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The oriental river prawn, Macrobrachium nipponense (De Haan, 1849), an economically important species, can live in freshwater and brackish water environments, but molecular mechanism of adaptation to varying salinity levels is still poorly understood. We aimed to investigate the effects of four different salinities (0, 6, 12, and 18‰) on metabolic responses, histological structure, and expression patterns of acid phosphatase (ACP) and alkaline phosphatase (AKP) in the hepatopancreas. The full-length cDNAs of ACP and AKP of M. nipponense (designated MnACP and MnAKP, respectively) were obtained by rapid-amplification of cDNA ends (RACE)-PCR. Immunofluorescence experiments confirmed that MnAKP is located in the cell membrane of the hepatopancreas. Both genes were expressed in all seven studied tissues, with highest expression in the hepatopancreas by quantitative real-time PCR. The mRNA expression levels and enzyme activities of MnACP and MnAKP in the hepatopancreas significantly increased in 18‰ salinity group compared to other groups. Measurements of glycogen, triglycerides, and total cholesterol levels suggested that prawns can adjust the energy metabolism in a way to provide additional energy under unsuitable salinity. Besides, prawns at high salinity produced higher B-cells and lower number of R-cells in hepatopancreatic tubules, also suggesting enhance the metabolic function of the hepatopancreas under high salinity. Our results provide a better understanding for the mechanism regulating salinity acclimation in the oriental river prawn.

Farmed caridean shrimps can experience starvation periods attributable to disease outbreaks or adverse environmental conditions. The hepatopancreas, or perigastric organ, of decapods, being the principal organ for storage of nutrients that can be mobilized during non-feeding periods, plays a fundamental role during starvation. We studied the ultrastructural changes in the perigastric organ of whiteleg shrimp Litopenaeus vannamei (Boone, 1931), a commonly farmed species, during starvation and refeeding. Starvation induced a progressive increase of cellular immunity (haemocytic infiltration, both hyaline and granular and/or semigranular haemocytes), necrosis (swelling of organelles and cytolysis), and autophagy (formation of phagophores, autophagosomes, autolysosomes, and multivesicular and residual bodies). The complete depletion of lipid droplets and a few signs of apoptosis (chromatin condensation and nuclear fragmentation) were also observed during starvation. Refeeding resulted in a partial recovery of the perigastric organ. Findings demonstrate the capacity of the perigastric organ to recover when refed for ten days after five days of starvation. Longer starvation periods severely affect the perigastric organ, causing potential economic losses to famers.

The trophic activity of freshwater decapod crustaceans varies throughout their ontogenetic process, molt cycle, and daily and annual rhythms. Among freshwater decapod crustaceans, Aegla uruguayana (Schmitt, 1942) is an omnivorous, generalist, and opportunistic species. The aim of this study was to analyze the metabolic profile of this species in relation to seasonal and ontogenetic variations. Specimens of different size and sex of A. uruguayana were collected in a stream in Entre Ríos province, Argentina during spring, summer, autumn, and winter. Hemolymph samples were collected to determine the levels of glucose, triglycerides, cholesterol, and total proteins. Hepatopancreas and muscle tissue were extracted to quantify glycogen, lipids, and proteins. Maximum concentrations of triglycerides and total proteins in hemolymph were those of spring and summer, respectively, being the size-season interaction significant in both cases. The interaction of the three factors (season, size, and sex) nevertheless showed no significant differences in the set of measurements obtained for glucose and cholesterol. The metabolic parameters analyzed in hemolymph showed no seasonal variations between males and females, except for summer measurements of glucose and cholesterol. The glycogen and lipid measurements in hepatopancreas were significantly affected by the size-season interaction. Instead, proteins showed only seasonal variation. The data obtained for muscle showed that the size-season interaction did not significantly affect the metabolites. The variations observed in the metabolites reflect the use and requirements of nutrients by the species, evidence that A. uruguayana is an opportunistic omnivorous species. These variations also reflect the dynamics of the communities and the environments where they live. Fil: Musin, Gabriela Eliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto Nacional de Limnología. Universidad Nacional del Litoral. Instituto Nacional de Limnología; Argentina Fil: Rossi, Andrea Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto Nacional de Limnología. Universidad Nacional del Litoral. Instituto Nacional de Limnología; Argentina Fil: Diawol, Valeria Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto Nacional de Limnología. Universidad Nacional del Litoral. Instituto Nacional de Limnología; Argentina Fil: Collins, Pablo Agustin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto Nacional de Limnología. Universidad Nacional del Litoral. Instituto Nacional de Limnología; Argentina Fil: Williner, Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto Nacional de Limnología. Universidad Nacional del Litoral. Instituto Nacional de Limnología; Argentina

We evaluated the effect of stocking density on the concentrations of oxy-hemocyanin and lactate in hemolymph, the antioxidant ability of the hepatopancreas, and the biochemical composition in large and small individuals of the prawn Macrobrachium nipponense (de Haan, 1849) cultured in a cage system. Juveniles (0.24 g initial weight) were stocked in 15 cages with stocking densities of 40, 80, 120, 160, and 200 individuals/m2 for 8 weeks. The results demonstrated that final mean weight, weight gain, gross feed conversion ratio, and survival rate were significantly lower in stocking densities of 160 and 200 individuals/m2 (P

