Xugan Wu

Insights into Hepatopancreatic Functions for Nutrition Metabolism and Ovarian Development in the Crab Portunus trituberculatus: Gene Discovery in the Comparative Transcriptome of Different Hepatopancreas Stages

The crustacean hepatopancreas has different functions including absorption, storage of nutrients and vitellogenesis during growth, and ovarian development. However, genetic information on the biological functions of the crustacean hepatopancreas during such processes is limited. The swimming crab, Portunus trituberculatus, is a commercially important species for both aquaculture and fisheries in the Asia-Pacific region. This study compared the transcriptome in the hepatopancreas of female P. trituberculatus during the growth and ovarian maturation stages by 454 high-throughput pyrosequencing and bioinformatics. The goal was to discover genes in the hepatopancreas involved in food digestion, nutrition metabolism and ovarian development, and to identify patterns of gene expression during growth and ovarian maturation. Our transcriptome produced 303,450 reads with an average length of 351 bp, and the high quality reads were assembled into 21,635 contigs and 31,844 singlets. Based on BLASTP searches of the deduced protein sequences, there were 7,762 contigs and 4,098 singlets with functional annotation. Further analysis revealed 33,427 unigenes with ORFs, including 17,388 contigs and 16,039 singlets in the hepatopancreas, while only 7,954 unigenes (5,691 contigs and 2,263 singlets) with the predicted protein sequences were annotated with biological functions. The deduced protein sequences were assigned to 3,734 GO terms, 25 COG categories and 294 specific pathways. Furthermore, there were 14, 534, and 22 identified unigenes involved in food digestion, nutrition metabolism and ovarian development, respectively. 212 differentially expressed genes (DEGs) were found between the growth and endogenous stage of the hepatopancreas, while there were 382 DEGs between the endogenous and exogenous stage hepatopancreas. Our results not only enhance the understanding of crustacean hepatopancreatic functions during growth and ovarian development, but also represent a basis for further research on new genes and functional genomics of P. trituberculatus or closely related species.

Current Trends in Hatchery Techniques and Stock Enhancement for Chinese Mitten Crab, Eriocheir japonica sinensis

Aquaculture of the Native Chinese mitten crab (Eriocheir japonica sinensis) has developed quickly in China to reach 570,000 mt in 2005, which exceeds all other worldwide production of crab from aquaculture and capture fisheries combined. We review the development of aquaculture techniques and stock enhancement that account for this successful production of Chinese mitten crabs. There are two models for mitten crab larviculture. In the first model, intensive larviculture occurs in indoor concrete ponds, with conditions of temperature control, aeration, and a plentiful supply of food (algae, egg yolk, rotifers, Artemia nauplii). In the second model, outdoor extensive larviculture occurs in earth ponds with no temperature control. Grow-out of the mitten crab focuses on release of cultured seed crabs into net enclosures in lakes. As a rule, the production of adult crabs is l50–450 kg/ha and as high as 975 kg/ha. Recently, Chinese researchers noticed deterioration of water quality and the exhaustion of natural food resources in many lakes due to over-stocking crabs, and many studies are now beginning to investigate a model for the sustainable aquaculture of Chinese mitten crab in these systems.

Meat Quality of Chinese Mitten Crabs Fattened with Natural and Formulated Diets

After 42 days feeding on either a Huayi formulated diet (HY) or trash fish (TF), the meat quality of Chinese mitten crab, Eriocheir sinensis, was compared. The meat yield of males (25 vs. 22%) was higher, but the total edible yield was lower than that of the females (p < 0.05). The proximate composition of both genders fed two diets was similar (p > 0.05), and there was no significant difference in the total collagen content of the meat (0.2–0.30%) between both dietary treatments and genders (p > 0.05). The diets had no significant effects on free amino acid content of the meat, while gender significantly affected the contents of some amino acids, such as taurine and glycine. Crabs fed TF had significantly lower contents of 20:5n-3 as well as higher arachidonic acid (ARA)/eicosapentaenoic acid (EPA) value compared with those of same sex crabs fed HY (p < 0.05). There were no significant dietary effects on a range of sensory traits evaluated by panelists, neither in female nor in male crabs. These results suggested no negative effects of formulated diet HY on the meat quality compared to trash fish. Therefore, diet HY could be used to replace the trash fish for fattening mitten crabs.

