Comprehensive mRNA and microRNA analysis revealed the effect and response strategy of freshwater fish, grass carp (Ctenopharyngodon idella) under geosmin exposure.

Geosmin is an environmental pollutant that causes off-flavor in water and aquatic products. The high occurrence of geosmin contamination in aquatic systems and aquaculture raises public awareness, however, few studies have investigated the response pathways of geosmin stress on freshwater fish. In this research, grass carp were exposed to 50 µg/L geosmin for 96 h, liver tissue was sequenced and validated using real-time qPCR. In total of 528 up-regulated genes and 488 down-regulated genes were observed, includes cytochrome P450 and uridine diphosphate (UDP)-glucuronosyltransferase related genes. KEGG analysis showed that chemical carcinogenesis-DNA adducts, metabolism of xenobiotics by cytochrome P450, drug metabolism-cytochrome P450 pathway was enriched. Common genes from the target genes of microRNAs and differential expression genes are enriched in metabolism of xenobiotics cytochrome P450 pathway. Two miRNAs (dre-miR-146a and miR-212-3p) down regulated their target genes (LOC127510138 and adh5, respectively) which are enriched cytochrome P450 related pathway. The results present that geosmin is genetoxic to grass carp and indicate that cytochrome P450 system and UDP-glucuronosyltransferase play essential roles in biotransformation of geosmin. MicroRNAs regulate the biotransformation of geosmin by targeting specific genes, which contributes to the development of strategies to manage its negative impacts in both natural and artificial environments.

Comparative digestive biology between the ponyfishes from the Pranburi River estuary, Thailand.

We investigated the digestive biology of two prevalent leiognathid species in Pranburi River estuary, Thailand: the decorated ponyfish (Nuchequula gerreoides) and the splendid polyfish (Eubleekeria splendens). A total of 632 samples collected from February to April and September to November 2017 were analysed using morphological and histological approaches. The overall structures were similar between the species: a short mucous-cell-rich oesophagus region, a well-developed gastric gland uniformly present across the stomach's mucosal layer, and three finger-like pyloric caeca between the stomach and intestine. However, there were marked differences in the mouth, gill raker, and intestinal coefficient (IC). N. gerreoides had a relatively longer mouth, smoother gill rakers, and an IC of 1.08 ± 0.01, similar to those of other carnivorous fish. In contrast, the gill raker of E. splendens had more villiform teeth that can filter-feed better, and their IC was 2.16 ± 0.02 (i.e., longer intestine). Although digestive structures were generally similar between the ponyfishes, these differences suggest that N. gerreoides is relatively carnivorous with stronger suction, whereas E. splendens may be an omnivorous or herbivorous filter-feeder.

Histological structure of the digestive tract, liver, and pancreas of Ambassis nalua (Hamilton, 1822) with ultrastructural details of the gastric gland.

The scalloped perchlet Ambassis nalua is one of the dominant fishes in the Estuarine Pranburi River, Thailand. It is suggested that this fish is in the secondary trophic level with a carnivorous nature. Studies on digestive system will help us further identify the niche of this species in the food web/food chain. The present study therefore aimed to report the detailed structure and ultrastructure of A. nalua digestive system. Fish samples (n = 30) with a total length of 5.7 ± 0.5 cm were obtained using beach seines from the Estuarine Pranburi River. Their digestive tract length and intestine coeficient were 3.6 ± 0.07 cm and 0.91, respectively. Light microscopic observation showed that the digestive wall comprised four layers, namely mucosa, submucosa, muscularis, and serosa. The prominent mucous-secreting cells were found in the mucosal oesophagus. The stomach had many gastric folds, with height and width being 649.76 ± 85.15 and 370.30 ± 68.56 µm, respectively. Gastric glands were found in the anterior stomach but not in the posterior stomach. Each gastric gland was made up of a single type of columnar cells. The gastric cells were ultrastructurally characterized by numerous mitochondria and well-developed secretory granules of varying sizes. A few small vacuoles were also identified in the apical area of the gastric cells. The intestine had two regions (anterior and posterior intestines), and pyloric caecum was absent. The density of the goblet cell was significantly higher in the posterior intestine. These results provide basic knowledge of the digestive system of A. nalua, and the low intestine coefficient and the absence of pyloric caecum suggest the carnivorous feeding habit of this species.