A full-length complementary DNA sequence of heat-shock protein 70 (HSP70) was cloned from the hepatopancreas of Charybdis japonica (A. Milne-Edwards, 1861) through reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The expression profile of HSP70 in different tissues of C. japonica and its expression upon exposure to ammonia-N, nitrite-N, and sulfide was also investigated. The full-length cDNA of HSP70 was 2203 bp long and contained a 107 bp 5′ untranslated region (UTR), a 143 bp 3′ UTR, and a 1953 bp open reading frame, which distributed 108-2060 bp and encoded a polypeptide of 650 amino acids (aa) with a calculated molecular mass of 71.24 kDa and an isoelectric point of 5.34. Three signature sequences of the HSP70 family, IDLGTT-S-V (residues 9-18), IFDLGGGTFDVSIL (197-210 aa), and IVLVGGSTRIPKIQK (334-348 aa) were detected in the predicted amino-acid sequence. The HSP70 gene of C. japonica showed the highest similarity (>90%) to HSP70 from other crustacean species. The HSP70 gene was expressed in all tissues tested, and its abundance in these tissues can be arranged as hepatopancreas > stomach > gill > heart > ova > muscle. The expression of HSP70 was significantly induced after the stress of ammonia-N, nitrite-N, and sulfide, and the induced degree showed a positively correlation with increasing concentrations. The HSP70 expression reached a peak after 3 or 6 h of stress, which suggests that the HSP70 expression was affected quickly by the changes of environmental factors.

Hemocyanin (HC) was purified from hemolymph of Fenneropenaeus merguiensis(de Man, 1888). Purified HC showed one band in non-denaturing PAGE with a molecular mass of 457 kDa, determined by gel filtration. Using an ELISA, HC content was 85.6% of total hemolymph protein. HC’s hexamer form contained no phenoloxidase activity under trypsin or SDS activation. FmHC, cloned from F. merguiensis, had a full-length cDNA comprised of 2128 bp with an open reading frame encoding a peptide of 661 amino acids with two Cu-binding sites. Expression of FmHCwas found only in the hepatopancreas where it was up-regulated after exposure to Vibrio harveyiinjection into shrimp and with tissue incubations. HC protein levels in the hemolymph of bacterial injected shrimp also increased. FmHCexpression was also up-regulated after shrimp were challenged with a lipopolysaccharide, which is a surface component of gram-negative bacteria. These results indicate that HC is inducible and up-regulated in response to pathogenic challenge, confirming that HC participates in the innate immune response in F. merguiensis.

An experiment was conducted to study the profiles and the dynamics of thyroxine in the hemolymph, hepatopancreas, ovary, sponge, and larvae of female mud crabs during ovarian maturation. The experiment consisted of three parts. First, we measured thyroxine concentrations in the hemolymph, hepatopancreas, and ovary. Second, we measured cholesterol, neutral lipid, phospholipid, and protein in the ovary. Third, we determined thyroxine concentrations in the sponge and larvae. During vitellogenesis, the highest thyroxine concentration was found in the hepatopancreas, followed by the hemolymph and ovary. Thyroxine concentrations in the hemolymph and the ovary increased with the increased stage of vitellogenesis. However, thyroxine concentrations in the hepatopancreas decreased with the increased stage of vitellogenesis. Cholesterol and phospholipid concentrations in the ovary increased from vitellogenic stage 1 to stage 2, but began to decline in vitellogenic stage 3. In contrast, neutral lipid concentrations decreased from vitellogenic stage 1 to stage 2, while the concentration of protein in the ovary increased with the advanced of vitellogenic stage. Concentration of thyroxine in the sponges was the highest during the early embryonal stage and continued to decrease with the completion of yolk absorption and during the transformation to the juvenile stage. The results imply that thyroxine plays a critical role in ovarian maturation and embryonic development of mud crabs.

Evaluation of hepatopancreas (HP), pincher claw muscle (PCM), crusher claw muscle (CCM), and abdominal muscle (AM) lipid content in 86 recently caught, adult, male and female American lobsters, Homarus americanus, in the Northumberland Strait, PE, Canada, using the Folch method identified lipid as a significant metabolic reserve. The HP was the primary lipid reservoir in male and non-berried females. Among five moult categories (late inter-moult (mc-1), early pre-moult (mc-2), mid pre-moult (mc-3), late pre-moult (mc-4) and post-moult (mc-5)), devised for the study, the prominent trend observed was a post-moult depletion of lipid reserves in the HP, PCM, and AM. An increased lipid index (LIn) in both CCM and PCM at late pre-moult suggested a differential distribution to provide needed energy in the immediate post-moult foraging period. Hemolymph plasma concentrations of triglyceride (TG), cholesterol (CHOL), and total protein (TP) were highly correlated (TG > CHOL > TP) with HP lipid content in male lobsters only, with the highest values obtained for inter-moult animals. Use of all three parameters in multiple linear regression model improved fit from 0.6656 using TP alone to 0.8226. Hemolymph evaluation presents a non-lethal alternative to assess hepatopancreas lipid content in American lobsters when sex and moult stage are taken into account. These data emphasize the importance of timing and subject selection when using blood chemistry to aid in the monitoring of lobster populations, e.g., for predicting growth on an individual or population basis, response to changing environmental conditions and reproductive capacity.

