Hepatopancreas Samples Research Articles

Celulasas presentes en el tracto digestivo del camarón Litopenaeus vannamei

Las celulasas han sido clasificadas como endoglucanasas responsables de degradar celulosa, lo que le proporciona gran importancia en los procesos de la cadena alimentaria. Las celulasas y enzimas relacionadas son utilizadas ampliamente en varios procesos industriales. La celulosa es utilizada como una fuente nutricional por varios organismos, inicialmente se pensaba que únicamente microorganismos podrían degradarla, pero en la actualidad se ha encontrado celulasas en varios grupos de animales. En este trabajo, presentamos la detección de celulasas en extractos obtenidos del camarón Litopenaeus vannamei. En los ensayos se utilizaron individuos adultos y de los diferentes subestadios de L. vannamei colectados en los estanques de cría de las camaroneras de la Península de Santa Elena y Muisne, en las provincias de Santa Elena y Esmeraldas, respectivamente. Los hepatopáncreas, los estadios larvarios y los intestinos fueron homogeneizados en un homogeneizador Braun-Melsungen. Para la identificación de la celulasa, se utilizó como sustrato carboximetil celulosa (CMC) de mediana viscosidad. Para medir su actividad se añadió ácido 3,5 dinitrosalicílico (DNS). La actividad de la celulasa se determinó en base en la cantidad de los grupos reductores liberados. Para intentar determinar el origen sea endógeno o microbiano de las celulasas identificadas, se sometieron las muestras de adultos a antibióticos.
 
 En el subestadio zoea 3, se identificó una celulasa con un rango de pH óptimo entre pH 6.5 y pH 9.5. En las muestras de hepatopáncreas del camarón adulto se determinaron dos pH óptimos: uno en el rango ácido (pH 4-6) y otro en el rango básico (pH 10-11) lo cual sugiere la presencia de dos celulasas diferentes. En el intestino se observó actividad únicamente en el pH ácido (pH 4-6). La actividad celulolítica detectada en el desarrollo ontogénetico del camarón fue baja e inestable, una condición reportada también en el análisis comparativo de enzimas digestivas en otras cuatro especies de crustáceos. La actividad relativa de las dos enzimas del hepatopáncreas es similar y cada una de estas tienen una actividad cuatro veces mayor a la encontrada en el intestino. Estos resultados indican la presencia de dos celulasas presentes en el hepatopáncreas y se sugiere que una de ellas (con pH óptimo ácido) podría ser transportada desde el hepatopáncreas al intestino, aunque los datos no son concluyentes. En el caso de la celulasa con actividad a pH básico (10.5) puede tratarse de una celulasa producida de manera endógena por el camarón. Los datos reportados en este estudio sobre las capacidades de producción enzimática del camarón puede ser la base para futuros proyectos de investigación cuyo objetivo sea optimizar los procedimientos de cultivo del camarón.

Dense populations of the microsporidian Enterocytozoon hepatopenaei (EHP) in feces of Penaeus vannamei exhibiting white feces syndrome and pathways of their transmission to healthy shrimp

White feces syndrome (WFS) is an emerging problem for penaeid shrimp farming industries in SE Asia countries, Thailand, Malaysia, Vietnam, Indonesia, China, and in India. This occurrence of this syndrome is usually first evidenced by the appearance of white fecal strings floating on surface of the shrimp ponds. The gross signs of affected shrimp include the appearance of a whitish hindgut and loose carapace, and it is associated with reduced feeding and growth retardation. To investigate the nature of the white feces syndrome, samples of white feces and shrimp hepatopancreas tissue were collected from Penaeus vannamei in affected farms in Indonesia, and these were examined histologically. Within the white feces, we found densely packed spores of the microsporidian Enterocytozoon hepatopenaei (abbreviated as EHP) and relatively fewer numbers of rod-shaped bacteria. From WFS ponds, hepatopancreas samples form 30 individual shrimp were analyzed by histology and in situ hybridization. The results showed that all of the shrimp examined were infected with EHP accompanied by septic hepatopancreatic necrosis (SHPN). Midgut epithelial cells were also infected and this increased the number of tissue types being affected by EHP. By PCR, EHP was detected in all the samples analyzed from WFS-affected ponds, but not in those sampled from healthy shrimp ponds. To determine the modes of transmission for this parasite, we performed feeding and cohabitation bioassays, the results showed that EHP can be transmitted through per os feeding of EHP-infected hepatopancreas tissue to healthy shrimp and through cohabitation ofinfected and healthy shrimp. In addition, we found the use of Fumagillin-B, an antimicrobial agent, was ineffective in either reducing or eliminating EHP in infected shrimp.

