. An immediate effect of the stress was a depression in complement and phagocytic activities, both of which recovered after 3 or 2 days, respectively, while respiratory burst remained unaffected. The depression of phagocytosis in head-kidney leucocytes seemed to correlate with stress-induced migration of active cells from the organ to circulating blood. The present results point to the importance of minimising intense short-term crowding stress in order to reduce possible states of immunodepression in farmed fish.     

Expression of Developmental-Stage-Specific Genes in the Gilthead Sea Bream Sparus aurata L.

The mechanism of early fish development as well as the control of egg quality is of great importance for the ability of the oocyte to develop after fertilization. Embryonic development is initially regulated by maternally provided mRNAs and later by the zygotic genome. Maternal mRNAs have an important role in initiating processes crucial to patterning the developing fish embryo. Furthermore, it has been shown that maternal RNA plays an important role in egg quality. The identification and characterization of candidate maternal genes in non-model fish species with important aquaculture interest like the gilthead sea bream Sparus aurata L. is of importance for future studies related to egg quality. The broodstock of the gilthead sea bream produces large quantities of eggs with a high and non-controllable quality variation. In the present study, we have studied the gene expression of 16 genes (gapdh 1 and 2, cathepsin D, L, S and Z, erk1, jnk1, p38 alpha and p38 delta, ppar alpha, beta and gamma, tubulin beta, ferritin M, cyclinA2) of different functional categories in seven developmental stages. The 16 genes were chosen based on their putative involvement in egg quality and regulation of early development. In total, 11 showed a characteristic gene expression pattern pinpointing to the possible function as maternal genes and thus may function as molecular biomarker for egg quality.     

Effects of injecting chitin particles on the innate immune response of gilthead seabream (Sparus aurata L.)

To determine the effects of chitin (poly [1-->4]-beta-N-acetyl-D--glucosamine) particles on the innate immune response of gilthead seabream (Sparus aurata L.), specimens were injected intravenously or intraperitoneally with the substance. Natural haemolytic complement activity, head-kidney leucocyte respiratory burst activity and phagocytic and cytotoxic activities were analysed in vitro 3, 5 or 10 days post-injection. All the immune parameters assayed remained unaffected when chitin was intravenously administered. However, the fish that had been intraperitoneally injected showed increased humoral and cellular immune responses. Natural haemolytic complement activity increased from 5 days post-injection although no statistically significant differences were observed. Respiratory burst and phagocytic activities peaked at 3 and 5 days post-injection, respectively, while cytotoxic activity had increased by 3 days post-injection and remained high until 10 days post-injection.     

Branchial osmoregulatory response to salinity in the gilthead sea bream, Sparus auratus

The branchial osmoregulatory response of gilthead sea bream (Sparus auratus L.) to short-term (2-192 hr) and long-term (2 weeks) exposure to different environmental salinities (5 per thousand, 15 per thousand, 25 per thousand, 38 per thousand and 60 per thousand) was investigated. A "U-shaped" relationship was observed between environmental salinity and gill Na+,K+ -ATPase activity in both long- and short-term exposure to altered salinity, with the increase in activity occurring between 24 and 96 hr after the onset of exposure. Plasma osmolality and plasma ions (sodium, chloride, calcium and potassium) showed a tendency to increase in parallel with salinity. These variables only differed significantly (P<0.05) in fish adapted to 60 per thousand salinity with respect to fish adapted to full-strength sea-water (SW). Plasma glucose remained unchanged whereas plasma lactate was elevated at 5 per thousand and 60 per thousand. Muscle water content (MWC) was significantly lower in fish adapted to 60 per thousand. Chloride cells (CC) were only present on the surface of the gill filaments and absent from the secondary lamellae. CC distribution was not altered by external salinity. However, the number and size of CC were significantly increased at salinity extremes (5 per thousand and 60 per thousand), whereas fish exposed to intermediate salinities (15 per thousand and 25 per thousand) had fewer and smaller cells. Furthermore, the CC of fish exposed to diluted SW became rounder whereas they were more elongated in fish in full-strength and hypersaline SW. This is consistent with previous reports indicating the existence of two CC types in euryhaline fish. At likely environmental salinities, gilthead sea bream show minor changes in plasma variables and the effective regulation of gill Na+,K+ -ATPase. However, at very low salinities both haemodilution and up-regulation of gill Na+,K+ -ATPase predict a poor adaptation most likely related to deficiency or absence of specific components of the CC important for ion xuptake.     