The aim of this study is to analyze the impact of food shortage on growth performance, by means 24 of energetic reserves (proteins, glycogen and lipids) mobilization and hepatopancreas cells 25 analysis in C. quadricarinatus juveniles maintained in groups, as well as the effect on culture 26 water quality. Two experiments were performed, each of them with two feeding regimes during 27 45 days. The Control feeding regime, in which crayfish were fed daily (once a day) throughout 28 the experimental period (DF), and the Cyclic feeding regime, in which juveniles were fed for 2 or 29 4 days (once a day) followed by 2 or 4 days of food deprivation (2F/2D and 4F/4D, respectively) 30 in repeated cycles. Cyclic feeding influenced growth, biochemical composition from 31 hepatopancreas and muscle, and water quality. Juveniles cyclically fed were unable to maintain a 32 normal growth trajectory during 45 days. Apparent feed conversion ratio, apparent protein 33 efficiency ratio, hepatosomatic index and relative pleon mass were similar in cyclic and daily fed 34 animals and no structural damage was found in the hepatopancreas of juveniles subjected to 35 cyclic feeding. The novelty of this study was the significant accumulation of proteins in pleonal 36 muscle in both cyclic feeding regimes (~18%) suggesting that the storage of this constitutive 37 material during food shortage may be an adaptation for a compensatory growth when food 38 becomes abundant again. The cyclic feeding regimes had a positive effect on water quality 39 decreasing inorganic nitrogen concentration. This was due to the reduction in the amount of 40 animal excretes and feces in the group that received ~50% less feed. Additionally, water pH was 41 higher in cyclic feeding tanks, as a result of lower organic matter decomposition and consequent 42 release of CO2. Accordingly, total ammonia in the water was significantly lower for the cyclic 43 feeding regimes compared to their respective controls. This study suggests that the protocol of 44 cyclic feeding could be applied at least 45 days in 1 g juveniles maintained in group conditions, 45 3 without affecting the energetic reserves and hepatopancreas structure, emphasizing the high 46 tolerance of this species to food restriction. Fil: Stumpf, Liane. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Biología de la Reproducción y Crecimiento de Crustáceos Decápodos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina Fil: Castillo Díaz, Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Biología de la Reproducción y Crecimiento de Crustáceos Decápodos; Argentina Fil: Viau, Veronica Elizabeth. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Biología de la Reproducción y Crecimiento de Crustáceos Decápodos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina Fil: Valenti, Wagner C.. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil Fil: Lopez, Laura Susana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Biología de la Reproducción y Crecimiento de Crustáceos Decápodos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina

The aim of this study was to characterize digestive proteases from wild and farmed morphotypes in males of Macrobrachium amazonicum (Heller, 1862). The hepatopancreas of wild and earthen pond reared specimens were collected and homogenized. Unspecific activities of trypsin, chymotrypsin and leucine aminopeptidase were determined using specific substrates. Serine-protease, trypsin, and chymotrypsin specific inhibitors were employed to assess enzyme inhibition. Optimum pH, temperature and thermal stability of the enzymes were also evaluated. Complementary study was carried out using electrophoresis and zymograms. Proteolytic activities were higher in farmed than in the wild prawns and differences were observed among the farmed male morphotypes, while no differences were observed in the wild ones. Both farmed and wild specimens showed similar values of amylase:protease ratio ranging from 0.06 to 0.12. Inhibition assays revealed the presence of serine proteases, trypsin, and chymotrypsin in the hepatopancreas. However, stronger trypsin inhibitions occurred in farmed morphotypes. The optimum temperature and pH were 65°C and 8.0 to 8.5, respectively. Electrophoresis revealed bands between 6 kDa and 205 kDa. Zymograms revealed bands with proteolytic activity. Using the wild animals as a reference, the results suggest that digestive proteases profile changes in farmed prawns, which may be an adaptation to the culture conditions.

The present study investigated the biochemical composition, including the amino acids and fatty acids profiles of the ovaries and hepatopancreas of the female marine crab, Charybdis japonica (A. Milne-Edwards, 1861), at different physiological stages (immature, mature, spawning and abortion), together with the dynamic changes of the Gonadosomatic Index (GSI) and Hepatosomatic Index (HSI) during gonad development. A significant inverse relationship between GSI and HSI (R=−0.82; P

A complete cDNA sequence of vitellogenin (Vg-H) was cloned from the hepatopancreas of Fenneropenaeus merguiensis (De Man, 1888). The full-length Vg-H gene consists of 7958 bp and contains an ORF of 7761 bp. It encodes a polypeptide of 2587 amino acids, comprising a predicted molecular mass of 283 kD and the theoretical pI is 6.5. The amino acid sequences of the mature vitellogenin (Vg) composed of a signal peptide, three possible O-glycosylation sites, three phosphorylation sites and putative possessing sites (R-X-K/R-R) recognized by subtilisin-like endoproteases. Cleavage at residues 725 to 728 will produce two Vg subunits (78 and 200 kD), which the N-terminal amino acid sequence of 78 kD subunit is identical to that of vitellin purified from the ovary. These properties suggest that Vg protein encoding by Vg-H undergoes processing at its synthetic sites prior to being transported to developing oocytes. The deduced primary structure of Vg-H showed the highest identity (98.8%) to the Vg-O previously cloned from the ovary of the same species. It is more related to the penaeid Vg sequences than that of nonpenaeids. In situ hybridization revealed that mRNA encoding Vg was expressed both in follicle cells in the ovary and parenchyma cells in the hepatopancreas. In the developing ovary, highest levels were detected during the early vitellogenic stage 2, declining thereafter. In the hepatopancreas, Vg mRNA levels reached a maximum at stage 3 of ovarian development. Profiles of Vg mRNA expression in the ovary and hepatopancreas suggest that the contribution of these tissues to vitellogenesis is inversely related during gonad maturation. Thus, Vg gene sequences expressed in the ovary and hepatopancreas are most likely identical and both tissues are responded for yolk protein synthesis in F. merguiensis.

