Hepatopancreas Transcriptome Analysis of Spinibarbus sinensis to Reveal Different Growth-Related Genes.

Spinibarbus sinensis, also known as Qingbo, is an important economic fish in China. However, the detailed mechanisms underlying its growth are still unknown. To excavate the genes and signaling pathways related to its growth, we compared the transcriptome profiles of the hepatopancreas tissues of S. sinensis, with two groups of growth rate for evaluation. An average of 66,304,909 and 68,739,585 clean reads were obtained in the fast growth (FG) and slow growth (SG) group, respectively. The differential gene expression analysis results showed that 272 differentially expressed genes (DEGs) were screened between the FG and SG groups, including 101 up-regulated genes and 171 down-regulated genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis results showed that GO terms related to metabolic process, organic substance metabolic process, and catalytic activity were enriched, pathway signals related to steroid biosynthesis and protein digestion and absorption were also detected. Meanwhile, the potential key regulatory genes sst2, fndc4, and cckra related to the growth of S. sinensis were screened. Reverse transcript fluorescence quantitative PCR (RT-qPCR) validation of 18 DEGs associated with growth differences showed that the RT-qPCR results were consistent with RNA-seq analysis, and nine genes, stk31, gpr149, angptl1, fstl1, sik1, ror2, nlrc3, pdlim2, and nav2 were significantly expressed in the FG group. bmp1, stc1, gpatch8, sstrt2, s100a1, ktf6, cckar6, sync1, bhlha15, a total of nine genes were significantly expressed in the SG group. This study provides basic information for improving the growth characteristics of S. sinensis and the functional research of candidate genes.

Functional Study of the Role of the Methyl Farnesoate Epoxidase Gene in the Ovarian Development of Macrobrachium nipponense.

Methyl farnesoate epoxidase (MFE) is a gene encoding an enzyme related to the last step of juvenile hormone biosynthesis. Mn-MFE cDNA has a total length of 1695 bp and an open reading frame (ORF) length of 1482 bp, encoding 493 amino acids. Sequence analysis showed that its amino acid sequence has a PPGP hinge, an FGCG structural domain, and other structural domains specific to the P450 family of enzymes. Mn-MFE was most highly expressed in the hepatopancreas, followed by the ovary and gill, weakly expressed in heart and muscle tissue, and barely expressed in the eyestalk and cranial ganglion. Mn-MFE expression remained stable during the larval period, during which it mainly played a critical role in gonadal differentiation. Expression in the ovary was positively correlated and expression in the hepatopancreas was negatively correlated with ovarian development. In situ hybridization (ISH) showed that the signal was expressed in the oocyte, nucleus, cell membrane and follicular cells, and the intensity of expression was strongest at stage O-IV. The knockdown of Mn-MFE resulted in a significantly lower gonadosomatic index and percentage of ovaries past stage O-III compared to the control group. However, no differences were found in the cumulative frequency of molting between the experimental and control groups. Moreover, the analysis of ovarian tissue sections at the end of the experiment showed differences between groups in development speed but not in subcellular structure. These results demonstrate that Mn-MFE promotes the ovarian development of Macrobrachium nipponense adults but has no effect on molting.

Hhex regulates the specification and growth of the hepatopancreatic ductal system.

Significant efforts have advanced our understanding of foregut-derived organ development; however, little is known about the molecular mechanisms that underlie the formation of the hepatopancreatic ductal (HPD) system. Here, we report a role for the homeodomain transcription factor Hhex in directing HPD progenitor specification in zebrafish. Loss of Hhex function results in impaired HPD system formation. We found that Hhex specifies a distinct population of HPD progenitors that gives rise to the cystic duct, common bile duct, and extra-pancreatic duct. Since hhex is not uniquely expressed in the HPD region but is also expressed in endothelial cells and the yolk syncytial layer (YSL), we tested the role of blood vessels as well as the YSL in HPD formation. We found that blood vessels are required for HPD patterning, but not for HPD progenitor specification. In addition, we found that Hhex is required in both the endoderm and the YSL for HPD development. Our results shed light on the mechanisms directing endodermal progenitors towards the HPD fate and emphasize the tissue specific requirement of Hhex during development.