Gilthead seabream (Sparus aurata L.) innate immune response after dietary administration of heat-inactivated potential probiotics

The effects of the dietary administration of two heat-inactivated whole bacteria from the Vibrionaceae family, singly or combined, on innate immune response of the seabream were studied. The two bacteria (Pdp11 and 51M6), which were obtained from the skin of gilthead seabream, showed in vitro characteristics that suggested they could be considered as potential fish probiotics. The fish were fed four different diets: control (non-supplemented), or diets supplemented with heat-inactivated bacteria at 10(8) cfu g(-1) Pdp11, 10(8) cfu g(-1) 51M6 or with 0.5 x 10(8) cfu g(-1) Pdp11 plus 0.5 x 10(8) cfu g(-1) 51M6 for 4 weeks. Six fish were sampled at weeks 1, 2, 3 and 4, when the main humoral (natural haemolytic complement activity and serum peroxidase content) and cellular innate immune responses (leucocyte peroxidase content, phagocytosis, respiratory burst and cytotoxicity) were evaluated. The serum peroxidase content and the natural haemolytic complement activity increased with time, reaching the highest values in the third and fourth weeks of feeding, respectively. The phagocytic ability of specimens fed the mixture of the two inactivated bacteria was significantly higher than in the controls after 2 and 3 weeks of treatment. The same activity increased significantly in seabream fed the Pdp11 diet for 2 weeks or the 51M6 diet for 3 weeks. Respiratory burst activity was unaffected by all the experimental diets at all times assayed. Cytotoxic activity had significantly increased after 3 weeks in fish fed the 51M6 diet. These results are discussed in terms of the usefulness of incorporating inactivated probiotic bacteria into fish diets.     

A study of the histology and morphology of the digestive tract of the sea-bream, Sparus aurata

The anatomy and the histology of the digestive tract of young and adult sea-bream is described from studies using light and scanning electron microscopy. The dentition in the juvenile (25–30 mm long) comprises all canine-like teeth, to which plate teeth and transition elements are added in the adult.
The oesophagus shows a multi-layered mucosa in the upper part, and single-layered regions in the lower part. The multi-layered regions are formed by epithelial cells, mucus-secreting cells and by cells rich with eosinophilic granules.
The Y-shaped stomach, clearly distinguishable, has a single-layered columnar epithelium under which, in the cardiac and fundic portion, gastric glands, comprised of all similar cells, are present. The pyloric region is characterized by four caeca, to the base of which the ductus pancreaticus and the ductus hepaticus discharge.
The pancreas is composed of small masses spread along the upper intestine; in the adult, pancreatic infiltrations can be seen in the liver.
The intestine is short (relative length 0.5–0.6). The intestine epithelium consists of columnar cells intercalated with mucus-secreting cells. A funnel-like valve marks the passage to the intestine terminal region, characterized by a mucosa of cells with an abundance of vacuoles full of eosinophilic granules.     

High dietary intake of alpha-tocopherol acetate enhances the non-specific immune response of gilthead seabream (Sparus aurata L.)

