Hepatopancreas

Improved methods of cell culture from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of shrimp (Penaeus vannamei) were established using synthetic media and shrimp muscle extract (SME). For hemocytes and ovarian cell cultures, Grace’s insect medium supplemented with 10% (v/v) fetal bovine serum and 10% SME (v/v) showed enhanced attachment and proliferation of the cells. The hemocyte and ovarian cell cultures could be maintained for 48 and 66 days, respectively, and have been sub-cultured four and six times, respectively. Both ovary and hemocyte cell cultures contained primarily epithelial-like cells. Cells derived from ovary tissue grew preferably between 26°C and 28°C with 5% CO2. Although the temperature preference of hemocyte cells was the same as ovarian cells, CO2 supplementation did not show any difference in the growth of hemocyte cells. When the shrimp were injected with lipopolysaccharide (8 μg/g of shrimp) and hemolymph was drawn 24 h post-injection, the in vitro multiplicity of hemocytes dramatically improved. The growth of eye stalk, hepatopancreas, and muscle-derived cells was much less compared to ovarian cells and hemocytes under the conditions described above. The optimal culture conditions for ovarian cells and hemocytes were also different from that for eye stalk, hepatopancreas, and muscle cell culture. The proliferation efficiencies of primary cultures of hepatopancreas, eyestalk, and muscle cells were about 30, 12, and <7 d, respectively. The improved culture conditions described here, particularly for hemocytes and ovary, will be very useful for in vitro studies involving viruses infecting shrimp and in shrimp genomic studies.

Carotenoprotein from shells of Pacific white shrimp (Litopenaeus vannamei) was extracted with the aid of proteases from hepatopancreas of the same species at various levels
(5–30 units/g shrimp shell) for different times (0–180 min). Recovery of carotenoprotein increased with increasing protease levels and hydrolysis times, but mainly reached the plateau at 120 min of hydrolysis. Carotenoprotein contained astaxanthin and astaxanthindiester as major carotenoids. Carotenoids extracted from carotenoprotein showed increasing
ABTS, DPPH radical scavenging activities, FRAP and metal chelating activity when the concentrations increased up to 5 mg/ml (po0.05). Carotenoprotein consisted of 73.58% protein, 21.87% lipids and 2.63% ash contents. It was rich in essential amino acids and Asp/Asn and Glu/Gln were found as dominant amino acids. Therefore, carotenoprotein from shell of Pacific white shrimp could be successfully extracted using shrimp hepatopancreas proteases and could serve as the value-added nutritive food ingredients or as the animal feed

Antioxidative activities of hydrolysates from skin of seabass (Lates calcarifer) with different degrees of hydrolysis (DH: 10–40%) prepared using an ammonium sulphate precipitated fraction (ASPF) from Pacific white shrimp hepatopancreas and commercial Alcalase were compared. The hydrolysate prepared using ASPF or Alcalase had increases in 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activities and ferric reducing antioxidative power (FRAP) as DH increased (P < 0.05). When the hydrolysate prepared using ASPF with 40% DH was subjected to a gastrointestinal model system (GIMs), ABTS radical scavenging activity and chelating activity increased, especially in the duodenal condition. The hydrolysate
(500–2000 mg/L) could inhibit lipid oxidation in a lecithin liposome system in a dose dependent manner. Based on gel filtration using a Sephadex G-15 column, peptide with
a molecular weight of 364 Da showed the strongest ABTS radical scavenging activity. Therefore, the extract from hepatopancreas could be used to increase DH of hydrolysate from seabass skin.

C. granulata is a semiterrestrial crab that lives in the mesolittoral and the supralittoral zones of estuaries and faces hypoxia and anoxia when exposed to atmospheric air. The carbohydrate or protein content of the diets administered to the crabs induced different metabolic adjustments during anoxia and post-anoxia recovery period. During the first hour in anoxia a marked increase in L-lactate concentration in hemolymph was induced, followed by a reduction in its levels accompanied by two peaks in hepatopancreas gluconeogenic capacity. Anoxia exposure did not induce a reduction in the hepatopancreas phosphoenolpyruvate carboxykinase activity in either dietary group. Our results suggest that in anaerobiosis this crab uses the conversion of lactate to glucose in hepatopancreas to maintain the acid-base balance and the glucose supply. In post-anoxia recovery, the fate of L-lactate is the hepatopancreas gluconeogenesis in high protein maintained crabs. On the other hand, in the crabs maintained on carbohydrate-rich diet the L-lactate levels decreased gradually in the hemolymph during the post-anoxia recovery; however, the hepatopancreas gluconeogenesis did not increase. In both dietary groups, an increase in the gluconeogenic capacity of hepatopancreas occurred at 30 h of post-anoxia recovery. J. Exp. Zool. 301A:240-248, 2004. © 2004 Wiley-Liss, Inc.