Histological characteristics of oocyte differentiation in the captive longnose seahorse Hippocampus trimaculatus (Leach, 1814).

This study aimed to explore the reproductive histology and oocyte differentiation of the longnose seahorse Hippocampus trimaculatus (Leach, 1814) in captivity. Five mature healthy females were histologically observed. The reproductive systems of the five specimens exhibited similar morphological characteristics with a pair of saccular creamy white ovaries merging caudally into a single gonoduct. There were two germinal ridges lined with a layer of germinal epithelium (GE). The ovarian maturation of this species was considered asynchronous. The oogenic cells were classified into oogonia and oocytes at several developmental phases based on their size and characteristics. Oogonia were identified among the connective tissue in the middle area of the GE. The stromal compartment contained oocytes that were classified into four distinct phases: the primary growth (PG) phase having two steps (perinucleolar and oil droplets-cortical alveolar steps) and the secondary growth (SG) phase with three oocyte types, including early SG oocytes, late SG oocytes, and fully grown oocytes. The atretic oocytes (AO) were observed in all stages of oogenesis. Postovulatory follicles were also seen among the ovarian connective tissue. The occurrence of postovulatory follicles suggested that the specimens analysed in this study were in the spawning period. This research provides new insights into the identification of the reproductive cycles and morphological characteristics of the ovary of H. trimaculatus.

Eicosapentaenoic acid (EPA) improves grass carp (Ctenopharyngodon idellus) muscle development and nutritive value by activating the mTOR signaling pathway.

Skeletal muscle is the mainly edible part of fish. Eicosapentaenoic acid (EPA) is a crucial nutrient for fish. This study investigated the effect of EPA on the muscle development of grass carp along with the potential molecular mechanisms in vivo and in vitro. Muscle cells treated with 50 µM EPA in vitro showed the elevated proliferation, and the expression of mammalian target of rapamycin (mTOR) signaling pathway-related genes was upregulated (P < 0.05). In vivo experiments, 270 grass carp (27.92 g) were fed with one of the three experimental diets for 56 days: control diet (CN), 0.3% EPA-supplement diet (EPA), and the diet supplemented with 0.3% EPA and 30 mg/kg rapamycin (EPA + Rap). Fish weight gain rate (WGR) was improved in EPA group (P < 0.05). There was no difference in the viscerosomatic index (VSI) and body height (BH) among all groups (P > 0.05), whereas the carcass ratio (CR) and body length in the EPA group were obviously higher than those of other groups (P < 0.05), indicating that the increase of WGR was due to muscle growth. In addition, both muscle fiber density and muscle crude protein also increased in EPA group (P < 0.05). The principal component analysis showed that total weight of muscle amino acid in EPA group ranked first. Dietary EPA also increased protein levels of the total mTOR, S6k1, Myhc, Myog, and Myod in muscle (P < 0.05). In conclusion, EPA promoted the muscle development and nutritive value via activating the mTOR signaling pathway.

Effects of thermal stress responses in goldfish (Carassius auratus): growth performance, total carotenoids and coloration, hematology, liver histology, and critical thermal maximum.

The present study aimed to investigate the effect of thermal stress on growth, feed utilization, coloration, hematology, liver histology, and critical thermal maximum (CTmax) in goldfish (Carassius auratus) cultured at three different acclimation temperatures including 27 °C, 30 °C, and 34 °C for 10 weeks. Goldfish were assigned randomly to tanks with a quadruplicate setup, accommodating 20 fish per tank. The result showed that fish acclimated to different temperatures did not significantly differ in weight gain (WG) and specific growth rate (SGR). However, increasing temperature significantly decreased feed efficiency ratio (FER), protein efficiency ratio (PER), and protein productive value (PPV), but significantly increased feed conversion ratio (FCR) (P < 0.05). The coloration parameters significantly decreased by high temperature in the trunk region with increasing temperature (L* and a* at week 5; L*, a*, and b* at week 10; P < 0.05). Total carotenoid contents in serum, fin, muscle, and skin also significantly decreased with increasing temperature (P < 0.05). Total protein, albumin, and globulin levels exhibited a notable decrease, while the albumin: globulin ratio showed a slight insignificant increase, with increasing temperature. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total cholesterol, and triglycerides significantly increased with increasing temperature (P < 0.05). While, high-density lipoprotein cholesterol (HDL-c) decreased linearly (P < 0.05). Glucose and cortisol levels linearly increased with increasing temperature, the highest levels being observed in the 34 °C group. Liver histology showed swollen hepatocytes, nuclei displacement, and infiltration of inflammation in fish cultured at 34 °C. Goldfish acclimated to 34 °C displayed a higher CTmax of 43.83 °C compared to other groups. The present study showed that temperature should be kept below 34 °C for goldfish culture to prevent high FCR, fading coloration, and liver damages.