Microbiological Quality Assessment by PCR and Its Antibiotic Susceptibility in Mangrove Crabs (Ucides cordatus) from Guanabara Bay, Rio de Janeiro, Brazil.

The bacteriological quality of crabs from three different mangroves (Itaóca, Suruí, and Piedade) from Rio de Janeiro state, Brazil, was investigated using conventional and molecular methods. The results revealed high counts for total coliforms in meat and hepatopancreas samples. PCR analyses identified 25 Escherichia coli colonies in the Itaóca, Piedade, and Suruí samples, detecting 13 enterotoxigenic colonies and 9 enteroaggregative colonies. Respectively, 12, 11, and 21 Vibrio parahaemolyticus strains were detected in the Itaóca, Piedade, and Suruí samples. Two V. cholerae strains were detected in the Piedade samples. The E. coli strains isolated in the present study showed resistance to gentamicin. E. coli strains from the Piedade samples showed 33% resistance to chloramphenicol and the strains also showed multiresistance to several antimicrobial agents with a MAR index ranging from 0.12 to 0.31. Vibrio strains from Piedade, Itaóca, and Suruí showed 86%, 78%, and 85% resistance, respectively, to ampicillin. The isolated Vibrio strains showed multiresistance to several antimicrobial agents, with a MAR index ranging from 0.12 to 0.25. The presence of these organisms in crab meat is an indication of microbial contamination, which may pose health risks to consumers when improperly cooked.

Antioxidant and oxidative damage responses in different organs of Pacific white shrimp Litopenaeus vannamei (Boone, 1931) reared in a biofloc technology system

Shrimp (Litopenaeus vannamei) reared in a conventional seawater (SW) aquarium system SW were compared with those raised in a biofloc technology (BFT) system. After 30 days, the L. vannamei shrimp were euthanized and samples of gills, hepatopancreas and muscle were dissected. Statistical analysis was performed using bi-factorial ANOVA, with the factors of the treatment (shrimp reared in SW or in a BFT system) and organs (gills, hepatopancreas and muscles). No differences (p > 0.05) in glutathione-S-transferase activity were observed between shrimp reared in SW and shrimp reared in BFT (p < 0.05). Glutathione levels were lower (p < 0.05) in the gills and hepatopancreas of shrimp reared in the BFT system, suggesting changes in the antioxidant composition of these organs. Lipid peroxidation levels were higher in the hepatopancreas than in muscle in shrimp reared in SW (p < 0.05) and this difference was not observed in organisms reared in the BFT system (p > 0.05). Overall, the results showed that rearing shrimp in the BFT system altered both their antioxidant and oxidative damage responses. This indicates that some microbial communities in BFT systems can influence the redox state of L. vannamei.