Thioredoxin, is a kind of ubiquitous small proteins with a redox active disulfide bridge, and has been found to play important regulatory roles in some vital biochemical reactions of metabolism. In the present study, a hepatopancreas cDNA library from the mud crab Scylla paramamosain Estampador, 1949 was constructed using the SMART technique. A complete cDNA sequence that showing high identity with the conserved sequence of the second thioredoxin gene (Trx2) was isolated from the cDNA library and subjected to further investigation. The full-length Trx2 of Scylla paramamosain consisted of 1336 bp and contained a complete open reading frame of 396 bp. The gene encoded a polypeptide of 131 amino acids and constituted a predicted molecular mass of 14.45 kDa and a theoretical isoelectric point of 8.08. Quantitative real-time PCR (qRT-PCR) analyses revealed that Trx2 expression was ubiquitous in various organs of Scylla paramamosain, including the gill, muscle, heart, haemocyte, testis, and hepatopancreas. The Trx2 expression level was the highest in the hepatopancreas and gill, and the lowest in the haemocyte. The result of the challenge experiment showed that Trx2 expression was up-regulated significantly after Vibrio parahaemolyticus challenge and reached the highest level at 6 h. This study provides useful information regarding the structure and functions of Trx2, suggesting that it may be a potential biomarker gene for evaluation of environmental stress in marine species.

Sperm and spermatophore production in Callinectes danae Smith, 1869 were examined by histochemistry and correlated with gonadosomatic (GSI) and hepatosomatic (HSI) indices. The GSI from developing (DE) and mature (MAT) males increased while the HSI decreased from DE to MAT, demonstrating that the maturation of the male reproductive system requires resources from the hepatopancreas. No histological or histochemical differences were observed between DE and MAT except for the larger amount of secretions produced in MAT. Spermatogenesis occurs in the seminiferous lobules surrounded by accessory cells. Each lobule is filled with cells at the same developmental stage. Spermatid maturation is characterized by an increase in the metachromatic acrosome. Sperm are released into seminiferous ducts, which moves them to the vas deferens divided into anterior (AVD), median (MVD), and posterior (PVD) regions. Spermatophore formation begins at the anterior part of AVD; sperm masses are separated and compacted in small packets by a basophilic and alcianophilic secretion. Small amounts of eosinophilic secretion, positive for proteins and neutral polysaccharides, are added around the sperm initiating the formation of the spermatophore wall. Mature round spermatophores are found in the posterior part of AVD and present a thick glycoproteinaceous wall, surrounded by acidic polysaccharides. The spermatophores are stored in MVD without size difference from DE to MAT. The MVD is filled with a granular secretion composed of glycoproteins. The secretion in PVD is fluid and homogeneous, facilitating the transference of the spermatophores. In conclusion, the hepatopancreas is related to the maturation of the male reproductive system in C. danae. DE males presented all histological conditions to fertilize females as MAT males, but the decrease in HSI and increase in GSI indices correlated with the vas deferens indicate that reserves are necessary to produce large amounts of seminal fluid in MAT males. Fil: Zara, Fernando J.. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil Fil: Toyama, Marcos H.. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil Fil: Caetano, Flávio H.. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil Fil: Lopez, Laura Susana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina

We measured concentrations of Ca2+ in tissues (hemolymph, hepatopancreas, cuticle, and muscle) of the whiteleg shrimp Litopenaeus vannamei (Boone, 1931) and recorded changes in tissue calcium levels at molting stages and different salinities. Calcium in tissues varied significantly with molting stage (p < 0.05). Salinity also affected Ca2+ concentrations in tissues (p < 0.05). The highest Ca2+ concentrations were found in the cuticle, followed by the hepatopancreas. Ratios of Ca2+/Na+ in tissues (except for muscle) varied with the molting stage and were higher in 4 g L-1 than in other salinities. Calcium entering the shrimp may initially be mineralized in the cuticle or stored in the hemolymph and hepatopancreas, after which it is released to support mineralization in the post-molt period. Muscles may take part in the growth of L. vannamei by storing calcium during the molting cycle.

Ferritin is an important protein for iron storage in cells. A hepatopancreas cDNA library from the mud crab Scylla paramamosain was constructed using the SMART technique. A complete cDNA sequence that showed high identity with the conserved sequence of the ferritin gene was cloned from the cDNA library and subjected to further investigation. The full-length ferritin gene of Scylla paramamosain (SpFer) consists of 767 bp and contains a complete open reading frame of 513 bp and a 26-bp iron-respective element in the 5′-untranslated region. The gene encodes a polypeptide of 170 amino acids, constituting a predicted molecular weight of 19.44 kDa and a theoretical isoelectric point of 5.24. The deduced protein shares 84% identity with the ferritin protein of the crab Eriocheir sinensis. Quantitative real-time PCR analyses revealed that the expression of ferritin was ubiquitous in different organs of S. paramamosain, including muscle, heart, ovary, testis, and hepatopancreas. The highest expression lev...

The biochemistry of fatty acids (FA) can be affected by a number of factors, including environmental temperature, which may affect the way FAs are physiologically metabolized. In this study, we investigated FAs in Pachygrapsus crassipes, in relation to environmental temperatures in southern California. Although there was a trend toward differences in FA abundances in the hepatopancreas of females compared with those found in the hepatopancreas of males, these differences were not significant through most of the year. The sampling month influenced changes in the abundances of both individual FAs (identities) and FA saturation categories (saturated = SAFA, monounsaturated = MUFA, polyunsaturated = PUFA). The abundances of palmitoleic acid, palmitic acid, and docosahexaenoic acid were found to fluctuate significantly over time, although this fluctuation did not appear to be directly influenced by temperature since an increase in FA abundance between February and April preceded an increase in t...