Effects of long-term exposure to high temperature on growth performance, chemical composition, hematological and histological changes, and physiological responses in hybrid catfish [♂Clarias gariepinus (Burchell, 1822) ×♀C. macrocephalus (Günther, 1864)].

The anthropogenic and climate-driven rise in water temperature is expected to have an effect on the physiological functions of ectothermic species. In the present study, hybrid catfish were subjected to three different temperatures (27, 32, and 37 °C) for 50 days to examine the effect of long-term exposure to high temperatures on growth and physiological parameters. The results showed that acclimation temperature improves growth and feed utilization with a quadratic effect (P < 0.05). The highest performance was observed at 32 °C, but fish acclimated at 37 °C decreased growth and feed utilization. In addition, skin darkening was observed in fish acclimated with increasing temperatures. Fat content of whole-body, liver, and dorsal muscle of fish was decreased by increasing temperatures (P < 0.05). Higher temperature levels significantly increased in all blood parameters (P < 0.05), except for high-density lipoprotein cholesterol, which was quadratically decreased (P = 0.004). Fish acclimated with increasing temperature also altered gill and liver histology such as gill shortening, hyperplasia and edema in the connective tissue, severe hyperplasia of epithelial cells, and desquamation, hepatocyte vacuolization, nuclei displacement, and pyknotic hepatic cells. While mucus cells were periphery distributed in the subcutaneous skin. In addition, cuboidal shape-like of club cells and melanophores were also observed in fish acclimated at 37 °C resulting in increased epithelial layer thickness. After fish subjected to increasing temperature exhibited an increase in the number of operculum movement and number of gasping for air (P < 0.05) in all acclimated groups. While fish challenged at 37 °C showed higher critical thermal maximum (CTmax, 41.33 °C) than those of the other groups. Overall, the maximum temperature (37 °C) may rick to hybrid catfish. To prevent physiological damage to the fish, as well as reduction of growth and productivity, the temperature in the aquaculture setting should be kept below 37 °C.

An update on the evolutionary origin of aglomerular kidney with structural and ultrastructural descriptions of the kidney in three fish species.

The kidney of fish contains numerous nephrons, each of which is divided into the renal corpuscle and renal tubules. This glomerular structure is the filtration unit of the nephron and is important for the kidney function, but it has been reported that the renal corpuscle was lost in at least four independent linages of fish (i.e., aglomerular kidney). In this study, the authors newly described renal structures for three species by histological and ultrastructural observations: two aglomerular kidneys from a seahorse Hippocampus barbouri and a toadfish Allenbatrachus grunniens and a glomerular kidney from a snake eel Pisodonophis boro. The renal development of H. barbouri was also described during 1-35 days after birth. In all species tested, the anterior kidney was comprised of haematopoietic tissues and a few renal tubules, whereas the posterior kidney contained more renal tubules. Although the glomerular structure was present in P. boro, light microscopic observations identified no glomeruli in the kidney of H. barbouri and A. grunniens. Ultrastructurally, abundant deep basal infoldings with mitochondria in the renal tubules were observed in A. grunniens compared to H. barbouri and P. boro, suggesting the possible role of basal infoldings in maintaining the osmotic balance. By integrating the results from the three species and comprehensive literature search, the authors further showed that 56 species have been reported to be aglomerular, and that the aglomerular kidney has evolved at least eight times in bony fishes.

Systemic effect of dietary lipid levels and α-lipoic acid supplementation on nutritional metabolism in zebrafish (Danio rerio): focusing on the transcriptional level.

