Pathological and ultrastructural changes of Bellamya bengalensis under chronic carboxylic acid exposure at environmentally relevant levels: Inferences from general unified threshold model for survival (GUTS) predictions and hepatopancreatic integrity assessment.

This study aimed to understand the effects of freshwater acidification, driven by industrial runoff, agricultural activities, and atmospheric deposition, on the freshwater mollusk Bellamya bengalensis. By systematically investigating the impact of two common carboxylic acids, acetic acid (AA) and benzoic acid (BA), this research employed diverse toxicological, pathological, and ecological assessments. We explored survival predictions through the generic unified threshold model of survival (GUTS-SD), examined oxidative stress responses, and investigated hepatopancreatic alterations. In the experimental design, Bellamya bengalensis were subjected to environmentally relevant sublethal concentrations (10%, 20% LC50) of AA (39.77 and 79.54 mg/l) and BA (31.41 and 62.82 mg/l) over 28 days. Acute toxicity tests revealed increased LC50 values, indicating heightened toxicity with prolonged exposure, particularly due to the greater potency of benzoic acid compared to acetic acid. The GUTS-SD model provided accurate predictions of time-specific effects on populations, presenting long-term exposure (100 days) LC50 values for AA (263.7 mg/l) and BA (330.9 mg/l). Sequentially, the integrated biomarker response (IBR) analysis across study intervals highlighted the 28-day interval as the most sensitive, with GST emerging as the most responsive enzyme to oxidative stress induced by AA and BA. Histopathological and ultrastructural assessments of the hepatopancreas showed severe alterations, including necrosis, vacuolation and disrupted micro-villi, which were especially pronounced in higher BA exposure concentrations. These findings highlight the health and survival impacts of carboxylic acid toxicity on Bellamya bengalensis, emphasizing the need for proactive measures to mitigate acidification in aquatic ecosystems. The broader ecological implications underscore the importance of effective management and conservation strategies to address ongoing environmental challenges.

Postembryonic development and differentiation of the midgut in the freshwater shrimp Neocaridina davidi (Crustacea, Malacostraca, Decapoda) larvae.

Neocaridina davidi is a freshwater shrimp that originates from Taiwan and is commonly bred all over the word. Like all decapods, which develop indirectly, this species has pelagic larvae that may differ entirely in their morphology and habits from adult specimens. To fill a gap of knowledge about the developmental biology of freshwater shrimps we decided to document the 3D-localization of the midgut inside the body cavity of larval stages of N. davidi using X-ray microtomography, and to describe all structural and ultrastructural changes of the midgut epithelium (intestine and hepatopancreas) which occur during postembryonic development of N. davidi using light and transmission electron microscopy. We laid emphasis on stem cell functioning and cell death processes connected with differentiation. Our study revealed that while the intestine in both larval stages of N. davidi has the form of a fully developed organ, which resembles that of adult specimens, the hepatopancreas undergoes elongation and differentiation. E-cells, which are midgut stem cells, due to their proliferation and differentiation are responsible for the above-mentioned processes. Our study revealed that apoptosis is a common process in both larval stages of N. davidi in the intestine and proximal region of the hepatopancreas. In zoea III, autophagy as a survival factor is activated in order to protect cells against their death. However, when there are too many autophagic structures in epithelial cells, necrosis as passive cell death is activated. The presence of all types of cell death in the midgut in the zoea III stage confirms that this part of the digestive tract is fully developed and functional. Here, we present the first description of apoptosis, autophagy and necrosis in the digestive system of larval stages of Malacostraca and present the first description of their hepatopancreas elongation and differentiation due to midgut stem cell functioning.

Ultraviolet B radiation affects epithelial cell morphology and ultrastructure in the hepatopancreas of the freshwater decapod Macrobrachium olfersii.