LvDJ-1 plays an important role in resistance against Vibrio alginolyticus in Litopenaeus vannamei

DJ-1 was first identified as an oncogene that transformed mouse NIH3T3 cells in cooperation with activated Ras. It has since exhibited a variety of functions in a range of organisms. In this study, the DJ-1 gene in Litopenaeus vannamei (LvDJ-1) was identified and characterized. A recombinant protein LvDJ-1 was produced in Pichia pastoris. LvDJ-1 expression in vivo was knocked down by dsRNA-mediated RNA interference (RNAi), which led to significantly decreased levels of LvDJ-1 mRNA and protein. When the L. vannamei were challenged with RNAi and Vibrio alginolyticus, the transcription and expression of copper zinc superoxide dismutase (LvCZSOD) in the hepatopancreas were dramatically lower in shrimp with knocked down LvDJ-1 than in controls. Transcription and expression of P53 (LvP53) were significantly higher in shrimp lacking LvDJ-1 than in controls. Hepatopancreas samples were analyzed using real time polymerase chain reaction and Western blot. Moreover, blood samples from the shrimp, assessed with flow cytometry, showed significant increases in respiratory burst and apoptosis in those lacking LvDJ-1 compared to the controls. Cumulative mortality in the shrimp lacking LvDJ-1 was significantly different from that in the control group after challenge with V. alginolyticus. Altogether, the results prove that LvDJ-1 regulates apoptosis and antioxidant activity, and that these functions play an important role in L. vannamei resistance against V. alginolyticus.

Effects of trichotecene mycotoxins on gene expression and activity of glutathione redox system in common carp (Cyprinus carpio L.)

Event Abstract Back to Event Effects of trichotecene mycotoxins on gene expression and activity of glutathione redox system in common carp (Cyprinus carpio L.) Csilla Pelyhe1*, Benjámin Kövesi1, Erika Zándoki2, Balázs Kovács3, Miklós Mézes1 and Krisztián Balogh1 1 Szent István University, Department of Nutrition, Hungary 2 Hungarian Academy of Sciences, Kaposvár University, Mycotoxins in the Food Chain Research Group, Hungary 3 Szent István University, Department of Aquaculture, Hungary The purpose of present study was to evaluate the short- and long-term effects of T-2 toxin and deoxynivalenol (DON) in common carp. Short-term effect was evaluated using single oral doses of DON or T-2 toxin of 0.25, 0.50 or 3.00 mg/kg b.w.. Experimentally contaminated or control complete feed was given by gavage directly into the stomach. Hepatopancreas samples were taken from 6 carps of each group at every 8th hour during a 24-hour long experimental period. The long-term effect was evaluated in 4 weeks in a feeding trial using 0.05 mg T-2 toxin/kg b.w. and 0.06 mg DON/kg b.w., where hepatopancreas samples were taken from 6 carps of each group weekly. Reduced glutathione (GSH) concentration and activities of glutathione-peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST) were measured. In addition, expressions of phospholipid hydroperoxide glutathione peroxidase (GPx4) genes (gpx4a and gpx4b) were analyzed. Lipid peroxidation was followed by the determination the amount of conjugated dienes (CDs) and -trienes (CTs) and malondialdehyde (MDA). Short-term exposure showed that both of the investigated mycotoxins caused rapid changes in the lipid peroxidation processes in the hepatopancreas, but the emerging free radical burden quickly activated the glutathione redox system, so the end-product of the lipid peroxidation (MDA) did not elevate significantly in the 24-hour period. Expression of the genes decreased after 8 hours and later increased during the 24-hour period. As effect of long-term exposure, the glutathione redox system showed decreased levels in T-2 toxin treated group, but the markers of lipid peroxidation processes did not elevate. Expression of the investigated genes showed dual response during the experiment. The results revealed that glutathione redox system eliminated effectively the peroxidative effect of the investigated mycotoxins in the applied doses and periods. Acknowledgements The research was supported by the National Scientific Research Fund (OTKA PD104823). Keywords: T-2 Toxin, Deoxynivalenol (DON), Antioxidant Enzymes, common carp, Glutathione redox system, gpx4 genes, Gene Expression Conference: XV European Congress of Ichthyology, Porto, Portugal, 7 Sep - 11 Sep, 2015. Presentation Type: Oral Presentation Topic: Physiology, Behavior and Toxicology Citation: Pelyhe C, Kövesi B, Zándoki E, Kovács B, Mézes M and Balogh K (2015). Effects of trichotecene mycotoxins on gene expression and activity of glutathione redox system in common carp (Cyprinus carpio L.). Front. Mar. Sci. Conference Abstract: XV European Congress of Ichthyology. doi: 10.3389/conf.fmars.2015.03.00028 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Oct 2015; Published Online: 04 Nov 2015. * Correspondence: Miss. Csilla Pelyhe, Szent István University, Department of Nutrition, Gödöllo, 2100, Hungary, pelyhe.csilla@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Csilla Pelyhe Benjámin Kövesi Erika Zándoki Balázs Kovács Miklós Mézes Krisztián Balogh Google Csilla Pelyhe Benjámin Kövesi Erika Zándoki Balázs Kovács Miklós Mézes Krisztián Balogh Google Scholar Csilla Pelyhe Benjámin Kövesi Erika Zándoki Balázs Kovács Miklós Mézes Krisztián Balogh PubMed Csilla Pelyhe Benjámin Kövesi Erika Zándoki Balázs Kovács Miklós Mézes Krisztián Balogh Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Distribution of Cellulase Activities in Acetes Shrimps Living in the Matang Mangrove Forest Reserve, Malaysia