Identification and relative abundances of mRNA for a gene encoding the vWD domain and three Kazal-type domains in the ovary of giant freshwater prawns, Macrobrachium rosenbergii.

Understanding Macrobrachium rosenbergii ovarian maturation control at the genome level is an important aspect for increasing larvae production. In this study, an ovarian maturation related gene, M. rosenbergii vWD domain and three Kazal-type domains of a gene (MrvWD-Kazal) have been studied. The MrvWD-Kazal gene was isolated using a rapid amplification of cDNA end (RACE) method and the relative abundances of MrvWD-Kazal mRNA in the ovary, hepatopancreas, stomach, intestine and gill were determined by using the quantitative PCR technique. The MrvWD-Kazal gene is composed of 2194 bp with an open reading frame (ORF) of 1998 bp encoding 665 amino acids and has great similarity to the M. nipponense vWD-Kazal gene (91%). The qPCR analyses indicated the relative abundance of MrvWD-Kazal mRNA transcript varied among different stages of ovarian function (P < 0.05), but there were no differences abundance in hepatopancreas, stomach, intestine and gill (P> 0.05). In the ovary, relative abundance of MrvWD-Kazal mRNA transcript gradually increased with ovarian maturation from Stages 1 (Spent; 1.00-fold), to 2 (Proliferative; 3.47-fold) to 3 (Premature; 6.18-fold) and decreased at Stage 4 (Mature; 1.31-fold). Differential relative abundances of MrvWD-Kazal mRNA transcript in the ovary indicate the MrvWD-Kazal protein may have an important function in ovarian maturation of M. rosenbergii. The results of this study also indicate the MrvWD-Kazal is not involved in regulation of the reproductive related function of the hepatopancreas, digestive system (stomach and intestine) and respiratory system (gill).

Integration of Biochemical, Cellular, and Genetic Indicators for Understanding the Aging Process in a Bivalve Mollusk Chlamys farreri.

The major causal factors for the irreversible decline in physical vitality during organismal aging are postulated to be a chronic state of cellular redox imbalance, metabolic toxicity, and impaired energy homeostasis. We assessed whether the relevant enzyme activity, oxidative stress, and intracellular ATP might be causally involved in the aging of short-lived Chlamys farreri (life span 4~5 years). A total of eight related biochemical and cellular indicators were chosen for the subsequent analysis. All the indicators were measured in seven different tissues from scallops aged one to four years, and our data support that the aging of C. farreri is associated with attenuated tissue enzyme activity as well as a decreased metabolic rate. Through principal component analysis, we developed an integrated vigor index for each tissue for comprehensive age-related fitness evaluation. Remarkably, all tissue-integrated vigor indexes significantly declined with age, and the kidney was observed to be the most representative tissue. Further transcriptional profiling of the enzymatic genes provided additional detail on the molecular responses that may underlie the corresponding biochemical results. Moreover, these critical molecular responses may be attributed to the conserved hierarchical regulators, e.g., FOXO, AMPKs, mTOR, and IGF1R, which were identified as potentially novel markers for chronic fitness decline with age in bivalves. The present study provides a systematic approach that could potentially benefit the global assessment of the aging process in C. farreri and provide detailed evaluation of the biochemical, cellular, and genetic indicators that might be involved. This information may assist in a better understanding of bivalve adaptability and life span.

Morphology and ultrastructure of the midgut gland ("hepatopancreas") during ontogeny in the common spider crab Maja brachydactyla Balss, 1922 (Brachyura, Majidae).