Compositions and yield of lipids extracted from hepatopancreas of Pacific white shrimp (Litopenaeus vannamei) subjected to autolysis at 60 C for different times (0, 30, 60, 90 120 and 150 min) were investigated. Extraction yield increased from 7.4% to 8.8% as autolysis time increased from 0 to 150 min. Coincidental increase in total carotenoid content was obtained with increasing autolysis time (p < 0.05). The increases in thiobarbituric acid-reactive substances (TBARS) and p-anisidine value (AV) of lipids were noticeable when autolysis time increased (p < 0.05). However, no changes in free fatty acid (FFA) content were observed within the first 60 min of autolysis (p > 0.05), but subsequently increased up to 150 min. (p < 0.05). No differences in fatty acid profiles of lipids extracted from hepatopancreas without and with 60 min prior autolysis were observed. Lipids extracted contained docosahexaenoic acid (DHA; C22:6(n  3)) as the most abundant fatty acid, followed by eicosapentaenoic acid (EPA; C20:5(n  3)). Therefore, prior autolysis at 60 C for 60 min increased the extraction yield without negative effect on lipid quality.

Hemocyanins are copper-containing glycoproteins in some molluscs and arthropods, and their best-known function is O2 transport. We studied the site of their biosynthesis in the gastropod Concholepas concholepas by using immunological and molecular genetic approaches. We performed immunohistochemical staining of various organs, including the mantle, branchia, and hepatopancreas, and detected C. concholepas hemocyanin (CCH) molecules in circulating and tissue-associated hemocytes by electron microscopy. To characterize the hemocytes, we purified them from hemolymph. We identified three types of granular cells. The most abundant type was a phagocyte-like cell with small cytoplasmic granules. The second type contained large electron-dense granules. The third type had vacuoles containing hemocyanin molecules suggesting that synthesis or catabolism occurred inside these cells. Our failure to detect cch-mRNA in hemocytes by reverse transcription with the polymerase chain reaction (RT-PCR) led us to propose that hemocytes instead played a role in CCH metabolism. This hypothesis was supported by colloidal gold staining showing hemocyanin molecules in electron-dense granules inside hemocytes. RT-PCR analysis, complemented by in situ hybridization analyses with single-stranded antisense RNAs as specific probes, demonstrated the presence of cch-mRNA in the hepatopancreas; this was consistent with the specific hybridization signal and confirmed the hepatopancreas as the site of CCH synthesis. Finally, we investigated the possibility that CCH catabolism in hemocytes was involved in the host immune response and in the generation of secondary metabolites such as antimicrobial peptides and phenoloxidase.

Recovery of proteases from hepatopancreas of Pacific white shrimp using three-phase partitioning (TPP) system in combination with aqueous two-phase system (ATPS) was investigated. TPP was performed using t-butanol with different crude protease extract (CPE)/t-butanol ratios. The interphase of system comprising CPE and t-butanol with a ratio of 1:1 in the presence of 30% ammonium sulphate yielded the highest purification fold (PF) (2.6-fold) with the recovery of 76.0%. Subsequently, TPP fraction was subjected to ATPS. Effects of phase compositions including PEG molecular weight (MW) and concentration as well as types and concentration of salts on partitioning of proteases were studied. ATPS comprising PEG1000 (15%, w/w) and magnesium sulphate (25%, w/w) provided the best condition for the maximal partitioning of proteases into the top phase and gave the highest PF (8.6-fold). The yield of 65.5% was obtained. ATPS fraction was further mixed with PEG8000 and several salts for back extraction (BE). BE using 25% PEG8000 and 10% MgSO4 gave the highest PF (9.9-fold) with the yield of 46.2%. Based on electrophoresis and activity staining, the fractionated proteases had the MW of 36 and 26 kDa. Therefore, the combined partitioning systems, TPP–ATPS–BE, could be effectively used to recover and purify
proteases from hepatopancreas of Pacific white shrimp.

ABSTRACTThe bacteria that cause necrotizing hepatopancreatitis inPenaeus vannameiadversely affect penaeid shrimp cultured in the western hemisphere. 16S rRNA and gyrase B gene analyses determined the taxonomic position of these bacteria. The name “CandidatusHepatobacter penaei” is proposed for these pathogenic bacteria, which are members of theRickettsialesorder.

Blue crabs from a contaminated estuary (Hackensack Meadowlands-HM) and a cleaner reference site (Tuckerton-TK) were analyzed for Cr, Cu, Hg, Pb, and Zn in muscle and hepatopancreas. Crabs from each site were taken into the laboratory and fed food from the other site, or in another experiment, transplanted to the other site for eight weeks. All crabs were analyzed for metals. Overall, tissue concentrations reflected environmental conditions. Tissue differences were found for Cu, Pb and Zn (all higher in hepatopancreas), and Hg (higher in muscle). HM muscle had more Hg than TK muscle, but did not decrease after transplanting or consuming clean food. HM crabs lost Cu, Pb and Zn in hepatopancreas after being fed clean food or transplanted. TK crabs increased Hg in muscle and Cr and Zn in hepatopancreas after transplantation or being fed contaminated (HM) food. Concentrations were variable, suggesting that blue crabs may not be fully reliable bioindicators of polluted systems.