To determine the effects of three high levels of dietary intake of alpha-tocopherol acetate (vitamin E) on the non-specific immune response of gilthead seabream (Sparus aurata L.), specimens were fed a commercial diet (100 mg alpha-tocopherol kg-1) as control, or vitamin E supplemented diets (600, 1200 or 1800 mg alpha-tocopherol acetate kg-1) for 15, 30 or 45 days. Growth, serum alpha-tocopherol levels, natural haemolytic complement activity and head-kidney leucocyte migratory, respiratory burst and phagocytic activities were studied at each of the assay times. A positive correlation between alpha-tocopherol acetate intake and serum alpha-tocopherol levels was observed, the increase being linked to both the dosage and length of treatment. Specimens fed the diet supplemented with 600 mg vitamin E kg-1 showed no enhancement in any of their immune parameters, while those fed the diet supplemented with 1200 mg vitamin E kg-1 presented a slightly higher (but not statistically significant) specific growth rate than fish fed the other diets. In addition, serum haemolytic activity and the phagocytosis of head-kidney leucocytes were enhanced by the dietary intake of 1200 mg vitamin E kg-1 after 30 and 45 days of treatment, although leucocyte migration and respiratory burst activity remained unaffected. The highest vitamin E dietary dose used, 1800 mg kg-1, unexpectedly provoked no immunostimulation. These results indicate that a moderate level of vitamin E in the diet (1200 mg kg-1) stimulates the seabream's non-specific immune system after 30 days of administration. Lower or higher vitamin E concentrations may not be so effective, because of an imbalance in the vitamin E ratio with other antioxidants. The proposed dietary levels of vitamin together with the indicated administration time could be useful for reducing the susceptibility of farmed fish to infectious diseases.     

Retinal development in the gilthead seabream Sparus aurata

The retinal development of the gilthead seabream Sparus aurata has been analysed from late embryonic development to juvenile stages using classical histological and immunohistological methods. Five significant phases were established. Phases 1 and 2 comprise the late embryonic and hatching stages, respectively. The results indicate that during these early stages the retina is composed of a single neuroblastic layer that consists of undifferentiated retinal progenitor cells. Phase 3 (late prolarval stage) is characterized by the emergence of the retinal layers and the appearance of neurochemical profiles in differentiating photoreceptors, amacrine and ganglion cells. Phases 4 and 5 comprise the late larval and juvenile stages. In these stages, all the retinal cell types can be detected immunohistochemically. All the maturational events described are first detected in the central retina and, as development progresses, spread to the rest of the retina following a central‐to‐peripheral gradient. The results of this study suggest that S. aurata is an altricial teleost species that hatches with a morphologically undifferentiated retina. The most relevant processes involved in retinogenesis occur during the late prolarval stage (phase 3).     

Effect of decreased environmental salinity on growth hormone cells in the gilthead sea bream (Sparus aurata)

The effect of decreased environmental salinity on growth hormone producing cells (GH cells) of the adenohypophysial proximal pars distalis has been studied in the gilthead sea bream ( Sparus aurata L.) adapted to sea water (SW, 980 mosmol kg ⁻¹) and brackish water (BW, 200 mosmol kg ⁻¹). A combined immunocytochemical, morphometric and electron microscopic study was carried out. GH cells offish adapted to BW occupied a greater hypophysial volume (about 21% of the total hypophysial volume in BW, 17% in SW) and had a larger nuclear area (mean 16 μm² in BW, 13 μm² in SW) than GH cells of SW-adapted fish. The immunoreactivity against a salmon GH-antiserum was lower in BW (mean optical density 142 in BW, 159 in SW). Ultrastructural characteristics of GH cells of BW-adapted fish were distended rough endo-plasmatic reticulum and large secretory granules (about 216 nm in diameter for BW, 209 nm in SW). Volumetric, densitometric and ultrastructural evidence suggested that the synthesis and release of GH were activated in S. aurata adapted to hypo-osmotic environment.     

The effect of dietary intake of vitamins C and E on the stress response of gilthead seabream (Sparus aurata L.)

