Posterior Intestine Research Articles

Maintenance of ionic gradients and tissue hydration in the intertidal sea cucumberHolothuria griseaunder hypo- and hyper-salinity challenges

Echinoderms are exclusively marine, osmoconformer invertebrates. Their distribution patterns are strongly influenced by salinity. Nevertheless, several species of the phylum inhabit the challenging intertidal zone, characterized by steep and fast salinity fluctuations. This study evaluated the response of coelomic fluid ionic concentrations (sodium, chloride, magnesium and potassium) of the intertidal sea cucumberHolothuria griseato hypo- and hypersaline challenges. A stepwise protocol was performed for the whole animal exposure to both anisosmotic conditions: from 35 to 15 psu along 8 h, and from 35 to 45 psu along 6 h, to simulate intertidal conditions. Tissue water regulation by the longitudinal muscle, oesophagus and posterior intestine was also evaluated, upon hypo- and hyper-osmotic shocks of 20 and 50% of intensity with respect to the isosmotic control. Ionic gradients were observed between coelomic fluid and external water, mainly for potassium and magnesium, but also sodium, and in a greater extent under hyposaline conditions than under hypersaline exposure. Consistently,H. griseashows retracted tube feet in 15 psu, but a more relaxed appearance and exposed tube feet in 45 psu. In addition,H. griseashowed greater capacity for tissue water maintenance during hyper- than in hyposmotic conditions.Holothuria griseashows an avoidance behaviour in low salinity (thus sustaining ionic gradients), preventing its tissues from intense swelling. This strategy allows it to dwell in the intertidal region.

Digestive Physiological Characteristics of the Gobiidae: - Characteristics of CCK-producing Cells and Mucus-secreting Goblet Cells of Stomach Fish and Stomachless Fish.

In this study, we investigated the characteristics of CCK-producing cells and mucus-secreting goblet cells with respect to stomach fish and stomachless fish of the Gobiidae in order to provide a basis for understanding the digestive physiology. Hairychin goby (Sagamia geneionema), which is stomachless fish, the numbers of mucus-secreting goblet cells is highest in the posterior intestine portion (P<0.05), while CCK-producing cells are scattered throughout the intestine. Gluttonous goby (Chasmichthys gulosus), which is stomach fish, mucus-secreting goblet cells are most abundant in the mid intestine portion (P<0.05), whereas CCK-producing cells are observed only in the anterior and mid intestine portion. Trident goby (Tridentiger obscurus) which is stomach fish, mucus-secreting goblet cells were most abundant in the mid intestine portion (P<0.05). CCK-producing cells are found in the anterior and mid intestine portion. Giurine goby, Rhinogobius giurinus which is also stomach fish, the largest number of mucus-secreting goblet cells showed in anterior intestine portion except for esophagus (P<0.05). CCK-producing cells are present only in the anterior and mid intestine portion. In S. geneionema, digestive action occurs in the posterior intestine portion to protect and functions to activate digestion. In contrast, in C. gulosus, T. obscurus and R. giurinus, their digestive action occurs in the anterior and mid intestine portion to protect and functions to activate digestion. Further studies of the modes of food ingestion by these fish, the contents of their digestive tracts, and the staining characteristics of the goblet cells need to be carried out.

Morpho-histological characterisation of the alimentary canal of an important food fish, Asian seabass (Lates calcarifer).

Asian seabass (Lates calcarifer) is a food fish of increasing aquaculture importance. In order to improve our understanding on the digestive system and feeding of this species, morphological and histological features of the gut were studied. Morphologically, the Asian seabass gut is defined by a short and muscular esophagus, well-developed stomach and comparatively short intestine. Mucous secreting goblet cells reactive to PAS (Periodic Acid Schiff) and AB (Alcian Blue) stain were present throughout the esophagus. The stomach was sac-like and could be distinguished into the cardiac, fundic and pyloric regions. Gastric glands and mucus cells were predominately present in the cardiac and fundic regions. Five finger-like pyloric caeca were present between the stomach and intestine. The intestine was a short, tubular structure with no morphological differences between the various regions. Histologically, the intestinal regions were similar, the main difference being in the number of goblet cells that increased from anterior to posterior intestine, with 114 ± 9, 153 ± 7 and 317 ± 21 goblet cells in the anterior, mid and posterior regions, respectively. The intestinal epithelium stained positively for PAS, but the staining was stronger for acidic glycoproteins. The rectum was similar to intestine, except for increased goblet cell numbers (anterior rectum: 529 ± 26; posterior rectum: 745 ± 29). Gut morpho-histology did not respond to salinity changes, however, there was a significant reduction of mucosal height, goblet cell numbers and muscularis thickness upon food deprivation.