The hepatopancreas is the digestive organ of crustaceans, and plays important roles also in the synthesis and secretion of sexual hormones, immunological defenses and xenobiotic detoxification. Although the importance of this organ in crustaceans cannot be underestimated, the effects of ultraviolet B (UVB) radiation on hepatopancreas are poorly understood. Moreover, Macrobrachium prawns, have a transparent carapace, which make them more susceptible to UVB radiation, since their internal organs, such as hepatopancreas, are easily reached by solar radiation. Therefore, we aimed to evaluate UVB radiation toxicity on the morphology and morphometry of hepatopancreatic epithelial cells, and to investigate these UVB effects in subcellular compartments of the ecologically-important freshwater decapod, Macrobrachium olfersii. Hepatopancreas from the UVB-irradiated group showed a granular cytoplasm, with non-defined cell limits. Morphometric analyses revealed that the UVB-irradiated group exhibited a higher frequency of fibrillar (F-cell), resorptive (R-cell) and midget (M-cell), and decreased the blister-like (B-cell). It was also observed increased vacuole frequencies and increased F-, B- and R-cell volumes in the UVB-irradiated group. In addition, it was observed increased B-cell vacuolar volumes and decreased R-cell vacuolar volumes. Ultrastructural alterations occurred in subcellular compartments in F- and R-cells, e.g. loss of mitochondrial crests, morphologically compatible with mitochondrial fission, rough endoplasmic reticulum cisternae dilation, dilation of Golgi lamellar sacs, and increased vacuole and concentric membrane formation in the UVB-irradiated group. Our data showed that the hepatopancreas is an important target of UVB radiation, as demonstrated by a series of organ-specific morphological and morphometric impairments. Therefore, cell damage caused by UVB radiation can compromise metabolic functions in epithelial cells from the hepatopancreas, potentially affecting absorption, secretion and digestion processes, vitellogenin synthesis, immune responses and xenobiotic detoxification.

The induced damage in the hepatopancreas of Orchestia species after exposure to a mixture of Cu/Zn-An ultrastructural study.

The hepatopancreas of crustaceans species has been recognized as an essential target organ to assess trace elements' effects. Due to its dynamic and capability of detoxifying trace metal, this organ often indicates distinct pathological disturbances. In the present work, we intend to evaluate the bioaccumulation of trace metal in three Orchestia species (Orchestia montagui, Orchestia gammarellus, and Orchestia mediterranea) living in symmetry in the banks of Bizerte lagoon (37°13'8″N 09°55'1″E) after their exposure during 14 days to a mixture of copper and zinc, and to highlight the effect of these metals on their hepatopancreas ultrastructure using transmission electron microscopy. At the end of the experiment, results showed that the mortality and the body mass varied according to the used nominal concentrations. Significant alterations were noted in all the treatment groups. The degree of these alterations depends on the used concentration, and they are represented especially by the cells remoteness and the border lyses, the reduction of the nuclear volume, the increase in the cytoplasm density with the presence of trace metal in the nucleus as well as in the vacuole, the disorganization and the destruction of microvilli, the condensation of the majority of cellular organelles and mitochondria swelling. Through this study, Orchestia genus could be an attractive candidate for the biochemical study of trace metal toxicity in Tunisian wetlands.

Morphostructural and immunohistochemical study for evaluation of nano-TiO2 toxicity in Armadillo officinalis Duméril, 1816 (Crustacea, Isopoda, Oniscidea).

Invertebrates are precious organisms in order to study environmental pollution. In particular, they appear to be suitable as a bioindicator species for pioneer ecotoxicity studies on new xenobiotics such as nanoparticles. In fact, they are able to absorb nanomaterials scattered in the environment in different ways and it's known the compartmentalization of nano-sized contaminants in selected tissues and intracellular organelles. Titanium dioxide represents the most used nanoparticulate, destined to become probably ubiquitous in the environment. Recently, some research has been published on the toxic potential of nano-TiO2 in several animal species. Among all invertebrates, Oniscidean Isopods are the only taxon of Crustaceans that has become completely terrestrial, known as excellent bioindicators and bioaccumulators. They have a digestive gland, the hepatopancreas, which is the location of election for the accumulation of pollutants. For this reason, they are considered efficient animal models to ecological studies. For this study, we collected Armadillo officinalis from Natural Oriented Reserve of "Vendicari" (Sicily, Italy), to evaluate the toxicity of titanium dioxide (TiO2 ) on their hepatopancreas, after a short period of exposure. We conducted morphostructural and immunohistochemistry assays. The results suggested a great capacity of the species of bioaccumulation of nanoparticles in the hepatopancreas, where a strong positivity to the metallothioneins was highlighted. Our study confirms that Oniscidean Isopods, in particular Armadillo officinalis, proved to be an appropriate indicator of pollution in terrestrial ecosystems from nanoparticles.

Effects and transcriptional responses in the hepatopancreas of red claw crayfish Cherax quadricarinatus under cold stress.