We studied the anatomy and cytology of the midgut gland (MGl) of the common spider crab Maja brachydactyla Balss, 1922 at several life stages (zoea, megalopa, first juvenile, and adult) using dissection, histology, electron microscopy, computed tomography, and micro-computed tomography (micro-CT). In newly hatched larvae, 14 blind-end tubules form the MGl. The length of the tubules increases during the larval development. In the late megalopa, the number of tubules also increases. In adults, 35,000 to 60,000 blind-ending tubules comprise the MGl. In all life stages, a square-net network of muscle fibers surround the tubules. We describe five cell types in the MGl in all larval stages, which have a similar location, histology, and ultrastructure in larvae and adults: embryonary (E-) cells, resorptive (R-) cells, fibrillar (F-) cells, blister-like (B-) cells, and midget (M-) cells. Major difference between larval and adult cells is the larger size of the adult cells. Microapocrine secretion occurs from the microvilli of the B-cells. No ultrastructural changes were observed during larval development, which suggests that the function of each cell type might be similar in all life stages. The role of each epithelial cell type in larvae and adults is discussed.

Role of Kruppel homolog 1 (Kr-h1) in methyl farnesoate-mediated vitellogenesis in the swimming crab Portunus trituberculatus.

Similar to the role of juvenile hormone (JH) in insects, methyl farnesoate (MF), the unepoxidized form of JH III, regulates many developmental processes in crustaceans, such as molting and reproduction. We have previously showed that the JH receptor, Methoprene-tolerant (Met), which is also a candidate receptor for MF, might be involved in the MF-mediated vitellogenesis in the swimming crab Portunus trituberculatus. In this study, the role of Kruppel homolog 1 (Kr-h1), a transcription factor downstream Met in JH signaling, was further investigated. The deduced protein of Pt-Kr-h1 contained seven repeats of zinc finger motifs, similar to Kr-h1s from other crustacean species, but differing from the eight zinc finger motifs found in insect Kr-h1s. MF treatment in vitro induced the expression of Pt-Kr-h1 in hepatopancreas but not ovary, which is similar to the MF-responsive pattern of Pt-Met as previously reported. Moreover, the expression of Pt-Kr-h1 decreased significantly after treating with Pt-Met dsRNA, strongly indicating that the Pt-Kr-h1 might be involved in the Met-mediated MF signaling pathway. RNAi of Pt-Met and Pt-Kr-h1 both led to a decrease in vitellogenin (Vg) expression, and the reduction cannot be rescued by adding MF, suggesting the regulation of vitellogenesis by MF may act through Met and Kr-h1. These results would help to enhance the current understanding of the regulatory mechanism of MF signaling, and provide a vital resource for further research into the evolution of hormonal pathways in arthropods.

Molecular cloning and functional expression of the 5-HT7 receptor in Chinese mitten crab (Eriocheir sinensis).

Serotonin (5-HT) regulates numerous physiological functions and processes, such as light adaptation, food intake and ovarian maturation, and plays the role through 5-HT receptors. To our knowledge, this is the first study to isolate and characterize the serotonin receptor 7 (5-HT7 receptor) cDNA encoded in Eriocheir sinensis, an economically important aquaculture species in China, by performing rapid-amplification of cDNA ends. The full-length of 5-HT7 receptor gene cDNA is 2328 bp and encodes a polypeptide with 590 amino acids that are highly homologous with other crustaceans 5-HT7 receptor genes. Analysis of the deduced amino acid sequence of the 5-HT7, including 7 transmembrane domains and some common features of G protein-coupled receptors (GPCRs), indicated that 5-HT7 receptor was a member of GPCRs family. A gene expression analysis of the 5-HT7 receptor by RT-PCR revealed that the 5-HT7 receptor transcripts were widely distributed in various tissues, in which high expression levels were observed in the cranial ganglia, thoracic ganglia and intestines. Further study about the effects of photoperiods on the 5-HT7 expression in the tissues showed that a significantly increasing expression of the 5-HT7 receptor was observed in the thoracic ganglia induced by constant light. In addition, in the eyestalks, the expression levels of 5-HT7 mRNA in constant darkness and constant light were lower than control treatment. Then, the expression levels of the 5-HT7 receptor in three feeding statuses displayed that there were significantly increasing expressions in the hepatopancreas and intestines after feeding, compared with before feeding and during the feeding period. Finally, the 5-HT7 mRNA expression levels in stage III and stage IV were higher than the levels in stage I of ovarian development. Our experimental results showed that the 5-HT7 receptor structurally belongs to GPCRs, and the thoracic ganglia and eyestalks are the important tissues of the 5-HT7 receptor for light adaptation. The 5-HT7 receptor may also be involved in the physiological regulation of the hepatopancreas and intestines after ingestion in E. sinensis. In addition, the 5-HT7 receptor is involved in the process of ovarian maturation. The study provided a foundation for further research of light adaptation, digestive functions and ovarian maturation of the 5-HT7 receptor in Decapoda.