For the purpose of investigating the cellulose degradation mechanism in mangrove estuaries,&nbsp;cellulase activities of three Acetes shrimps (Acetes sibogae, A. japonicus and A. indicus) were examined in the Matang&nbsp;Mangrove Forest Reserve (MMFR). As a result of cellulose plate assay measurement, we detected the activities&nbsp;from all three species. SDS-PAGE zymographic analysis revealed that all Acetes shrimps demonstrated active bands&nbsp;at approximately 25 kDa and higher than 100 kDa. In addition, by quantifi cation of reducing sugar production,&nbsp;hepatopancreas samples of Acetes showed signifi cantly higher cellulase activity compared to whole body samples&nbsp;suggesting their endogenous origin. Our fi ndings that cellulase activities are widely distributed among the Acetes&nbsp;shrimps living in the MMFR, suggests that mangroves are indirectly utilized by higher trophic consumers.(Keywords: cellulase, mangrove, Acetes, hepatopancreas)

Accumulation trends of metals and a metalloid in the freshwater crayfish Astacus leptodactylus from Lake Yeniçağa (Turkey)

This study aims to determine the extent of metal pollution in Lake Yeniçağa (Bolu, Turkey) by investigating the accumulation trends of five metals (Al, Cu, Fe, Ni and Zn) and a metalloid (As) in gills, exoskeleton, hepatopancreas and abdominal muscles of the freshwater crayfish Astacus leptodactylus. Principal component analysis (PCA), cluster analysis (CA), correlation analysis and analysis of variance (ANOVA) were utilised to determine the accumulation profiles of each element over four seasons. The greatest element accumulation was found to occur in the gills. All elements in exoskeletal tissue displayed positive correlations with each other, a similar trend was also observed in the hepatopancreas samples. Strong (r=0.868) and very strong (r=0.960) positive correlations were found between the accumulations of Al and Fe in gills and the exoskeleton, respectively. Correlations in tissue accumulation rates are discussed in the context of metabolic roles and impacts associated with the elements tested. Elemental compositions of Yeniçağa water and sediment samples were also investigated to determine whether the composition of the surrounding environment matches the metal accumulation trends of tissue samples. We demonstrate that, by the criteria set by the United States Environmental Protection Agency, Lake Yeniçağa is heavily polluted in terms of As and Ni.