Immunorecognition and Distribution of Progesterone Receptors in the Chinese Mitten Crab Eriocheir sinensis During Ovarian Development

ABSTRACT Progesterone is an important sex steroid that plays a vital role during ovarian development in crustaceans. In vertebrates, progesterone mediates reproduction via the progesterone receptor (PR). Previous studies have shown that PR is present in the ovary, hepatopancreas, and nerve tissues of some crustacean species. The Chinese mitten crab, Eriocheir sinensis, is an important aquaculture species in China and has become an invasive species in Europe and North America. A better understanding of the relevant reproductive mechanisms could potentially benefit artificial propagation and production of E. sinensis. Our intention was to immunorecognize and immunolocalize PR in the ovary, hepatopancreas, optic ganglion, brain ganglion, and thoracic ganglion of female E. sinensis using Western blot and immunohistochemistry. Changes in the distribution of PR were also investigated in these tissues during ovarian development. With an apparent molecular weight of 70 kDa, PR was identified in the optic, brain, and thoracic ganglion of female E. sinensis. During ovarian development, follicle cells were stained with positive PR at each ovarian stage. In germinal cells, positive PR was found in the cytoplasm only during the early ovarian development stages (I–III), whereas positive PR stained in the nucleus of germinal cells from stage III—stage V. In the hepatopancreas, PR was localized in the nucleus of resorptive cells as well as in the cytoplasm and nucleus of fibrillar cells for all stages of ovarian development. On the contrary, no PR-like substance was found in the other types of hepatopancreatic cells, such as blisterlike cells and embryonic cells, during ovarian development. However, a PR-like substance was detected in the nerve tissues of female E. sinensis. In the optic ganglion, PR was localized in the nucleus only of nerve cells. In the thoracic ganglion, PR was detected in the cytoplasm and nuclei of nerve cells during all ovarian development stages, with stronger detection during late ovarian development (stages III–V) rather than early (stages I and II). In addition, PR was localized in the brain ganglion, which is supported by evidence that the nuclei of nerve cells stained positively for PR antibody during all ovarian development stages. We suggest that progesterone not only regulates vitellogenesis and ovarian development directly by binding PR in the ovary and hepatopancreas, but also modulates indirectly ovarian development through nerve tissue.

Effects of salinity on gonadal development, osmoregulation and metabolism of adult male Chinese mitten crab, Eriocheir sinensis

As a catadromous species, salinity is a key parameter that affects gonadal development of Chinese mitten crab Eriocheir sinensis during reproductive migration. It is however unclear the effects of salinity on the gonadal development of male E. sinensis as well as their physiological responses to salinity during reproductive migration. This study investigated the effects of four salinities (0 ‰, 6 ‰, 12 ‰ and 18 ‰) on gonadal development, osmoregulation and metabolism of adult male E. sinensis over a 40-day period. The results showed that elevating salinity promote gonadal development, increase hemolymph osmolality and K+ and Mg2+ concentrations (P < 0.05). The 12 ‰ salinity resulted in the highest contents of taurine and arginine in the hemolymph while the highest contents of threonine, phenylalanine, lysine, ß-alanine, tryptophan, ornithine and total free amino acids were found for 0 ‰ treatment (P < 0.05). A decreasing trend was detected for the Na+/K+-ATPase activity and its mRNA expression level in the posterior gills with salinity (P < 0.05). Total saturated fatty acids in the anterior gills decreased with increasing salinity (P < 0.05); the 0 ‰ treatment had the highest total polyunsaturated fatty acids in the posterior gills while total n-6 polyunsaturated fatty acids increased with salinity (P < 0.05). The hemolymph glucose and uric acid showed a decreasing trend as salinity while an increasing trend was found for the hemolymph triglyceride and high-density lipoprotein cholesterol (P < 0.05). The 12 ‰ treatment had the highest levels of hemolymph malonaldehyde and hepatopancreatic γ-glutamyltranspeptidase (P < 0.05). In conclusion, these results suggested that the brackish water promote gonadal development of male E. sinensis, and increase osmolality and ionic concentrations in hemolymph while reduced the activity of Na+ /K+- ATPase and its mRNA expression in the posterior gills as well as metabolism.