Myosin Va from Eriocheir sinensis: cDNA cloning, expression and involvement in growth and development.

Myosin Va, a member of the myosin superfamily, has been widely identified associated with processes of cellular motility, which include neurotransmitter release and synaptic plasticity during neurodevelopment. However, the function of myosin Va in the growth and development of crustaceans has not yet been reported. In this study, a full-length cDNA of myosin Va (named as EsMyoVa) was cloned from the Chinese mitten crab, Eriocheir sinensis, and the expression patterns were detected in different tissues and larval developmental stages. The full-length cDNA of EsMyoVa was 6037 bp in length. Real time quantitative reverse transcription PCR (qRT-PCR) analysis showed that EsMyoVa transcript has a wide tissue distribution pattern and is expressed in zoeae, megalopa, juvenile crab stages and adults. In order to further study the function of this gene, we used RNAi technology in the muscle, hepatopancreas, gill, and gonad. After double-stranded RNA (dsRNA) injection, the expression level of EsMyoVa was significantly decreased in all tissues in both sexes and the gene knockdown effects of dsRNA persisted for at least 6 days. Subsequently, the role of EsMyoVa was revealed by silencing the transcript through one month injections of Myosin Va dsRNA. Crabs with reduced levels of EsMyoVa transcripts displayed a dramatic slowing in growth rate and considerably higher mortality compared to control groups, which indicated that this gene had important role of regulating growth and development.

Phosphoenolpyruvate carboxykinase cytosolic and mitochondrial isoforms are expressed and active during hypoxia in the white shrimp Litopenaeus vannamei.

Hypoxic zones in marine environments are spreading around the world affecting the survival of many organisms. Marine animals have several strategies to respond to hypoxia, including the regulation of gluconeogenesis. Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme of gluconeogenesis. The objective of this work was to study two isoforms of PEPCK, one mitochondrial (PEPKC-M) and one cytosolic (PEPCK-C), from the white shrimp Litopenaeus vannamei and the response to hypoxia. Both PEPCK isoforms are 72 kDa proteins and have 92% identity at the amino acid level. The mitochondrial isoform has a N-terminal signal peptide for mitochondrial import. Gene expression and enzymatic activity in subcellular fractions were detected in gills, hepatopancreas and muscle in normoxic and hypoxic conditions. Expression of PEPCK-C was higher than PEPCK-M in all the tissues and induced in response to hypoxia at 48 h in hepatopancreas, while the enzymatic activity of PEPCK-M was higher than PEPCK-C in gills and hepatopancreas, but not in muscle and also increased in response to hypoxia in hepatopancreas but decreased in gills and muscle. During limiting oxygen conditions, shrimp tissues obtain energy by inducing anaerobic glycolysis, and although gluconeogenesis implies energy investment, due to the need to maintain glucose homeostasis, these gluconeogenic enzymes are active with contrasting behaviors in the cytosol and mitochondrial cell compartments and appear to be up-regulated in hepatopancreas indicating this tissue pivotal role in gluconeogenesis during the response to hypoxia.