Prevalence, Serovars, and Antimicrobial Susceptibility of Salmonella Isolated from Blue Land Crabs (Cardisoma guanhumi) in Grenada, West Indies

Samples of intestine and hepatopancreas from 65 blue land crabs (Cardisoma guanhumi), a crustacean commonly consumed as a food item in Grenada, were collected from six geographic sites in Grenada and tested for Salmonella by enrichment and selective culture. The individual animal prevalence of Salmonella based on isolation was 17% (11 of 65), and all infected crabs were from three of the six sampled locations. Isolates were identified by serotyping as Salmonella enterica serovars Saintpaul (n = 6), Montevideo (n = 4), and Newport (n = 1). The intestines of all 11 infected crabs were positive for Salmonella, but only 7 of 11 hepatopancreas samples were positive for Salmonella, and these isolates were the same serovar as isolated from the matching intestine. These three Salmonella serovars are known to cause human illness in many countries, and in the Caribbean Salmonella Saintpaul has been frequently isolated from humans. In a disc diffusion assay, all isolates were susceptible to all 11 drugs tested: amoxicillin-clavulanic acid, ampicillin, cephalothin, chloramphenicol, ciprofloxacin, gentamicin, imipenem, neomycin, streptomycin, tetracycline, and trimethoprim-sulfamethoxazole. To our knowledge, this report is the first concerning isolation and antimicrobial susceptibilities of Salmonella serotypes from the blue land crab.

A vortex-assisted MSPD method for the extraction of pesticide residues from fish liver and crab hepatopancreas with determination by GC–MS

A method based on matrix solid-phase dispersion (MSPD) and gas chromatography-mass spectrometry to determine pesticides in fish liver and crab hepatopancreas was optimized. Ethyl acetate and acetonitrile were evaluated as elution solvents and their volumes were also checked. The best results were obtained with 1.0g reused C18 as sorbent, using 5mL acetonitrile as the elution solvent. Analytical recoveries ranged between 57 and 107% with RSD lower than 26% in fish liver and between 56 and 122% with RSD lower than 21% in crab hepatopancreas. The LOQ values for these compounds ranged from 0.05 to 0.5mgkg−1 for crab hepatopancreas and from 0.125 to 1.25mgkg−1 for fish liver. MPSD was shown to be easy and fast to use, with a clear advantage regarding costs because it does not need any expensive instrument. The proposed method was successfully applied to determine dimethoate, atrazine, clomazone, fenitrothion, malathion, fipronil and tebuconazole in fish liver and crab hepatopancreas samples.

Daily variations of the antioxidant defense system of the lithodid crab Lithodes santolla

Several physiological processes can induce daily variations in aerobic metabolism. Lithodes santolla is a decapod crustacean of special concern since it is a sub-Antarctic species of commercial interest. The aim of this work was to study in L. santolla the daily variations in levels of enzymatic and non-enzymatic antioxidants, lipid peroxidation and protein oxidation, and haemolymphatic pH. Males of L. santolla of commercial size were randomly dissected every 4h during a period of 24h. Enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase and glutathione peroxidase were determined in samples of gills, muscle, hepatopancreas and haemolymph. Ascorbic acid, total glutathione, lipid peroxidation and protein oxidation were also determined in all tissues. Gills showed the highest enzymatic activity and hepatopancreas the highest concentration of non-enzymatic antioxidants. Maximum antioxidant activity was during the dark phase in gills and during the photophase in the haemolymph. Muscle showed significant daily variations, with peaks during the photophase and scotophase. Overall, an antioxidant protective mechanism is present in all tissues, where SOD and CAT represent the first line of defense. The defense mechanism in L. santolla seems to be more active during the dark phase, with slight differences among the analyzed tissues, indicating a higher metabolic rate.

Functional cytology of the hepatopancreas of Palaemonetes argentinus (Crustacea, Decapoda, Caridea) under osmotic stress