Developmental transcriptome analysis and identification of genes involved in formation of intestinal air-breathing function of Dojo loach, Misgurnus anguillicaudatus.

Dojo loach, Misgurnus anguillicaudatus is a freshwater fish species of the loach family Cobitidae, using its posterior intestine as an accessory air-breathing organ. Little is known about the molecular regulatory mechanisms in the formation of intestinal air-breathing function of M. anguillicaudatus. Here high-throughput sequencing of mRNAs was performed from six developmental stages of posterior intestine of M. anguillicaudatus: 4-Dph (days post hatch) group, 8-Dph group, 12-Dph group, 20-Dph group, 40-Dph group and Oyd (one-year-old) group. These six libraries were assembled into 81300 unigenes. Totally 40757 unigenes were annotated. Subsequently, 35291 differentially expressed genes (DEGs) were scanned among different developmental stages and clustered into 20 gene expression profiles. Finally, 15 key pathways and 25 key genes were mined, providing potential targets for candidate gene selection involved in formation of intestinal air-breathing function in M. anguillicaudatus. This is the first report of developmental transcriptome of posterior intestine in M. anguillicaudatus, offering a substantial contribution to the sequence resources for this species and providing a deep insight into the formation mechanism of its intestinal air-breathing function. This report demonstrates that M. anguillicaudatus is a good model for studies to identify and characterize the molecular basis of accessory air-breathing organ development in fish.

Acid and alkaline phosphatase localization in the digestive tract mucosa of the Hemisorubim platyrhynchos

This cytochemical study investigated the acid and alkaline phosphatase of the digestive tract of Hemisorubim platyrhynchos. Acid phosphatase was detected in the lining epithelium throughout the digestive tract, whereas alkaline phosphatase was only observed in the intestine. In the esophagus, an acid phosphatase reaction occurred in the apical cytoplasm of the epithelial cells and was related to epithelial protection and freeing of superficial cells for sloughing. Similar results were also observed in epithelial cells of gastric epithelium. In the gastric glands, acid phosphatase occurred in lysosomes of the oxynticopeptic cells acting in the macromolecule degradation for use as an energy source, whereas in the vesiculotubular system, its presence could be related to secretion processes. Furthermore, acid phosphatase in the intestine occurred in microvilli and lysosomes of the enterocytes and was correlated to absorption and intracellular digestion. However, no difference was reported among the regions of the intestine. However, alkaline phosphatase reaction revealed a large number of reaction dots in the anterior intestine, with the number decreasing toward the posterior intestine. This enzyme has been related to several functions, highlighting its role in the nutrient absorption primarily in the anterior intestine but also being essential in pH regulation because this is a carnivorous species with many gastric glands with secretions that could damage the intestine.

Transcriptomic Analysis of Compromise Between Air-Breathing and Nutrient Uptake of Posterior Intestine in Loach (Misgurnus anguillicaudatus), an Air-Breathing Fish.