Silencing of HIF-1 in WSSV-infected white shrimp: Effect on viral load and antioxidant enzymes.

Hypoxia inducible factor-1 (HIF-1) is a transcriptional factor that induces genes involved in glucose metabolism. HIF-1 is formed by a regulatory α-subunit (HIF-1α) and a constitutive ß-subunit (HIF-1ß). The white spot syndrome virus (WSSV) induces a shift in glucose metabolism and oxidative stress. HIF-1α is associated with the induction of metabolic changes in tissues of WSSV-infected shrimp. However, the contributions of HIF-1 to viral load and antioxidant responses in WSSV-infected shrimp have been not examined. In this study, the effect of HIF-1 silencing on viral load and the expression and activity of antioxidant enzymes (superoxide dismutase-SOD, glutathione S-transferase-GST, and catalase) along with oxidative damage (lipid peroxidation and protein carbonyl) in tissues of white shrimp infected with the WSSV were studied. The viral load increased in hepatopancreas and muscle after WSSV infection, and the accumulative mortality was of 100% at 72 h post-infection. The expression and activity of SOD, catalase, and GST decreased in each tissue evaluated after WSSV infection. Protein carbonyl concentrations increased in each tissue after WSSV infection, while lipid peroxidation increased in hepatopancreas, but not in muscle. Silencing of HIF-1α decreased the WSSV viral load in hepatopancreas and muscle of infected shrimp along with shrimp mortality. Silencing of HIF-1α ameliorated the antioxidant response in a tissue-specific manner, which translated to a decrease in oxidative damage. These results suggest that HIF-1 is essential for restoring the antioxidant response, which counters the oxidative injury associated with WSSV infection.

Morphological, biochemical and histological analysis of mud crab ovary and hepatopancreas at different stages of development.

This study describes the fatty acids, total carotenoids, and cell diameter characteristics of the female ovary and hepatopancreas of the mud crab, Scylla olivacea, with comparisons at different ovarian maturation stages. Seventy-one S. olivacea individuals at all stages of ovarian maturation were sampled from the Setiu wetlands, Terengganu, Malaysia. The ovary and hepatopancreas of each crab were used for morphological studies, histological and biochemical analyses (fatty acid composition and total carotenoids). Morphological observations indicated there was an increase in ovarian gonado-somatic index (GSI), with color changes from translucent to dark red; however, a relatively consistent hepato-somatic index (HSI) in the hepatopancreas, with the color ranging from yellow to yellowish-brown. Histological analysis indicated that oocyte diameter was positively correlated with GSI. Hepatopancreatic tubules had a relatively constant diameter from Stage 2 to 4, with increased proportions of R- and B-cells. Biochemical analysis indicated there was a significant increase in total carotenoids in the ovary during maturation. The hepatopancreas, however, had relatively consistent total carotenoid concentrations that were greater than those of the ovary. Overall, the lipid analysis results indicated there were lesser concentrations of fatty acids in the hepatopancreas, while in the ovary there were increasing concentrations during maturation. The lesser concentrations of fatty acids in the hepatopancreas than ovary suggested that energy was transferred to the ovary for future embryonic and larval development. The relationship between the hepatopancreas and the ovary in nutrient content is an important finding in providing a baseline to formulate an optimal diet for improved mud crab hatchery practices.

Molecular characterization and function of ß-N-acetylglucosaminidase from ridgetail white prawn Exopalaemon carinicauda.