Considering the excessive lipid accumulation status caused by the increased dietary lipid intake in farmed fish, this study aimed to investigate the systemic effect of dietary lipid levels and α-lipoic acid supplementation on nutritional metabolism in zebrafish. A total of 540 male zebrafish (0.17 g) were fed with normal (CT) and high lipid level (HL) diets for 6 weeks, then fed on 1000 mg/kg α-lipoic acid supplementation diets for the second 6 weeks. HL diets did not affect whole fish protein content, but increased ASNS expression (P < 0.05). Dietary α-lipoic acid increased whole fish protein content, and decreased the expressions of protein catabolism-related genes in muscle of high lipid level groups (P < 0.05). Furthermore, HL diets increased the whole fish lipid content and the expressions of gluconeogenesis and lipogenesis-related genes (P < 0.05), and α-lipoic acid counteracted these effects and decreased the whole fish triglyceride and cholesterol contents and expressions of lipogenesis-related genes, with the enhanced expressions of lipolytic genes, especially in high lipid groups (P < 0.05). HL diets did not affect hepatocyte mitochondrial quantity or the mRNA expressions of mitochondrial biogenesis and electron transport chain-related genes; they were significantly increased by dietary α-lipoic acid (P < 0.05). These results indicated that high dietary lipid promotes lipid accumulation, while α-lipoic acid increases protein content in association of enhanced lipid catabolism. Thus, dietary α-lipoic acid supplementation could reduce lipid accumulation under high lipid, which provides a promising new approach in solving the problem of lipid accumulation in farmed fish.

Molecular characterization and homology modeling of liver X receptor in Asian seabass, Lates calcarifer: predicted functions in reproduction and lipid metabolism.

Liver X receptor (LXR) is a ligand-activated transcription factor that plays vital roles in maintaining cholesterol and lipid homeostasis. Much work has been done on mammalian LXRs, but the role of LXR in fish remains unclear. In the present study, LXR gene was identified from adult Asian seabass, Lates calcarifer, and its predicted protein structure was docked with several cholesterol derivatives at the binding site. The LXR cDNA consisted of 1495 bp encoding a putative LXR protein of 494 amino acids. The Asian seabass LXR retained many important structural features found in LXRs of other fishes and mammals, such as putative signal peptide, activation function-1 (AF-1) domain, DNA-binding domain (DBD), ligand-binding domain (LBD), activation function-2 (AF-2) domain, and eight conserved cysteine residues. The deduced amino acid sequence of LXR shared significant identity with those of other species ranging from 65.7 to 95.8%. The homology modeling and in silico molecular docking demonstrated that Asian seabass LXR could interact with cholesterol derivatives at amino acid residues Phe274 and Ile312. Real-time PCR further revealed that LXR transcripts are ubiquitously expressed in all tissues examined, with the highest levels detected in the gonad followed by the liver. Given the well-known importance of cholesterol-mediated signaling in these tissues, Asian seabass LXR may reasonably be involved in reproduction and lipid metabolism.

Comparative analysis of effects of dietary arachidonic acid and EPA on growth, tissue fatty acid composition, antioxidant response and lipid metabolism in juvenile grass carp, Ctenopharyngodon idellus.

Four isonitrogenous and isoenergetic purified diets containing free arachidonic acid (ARA) or EPA (control group), 0·30 % ARA, 0·30 % EPA and 0·30 % ARA+EPA (equivalent) were designed to feed juvenile grass carp (10·21 (sd 0·10) g) for 10 weeks. Only the EPA group presented better growth performance compared with the control group (P<0·05). Dietary ARA and EPA were incorporated into polar lipids more than non-polar lipids in hepatopancreas but not intraperitoneal fat (IPF) tissue. Fish fed ARA and EPA showed an increase of serum superoxide dismutase and catalase activities, and decrease of glutathione peroxidase activity and malondialdehyde contents (P<0·05). The hepatopancreatic TAG levels decreased both in ARA and EPA groups (P<0·05), accompanied by the decrease of lipoprotein lipase (LPL) activity in the ARA group (P<0·05). Fatty acid synthase (FAS), diacylglycerol O-acyltransferase and apoE gene expression in the hepatopancreas decreased in fish fed ARA and EPA, but only the ARA group exhibited increased mRNA level of adipose TAG lipase (ATGL) (P<0·05). Decreased IPF index and adipocyte sizes were found in the ARA group (P<0·05). Meanwhile, the ARA group showed decreased expression levels of adipogenic genes CCAAT enhancer-binding protein α, LPL and FAS, and increased levels of the lipid catabolic genes PPAR α, ATGL, hormone-sensitive lipase and carnitine palmitoyltransferase 1 (CPT-1) in IPF, whereas the EPA group only increased PPAR α and CPT-1 mRNA expression and showed less levels than the ARA group. Overall, dietary EPA is beneficial to the growth performance, whereas ARA is more potent in inducing lipolysis and inhibiting adipogenesis, especially in IPF. Meanwhile, dietary ARA and EPA showed the similar preference in esterification and the improvement in antioxidant response.