The ovarian development pattern of pond-reared Chinese mitten crab, Eriocheir sinensis H. Milne-Edwards, 1853

Although pond culture is the major culture method for Chinese mitten crab (Eriocheir sinensis) in China, the pattern of ovarian development in pond-reared E. sinensis remains unclear. This study investigated the changes in ovarian morphology and histology, gonadosomatic index (GSI), hepatosomatic index (HSI), and monthly variation of the ovarian development pattern during the ovarian maturation of pond-reared female E. sinensis. Based on the pubertal moult, and ovarian morphology and histology, the ovarian development cycle of E. sinensis could be divided into five stages, i.e., Stage I: the ovary appears thin ribbon-like and translucent, dominated by oogonia (OG) and previtellogenic oocytes (PRO); Stage II: the ovary appears milk white or buff, dominated by endogenous vitellogenic oocytes (EN); Stage III: the ovary appears orange or light brown, dominated by exogenous vitellogenic oocytes (EX); Stage IV: the ovary appears crimson red or brown and ovarian lobes occupy most available body cavities. The major oocytes were nearly mature oocytes (NO); Stage V: the ovary appears deep purple and is filled with mature oocytes (MO). During ovarian maturation, the GSI increased significantly and a significant, positive correlation was found between the GSI and the mean long diameter of the oocyte from stage III to stage V. However, a significantly negative correlation was found between GSI and HSI. The ovarian development of pond-reared female E. sinensis was not synchronous, most of the pubertal moults were found until mid-late August. At the end of August, the percentages of females that reached ovarian stages II and III were 55 and 24%, respectively. After that, the ovarian development of pond-reared female E. sinensis became fast, and females with stage III or more advanced ovaries were found to be in excess of 80% by the end of September and onward. From the end of November to late December, the GSI of female E. sinensis did not increase significantly, which indicated pond-reared females had reached mature or nearly-mature ovaries by the end of November.

Physiological Responses and Ovarian Development of Female Chinese Mitten Crab Eriocheir sinensis Subjected to Different Salinity Conditions

Salinity plays a key role affecting ovarian development, osmoregulation and metabolism of female Chinese mitten crab, Eriocheir sinensis during reproductive migration. In this study, female E. sinensis after their puberty molt were subjected to four salinities of 0, 6, 12, and 18‰ for 40 days to investigate the salinity effects on their ovarian development as well as a range of important physiological parameters. Elevated salinity accelerated the ovarian development with ovigerous crabs found at salinity treatments of 12 and 18‰ despite no copulation had occurred. Meanwhile the survival rate of female crabs showed a decreasing trend with increasing salinity. Higher salinity also led to increased hemolymph Na+, K+, Ca2+, Cl−, and Mg2+ concentrations. The 6‰ treatment had the highest contents of hemolymph total and major free amino acids while the Na+/K+ -ATPase activity in the posterior gills was the lowest among treatments. Total n-3 polyunsaturated fatty acids (∑n-3PUFA) and n-3/n-6 PUFA ratio in the anterior gills showed a decreasing trend with salinity while 18‰ had the highest ∑PUFA and ∑n-6PUFA. The ∑n-3PUFA content and n-3/n-6 PUFA ratio of the posterior gills showed a fluctuating pattern and the highest value was detected at 0‰, while an increasing trend was found for the ∑n-6PUFA with increasing salinity. The hemolymph glucose showed a decreasing trend with increasing salinity and the highest total cholesterol in hemolymph was detected at 12‰. The 18‰ treatment had the highest levels of hemolymph γ-glutamyltransferase, alkaline phosphatase and acid phosphatase, as well as glucose, urea and acid phosphatase in hepatopancreas while the highest hemolymph superoxide dismutase and malondialdehyde were detected at 0‰. Overall, the results showed that salinity increase from freshwater to brackish conditions led to lower metabolism, accelerated ovarian development, and the appearance of ovigerous crabs without copulation in female E. sinensis post puberty molt.