Chitin degradation is catalyzed by a two-component chitinolytic enzyme system, chitinase and ß-N-acetylglucosaminidase (NAGase). In this paper, the full-length cDNA sequence encoding NAGase (EcNAG) was obtained from Exopalaemon carinicauda. The deduced amino acid sequence of EcNAG open reading frame (ORF) contained one Glycohydro_20b2 domain and one Glyco_hydro_20 domain. Based on the cDNA sequence, the genomic structure of EcNAG was characterized and it was composed of six exons and five introns. EcNAG mRNA majorly expressed in the hepatopancreas and epidermis. During the molting stages, EcNAG mRNA expression was well-regulated and its expression reached the highest level at the molting stage E. In addition, EcNAG was recombinant expressed in Pichia pastoris and the partial enzymatic characterization of recombinant EcNAG was confirmed. After being challenged with Vibrio parahaemolyticus and Aeromonas hydrophila, the expression of EcNAG was up-regulated significantly at 6 h and reached the peak at 12 h. And then, the expression began to down-regulated and came to the normal level at 72 h. It is helpful to research the relationship between the molt-related hormones and chitinlytic enzymes.

Molecular cloning and characterization of glycogen synthase in Eriocheir sinensis.

Glycogen plays an important role in glucose and energy homeostasis at cellular and organismal levels. In glycogen synthesis, glycogen synthase (GS) is a rate-limiting enzyme catalysing the addition of α-1,4-linked glucose units from (UDP)3-glucose to a nascent glycogen chain using glycogenin (GN) as a primer. While studies on mammalian liver GS (GYS2) are numerous, enzymes from crustaceans, which also use glycogen and glucose as their main energy source, have received less attention. In the present study, we amplified full-length GS cDNA from Eriocheir sinensis. Tissue expression profiling revealed the highest expression of GS in the hepatopancreas. During moulting, GS expression and activity declined, and glycogen levels in the hepatopancreas were reduced. Recombinant GS was expressed in Escherichia coli Rosetta (DE3), and induction at 37°C or 16°C yielded EsGS in insoluble inclusion bodies (EsGS-I) or in soluble form (EsGS-S), respectively. Enzyme activity was measured in a cell-free system containing glucose-6-phosphate (G6P), and both forms possessed glycosyltransferase activity, but refolded EsGS-I was more active. Enzyme activity of both GS and EsGS-I in the hepatopancreas was optimum at 25°C, which is coincident with the optimum growth temperature of Chinese mitten crab, and higher (37°C) or lower (16°C) temperatures resulted in lower enzyme activity. Taken together, the results suggest that GS may be important for maintaining normal physiological functions such as growth and reproduction.

Serotonin induces ecdysteroidogenesis and methyl farnesoate synthesis in the mud crab, Scylla serrata.

In the current study, we have examined the role of serotonin in regulating the levels of methyl farnesoate and ecdysteroids in the giant mud crab Scylla serrata and validated that serotonin indeed is a reproductive hormone. Administration of serotonin elevated circulatory levels of methyl farnesoate and ecdysteroids in crabs. Since methyl farnesoate and ecdysteroid act through retinoid X receptor (RXR) and ecdysteroid receptor (EcR) respectively and these receptors are involved in the regulation of reproduction in crustaceans, we have determined the mRNA levels of RXR and EcR in hepatopancreas and ovary after serotonin administration. The expression levels of both RXR and EcR increased significantly in the hepatopancreas and ovary of serotonin injected crabs when compared to the controls. In vitro organ culture studies revealed that incubation of Y-orgas and mandibular organ explants in the presence of serotonin resulted in a significant increase in the secretion of ecdysteroids by Y-organs, but without alterations in MF synthesis in mandibular organs. From the above studies it is evident that serotonin stimulates Y organs resulting in increased ecdysteroidogenesis. Though the circulatory levels methyl farnesoate elevated after serotonin administration, organ culture studies revealed serotonin mediated methyl farnesaote synthesis is indirect probably by inhibiting release of mandibular organ inhibiting hormone from eyestalks.

The physiological role and toxicological significance of the non-metal-selective cadmium/copper-metallothionein isoform differ between embryonic and adult helicid snails.