Diversity of Lipid Distribution in Fish Skeletal Muscle.

Adipose tissue is a lipid storage organ characterized by the pronounced accumulation of adipocytes. Although adipose tissues are found in various parts of the vertebrate body, it is unclear whether these tissues have a common ancestral origin or have evolved in several phylogenetic lineages by independent adipocyte accumulation events. To gain insight into the evolutionary history of vertebrate adipose tissues, we determined the distribution of adipocytes by oil red O staining in skeletal muscle of 10 teleost species spanning eight orders: Tetraodontiformes, Pleuronectiformes, Spariformes, Salmoniformes, Clupeiformes, Beloniformes, Osmeriformes, and Cypriniformes. Accumulation of adipocytes in the myoseptum was observed in many species, including red seabream, rainbow trout, Pacific herring, Pacific saury, zebrafish and giant danio. We also found some order-, species-, and swimming mode-specific distribution patterns of adipocytes: 1) almost complete absence of intramuscular adipocytes in the order Tetraodontiformes (torafugu and spotted green pufferfish), 2) clear adipocyte accumulation in the inclinator muscles of fin in Japanese flounder, 3) a large intramuscular adipose tissue at the root of the dorsal fin in ayu, and 4) thick lipid layers consisting of subcutaneous adipose tissue and red muscle lipids in pelagic migratory fish (Pacific herring and Pacific saury). Of note, Pacific herring and Pacific saury are phylogenetically distinct species sharing a similar niche and swimming mode, suggesting that their analogous adipocyte/lipid distribution patterns are the consequence of convergent evolution. The potentially heterogeneous origin of adipose tissues has significant implications for the interpretation of their functional diversity.

Different effects of growth hormone and fasting on the induction patterns of two hormone-sensitive lipase genes in red seabream Pagrus major.

Growth hormone (GH) increases phosphorylation and mRNA levels of hormone-sensitive lipase (HSL) in the livers of some marine teleosts. The hepatic GH-HSL axis appears to play important roles in fasting-induced lipolysis. However, it is not known whether GH exerts similar effects on HSL in fish adipose tissues. Functional differentiation of two fish-specific HSL isoforms (HSL1 and HSL2) also remains unclear. The present study seeks to address two unanswered questions about fish lipolysis using red seabream (Pagrus major): (1) Does GH increase phosphorylation and mRNA levels of HSL in adipose tissue? (2) How do GH and fasting affect mRNA levels of two HSL isoform genes in the liver and adipose tissue? To this end, we first cloned HSL1 and HSL2 cDNAs and investigated their tissue distribution. Transcripts of both HSLs and HSL1 proteins were abundant in the visceral adipose tissue, gonads, and liver, suggesting the important role of HSL in adipose tissue lipolysis. HSL2 transcript levels were 20-65% those of HSL1 except in the skin, and HSL2 proteins were not detected by our in-house antisera. Ex vivo administration of GH increased HSL1 phosphorylation, non-esterified fatty acid (NEFA) release, and levels of HSL1 and HSL2 mRNA in both the liver and visceral adipose tissue. Hepatic HSL2 mRNA was particularly sensitive to GH administration and sometimes exceeded HSL1 mRNA levels with up to 13-fold induction. In contrast, fasting for 4 and 7d increased HSL1 mRNA levels, but had only marginal effects on HSL2 mRNA levels in both adipose tissue or liver. We concluded that GH would increase HSL mRNAs during adipose tissue lipolysis in red seabream; however, GH and fasting result in different induction ratio of two HSL isoform genes, suggesting that other hormone(s) also contributes to fasting-induced lipolysis.

Docosahexaenoic acid (DHA) inhibits abdominal fat accumulation by promoting adipocyte apoptosis through PPARγ-LC3-BNIP3 pathway-mediated mitophagy.