High dietary doses of the antioxidant vitamins C and E were administered to gilthead seabream (Sparus aurata L.) in an attempt to reduce the stress response in specimens exposed to a multiple stress situation. Fish were fed four different diets for 6 weeks: a commercial feed containing 0.1g vitamin C and 0.1g vitamin E kg(-1) acted as control diet, while experimental diets consisted of the same feed supplemented with 3g vitamin C kg(-1), 1.2g vitamin E kg(-1) or both 3g vitamin C and 1.2g vitamin E kg(-1). After 2, 4 and 6 weeks fish were exposed to stressors typical of aquacultural practices, and serum cortisol levels, complement activity (measured by the alternative pathway), blood glucose level and respiratory burst activity of head-kidney leucocytes were evaluated. The results showed that all stress-induced increases in blood glucose concentration were lower in fish fed the vitamin C and/or E-supplemented diet than in fish fed the control diet after 2 weeks of treatment, although no other differences were found at the rest of the times. Cortisol levels increased in stressed fish and did not suffer depletion as a consequence of administering vitamins C and/or E as a supplement. The natural haemolytic complement activity was not affected by the stressors but enhanced in specimens fed vitamin-supplemented diets at week 6. The respiratory burst activity was depressed by the stressors in fish fed the control diet, although only after 6 weeks of treatment were the differences statistically significant. These results suggest that vitamins C and E are involved in the hypothalamic-sympathetic-chromaffin cell axis and also interfere in tertiary stress responses such as immunodepression, where they protect the leucocyte functions.     

Gill area and dimensions of gilthead sea bream Sparus aurata L.

Detailed measurements of gill area and constituent variables (total filament length, lamellar frequency and bilateral area) were performed on both hemibranchs of all eight arches in six specimens of gilthead sea bream Sparus aurata (mean ±s.e . 49·9 ± 0·2 g). Shrinkage was also quantified and results were corrected accordingly. Filament number decreased from the first to the fourth gill arch, and average bilateral area of secondary lamellae was higher in the second and third arches. Total and mean filament length, total number of secondary lamellae and total gill area (A_TG) were lower in posterior than in anterior hemibranchs of the second, third and fourth gill arches; while the opposite was observed for the first arch. Lamellar frequency was increased in posterior hemibranchs of all arches compared to that in anterior hemibranchs, especially at the fourth arch. Comparison of the actually measured A_TG and constituent variables with estimates revealed that the third gill arch is the most representative for appropriate measurements and that any of its components (even one hemibranch) approximates the best A_TG (within the range of 0·2–4·3%, P > 0·05) and related dimensions. Consequently, necessary measurements were restricted to the posterior hemibranch of the third gill arch, and A_TG and dimensions (y) were estimated in 21 specimens (23·5–217·6 g) and correlated to body mass (M) according to the allometric equation y = aM^b. As fish increased in size, A_TG (b= 0·664), total (b= 0·425) and mean (b= 0·323) filament length, total number of filaments (b= 0·103) and secondary lamellae (b= 0·377), as well as average lamellar bilateral area (b= 0·288), increased, while the opposite was observed for lamellar frequency (b=?0·049) and mass‐specific area (b=?0·336). Data obtained are discussed in relation to S. aurata activity and living ethology.     

Transcriptomic differences between euryhaline and stenohaline malaria vector sibling species in response to salinity stress

Evolution of osmoregulatory systems is a key factor in the transition of species between fresh‐ and saltwater habitats. Anopheles coluzzii and Anopheles merus are stenohaline and euryhaline malaria vector mosquitoes belonging to a larger group of sibling species, the Anopheles gambiae complex, which radiated in Africa within the last 2 million years. Comparative ecological genomics of these vector species can provide insight into the mechanisms that permitted the rapid radiation of this species complex into habitats of contrasting salinity. Here, we use RNA‐Seq to investigate gene expression differences between An. coluzzii and An. merus after briefly exposing both young and old larval instars of each species to either saltwater (SW) or freshwater (FW). Our study aims to identify candidate genes and pathways responsible for the greater SW tolerance of An. merus. Our results are congruent with the ability of gene induction to mediate salinity tolerance, with both species showing increasing amounts of differential gene expression between SW and FW as salt concentrations increase. Besides ion transporters such as AgAE2 that may serve as effectors for osmoregulation, we also find mitogen‐activated protein kinases that may serve in a phosphorylation signalling pathway responding to salinity, and report potential cross‐talk between the mosquito immune response and osmoregulation. This study provides a key step towards applying the growing molecular knowledge of these malaria vectors to improve understanding of their ecological tolerances and habitat occupancy.