Metal regulation is essential for terrestrial gastropods to survive. In helicid snails, two metal-selective metallothionein (MT) isoforms with different functions are expressed. A cadmium-selective isoform (CdMT) plays a major role in Cd2+ detoxification and stress response, whereas a copper-selective MT (CuMT) is involved in Cu homeostasis and hemocyanin synthesis. A third, non-metal-selective isoform, called Cd/CuMT, was first characterized in Cantareus aspersus. The aim of this study was to quantify the transcriptional activity of all three MT genes in unexposed and metal-exposed (Cd, Cu) embryonic Roman snails. In addition, the complete Cd/CuMT mRNA of the Roman snail (Helix pomatia) was characterized, and its expression quantified in unexposed and Cd-treated adult individuals. In embryos of Helix pomatia, the Cd/CuMT gene was induced upon Cu exposure. Its transcription levels were many times higher than that of the other two MT genes, and also exceeded by far the Cd/CuMT mRNA concentrations of adult snails. In the hepatopancreas of adult Roman snails, no Cd/CuMT could be detected at the protein level, irrespective of whether the snails had been exposed to Cd or not. This contrasts with the situation in the near relative, Cantareus aspersus. It appeared that the 3'-UTR of the Cd/CuMT mRNA differed largely between Cantareus aspersus and Helix pomatia, being larger in the latter species, with a number of putative binding sites for proteins and miRNAs known to inhibit mRNA translation. We suggest this as a possible mechanism responsible for the lack of Cd/CuMT protein expression in adult Roman snails.

Laboratory studies on the thermal tolerance and response of enzymes of intermediate metabolism in different land snail species.

Land snails species occur in a range of habitats from humid to semi-arid and arid ones and seasonal variations in their physiology and biochemical composition have been linked to annual cycles of photoperiod, temperature, humidity and water availability. In an effort to understand the thermal tolerance and the impact of temperature elevation on tissue metabolism of land snails we determined the mortality, heamolymph PO2 and the activities of enzymes of intermediary metabolism in three land snail species (Helix lucorum, Helix pomatia and Cornu aspersum) differing in their geographical distribution and inhabiting areas with different climatic characteristics. No mortality was observed in both population of Cornu aspersum, while Helix pomatia exhibited higher mortality than Helix lucorum. PO2 dropped within the first 10days of exposure to elevated temperature in all species, although in Cornu aspersum this decrease was significantly lower. No significant reduction in the enzymatic activities of all glycolytic enzymes studied, as well as of citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HOAD) was observed in the more thermal tolerant species C. aspersum from both populations studied. Significant reductions of enzymatic activity of the glycolytic enzymes phosphofructokinase (PFK), pyruvate kinase (PK) and d-Lactate dehydrogenase (d-LDH) was observed in Helix lucorum and Helix pomatia. The observed inter-specific differences seem to be in accordance with the life cycle characteristics of each species and may be attributed to climatic differences among habitats within their distribution range.

Expression patterns of two heat-shock cognate 70 genes during immune responses and larval development of the Chinese mitten crab Eriocheir sinensis.

Two heat-shock protein (HSP) 70 family transcripts, heat-shock protein 70 cognate 5 and heat-shock protein 70 cognate 3 (designated as EsHSC70-5 and EsHSC70-3, respectively), were isolated from the Chinese mitten crab Eriocheir sinensis and their expression profiles were evaluated for their responsiveness to larval development and immune challenge in adult crabs. The HSPs exhibited 45-89% identity with other heat-shock proteins, and they shared similar structural features. EsHSC70 mRNA expression was detected not only during infection but also during the developmental larval stages. The EsHSC70s were enriched, and their expression fluctuated during early development. EsHSC70 mRNA expression was significantly induced by Vibrio parahaemolyticus challenge in all of the tissues studied (P < 0.05). Expression of EsHSC70 mRNA in the hepatopancreas and at the early zoeal stages was particularly pronounced, and the two EsHSC70s exhibited differential expression patterns both chronologically and spatially. The EsHSC70-5 mRNA level was significantly downregulated in the intestine and gills compared to that in controls at nearly all time points, and was expressed at a lower level after the bacterial challenge, indicating that EsHSC70-5 and EsHSC70-3 respond to immune challenges. The stage-specific enrichment of EsHSC70 transcripts in crabs suggests that these stress proteins play an essential role during brachyurization events.