The red claw crayfish, Cherax quadricarinatus, is an economically important freshwater crustacean that cannot tolerate low temperature, which diminishes survival via unknown mechanisms. Herein, physiological regulation of C. quadricarinatus was investigated following exposure to low temperature stress at 9 ±â€¯2 °C for 4 weeks. Hepatopancreas tissue was tested for nonspecific enzyme activity, histological structure, and transcriptome sequencing analyses. The results showed that the activities of nonspecific enzymes were inhibited following low temperature stress. Ultrastructural observation revealed that the hepatopancreas structure was oxidatively damaged at low temperature, with numerous autophagic vesicles visible. Apoptosis in the hepatopancreas was significantly increased in the cold stress group, indicating diminished function. Transcriptome sequencing identified 2615 differentially expressed genes (DEGs) following low temperature stress, of which 1147 and 1468 were up- and down-regulated, respectively. Functional analysis of DEGs indicated involvement in substance metabolism, antioxidant defences, signal transduction, and immune responses. Therefore, chronic cold stress can suppress metabolism and cause oxidative damage and immune deficiency in crayfish. The findings provide fundamental molecular information for further study of the regulatory mechanisms of cold tolerance in red claw crayfish.

The effect of exuviae ingestion on lysosomal calcium accumulation and the presence of exosomes in the hepatopancreas of Porcellio scaber.

The hepatopancreas of isopods has major functions in food digestion and storage of carbohydrates and lipids. Also, it stores essential and accumulates xenobiotic metals in lysosomal granules within the two major cell types, the S- and B-cells of the tissue. A µCT study on moulting Porcellio scaber has shown mineral within the hepatopancreas lumen, when the animal has ingested their shed cuticle after moulting, suggesting recycling of mineral from the exuviae. This study aims to reveal if the lysosomal metal containing granules store calcium originating from the ingested exuviae. Therefore, we investigated the effect of cuticle ingestion on the elemental composition of the hepatopancreas granules of P. scaber, using electron probe X-ray microanalysis. For the preservation of diffusible elements, samples were high pressure frozen and freeze substituted in acetone and we used Propane-1,3-diol as a floatation medium for sections. We analyzed S- and B-cells of animals in the postmoult and intermoult stage that have ingested their exuviae and, as a negative control, cells from postmoult animals that have not ingested their exuviae. STEM and TEM were used for the investigation of the ultrastructure. Unexpectedly, the cryo-fixed samples contain numerous extracellular vesicles (exosomes) and many multivesicular bodies containing pro-exosomes. We show a significant increase of calcium, copper, zinc and sulphur within the metal granules upon exuviae ingestion, and, after 9 days, a reduction of calcium and zinc. The results indicate transitory storage of calcium from the exuviae within the metal granules and its subsequent utilization in cuticle mineralization.

Functional cytology of the hepatopancreas of decapod crustaceans.

This article reviews the morphogenesis, morphology, histology, ultrastructure, and structural-functional relationships of the hepatopancreas, the main metabolic organ of the Decapoda. The hepatopancreas develops in early larval stages from a pair of lateral lobes of the midgut anlage. In adults, it consists of hundreds of blindly ending tubules that are enveloped by a muscle net consisting of longitudinal and circular fibers. Stem cells at the distal ends of the tubules give rise to three ultrastructurally different epithelial cell types, the R-, F-, and B-cells. Histochemistry, immunohistochemistry, in situ hybridization, and monitoring of ultrastructural changes under different experimental conditions allowed the attribution of functions to these cell types. R-cells serve for the absorption and metabolization of nutrients, storage of energy reserves and minerals, synthesis of lipoproteins for export to other organs, detoxification of heavy metals, and excretion of uric acid. F-cells synthesize digestive enzymes and blood proteins involved in oxygen transport and immune defense. They also detoxify some heavy metals and probably organic xenobiotics. B-cells are assumed to produce and recycle fat emulsifiers. The hepatopancreas tubules lack nerves. The presence of scattered M-cells with putative endocrine function in the epithelium suggests that the hepatopancreas is mainly hormonally controlled. M-cells probably represent a self-perpetuating cell lineage independent from E-cells. The interstitium between the tubules contains connective tissue, arterioles, hemolymph with circulating hemocytes, and fixed phagocytes that eliminate pathogens. The hepatopancreas is histologically and ultrastructurally uniform throughout the Decapoda, despite their broad variety in body size, morphology, life style, and ecology. However, in a few cavernicolous and deep-sea shrimps parts of the hepatopancreas are transformed into large oil storing and bioluminescent compartments. Within the malacostracan crustaceans, the hepatopancreas of the Decapoda is most similar to the digestive gland of the Euphausiacea, supporting close taxonomic relationship of these two taxa.