Dojo loach (Misgurnus anguillicaudatus) is an air-breathing fish species by using its posterior intestine to breathe on water surface. So far, the molecular mechanism about accessory air-breathing in fish is seldom addressed. Five cDNA libraries were constructed here for loach posterior intestines form T01 (the initial stage group), T02 (mid-stage of normal group), T03 (end stage of normal group), T04 (mid-stage of air-breathing inhibited group), and T05 (the end stage of air-breathing inhibited group) and subjected to perform RNA-seq to compare their transcriptomic profilings. A total of 92,962 unigenes were assembled, while 37,905 (40.77%) unigenes were successfully annotated. 2298, 1091, and 3275 differentially expressed genes (fn1, ACE, EGFR, Pxdn, SDF, HIF, VEGF, SLC2A1, SLC5A8 etc.) were observed in T04/T02, T05/T03, and T05/T04, respectively. Expression levels of many genes associated with air-breathing and nutrient uptake varied significantly between normal and intestinal air-breathing inhibited group. Intraepithelial capillaries in posterior intestines of loaches from T05 were broken, while red blood cells were enriched at the surface of intestinal epithelial lining with 241 ± 39 cells per millimeter. There were periodic acid-schiff (PAS)-positive epithelial mucous cells in posterior intestines from both normal and air-breathing inhibited groups. Results obtained here suggested an overlap of air-breathing and nutrient uptake function of posterior intestine in loach. Intestinal air-breathing inhibition in loach would influence the posterior intestine's nutrient uptake ability and endothelial capillary structure stability. This study will contribute to our understanding on the molecular regulatory mechanisms of intestinal air-breathing in loach.

Effects of dietary waterlily (Nymphaea pubescens) stamen extract on growth performance and intestinal morphology of common lowland frog (Rana rugulosa)

The effects of diets containing various levels (0, 1, 3 and 5%) of waterlily (Nymphaea pubescens) stamen extract (NPSE) on growth performance and intestinal morphology were investigated in common lowland frog (Rana rugulosa) with an initial weight of 16.09±0.50 g. After 11 weeks of feeding, growth parameters were evaluated. The results showed that the frogs fed with diets supplemented with 3 and 5% of NPSE exhibited significantly higher weight gain, specific growth rate and feed conversion ratio than frogs fed diets supplemented with 0 and 1% of NPSE (P 0.05). Villi heights, villi widths and the thickness of intestinal muscle layers in posterior intestine were significantly increased in frogs fed the diets containing NPSE (P 0.05). Feeding behavior and feed acceptability of the experimental groups were the same as the control group. The optimal levels of NPSE observed in this present study were ranged between 3-5%. Our findings suggest that NPSE can be applied in the diets as a growth promoter in common lowland frog.

Histomorphological changes in digestive tract of golden mahseer (Tor putitora) during different developmental stages.

Histomorphological changes in digestive tract of golden mahseer (Tor putitora) were examined in larvae [starting from hatching to 45days post-hatching (dph)], fry, fingerling, and adult. Digestive tract appeared during hatching, on the dorsal side of yolk sac, as a straight tube with a narrow lumen. Mouth opening and appearance of liver and pancreas were observed at 2dph, and subsequently anal opening, appearance of goblet cells in esophagus, and posterior intestine were evident at 3dph. The remodeling of oral cavity in terms of epithelial stratification, appearance of taste buds, and goblet cells were observed in a window of 4-5dph. Intestinal folding was found to be initiated at 8dph. From 12 to 45dph, thickening of oral and esophageal mucosal/extramucosal layers, increase in intestinal folding, increases in the density of goblet cells in entire gut were observed. Within the same time window, other histological changes such as disappearance of vacuoles in liver, and abundance of zymogen granules in pancreas were also observed. Supranuclear vesicles in mid-to-posterior intestine were found to be prominent from first feeding to 45dph; however, this phenomenon was no longer evident in fry and fingerling. Overall, the increase in intestinal folding and complexity of extramucosal layer were found to be continuous from the first appearance to adult, and this inferred the fact that the nutritional physiology, in terms of digestion and assimilation, progressively changes throughout the life stages of golden mahseer. Findings of this study will, therefore, help in preparing diets for different life stages of this fish, and in addition, the present information widens the understanding of digestive physiology of golden mahseer.

Molecular characterization and expression of Na+/K+-ATPase α1 isoforms in the European sea bass Dicentrarchus labrax osmoregulatory tissues following salinity transfer.