The present work describes the effect of different salinities on the functional morphology of the P. argentinus hepatopancreas and analyses the tissue recovery after re-acclimation to freshwater. Adult prawns of both sexes at sexual rest were collected from a tributary of the Mar Chiquita coastal lagoon. The prawns were acclimated in aquaria to four salinity conditions: 0 (control), 8, 16 and 24‰. To evaluate the possible tissular recovery, after 60 days individuals from all the treatments were gradually acclimated to freshwater and maintained for other 30 days. Hepatopancreas samples were processed at the beginning of the trial and every 30 days using standard histological techniques for OM and TEM. The individuals from all the treatments, except the controls, showed a continuous weight decrease, and survival was lower when higher the salinity. At 30 days from the beginning of the experiment, hepatopancreas from 16 and 24‰ salinities showed an enlarged tubular lumen and an infolded basal lamina. Ultratructurally, nuclear retraction, cytoplasmolysis, and RER membranes separated with electron-dense content were observed in all the treatments except 0‰. After 60 days, profound alterations were observed with the three treatments. After the re-acclimation period, there was no reestablishment of the functional cytology. The tolerance to short-term salinity changes explains the capability of this prawn to inhabit in estuarine environments.

Density of Vibrios in Hemolymph and Hepatopancreas of Diseased Pacific White Shrimp, Litopenaeus vannamei, from Northwestern Mexico

Vibriosis is one of the most widespread infectious diseases of cultured shrimp in Mexico; as a result, the Research Centre for Food and Development (CIAD, for the Spanish wording) offers a diagnostic service to the shrimp farmers where they bring samples of diseased shrimp as judged by themselves. Hemolymph (HL) and hepatopancreas (HP) of diseased Litopenaeus vannamei were analyzed from five regions of Northwestern Mexico (from 2001 to 2006) in tiosulphate citrate bile sacharose (TCBS) agar; a total of 5912 shrimp were examined. The results showed a mean vibrios density of 6.45 × 103, SD = 6.90 × 104 CFU/mL for HL and 1.40 × 105, SD = 3.20 × 105 CFU/g for HP (n = 5912). HL samples had 12% (SD = 26%) of green colonies (GCs) in TCBS, and 23% (SD = 34%) of GCs in HP samples. Of the total shrimp analyzed, 46% were not found to possess vibrios in HL, whereas 60% of the shrimp examined contained more than 105 CFU/g of HP. More than 60 and 40% of the shrimp did not have GC in HL and HP, respectively. The highest Pearson coefficient correlation was observed between vibrios HL and GCs in HL for 2006 (r = 0.611, n = 712, P &lt; 0.001).

Hepatopancreatic and muscular distribution of oxytetracycline antibiotics in farmed pacific white shrimp (Penaeus vannamei): a physiological-based pharmacokinetic model approach

Oxytetracycline (OTC) pharmacokinetic models previously used to investigate Penaeus vannamei have not addressed the specific problems related to drug distribution/disposition in particular tissues. This study aimed to provide an insight into OTC kinetics in the hepatopancreas and muscle based on a physiological model approach. Adult male P. vannamei at the C-D0 inter-moulting stage were randomly assigned to intra-sinus and oral administrations. In the intra-sinus group, shrimps were dosed via the ventral sinus at an OTC level of 10.0 μg g−1 body weight, while in the oral one, they were force fed at a dose level of 50.2 μg g−1. The medicated animals were sampled at various time intervals until 170 h after dosing. Haemolymph, muscle and hepatopancreas samples were taken and OTC levels were determined using the validated HPLC method. A model focused on the hepatopancreas and muscle was developed. Oxytetracycline pharmacokinetic profiles in particular tissues were fitted into the model with an R2 of between 0.6568 and 0.9904. Oxytetracycline muscular distributions were essentially identical for both groups and the drug did not accumulate in muscle. The distributions in the hepatopancreas for both groups were extensive, whereas that for oral administration was approximately 2.3 times greater than that for the intra-sinus one. It was demonstrated that hepatopancreatic OTC may undergo significant first-pass elimination with non-linear kinetics.