Genetic diversity and genetic structure of farmed and wild Chinese mitten crab (Eriocheir sinensis) populations from three major basins by mitochondrial DNA COI and Cyt b gene sequences

Abstract The Chinese mitten crab, Eriocheir sinensis, is one of the important native crab species in East Asian region, which has been widely cultured throughout China, particularly in river basins of Yangtze, Huanghe and Liaohe. This study was designed to evaluate the genetic diversity and genetic structure of cultured and wild E. sinensis populations from the three river basins based on mitochondrial DNA (mtDNA) cytochrome oxidase subunit I (COI) and cytochrome b (Cyt b). The results showed that there were 62 variable sites and 30 parsimony informative sites in the 647 bp of sequenced mtDNA COI from 335 samples. Similarly, a 637 bp segment of Cyt b provided 59 variable sites and 26 parsimony informative sites. AMOVA showed that the levels of genetic differentiation were low among six populations. Although the haplotype diversity and nucleotide diversity of Huanghe wild population had slightly higher than the other populations, there were no significant differences. There was no significant differentiation between the genetic and geographic distance of the six populations, and haplotype network diagram indicated that there may exist genetic hybrids of E. sinensis from different river basins. The results of clustering and neutrality tests revealed that the distance of geographical locations were not completely related to their genetic distance values for the six populations. In conclusion, these results have great significance for the evaluation and exploitation of germplasm resources of E. sinensis.

Hypoxia induces changes in AMP-activated protein kinase activity and energy metabolism in muscle tissue of the oriental river prawn Macrobrachium nipponense

Hypoxia has important effects on biological activity in crustaceans, and modulation of energy metabolism is a crucial aspect of crustaceans’ ability to respond to hypoxia. The adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) enzyme is very important in cellular energy homeostasis; however, little information is known about the role of AMPK in the response of prawns to acute hypoxia. In the present study, three subunits of AMPK were cloned from the oriental river prawn, Macrobrachium nipponense. The full-length cDNAs of the α, β, and γ AMPK subunits were 1,837, 3,174, and 3,773 bp long, with open reading frames of 529, 289, and 961 amino acids, respectively. Primary amino acid sequence alignment of these three subunits revealed conserved similarity between the functional domains of the M. nipponense AMPK protein with AMPK proteins of other animals. The expression of the three AMPK subunits was higher in muscle tissue than in other tissues. Furthermore, the mRNA expression of AMPKα, AMPKβ, and AMPKγ were significantly up-regulated in M. nipponense muscle tissue after acute hypoxia. Probing with a phospho-AMPKα antibody revealed that AMPK is phosphorylated following hypoxia; this phosphorylation event was found to be essential for AMPK activation. Levels of glucose and lactic acid in hemolymph and muscle tissue were significantly changed over the course of hypoxia and recovery, indicating dynamic changes in energy metabolism in response to hypoxic stress. The activation of AMPK by hypoxic stress in M. nipponense was compared to levels of muscular AMP, ADP, and ATP, as determined by HPLC; it was found that activation of AMPK may not completely correlate with AMP:ATP ratios in prawns under hypoxic conditions. These findings confirm that the α, β, and γ subunits of the prawn AMPK protein are regulated at the transcriptional and protein levels during hypoxic stress to facilitate maintenance of energy homeostasis.

Ontogenetic patterns of growth and lipid composition changes of blue swimmer crab larvae: insights into larval biology and lipid nutrition

Blue swimmer crab, Portunus pelagicus, is a commercially important crab throughout the Indo-Pacific region, that plays an important role in the food chains of coastal ecosystems. P. pelagicus undergoes four pelagic zoeal stages and a post-larval megalopal stage before settling as benthic juvenile; however, very limited information is available on larval biology and lipid nutrition of this species. The present comprehensive study investigated survival, growth and ontogenetic changes in lipid composition of P. pelagicus larvae under laboratory conditions. Larvae were fed rotifers (zoea I), rotifers and Artemia (zoea II) and enriched Artemia (zoea III – megalopa), consecutively. Among five larval stages of P. pelagicus, zoea I and II had the shortest durations (2.2–2.3 days), whereas megalopa had the longest duration (4.7 days). As larvae developed, their wet weight, dry weight and carapace length increased exponentially. Lower mortality occurred during zoea II (12.5%) and zoea III (14.3%), coincidental with the lower percentage increments in dry weight and specific growth rate of dry weight during these two stages. Generally, fatty acid composition of larvae can reflect that of their diets. However, increases in mono-unsaturated fatty acids, 20 : 4n-6 and 22 : 6n-3 in megalopa and the first crab compared with zoea IV might suggest that the later larvae were still deficient in essential fatty acids, and that 20 : 4n-6 and 22 : 6n-3 were preferentially accumulated. Therefore, measures of ontogenetic patterns of growth and lipid composition will provide valuable insights for better understanding of larval biology and lipid nutrition of P. pelagicus.