Two glutamate dehydrogenase (GDH) cDNA sequences encoding 474 (GDH A, a truncated gene) and 552 amino acids (GDH B) were discovered in the Pacific white shrimp, Litopenaeus vannamei. Both GDH A and GDH B were fairly conserved as shown by the amino acid alignment. The two cDNAs differed only at the C-terminal sequences. The quantitative real time polymerase chain reaction (qPCR) assay was developed to estimate the specific expression on different tissues. Both GDH A and B were mainly expressed in shrimp muscle, followed by in the gill and epithelium, while the expression in shrimp hepatopancreas was the lowest when compared with other tissues. Though no significant difference was observed among all the test tissues, the ratio of GDH B to GDH A transcription was kept at a very high level ranging from 32.04 in the eyestalk to 64.52 in the muscle, indicating that GDH B was largely expressed in L.vannamei. The GDH activity in the muscle of L.vannamei was also the highest followed by in the epithelium, eyestalk and gill. The GDH activity in hepatopancreas was significantly lower than that in other tissues. As a control, the tissue expression of Na + -K + ATPase was also determined. This enzyme was mainly expressed in the gill, followed by in the muscle and epithelium. Our results provide baseline data in physiological expressions of GDH and Na + -K + ATPase for osmoregulation in L.vannamei. The research protocol offers a practical guideline for the analysis of gene expression in shrimp. The sequences and techniques developed in this study will also contribute to the GDH gene research in other crustacean species.

We studied the morphology of the male reproductive system and spermatophore formation in Astacus leptodactylus. The testis has two anterior lobules and only one posterior lobule, which lie dorsal to the gut on the large hepatopancreas. Collecting tubule cells comprise a simple cuboidal epithelium with synthetic activity. Vasa deferentia are laterally connected with the testis and have three parts: proximal vas deferens (PVD), middle vas deferens (MVD) and distal vas deferens (DVD). While the PVD is off-white in color similar to the testis and has no convolutions, the MVD is intensely white in color and convoluted. On the other hand, the DVD is the widest of all and an intensely white structure. The spermatophore, which is non-pedunculate and tubular, extrudes an uninterrupted column and consists of a sperm mass covered with primary and secondary layers. The primary layer is stained with bromephenol blue, while secondary layer gives positive reaction with Alcian blue and aldehyde fuchsin. The histochemical results indicate that the functions of layers are different.

The export of cellular glucose depends on the endoplasmic reticulum bound enzyme glucose-6-phosphatase (G-6-Pase), which catalyzes the hydrolysis of glucose-6-phosphate to glucose-6, the final step required for the release of new glucose into the blood or hemolymph. The current study examined the effect of chloride on G-6-Pase activity in two marine crustaceans: the ion-regulating crab Menippe mercenaria, and the ion-conforming crab Libinia dubia. Elevated chloride (from 0 to 250 mmol L−1 Cl−) stimulated hepatopancreas G-6-Pase activity in both crabs (2 times in M. mercenaria and up to 1.6 times in L. dubia). Elevated chloride also increased glucose export from isolated M. mercenaria hepatopancreas. These results are in stark contrast to G-6-Pase chloride inhibition previously reported in vertebrate liver. The sensitivity of G-6-Pase to changes in environmental chloride was determined in crabs acclimated to low (20‰) and high (45‰) salinity. M. mercenaria acclimated to high salinity became less s...

An actin-encoding cDNA of the kuruma shrimp (Marsupenaeus japonicus) was cloned from a hemocyte cDNA library. To obtain the full length of actin cDNA, a partial cDNA fragment of 517 bp was isolated by reverse transcriptase-polymerase chain reaction (RT-PCR) from poly (A)+ RNA derived from hemocytes and used as a probe for plaque hybridization. The full actin cDNA was 1,327 bp in length, encoding 376 amino acid residues. The deduced amino acid sequence had 100% identity to that of β-actin from the Pacific white shrimp Litopenaeus vannamei, and extremely high similarity to the sequences of actins in other vertebrates and invertebrates. The cDNA fragments of the actin were amplified by RT-PCR from all organs tested: lymphoid organ, stomach, hepatopancreas, heart, kidney, muscle, eye, ovary, and testis.

Enzyme extracts were used to characterize α-amylase-, trypsin-, and nonspecific-esterase-specific and total activities during the early life-history stages of the red swamp crayfish Procambarus clarkii. Significant increases in the specific activities of all three digestive enzymes were observed prior to the onset of feeding, suggesting that digestive competence is attained prior to feeding and appears to be controlled genetically. After the onset of feeding, the digestive-enzyme-specific and total activity increased steadily through the 42nd day of development, after which point activity did not change significantly. We hypothesize that the observed increase in specific activity reflects the maturation of the hepatopancreas, as greater amounts of digestive enzymes are being produced per unit weight of tissue. As individuals exceeded approximately 100 mg in total weight, specific activity was variable and did not change substantially. Further increases in total enzyme activity (total digestive ca...