Seasonality of Dinophysis spp. and Prorocentrum lima in Black Sea phytoplankton and associated shellfish toxicity

Plankton surveys, between 2001 and 2005 along the Russian Caucasian Black Sea Coast, revealed Dinophysis rotundata, D. caudata and Prorocentrum lima as the most ubiquitous of the known dinoflagellates associated with diarrhetic shellfish poisoning (DSP). Dinophysis spp. were first observed during the spring phytoplankton succession and persist throughout the late summer phytoplankton peak. The highest total concentration, 3000 cells/L, of D. rotundata and D. caudata was observed in April 2001. Unlike Dinophysis, P. lima was rarely observed in plankton samples but closely followed storm events with maximum cell counts of P. lima occurred in July 2002. The presence of Dinophysis in mussel ( Mytilus galloprovincialis) hepatopancreas correlated with concentration with Dinophysis observed in the plankton samples. Conversely, P. lima could be found in most hepatopancreas samples collected during the May to October period. Therefore, planktonic concentration of P. lima does not reflect its availability for and consumption by shellfish. Samples of mussel hepatopancreas, from August 2002, with a corresponding Dinophysis concentration of 250 cells/L and no observable P. lima, were found to contain 0.03 ng OAE/g. This sample analyses by LC-MS/MS displayed okadaic acid (OA) and related congeners (DTX1) along with the pectinotoxins (PTX2 and PTX2sa). Highest observed levels of P. lima-induced DSP-toxicity in hepatopancreas was 0.41 g OA-equivalents/g corresponded to the highest observed planktonic cell counts of P. lima, 300 cell/L in August 2001. Cultures isolated from this sample were found to produce OA, DTX1 and their related diol esters. These data reveal a threat, represented by DSP-toxic species, at Black Sea coasts, and provide grounds for the introduction of phycotoxin control measures in the region.

Study on the pharmacokinetics of enrofloxacin in the Chinese mitten-handed crab,Eriocheir sinensis, after different administration regimes

A pharmacokinetic study on enrofloxacin (ENR) following different administration regimes (i.e. intramuscular, oral and bath administrations) was carried out for the Chinese mitten-handed crab (Eriocheir sinensis) at 17 °C water temperature. Muscle and hepatopancreas samples were collected at each sampling time of 1 and 10 min and 0.5, 1, 2, 8, 16, 24, 48, 72 and 168 h for intramuscular administration and 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72 and 168 h for oral and bath administration. The levels of ENR and ciprofloxacin (CIP) were simultaneously determined (n=5) by high-performance liquid chromatography. The results showed that similar absorption and elimination patterns of ENR level in muscle and hepatopancreas were observed for oral administration and bath treatment; however, the trend in ENR level changes in muscle and hepatopancreas after intramuscular administration was quite different from oral and bath treatments. Meanwhile, the level of ENR in hepatopancreas was much higher than that in muscle for the same administration regimes. The concentration–time data were analysed with the Practical Pharmacokinetics Program (3P97), and the kinetic profiles of ENR in the hepatopancreas and muscle conformed to two-compartment models. The absorption and elimination of ENR in hepatopancreas for bath treatment were faster than those for oral and intramuscular administrations. However, the pattern was quite different in muscle. The fastest absorption and elimination of ENR in muscle were seen for intramuscular administration.

Oxytetracycline (OTC) accumulation and elimination in hemolymph, muscle and hepatopancreas of white shrimp Litopenaeus vannamei following an OTC-feed therapeutic treatment

The white shrimp, Litopenaeus vannamei, has become a very important species for the development of shrimp aquaculture in Northwest Mexico. However, viral and bacterial diseases are considered a major threat to the development of this industry. In the present study a trial was conducted to evaluate the tissue distribution, maximum concentration, and elimination of the widely used antibiotic oxytetracycline (OTC) in L. vannamei using indoor tanks under laboratory-controlled conditions. OTC was given to shrimp simulating a therapeutic treatment through medicated feed for 14 days followed by a period of feeding without antibiotic for another 14 days to evaluate the elimination pattern. Samples of hemolymph, muscle, and hepatopancreas were taken from medicated animals every two days for 28 days. All tissues were removed and frozen immediately in liquid nitrogen. OTC levels were analyzed by High Performance Liquid Chromatography (HPLC). Results showed an important OTC increase during consumption of medicated feed in all examined tissues. OTC maximum concentrations were 33.54±11.19, 194.37±16.11, and 18.79±5.87 µg g−1 for muscle, hepatopancreas and hemolymph, respectively. Although the highest OTC level was found in the hepatopancreas, it required only two days after the start of dosing to reach this value, whereas the maximum OTC for muscle and hemolymph was detected after eight days of dosing. Ten days after the cessation of medicated feeding, the drug content in the shrimp tail muscle was under the detectable limit for the method (0.01 µg g−1 of OTC).