The Na+/K+-ATPase (NKA) is considered as the main pump involved in active ion transport. In the European sea bass, Dicentrarchus labrax, we found two genes encoding for the alpha 1 subunit isoforms (NKA α1a and NKA α1b). NKA α1a and NKA α1b isoform amino acid (aa) sequences were compared through phylogeny and regarding key functional motifs between salmonids and other acanthomorph species. Analysis of aa sequences of both isoforms revealed a high degree of conservation across teleosts. The expression pattern of both nka α1a and nka α1b was measured in the gill, kidney and posterior intestine of fish in seawater (SW) and transferred to fresh water (FW) at different exposure times. Nka α1a was more expressed than nka α1b whatever the condition and the tissue analyzed. After long-term salinity acclimation (2.5years) either in FW or SW, transcript levels of nka α1a were higher in the kidney followed by the posterior intestine and the gill. Compared to SW conditions, expression of nka α1a in FW was significantly increased or decreased, respectively, in gill and posterior intestine. In contrast, branchial nka α1b was significantly decreased in FW-acclimated fish. Short-term FW acclimation seems to rapidly increase nka α1a transcript levels in the kidney unlike in gill tissues where different gene expression levels are detected only after long-term acclimation.

Dietary replacement of fish meal by soy products (soybean meal and isolated soy protein) in silvery-black porgy juveniles (Sparidentex hasta)

A two month feeding trial was conducted in silvery-black porgy (Sparidentex hasta) juveniles (initial mean body weight, 16.7g) to determine the amount of soy products (SP) that could replace fishmeal (FM) in formulated diets without reducing growth performance. Six isoproteic (ca. 50%) and isoenergetic (ca. 22.4MJkg−1) diets were formulated in which 15% (SP15), 30% (SP30), 45% (SP45), 60% (SP60) and 75% (SP75) of FM was replaced by SP, whereas the control diet (FM) was prepared with FM as the major source of protein. Feed intake, growth performance, feed utilization and digestibility of dry matter, protein and lipid significantly decreased by increasing dietary SP supplementation (P<0.05). Fish fed with the SP75 diet showed signs of hyperlipidemia such the highest hepatosomatic index (HSI), high whole body lipid content as well as high plasma cholesterol, triglyceride and very low density lipoprotein levels (P<0.05). Whole body lysine, histidine, methionine and phenylalanine concentrations were lowest in fish fed with the SP75 diet (P<0.05). Concentrations of glycine and taurine decreased in the whole body with increasing dietary SP level. The activity of the trypsin, lipase and α-amylase significantly decreased in pyloric caeca, anterior, mid and posterior intestine of fish with increasing dietary SP inclusion (P<0.05). Fish fed with the SP75 diet showed signs of anemia such as decrease in red blood cell counts and hematocrit, but increase in the mean cell volume. Plasma inorganic phosphorous and alkaline phosphatase decreased and increased, respectively with increasing dietary SP level (P<0.05). The broken-line regression method using feed conversion ratio (FCR) and weight gain (WG) data allowed us to estimate that the maximum replacement of FM with SP in silvery-black porgy diets was between 16.5 and 27.3%. Statements of relevanceSilvery-black porgy (Sparidentex hasta, Valenciennes 1830) is a commercially valuable carnivorous fish species in Oman Sea and Persian Gulf region for supporting marine aquaculture activities. Thus, the aim of present study was to provide insight into the potential ability of silvery-black porgy in using plant proteins sources, by evaluating the effects of dietary replacement of fish meal by soy products (soybean meal and isolated soy protein) in this fish.

Organogenesis of the digestive system in Pacific red snapper (Lutjanus peru ) larvae

This study reports the ontogenetic development of the digestive system of larval Pacific red snapper (Lutjanus peru), an important candidate species for aquaculture on the Pacific coast of Mexico. Histological sections of larvae were cut and dyed using the haematoxylin–eosin technique. The development of the digestive tract of Pacific red snapper larvae follows a general pattern of differentiation that can be divided into three stages. Stage I lasted from 1–3 days post hatching (DPH) and included the endogenous nutrition period; it was characterized by the initial differentiation of the digestive tract in preparation for the onset of exogenous feeding (3 DPH). At this time, the digestive tract was differentiated into buccopharynx, oesophagus, stomach anlage, anterior intestine, posterior intestine and a short rectum. The liver, pancreas and kidney were also present. The mouth and anus were open. Stage II occurred after first feeding, lasted for 16 days (4–23 DPH) and included both preflexion and flexion larvae. The main changes that occurred during this stage reflected the adaptation to exogenous feeding and the concomitant growth. Stage III (24–30 DPH) included post-flexion larvae and started with the appearance of the gastric glands and pyloric caeca. The presence of the gastric glands suggests that early weaning during culture trials of the Pacific red snapper larvae may be possible at this early age.