Threonine affects digestion capacity and hepatopancreatic gene expression of juvenile blunt snout bream (Megalobrama amblycephala).

The present study conducted a 9-week feeding trial to investigate the effects of threonine (Thr) on the digestion capacity and hepatopancreas gene expression of juvenile blunt snout bream (Megalobrama amblycephala). For this purpose, three tanks (300 litres/tank) were randomly arranged and assigned to each experimental diet. Juvenile fish were fed with diets containing graded Thr levels (0·58, 1·08, 1·58, 2·08 or 2·58 % of the diet) to apparent satiation four times daily. At the end of the feeding trial, the results indicated that hepatopancreas weight, hepatosomatic index, hepatopancreatic protein content, intestinal weight, intestosomatic index and intestinal protein content increased with increasing dietary Thr levels up to 1·58 % and thereafter decreased (P< 0·05). The activities of chymotrypsin, trypsin, amylase and lipase elevated as dietary Thr levels increased up to 1·58 % (P< 0·05), while these activities decreased in most cases after 1·58 % dietary Thr except for chymotrypsin and trypsin in the hepatopancreas (plateau 1·58-2·08 % Thr). The relative gene expression levels of chymotrypsin, trypsin, amylase, lipase, target of rapamycin and insulin-like growth factor-I were up-regulated, and the highest values were observed with 1·58 % dietary Thr or 1·58 and 2·08 % dietary Thr, whereas the relative gene expression levels of eukaryotic translation initiation factor 4E-binding protein 2 gradually decreased (P< 0·10) as dietary Thr levels increased up to 1·58 % and thereafter significantly increased (P< 0·05), which could explain that about 1·58 % dietary Thr could improve the growth and development of digestive organs and activities of digestive enzymes of juvenile blunt snout bream.

Molecular characterization and developmental expression of vitellogenin in the oriental river prawn Macrobrachium nipponense and the effects of RNA interference and eyestalk ablation on ovarian maturation.

Vitellogenin (Vg) is the precursor of yolk protein, which functions as a nutritive resource that is important for embryonic growth and gonad development. In this study, the cDNA encoding the Vg gene from the oriental river prawn Macrobrachium nipponense was cloned using expressed sequence tag (EST) analysis and the rapid amplification of cDNA ends (RACE) approach. The transcript encoded 2536 amino acids with an estimated molecular mass of 286.810 kDa. Quantitative real-time PCR indicated high expression of Mn-Vg in the female ovary, hemocytes, and hepatopancreas. As ovaries developed, the expression level of Mn-Vg increased in both the hepatopancreas and ovary. In the hepatopancreas, the expression level rose more slowly at the early stage of vitellogenesis and reached the peak more rapidly compared to the expression pattern in ovary. The observed changes in Mn-Vg expression level at different development stages suggest the role of nutrient source in embryonic and larval development. Eyestalk ablation caused the Mn-Vg expression level to increase significantly compared to eyestalk-intact groups during the ovary development stages. Ablation accelerated ovary maturation by removing hormone inhibition of Mn-Vg in the hepatopancreas and ovary. In adult females, Mn-Vg dsRNA injection resulted in decreased expression of Mn-Vg in both the hepatopancreas and ovary, and two injection treatment dramatically delayed ovary maturation. Vg RNA interference down-regulated the vitellogenin receptor (VgR) expression level in the ovary, which illustrates the close relationship between Vg and VgR in the process of vitellogenesis.