The effect of 16 weeks starvation and 6 weeks refeeding on the size and chemical composition of the hepatopancreas of 2 size classes of C. destructor (20–40 g and 40–60 g) maintained at 2 temperatures (10°C and 25°C) was investigated under laboratory conditions. The response to starvation was not influenced by size class. In fed animals, the wet hepatosomatic index (HSIw-%) and dry hepatosomatic index (HSId-mg/g) increased to 7% at week 2 and 36 mg/g at week 10 respectively, whereas in starved animals HSIw and HSId decreased to 2.6% and 3.6 mg/g at week 10 respectively. Hepatopancreas moisture (HM-%) of fed animals decreased to 51% after 10 weeks, whereas in starved animals it increased to a maximum of 81% after 2 weeks. In fed animals, hepatopancreas lipid (HL-mg/g), protein (HP-mg/g), and ash (HA-mg/g) reached a maximum of 30 mg/g at week 10, 7.8 mg/g at week 6, and 2 mg/g at week 4 respectively. Hepatopancreas carbohydrate (HNFE-mg/g) in fed animals fluctuated considerably. In starved animals,...

We evaluated the usefulness of immersion in neutral red solution as a marking technique for studies of movement of the amphipod Corophium volutator. Amphipods were immersed for 20 minutes in solutions of neutral red at different concentrations (25, 50 and 100 mg L−1). Amphipods incorporated dye at all concentrations, and the hepatopancreas remained distinctly stained red for at least 7 days. No increase in mortality was detected at any concentration when compared to a control treatment (0 mg L−1). In a separate field experiment, staining did not significantly affect tendency of C. volutator to move. Overall, immersion in neutral red seems to be an appropriate technique to rapidly mark large numbers of C. volutator and is suitable for short-term mark-recapture experiments. This technique may also apply to other small-sized crustacean species with relatively transparent exoskeletons.

Vitellogenesis in the mud crab Scylla serrata was investigated in vivo using the radiolabeled amino acid ['4C]phenylalanine to trace the synthetic site(s) of the major yolk protein lipovitellin. The incorporation of [14C]phenylalanine into the synthesized proteins of different tissues, such as ovary (OV), hepatopancreas (HP), subepidermal adipose tissue (SAT), and hemolymph (HL) of vitellogenic phases I and II, provided the first in vivo demonstration of possible extraovarian synthesis of lipovitellin precursor, vitellogenin, in S. serrata. The results indicated that among different tissues of vitellogenic phase I, SAT and HP actively take part in synthesizing the yolk proteins which are immediately released into the HL. This is evidenced by a high level of ['4C]phenylalanine incorporation into the protein fraction of the HL. The incorporation of ['4C]phenylalanine into the synthesized proteins of the OV indicates the fact that OV is also capable of synthesizing the precursors to some extent. However, a dramatic increase of ['4C]phenylalanine incorporation into the synthesized ovarian proteins of vitellogenic phase II, concurrent with a decline in the level of ['4C]phenylalanine incorporation into the HL proteins, suggests the probable sequestration of the synthesized proteins from an extraovarian site. The data also suggest a dual role for the HP, in-asmuch as it synthesizes and stores the vitellogenin prior to its release into the OV for sequestration during intense vitellogenesis.

Female blue crabs (Callinectes sapidus) were assigned to 1 of 6 stages based on oocyte development. An immunohistochemical study showed vitellin (Vn)-immunoreactivity was absent from ovaries in stages I and II, and was present in stages III, IV, V, and VI (postspawning). Vn-immunoreactivity was not detected in hepatopancreas, regardless of stage. Hemolymph vitellogenin (Vg) was quantified by enzyme-linked immunosorbent assay. Vg was not detected in hemolymph of stage I and II animals, but was detected in some animals in stages III, IV, V, and VI. The percentage of crabs having detectable hemolymph Vg was 17% (III), 32% (IV), 73% (V), and 100% (VI). Mean Vg levels were 0.02 ± 0.01 mg/ml (III), 0.02 ± 0.04 mg/ml (IV), 0.12 ± 0.15 mg/ml (V), and 0.14 ± 0.04 mg/ml (VI). The facts that Vg was present at low concentration in only a portion of the vitellogenic crabs, and that the level of Vg did not reach maximum until after spawning, are not consistent with the hypothesis that Vg is synthesized by extraovarian tissues and transported via hemolymph to the ovaries. It is possible that the presence of Vg in hemolymph may be a result of leakage of yolk proteins from the ovary due to oocyte reabsorption. The existence of atypical, possible atretic, oocytes in stage V and VI ovaries is consistent with this hypothesis. Considered together with the results of previous studies, these findings suggest that the ovary is the exclusive site of Vn synthesis in C. sapidus.

The morphology of the foregut of the spider crab Notomithrax ursus is described from stereomicroscope and SEM examination. The foregut is divided into 2 parts: a large, anterior, cardiac chamber and a smaller, posterior, pyloric chamber. The cardiac chamber is well supplied with muscles and is lined with chitin thickened in places to form a complex, articulating set of ossicles. Some of these ossicles are developed into teeth which grind up the food. Other ossicles and setae inside the cardiac chamber appear to direct the movement of food through the cardiac chamber and sort the food according to particle size as digestion takes place. The pyloric chamber acts as a filter to separate digested liquid, which is directed to the hepatopancreas for absorption, from solid particles which are directed to the midgut for excretion. The morphology of the foregut of N. ursus, in particular, ossicle structure, supraampullary folds, and degree of setation, suggests that it is adapted to an unspecialized, macrophagous diet.