The role of the rectum in osmoregulation and the potential effect of renoguanylin on SLC26a6 transport activity in the Gulf toadfish (Opsanus beta).

Teleosts living in seawater continually absorb water across the intestine to compensate for branchial water loss to the environment. The present study reveals that the Gulf toadfish (Opsanus beta) rectum plays a comparable role to the posterior intestine in ion and water absorption. However, the posterior intestine appears to rely more on SLC26a6 (a HCO3 (-)/Cl(-) antiporter) and the rectum appears to rely on NKCC2 (SLC12a1) for the purposes of solute-coupled water absorption. The present study also demonstrates that the rectum responds to renoguanylin (RGN), a member of the guanylin family of peptides that alters the normal osmoregulatory processes of the distal intestine, by inhibited water absorption. RGN decreases rectal water absorption more greatly than in the posterior intestine and leads to net Na(+) and Cl(-) secretion, and a reversal of the absorptive short-circuit current (ISC). It is hypothesized that maintaining a larger fluid volume within the distal segments of intestinal tract facilitates the removal of CaCO3 precipitates and other solids from the intestine. Indeed, the expression of the components of the Cl(-)-secretory response, apical CFTR, and basolateral NKCC1 (SLC12a2), are upregulated in the rectum of the Gulf toadfish after 96 h in 60 ppt, an exposure that increases CaCO3 precipitate formation relative to 35 ppt. Moreover, the downstream intracellular effects of RGN appear to directly inhibit ion absorption by NKCC2 and anion exchange by SLC26a6. Overall, the present findings elucidate key electrophysiological differences between the posterior intestine and rectum of Gulf toadfish and the potent regulatory role renoguanylin plays in osmoregulation.

Effect of temperature on growth performance and immunological parameters of greater amberjack Seriola dumerili juveniles

The greater amberjack Seriola dumerili (Risso 1810) is a marine pelagic species with a high interest for aquaculture due to its rapid growth and high commercial value. However, during grow out, monogenean parasites cause occasional mass mortalities in farmed fish and there is not so much published data on the optimum temperature for growth and immunological parameters of this fish species fingerlings. Two hundred and twenty five juveniles were kept in triplicated in three experimental recirculated aquaculture system (RAS) at 17, 22 and 26 oC, each RAS controlling 3 tanks. Fish were fed a commercial diet to apparent satiety during four months. Growth was monitored each month. At the end of the experimental period samples of intestine, skin, blood and skin mucus were obtained. Growth, feed efficiency and immunological parameters, such as lysozyme and bactericidal activity have been studied to observe the relations between the different growth rates produced by the temperature and the immune system in serum and skin mucus. Besides, samples form skin and intestine were also obtained for histological studies, including the mucus production potential by analysing the goblet cells and the distribution of GALT in posterior intestine. Fish held at 26oC presented the best growth and feed utilization, being the results obtained for immune potential discussed also in terms of temperature influence.

Comparison of the effects of inosine and inosine monophosphate on growth, immune response, stress resistance and gut morphology of juvenile red sea bream, Pagrus major