Obesity has always been an overwhelming health concern worldwide. Docosahexaenoic acid (DHA) reduces abdominal fat accumulation by inducing adipocyte apoptosis, but the underlying mechanism remains unclear. Mitophagy, the process of maintaining mitochondrial homeostasis, has a double-edged sword effect that positively or negatively regulates apoptosis. In this study, grass carp (Ctenopharyngodon idellus) was used as an animal model to investigate the role of mitophagy in regulating apoptosis and the potential molecular mechanisms for DHA-induced mitophagy in vivo and in vitro. Firstly, we found that DHA induced the intrinsic apoptosis in grass carp adipocytes, accompanying by activating BNIP3/NIX-mediated mitophagy. Then, suppression of mitophagy alleviated apoptosis and eliminated the inhibition of lipid accumulation induced by DHA in vivo and in vitro. Mechanistically, the DHA-induced mitophagy was caused by activating PPARγ and its DNA binding capacity to the LC3 promoter, which promoted the interaction of BNIP3 (rather than NIX) with LC3. However, the inhibition of PPARγ in vitro significantly decreased the expression of autophagy-related genes (P < 0.05), reducing the colocalization of mitochondria and lysosomes while preventing BNIP3/NIX-mediated mitophagy-mediated apoptosis and subsequently alleviating the inhibition of lipid accumulation in adipocytes induced by DHA. For the first time, we demonstrated that DHA activates mitophagy by regulating the PPARγ-LC3-BNIP3 pathway, consequently inducing apoptosis, which decreases adipocytes, inhibiting lipid accumulation in grass carp. These findings provide new insight into the mechanism of DHA-induced apoptosis mediated by mitophagy as the potential therapeutic target of inhibiting abdominal fat accumulation in vertebrates.

A rapid and simple procedure for the whole-mount bone staining of small fish.

The cartilage and bone structure has provided insightful knowledge about evolution and ecology of fish, which is an important component of biological oceanography. However, the whole-body bone staining is a lengthy and complicated process that typically takes five days to several months, and the improvement of the conventional method has been one of the important issues in this field. Here we report a quick and easy whole-mount bone staining method for small fish, in which a newly designed fixative is applied. Compared to conventional methods, this novel protocol is a straightforward process that could be adopted for small estuarine fish and other small vertebrates.

Integration of Multi-Omics, Histological, and Biochemical Analysis Reveals the Toxic Responses of Nile Tilapia Liver to Chronic Microcystin-LR Exposure.

Microcystin-LR (MC-LR) is a cyanobacterial metabolite produced during cyanobacterial blooms and is toxic to aquatic animals, and the liver is the main targeted organ of MC-LR. To comprehensively understand the toxicity mechanism of chronic exposure to environmental levels of MC-LR on the liver of fish, juvenile Nile tilapia were exposed to 0 µg/L (control), 1 µg/L (M1), 3 µg/L (M3), 10 µg/L (M10), and 30 µg/L (M30) MC-LR for 60 days. Then, the liver hepatotoxicity induced by MC-LR exposure was systematically evaluated via histological and biochemical determinations, and the underlying mechanisms were explored through combining analysis of biochemical parameters, multi-omics (transcriptome and metabolome), and gene expression. The results exhibited that chronic MC-LR exposure caused slight liver minor structural damage and lipid accumulation in the M10 group, while resulting in serious histological damage and lipid accumulation in the M30 group, indicating obvious hepatotoxicity, which was confirmed by increased toxicity indexes (i.e., AST, ALT, and AKP). Transcriptomic and metabolomic analysis revealed that chronic MC-LR exposure induced extensive changes in gene expression and metabolites in six typical pathways, including oxidative stress, apoptosis, autophagy, amino acid metabolism, primary bile acid biosynthesis, and lipid metabolism. Taken together, chronic MC-LR exposure induced oxidative stress, apoptosis, and autophagy, inhibited primary bile acid biosynthesis, and caused fatty deposition in the liver of Nile tilapia.

Differences in lipid distribution and expression of peroxisome proliferator-activated receptor gamma and lipoprotein lipase genes in torafugu and red seabream.