Effects of microcystin-LR on the immune dysfunction and ultrastructure of hepatopancreas in giant freshwater prawn Macrobrachium rosenbergii.

Microcystins (MCs), produced by cyanobacteria, can strongly inhibit the activity of protein phosphatase, and exhibit strong hepatotoxicity. Macrobrachium rosenbergii is an important aquaculture economic species. Cyanobacterial blooms occur frequently during the culture of M. rosenbergii. However, the effects of MCs on the M. rosenbergii immune function have not been studied. In the present study, M. rosenbergii were exposed to environment-related concentrations of MC-LR type (0.5 and 5 µg/L) for 3 weeks. Hepatopancreatic histology was investigated, and antioxidant enzymes activity, acid phosphatase, alkaline phosphatase and lysozyme activity in hepatopancreas were also analyzed. Results showed that MC-LR could damage M. rosenbergii hepatopancreas, induce hepatopancreatic apoptosis and antioxidant dysfunctions. The expression profiles of major immune-related genes after MC-LR exposure were also detected. Some genes with antibacterial functions were suppressed, and the expression of apoptosis-related genes were up-regulated. After MC-LR exposure, the cumulative mortality of M. rosenbergii infected with Vibrio vulnificus and Aeromonas hydrophila were much higher than the control in a time-dose dependent manner. These results indicated the potential negative influence of MC-LR on the immune function of M. rosenbergii.

Histopathological and ultrastructural indices for the assessment of glyphosate-based herbicide cytotoxicity in decapod crustacean hepatopancreas.

Glyphosate-based herbicides (GBH), including Roundup, are the most widely used pesticides in the world. Glyphosate residues have been detected in surface and groundwater, in food, and in human blood and urine. The effects of this herbicide on different levels of biological organization are an important concern that needs to be investigated. In general, the toxicity of GBH in invertebrates is poorly understood, and it is the motivation of this study. Thus, the aim of this study was to evaluate cellular responses of the hepatopancreas, an organ involved in the detoxification process in invertebrates, after exposure to environmentally relevant concentrations of GBH, using prawn Macrobrachium potiuna as a model. Prawns were exposed to three concentrations of GBH (0.0065, 0.065 and 0.28 mg L-1) for 7 or 14 days. Alterations in the morphology of the hepatopancreas and in subcellular components of R cells, which are responsible for the detoxification process, were analyzed, and an index for subcellular alterations was standardized. GBH exposure induced tissue commitments on the hepatopancreas, as well as important impairments of R cells that could compromise the normal functioning of the cells, especially in the detoxification processes. The major cellular impairments were intense vacuolization, dilatation of the cisterns of the rough endoplasmic reticulum and Golgi bodies, increase of perinuclear space, necrosis, concentric membrane formation and mitochondria crest loss. Our data contribute to the knowledge of the cytotoxic effects of low GBH concentrations on aquatic invertebrates, specifically their effects on the hepatopancreas, an important organ for the metabolism of crustaceans. These results also indicate that concentrations considered safe by regulatory agencies should be reviewed to minimize the effects on non-target organisms. This study also contributes to the standardization of an ultrastructure index for the assessment of GBH in palaemonids, which could be used for the assessment of contaminants in crustaceans and other species with hepatopancreas.

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

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

Effect of cadmium exposure on hepatopancreas and gills of the estuary mud crab (Scylla paramamosain): Histopathological changes and expression characterization of stress response genes.