Inosine and inosine monophosphate (IMP) are the most extensively studied specific nucleotide for feeding stimulation research rather than using as functional nutrients for potential growth and health benefit of aquatic species. Therefore, a 10-week feeding trial was conducted to determine comparative effects of inosine and IMP as functional nutrients on growth performance, immune responses, stress resistance and intestinal morphology of juvenile red sea bream (Pagrus major). Casein based semi-purified basal diet was formulated to contain 54% protein, 11% lipid, without inosine and IMP supplementation (Control). Four levels of inosine nucleoside (0.2, 0.4, 0.6 and 0.8% for diet groups INO-0.2, INO-0.4, INO-0.6 and INO-0.8 respectively) and IMP nucleotide (0.2, 0.4, 0.6 and 0.8% for diet groups IMP-0.2, IMP-0.4, IMP-0.6 and IMP-0.8 respectively) were added to the basal diet. Each diet was randomly allocated to triplicate groups of fish with initial average weight of 6.6g. The results indicated that dietary inosine and IMP supplementations tended to improve growth performances, in which one of the best ones was found in diet group IMP-0.6, but the values did not significantly differ from those in diet groups IMP-0.4 and INO-0.4, respectively. Superoxide dismutase, peroxidase and bactericidal activity were significantly influenced by dietary supplementation of inosine and IMP. However, the values of these parameters were not significantly different among diet groups INO-0.4, IMP-0.4 and IMP-0.6. Total serum protein, catalase and lysozyme activity were also improved (P>0.05) by dietary supplementation of inosine and IMP. Supplementations improved both freshwater stress and oxidative stress resistances. Fish fed diet groups INO-0.4 and IMP-0.6 showed the least oxidative stress condition. Inosine and IMP supplementations significantly increased enterocyte height (hE), fold height (hF) of both anterior and posterior intestine and microvillus height (hMV) of posterior intestine compared to those of the control. The highest hF observed in diet group IMP-0.4 and the mean value was not significantly different from those in other IMP diets, INO-0.4 and INO-0.6, respectively. Significantly highest anterior hE and posterior hF, hE and hMV observed in diet group INO-0.4. However, no significant difference of posterior hF observed between INO-0.4 and IMP supplemented diet groups. Fish fed supplemented diets also showed improved survival compared to control (P>0.05). Considering overall performances, it can be concluded that diet groups INO-0.4 and IMP-0.4 showed relatively better performance among inosine and IMP supplemented diet groups respectively compared to control. Furthermore, diets supplemented with either inosine or IMP at 0.4% are beneficial to promote growth, immune responses, stress resistance and intestinal health condition of juvenile red sea bream. Statement of relevanceIn this study inosine and inosine monophosphate has been evaluated as potential functional nutrients for marine fish such as red sea bream. Since supplementations of such immunostimulant-like functional nutrients are very important for the development of functional aquafeeds, I hope findings of this paper will help to develop low fishmeal based functional aquafeeds in near future.

High-Throughput Sequencing Identifies MicroRNAs from Posterior Intestine of Loach (Misgurnus anguillicaudatus) and Their Response to Intestinal Air-Breathing Inhibition.

MicroRNAs (miRNAs) exert important roles in animal growth, immunity, and development, and regulate gene expression at the post-transcriptional level. Knowledges about the diversities of miRNAs and their roles in accessory air-breathing organs (ABOs) of fish remain unknown. In this work, we used high-throughput sequencing to identify known and novel miRNAs from the posterior intestine, an important ABO, in loach (Misgurnus anguillicaudatus) under normal and intestinal air-breathing inhibited conditions. A total of 204 known and 84 novel miRNAs were identified, while 47 miRNAs were differentially expressed between the two small RNA libraries (i.e. between the normal and intestinal air-breathing inhibited group). Potential miRNA target genes were predicted by combining our transcriptome data of the posterior intestine of the loach under the same conditions, and then annotated using COG, GO, KEGG, Swissprot and Nr databases. The regulatory networks of miRNAs and their target genes were analyzed. The abundances of nine known miRNAs were validated by qRT-PCR. The relative expression profiles of six known miRNAs and their eight corresponding target genes, and two novel potential miRNAs were also detected. Histological characteristics of the posterior intestines in both normal and air-breathing inhibited group were further analyzed. This study contributes to our understanding on the functions and molecular regulatory mechanisms of miRNAs in accessory air-breathing organs of fish.

Histoenzymological Observations on Acid Phosphatase Activity in the Posterior Intestine of HGCL2–Treated Fish, Channa striatus

Present studies incorporate enterotoxic effects of HgCl2 on the relative distribution of acid phosphatase (ACP) activity in the posterior intestine of fish, Channa striatus. It was observed that there appears to be slightly increased ACP activity accompanied by histolytic changes in the columnar epithelial cells lining of villi and intestinal glands of the posterior intestine of treated carnivorous fish. However, in control, these changes were invisible.