Quantitative Assessment of Effects of Zinc on the Histological Structure of the Hepatopancreas of Terrestrial Isopods

The objective of this study was to quantify the effects of zinc exposure on the histological structure of the hepatopancreas of Porcellio laevis. Woodlice were experimentally exposed to various concentrations (1000, 4000, and 8000 mg. kg(-1)) of zinc sulphate. Hepatopancreas samples of exposed isopods were histologically prepared and analysed with Leica QWin image analysis software. The B-cells in hepatopancreases of zinc sulphate - exposed woodlice were reduced in size to varying degrees, compared to that of the control, Percentage Cellular Area (PCA) of the hepatopancreas samples revealed that zoning occurred through the length of hepatopancreas lobes. Analysis of the PCA data of hepatopancreases of P. laevis exposed to the zinc sulphate revealed that there was a decline in PCA, in all the zones, compared to those in the control. The Z3 zone (the part from the middle to three quarters to the back of the tubule) was shown to be the best suited, if PCAs are to be considered as a biomarker in woodlouse toxicity studies.

The use of a metallothionein-like-proteins (MTLP) kinetic approach for metal bioavailability monitoring in dredged material

Ecotoxicological effects associated with contaminants present in dredged material from Spain were determined using a marine biotest based on the determination of metallothionein-like-protein concentrations (MTLPs) in the shore crab Carcinus maenas. Intermoult female C. maenas were exposed in the laboratory to sediments from three Spanish ports, Ría de Huelva (SW, Spain), La Coruña (NW, Spain) and Bahía de Cádiz (SW, Spain) per replicate during 21 days. Hepatopancreas samples from crabs were taken for metallothioneins analysis on days 0, 7 and 21. Furthermore, chemical analysis was performed in the stations to determine the degree and nature of sediment contamination (Cr, Ni, Cu, Zn, Cd, Pb, Hg, As, PAHs and PCBs). A significant increase ( p < 0.05) in metallothionein concentration was observed over time in individuals exposed to sediment from the port Ría de Huelva characterized by high concentrations of metals. A toxicokinetic approach is proposed in this study related to the use of this biomarker in C. maenas to evaluate bioavailability associated with metals present in dredged material. As a first step, this toxicokinetic approach might reveal as a sensitive tool for evaluating bioavailability of contaminants present in dredged material.

Alexandrium in the Black Sea — identity, ecology and PSP toxicity

Alexandrium cf. tamarense was recorded for the first time along the north-eastern coast of the Black Sea in July 2001. Since then, it has been observed annually between May and October. A maximum density of Alexandrium spp. of 9 000 cells l−1 was recorded during August 2004. Examination of the Black Sea Alexandrium spp. by scanning electron microscopy showed both A. tamarense and A. minutum to be present. Monitoring during 2000–2004 included the collection of mussel hepatopancreas samples and subsequent analysis for paralytic shellfish poisoning (PSP) toxins. A receptor-binding assay showed a maximum value of 14μg STXdiHCl equivalents 100g−1 tissue to correspond to ambient concentrations of A. tamarense of 300 cells l−1. Analysis of the PSP toxins by liquid chromatography with fluorescence detection showed the presence of GTX2, GTX3 and GTX5 in mussel hepatopancreas extracts. PSP in humans has not been reported in the Black Sea region of Russia. Nevertheless, the presence of Alexandrium spp and of PSP toxins in shellfish are compelling reasons for the introduction of phycotoxin control methods and monitoring in Russian waters.