Lipid content is one of the major determinants of the meat quality in fish. However, the mechanisms underlying the species-specific distribution of lipid are still poorly understood. The present study was undertaken to investigate the mechanisms associated with lipid accumulation in two species of fish: torafugu (a puffer fish) and red seabream. The lipid content of liver and carcass were 67.0% and 0.8% for torafugu, respectively, and 8.8% and 7.3% for red seabream, respectively. Visceral adipose tissue was only apparent in the red seabream and accounted for 73.3% of its total lipid content. Oil red O staining confirmed this species-specific lipid distribution, and further demonstrated that the lipid in the skeletal muscle of the red seabream was mainly localized in the myosepta. We subsequently cloned cDNAs from torafugu encoding lipoprotein lipase 1 (LPL1) and LPL2, important enzymes for the uptake of lipids from blood circulation system into various tissues. The relative mRNA levels of peroxisome proliferator-activated receptor gamma (PPARγ) and the LPLs of torafugu were determined by quantitative real-time PCR together with their counterparts in red seabream previously reported. The relative mRNA levels of PPARγ and LPL1 correlated closely to the lipid distribution of both fish, being significantly higher in liver than skeletal muscle in torafugu, whereas the highest in the adipose tissue, followed by liver and skeletal muscle in red seabream. However, the relative mRNA levels of LPL2 were tenfold lower than LPL1 in both species and only correlated to lipid distribution in torafugu, suggesting that LPL2 has only a minor role in lipid accumulation. In situ hybridization revealed that the transcripts of LPL1 co-localized with lipids in the adipocytes located along the myosepta of the skeletal muscle of red seabream. These results suggest that the transcriptional regulation of PPARγ and LPL1 is responsible for the species-specific lipid distribution of torafugu and red seabream.

A genome-wide screening of the 70 kDa heat shock protein (HSP70) genes in the rotifer Brachionus plicatilis sensu stricto with a characterization of two heat-inducible HSP70 genes.

The 70 kDa heat shock proteins (HSP70s) and the constitutive members of the HSP70 family (heat shock cognates; HSC70s) play essential roles in various biological processes. The number of hsp70/hsc70 in the database is rapidly increasing because of their importance and the automatic annotation of newly sequenced genomes. However, accumulating evidence indicates that neither hsp70 nor hsc70 forms a monophyletic gene family, raising the need to reconsider the annotation strategy based on the traditional concept of the inducible HSP70 and constitutive HSC70s. The main aim of this study is to establish a systematic scheme to annotate hsp70-like genes taking the latest phylogenetic insights into account. We cloned two hsp70s from the rotifer Brachionus plicatilis sensu stricto (s.s.), an emerging model in evolutionary genetics, and conducted a genome-wide screening of B. plicatilis s.s. hsp70s using the two sequences as queries. A total of 15 hsp70-like genes were found, and 7 of them encoded distant members of the HSP70 family, the function of which largely remains unknown. Eight canonical hsp70s were annotated according to a recently proposed nomenclature based on the molecular evolution: e.g., HSP70cA1/B1 for the cytosolic lineage, HSP70er1 for the endoplasmic reticulum lineage, and HSP70m1 for the mitochondrial lineage. The two cloned hsp70s, HSP70cB1 and HSP70cB2, ubiquitously increased their mRNA levels up to 7.5 times after heat treatment as assessed by semi-quantitative PCR, real-time PCR, and in situ hybridization. This systematic screening incorporating a reasonable update to the annotation strategy would provide a useful example for future HSP70 studies, especially those in non-traditional model organisms.

Effects of heat stress on the chemical composition, oxidative stability, muscle metabolism, and meat quality of Nile tilapia (Oreochromis niloticus).

The present study investigated the effects of chronic heat stress (HS) on the chemical composition, oxidative stability, muscle metabolism, and meat quality of Nile tilapia (Oreochromis niloticus). Compared with the control (26 °C), chronic HS (32 °C) lowered growth performance, the contents of whole-body lipid, muscle protein, and muscle lipid. Also, HS significantly increased the contents of reactive oxygen species (ROS) and decreased antioxidative status, causing a decline in meat quality, including increased lipid and protein oxidation, the centrifugal water loss, and cooking loss as well as decreased the fragmentation index and pH at 24 h, which may be attributed to induced apoptosis by excessive ROS in Nile tilapia meat. Moreover, metabolomic analysis showed HS lowered flavor and nutritional value by affecting amino acid, lipid, and nucleotide metabolism. These results reveal that HS adversely affects oxidative stability, meat quality, flavor, and nutrition, warranting its recognition and prevention.