Changes in weight and water content of the hepatopancreas and ovary of Crangon crangon are described in relation to body weight and ovarian development. Percentage of water content of the organs in relation to ovarian stage was determined for 43 females; organ wet weight data were obtained from another 137 animals. All stages ofgonadal development (oogenesis, previtellogenesis, vitellogenesis, and depleted) were represented in the sample population of ovigerous, nonovigerous, and postovigerous females. The ovary and hepatopancreas progress through cycles of progressive and retrogressive changes in weight and volume which are characterized by a phase shift between the two organs. This resembles a Volterra-Lotka system, for which equations were derived. Wet weight of the ovary increases by 69% through previtellogenesis, and by 282% from preto secondary vitellogenesis. This represents an increase of 5.5% of body weight occupied by the ovary during development. The hepatopancreas nearly doubles in size from early oogonial development through previtellogenesis, but decreases markedly during vitellogenesis. The net result is an increase from a minimum value of 4% to a combined organ volume occupying 9.5% of total body weight when the ovary is fully vitellogenic. Increase in ovarian mass during development is not due to water increase. Water content decreases from > 70% in immature ovaries to 46% in vitellogenic organs. The hepatopancreas also loses water, but to a lesser extent: from 73% in oogenic individuals to 67% in vitellogenic animals. Female decapod crustaceans progress through a cycle that includes proliferation of gonial cells (gametogenesis), growth of gametes to maturation (gamete production), ovulation and oviposition (egg extrusion), and incubation of embryos until hatching (Sastry, 1983). Numerous papers contain descriptions of various aspects of crustacean reproduction, with emphasis on oogenic and ovarian cycles (see, e.g., Adiyodi and Adiyodi, 1970; Aiken and Waddy, 1980). Quantitative studies of the period and amplitude of ovarian growth of crustaceans are relatively scarce, and even fewer have adequately correlated ovarian development with changes in the hepatopancreas (physically, in terms of mass and water content, and biochemically) (Chang and O'Connor, 1983; Sastry, 1983). Such is the case for the common brown shrimp Crangon crangon, which is exploited commercially in the North Sea (Tiews, 1970). The reproductive cycle of coastal populations has been well documented (Boddeke, 1989), and increase in the size of the ovary during its development has been described (Meredith, 1952). This paper documents changes in weight and water content of the ovary and hepatopancreas relative to ovarian develpment in C. crangon, and derives mathematical equations for the phase shift between the two organs. MATERIALS AND METHODS Specimens of Crangon crangon were collected from the Waddenzee from March through June 1992 and held in an aquarium supplied with running sea water at ambient salinity (n28%oo) and temperature (= 15?C). They were fed frozen shrimp ad libitum until processed. The percentage of water content of the ovary and the hepatopancreas was determined for 43 females. These individuals were blotted dry and weighed to the nearest mg; the ovigerous condition and the presence of egg remnants on the pleopods were noted. The stage of the ovary was identified according to Meredith's (1952) illustrations of size and shape in proportion to the gastric mill and thoracic cavity (Table 1). The ovary and hepatopancreas were removed intact, weighed, dried at 60?C for 16-18 h and weighed again. From these data, the percentage of water content of each organ was determined. Regression equations were derived for the relationships of dry weight to wet weight for the ovary and the hepatopancreas. Wet weights of the entire specimen, ovary, and hepatopancreas were also obtained from another 137 individuals (dry weights were not obtained from these animals). The percentage of the body wet weight (BW)

Motility of the gut in developmental stages of Penaeus setiferus (Linnaeus) was monitored in vivo with video microscopy. Oral drinking, anal drinking, and propagation of contractile waves in the midgut trunk (MGT) were observed in larval and postlarval stages. A gastric mill and functional gland filter were absent in larvae. In early postlarval stages, the gastric mill did not function in trituration. The gland filter appeared to function in emulsification rather than in exclusion of large-sized particles from the hepatopancreas. Chyme from within the peritrophic membrane, as far posteriad as abdominal segment 2, was moved at times anteriad into the hepatopancreas. Both antiperistaltic and peristaltic waves mixed and transported chyme within the midgut. Substantial ontogenetic change in function of the foregut and midgut occurred (1) at metamorphosis, (2) at onset of motility in the median tooth, and (3) with differentiation of hepatopancreatic tubules. We propose that antiperistalsis along the MGT, anal drinking, oral drinking, and contractions of the foregut maintain positive hydraulic pressure within the midgut. This pressure expands regions of the midgut following contractions of intrinsic muscles. Hydraulic pressure, particularly that propagated by antiperistalsis of the MGT, is responsible for expansion of contracted hepatopancreatic tubules. We further propose that the peritrophic membrane separates stored chyme and rejected fecal matter from water taken up by anal drinking and pumped anteriad by antiperistaltic waves. In P. setiferus hydraulic pressure in the extraperitrophic space of the midgut lumen, together with pressure exerted by constriction of the MGT in abdominal segments 3 and 4, compresses the peritrophic membrane around its contents. This compression expels fluids anteriad and compacts feces before the fecal strand reaches the hindgut.