Cloning, tissue expression, and nutritional regulation of the α-amylase gene in the herbivorous marine teleost Siganus canaliculatus

Alpha-amylase (α-amylase) is a carbohydrase that plays a major role in carbohydrate metabolism. However, little is known about its molecular and biochemical characterisation in herbivorous marine teleosts. In the present study, an α-amylase gene was cloned and its tissue expression was determined in rabbitfish Siganus canaliculatus, a crucial cultured herbivorous marine teleost in China. The nutritional regulation of the gene, including its mRNA expression and enzymatic activity, was also investigated. The full length of its cDNA was 1903bp, containing a 1539bp open reading frame encoding a polypeptide of 512 amino acids, which possessed all the characteristic features of the α-amylase family. Its mRNA expression was detected in hepatopancreas, anterior intestine, middle intestine and posterior intestine, but not in other parts of the alimentary system. Four dietary groups of rabbitfish were each fed one of the following: raw fish (RF), formulated diet (FD), seaweed Enteromorpha prolifra (EP), or seaweed Gracilaria lemaneiformis (GL). After 8weeks of feeding, the expression level of α-amylase mRNA in hepatopancreas, anterior intestine, middle intestine and posterior intestine showed no significant differences among the four dietary groups, whereas the α-amylase activity in the RF group was significantly lower than that in the other dietary groups (P<0.001), and significantly higher in the mid and posterior intestine of fish fed GL than those in fish fed with EP (P<0.01). These findings suggest that the nutritional regulation of α-amylase synthesis in rabbitfish may occur at the posttranscriptional level. This study is the first characterisation of the α-amylase gene from a herbivorous marine culture teleost. Statement of relevanceAlpha-amylase (α-amylase) is an important carbohydrase that plays a major role in carbohydrate metabolism. This study provides the first investigation of cloning and characterisation of α-amylase gene from a herbivorous marine-cultured teleost rabbitfish, and provides a foundation for the development of low-cost and effective formulated feed with seaweed as a dietary ingredient for rabbitfish. The work described has not been published or is under consideration for publication elsewhere. The study was reviewed and approved by the Ethics Committee of Animal Experiments of Shantou University.

Morphological and histological changes in digestive tract development during starvation in the miiuy croaker.

A histological method was used to describe the ontogenetic development of the digestive tract of laboratory-reared miiuy croaker (Miichthys miiuy) and to evaluate the effects of short-term food deprivation on the morphology and histology of the digestive tract. Larvae and juveniles were maintained at 24 °C in a thermostatically controlled system. Three starvation experiments were conducted during different developmental stages: 1-7 days after hatching (dah; prior to benthic swimming); 26-35 dah (during settling); and 42-53 dah (after benthic swimming). According to the structural changes in the ontogenetic development of the digestive tract, three stages were observed. The first stage was from hatching to 3 dah; the digestive tract was undifferentiated in newly hatched larvae and then showed remarkable morphological changes and differentiation. During this period, larvae depended on endogenous nutrition. The second stage (4-20 dah) was a critical period in which larvae transitioned from endogenous feeding to exogenous feeding and the digestive tract fully differentiated into the buccopharynx, oesophagus, stomach, anterior intestine and posterior intestine. Goblet cells and vacuoles appeared in the digestive tract, and pharyngeal teeth and taste buds developed. During the third stage (20-36 dah), the gastric glands developed and the stomach differentiated into the fundic, cardiac and pyloric regions. At 25 dah, pyloric caeca developed and mucosal folds and spiral valves were clearly distinguishable. After 30 dah, the digestive tract did not undergo any noticeable differentiation, indicating the complete development of the digestive system. The wet weight and SGR (specific growth rate) of miiuy croaker larvae and juveniles greatly decreased when they were deprived of food, and compensatory growth was observed in re-feeding juveniles. The livers of starved larvae and juveniles were atrophied and dark coloured, the intestines were transparent and thin, and the stomach cubages were reduced. The histological effects of starvation were mainly evident in the degeneration of cells in digestive organs, as seen in the shrinkage and separation of cells and the loss of intercellular substances in the liver, pancreas, intestine and stomach. These changes became more severe with increased duration of starvation. In addition, the histological structure of the digestive tracts of starved larvae and juveniles partly recovered after re-feeding, and the effects of starvation on miiuy croaker were age dependent.