Cadmium (Cd) is a heavy metal that accumulates easily in organisms and causes several detrimental effects, including tissue damage. Cd contamination from anthropogenic terrestrial sources flows into rivers, and through estuaries to the ocean. To evaluate the toxic effects of Cd on estuary crustaceans, we exposed the mud crab Scylla paramamosain to various Cd concentrations (0, 10.0, 20.0, and 40.0mg/L) for 24h. We also exposed mud crabs to a fixed Cd concentration (20.0mg/L) for various periods of time (0, 6, 12, 24, 48, and 72h). We observed that after exposure to Cd, the surfaces of the gill lamellae were wrinkled, and the morphologies of the nuclei and mitochondria in the hepatopancreas were altered. We analyzed the expression profiles of 36 stress-related genes after Cd exposure, including those encoding metallothioneins, heat shock proteins, apoptosis-related proteins, and antioxidant proteins, with quantitative reverse transcription PCR. We found that exposure to Cd altered gene expression, and that some genes might be suitable bioindicators of Cd stress. Gene expression profiles were organ-, duration-, and concentration-dependent, suggesting that stress-response genes might be involved in an innate defense system for handling heavy metal exposure. To the best of our knowledge, this study is the first one of histopathology and stress-response gene expression pattern of Scylla paramamosain after Cd exposure. Our work could increase our understanding of the effect of environmental toxins on estuary crustaceans.

The oxidative stress and antioxidant responses of Litopenaeus vannamei to low temperature and air exposure.

Low temperature and air exposure were the key attributes for waterless transportation of fish and shrimp. In order to investigate the oxidative stress and antioxidant responses of the live shrimp Litopenaeus vannamei in the mimic waterless transportation, live shrimp were cooled at 13 °C for 3 min, stored in oxygen at 15 °C for 12 h, and then revived in water at 25 °C. The survival rate of shrimp under this waterless transportation system was over 86.67%. The ultrastructure of hepatopancreas cells were observed while activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutathione peroxidase (GSH-Px), antisuperoxide anion free radicals (ASAFR), total antioxidant capacity (TAOC), reactive oxygen species (ROS) production, content of malondialdehyde (MDA) and relative mRNA expressions of CAT and GSH-Px in the hemolymph and hepatopancreas were determined. Slight distortions of some organelles in hepatopancreas cells was reversible upon the shrimp revived from the cold shock. The activities of SOD, POD, CAT, GSH-Px, TAOC, ROS production and relative mRNA expressions of CAT and GSH-Px increased following the cold shock and reached peak levels after 3 or 6 h of storage, and then decreased gradually. There was no significant difference between the fresh and the revived shrimp in SOD, POD, GSH-Px, TAOC, ROS, MDA and relative mRNA expressions of CAT and GSH-Px. The oxidative stress and antioxidant responses were tissue-specific because hepatopancreas seemed to have a greater ability to defend against organelle damage and was more sensitive to stress than hemolymph based on the results of SOD activity, MDA content and GSH-Px mRNA expression. These results revealed that low temperature and air exposure caused significant oxidative and antioxidant responses, but did not lead to irreversible damages in this waterless system.

'Venus trapped, Mars transits': Cu and Fe redox chemistry, cellular topography and in situ ligand binding in terrestrial isopod hepatopancreas.

Woodlice efficiently sequester copper (Cu) in 'cuprosomes' within hepatopancreatic 'S' cells. Binuclear 'B' cells in the hepatopancreas form iron (Fe) deposits; these cells apparently undergo an apocrine secretory diurnal cycle linked to nocturnal feeding. Synchrotron-based µ-focus X-ray spectroscopy undertaken on thin sections was used to characterize the ligands binding Cu and Fe in S and B cells of Oniscus asellus (Isopoda). Main findings were: (i) morphometry confirmed a diurnal B-cell apocrine cycle; (ii) X-ray fluorescence (XRF) mapping indicated that Cu was co-distributed with sulfur (mainly in S cells), and Fe was co-distributed with phosphate (mainly in B cells); (iii) XRF mapping revealed an intimate morphological relationship between the basal regions of adjacent S and B cells; (iv) molecular modelling and Fourier transform analyses indicated that Cu in the reduced Cu(+) state is mainly coordinated to thiol-rich ligands (Cu-S bond length 2.3 Å) in both cell types, while Fe in the oxidized Fe(3+) state is predominantly oxygen coordinated (estimated Fe-O bond length of approx. 2 Å), with an outer shell of Fe scatterers at approximately 3.05 Å; and (v) no significant differences occur in Cu or Fe speciation at key nodes in the apocrine cycle. Findings imply that S and B cells form integrated unit-pairs; a functional role for secretions from these cellular units in the digestion of recalcitrant dietary components is hypothesized.

Cell Death in the Epithelia of the Intestine and Hepatopancreas in Neocaridina heteropoda (Crustacea, Malacostraca).