Protein, lipid, and carbohydrate content were determined for hemolymph, ovary, and hepatopancreas of the brown shrimp Crangon crangon (Linnaeus) relative to ovarian development. Total organic content (pooled proteins, lipids, and carbohydrates in all three tissues) increases from oogenesis through vitellogenesis. The protein component accounts for the greatest increase, followed by lipid and carbohydrate. All measured chemical species in the depleted ovary (following oviposition) decrease to the values observed in oogenic ovaries. Increase in total protein content is associated with the increase in ovarian protein content; hemolymph and hepatopancreatic protein increase only slightly prior to previtellogenesis, and decrease during vitellogenesis. Total lipid increase is traceable to hepatopancreatic accumulation prior to vitellogenesis; subsequent decrease during vitellogenesis is associated with increases in hemolymph and ovarian lipid contents. Changes in total carbohydrate content are also due to hepatopancreatic accumulation during previtellogenesis, and subsequent decrease during vitellogenesis. Hemolymph carbohydrate content increases when hepatopancreatic content decreases. The ovary never accumulates large quantities of carbohydrates. Although the concentrations of proteins, lipids, and carbohydrates vary considerably, certain patterns related to ovarian development and maturation are evident. These trends, and shifts in chemical content associated with changes in the size (volume) of the tissues and organs (hemolymph, ovary, hepatopancreas), are discussed in terms of biochemical conversion and transport of the compounds between organs during the reproductive cycle.

Adult males of the fresh-water prawn Macrobrachium rosenbergii in a single-age population can be categorized into three morphotypes. Each morphotype represents a different stage in the development of the adult males from small males (SM) through orange-claw (OC) males to blueclaw (BC) males (Cohen et al., 1981; Ra'anan, 1982). All males are capable of developing through all of the above stages, but individual males largely differ in the rate in which they transform from one morphotype to another. It was observed that SM and BC males are more sexually active than OC males, while the OC males grow more rapidly than SM and BC males (Ra'anan and Sagi, 1985). To examine further this behavioral observation, two internal organs in each of the morphotypes were measured and compared. The development of the reproductive system (testes, sperm ducts, and ampullae), as an indication of sexual activity, was compared with the development of the midgut gland (hepatopancreas), an organ which is suggested to be in correlation with somatic growth. The hepatopancreas is significantly larger and the reproductive system is relatively smaller, in relation to body size, in OC males. The opposite is true of SM and BC males, in which the reproductive system is significantly larger while the hepatopancreas is much smaller in relation to body size. Thus, the relative sizes of the two organs of an individual prawn are closely associated with its position in the male developmental pathway from the SM through OC to the BC morphotype. Further, the changes in specific ratio between the two organs reflect the inverse relationship between reproduction and growth.

The organization of the muscle cell layer surrounding the tubules of the midgut gland in Homarus americanus was examined with transmission and scanning electron microscopy. This layer contains a uniform network of broad circular myofilaments interconnected by smaller longitudinal connectives. The primary means of constriction of the lumen of the tubule appears to be by contraction of the circular muscle fibers. The longitudinal myofilaments may provide both structural support and a means for conducting the contractile impulse along the tubule. The decapod hepatopancreas, or midgut gland, is a multilobed organ comprised of numerous blindly ending tubules, connected by a graduated series of collecting ducts. The collecting ducts coalesce into two main ducts which articulate at the pyloric-intestinal junction. As the primary organ for digestion and absorption of nutrients, both digestive enzymes and the products of digestion are moved through the tubules; the food brei moves from the stomach to the tubules and the enzymes and digestive waste products return from the tubules to the stomach. In a general sense, the midgut gland may be equated to the vertebrate small intestine with associated digestive organs, while the true decapod intestine may be equated with the large bowel. Although Gibson and Barker (1979) have published a thorough review on the hepatopancreas, including the basic cell types, sequence of digestive epithelial cell development, and enzymatic activity of the cells involved in both extracellular and intracellular digestion, the mechanism for moving the enzymatic secretions out and the food solution into the tubules of the hepatopancreas is poorly known and often casually dismissed. For example, Stanier et al. (1968) described only a discontinuous stellate network of contractile fibrils around each tubule of Carcinus maenas, and Young (1959) did not mention muscular activity associated with the hepatopancreas of Penaeus setiferus. The musculature associated with the tubules of the midgut gland in Astacus fluviatilis were reported as a series of parallel circular fibers of striated muscle with smaller longitudinal connectives (Pump, 1914). The striated muscle found in the hepatopancreas of crayfish was described as tonic muscle (Loizzi, 1971). The contractile function of tonic invertebrate striated muscle can be equated to that observed in vertebrate intestinal smooth muscle (Prosser, 1973). Van Weel (1974) speculated that the action of the muscle fibers surrounding each tubule of the midgut gland was similar to the squeezing and releasing of a rubber syringe during filling and emptying. This study examines the musculature around the tubule of the midgut gland in the American lobster Homarus americanus, confirming Pump's (1914) work at the level of scanning electron microscopy and provides indirect evidence for a peristaltic transport mechanism to move materials within the tubules of the hepatopancreas.

Yolk production of the marine shrimp Penaeus vannamei was measured using an antibody to a subunit of the yolk protein complex. The effects of unilateral eyestalk ablation on protein synthesis of the hepatopancreas and ovary were measured in vitro. Initially, activity in both tissues increased; 21 days after eyestalk ablatior activity levels in both tissues returned to levels equal to or lower than preablation levels. The yolk protein content of the tissues increased shortly after eyestalk ablation and reached a maximum of 8% in ovarian tissue, but less than 1% in the hepatopancreas. Yolk proteins in the hemolymph were detected using an enzymelinked immunoassay. Both intact controls and eyestalk-ablated shrimp had as much as 1 mg/ ml yolk protein in the hemolymph during ovarian maturation. Newly released shrimp eggs had an average of 53 ng yolk protein per egg. Eyestalk ablation induced a rapid increase in protein synthesis in both ovary and hepatopancreas. During ovarian maturation, yolk proteins were transported through the hemolymph in this marine shrimp.