Hydrolysed wheat gluten as part of a diet based on animal and plant proteins supports good growth performance of Asian seabass (Lates calcarifer), without impairing intestinal morphology or microbiota

A trial was conducted to determine the effect of 6% hydrolysed wheat gluten (HWG) inclusion in a low-fishmeal diet on growth performance and intestinal microbiota and morphology of Asian seabass (Lates calcarifer). Fish (initial average weight of 36.3g) were allocated into floating cages (eight replicates per diet, 30 fish per cage). They were fed either a fishmeal-based diet (positive, diet code: POS; 36% fishmeal), or a diet based on a mix of animal and plant proteins (negative, diet code: NEG). The fishmeal inclusion rate in this diet was reduced to 6%, with the major protein sources being soybean meal and animal by-products. The third diet (diet code: HWG) was similar to the NEG diet with the exception that 6% HWG was included in replacement of the other proteins. At the end of the 48day trial, fish were sampled for intestinal microbiology and histology. Growth parameters were also assessed. Final body weight, SGR and daily feed intake were not significantly different across diets. However, the feeding rate was significantly affected, with the lowest rate observed in the POS treatment and the highest with the NEG treatment. The FCR was significantly lower for both POS and HWG fed fish than for the NEG fed fish. High throughput sequencing revealed that the majority of reads derived from the mucosa samples belonged to members of Proteobacteria (70.3% of the reads), Cyanobacteria (10.0%) and Firmicutes (7.6%). In the digesta reads were mainly assigned to Proteobacteria (34.5%), Fusobacteria (34.5%), and Firmicutes (22.6%). The alpha diversity did not differ among dietary treatments. Some differences in OTU relative abundances were obtained between diets, however, the overall community was not modified to a large extent by HWG. Histological appraisal revealed that the HWG fed fish exhibited significantly higher posterior intestinal perimeter ratio than that of the POS treatment. Overall, including HWG in a low fishmeal diet positively affects feed efficiency. Concomitantly the absorptive surface area of the posterior intestine was improved while the intestinal microbiota, described comprehensively here for the first time in Asian seabass, was similar to the microbiota of other healthy carnivorous marine fish species. The mechanisms involved in these changes may be related to the high glutamine content and to the high protein digestibility of HWG. Statement of relevanceFishmeal used to be the major source of proteins, especially for marine fish and salmonids. Nevertheless, its inclusion in diets has been reduced. Some commercial fish feeds may contain less than 10% fishmeal, largely replaced by a mix of animal and plant proteins. Our manuscript demonstrates that hydrolysed wheat gluten may be used to formulate high quality low fishmeal diets.

Influence of long-term exposure to lead on its accumulation and elimination from tissues and on selected reproductive parameters in the Prussian carp (Carassius gibelio B.) in pond environment

The bioaccumulation of lead in selected tissues of Prussian carp kept in pond condition during 12 and 24 months of exposure to different doses (8, 13, 24, and 49 mg/kg) of this metal in feed and its elimina - tion from tissues during the following 12-month depuration period was studied. Additionally gonadosomatic index and luteinizing hormone (LH) secretion, as the effect of exposure to Pb, were examined. The concentra - tion of lead in all the studied tissues, except for the gonads, correlated positively with the metal concentration in the diet, and the maximum level was usually achieved after 3 months of the exposure. The highest levels of lead, i.e. 2.1 ± 0.14 mg/kg, were found in the posterior intestine in the 15 th month of the exposure, followed by bones, scales and kidneys, in which the level of lead amounted to 1.8 ± 0.20, 1.22 ± 0.07, and 1.17 ± 0.17 mg/kg, respectively. The negative effect of chronic exposure to lead was manifested by a significantly higher spontane - ous LH secretion in groups exposed to 24 and 49 mg/kg of lead and a higher LH secretion level 6 h after the stimulating secretion. After 12 months of exposure and 12 months of depuration, as well as after 24 months of exposure, the effects of lead on LH secretion were not observed. Environmental lead can be a potent endocrine disruptor, which may have an adverse impact on fish reproduction. Prussian carps become resistant to the negative effects of lead with age and their organisms cope by reaching a state of homeostasis.