The endodermal region of the digestive system in the freshwater shrimp Neocaridina heteropoda (Crustacea, Malacostraca) consists of a tube-shaped intestine and large hepatopancreas, which is formed by numerous blind-ended tubules. The precise structure and ultrastructure of these regions were presented in our previous studies, while here we focused on the cell death processes and their effect on the functioning of the midgut. We used transmission electron microscopy, light and confocal microscopes to describe and detect cell death, while a quantitative assessment of cells with depolarized mitochondria helped us to establish whether there is the relationship between cell death and the inactivation of mitochondria. Three types of the cell death were observed in the intestine and hepatopancreas-apoptosis, necrosis and autophagy. No differences were observed in the course of these processes in males and females and or in the intestine and hepatopancreas of the shrimp that were examined. Our studies revealed that apoptosis, necrosis and autophagy only involves the fully developed cells of the midgut epithelium that have contact with the midgut lumen-D-cells in the intestine and B- and F-cells in hepatopancreas, while E-cells (midgut stem cells) did not die. A distinct correlation between the accumulation of E-cells and the activation of apoptosis was detected in the anterior region of the intestine, while necrosis was an accidental process. Degenerating organelles, mainly mitochondria were neutralized and eventually, the activation of cell death was prevented in the entire epithelium due to autophagy. Therefore, we state that autophagy plays a role of the survival factor.

Effect of desiccation and resubmersion on the oxidative stress response of the kuruma shrimp Marsupenaeus japonicus.

In the present study, the oxidative stress response in hepatopancreas of Marsupenaeus japonicus to desiccation stress and resubmersed in seawater were studied, such as respiratory burst, ROS production ( [Formula: see text] ), activities of antioxidant enzymes (CAT, GPx, SOD, POD and GST) and oxidative damage to lipid and protein (indexed by contents of MDA). The duration of desiccation significantly influenced shrimp survival, and the mortality rates were 37.5% and 87.5% after desiccation 5 h and 10 h, respectively. After desiccation stress 3 h, the respiratory burst, ROS production, and the activity of SOD and CAT were up-regulated significantly. The activity of GPx and POD, and the content of MDA decreased significantly at 0.5 h and 1 h, and then increased significantly at 3 h. But GST activity was no significant change after desiccation. During the resubmersion period, most of the antioxidant enzymes activities could recover to the control level at 24 h, but a small quantity of the oxidative stress still existed in tissues. HE staining showed that desiccation stress induced damage symptoms in hepatopancreas of M. japonicus. These results revealed that desiccation influenced the antioxidative status and caused oxidative stress and tissue damage via confusion of antioxidant enzymes in M. japonicus, but the oxidative stress could be eliminated within a certain range after the shrimps were resubmersed in seawater.

Hepatoprotective effects of a Chinese herbal formulation, Yingchen decoction, on olaquindox-induced hepatopancreas injury in Jian carp (Cyprinus carpio var. Jian).

In order to identify effective hepatoprotective herbs for clinical application in fish farming, 200 mg/kg olaquindox (OLA) was added to a basal diet (group 1, control) to form OLA diet (group 2), then 1.35, 2.7 and 5.4 % (w/w) of a Chinese herbal formulation, Yingchen decoction (YCD), were added to the OLA diet to form three additional diets for groups 3, 4 and 5, respectively. A total of 375 juvenile Jian carp (Cyprinus carpio var. Jian) (52.12 ± 2.95 g/tail) were divided into five groups (triplicates per group) and fed the five diets mentioned above, respectively, for 6 weeks. At the termination of feeding experiment, serum biochemical indexes, viability of hepatocytes and the hepatopancreas microstructure for each group were detected and observed. The results showed that serum ALT and AST in group 2 were significantly higher than the control (P < 0.05). Plasma membranes hepatocyte nuclei in group 2 were found to be mostly indistinct, compared to group 1, and gradually recovered with the increasing supplementation of YCD in group 3, 4 and 5. The viability of isolated hepatocytes in group 2 was the lowest and gradually recovered with the increasing supplementation of YCD in group 3, 4 and 5. The results suggest that YCD protected the Jian carp hepatopancreas against injury from OLA, and that 5.4 % YCD would be the optimum dosage in a Jian carp diet.

Bioaccumulation of cadmium and lead and its effects on hepatopancreas morphology in three terrestrial isopod crustacean species.

This study was designed to compare cadmium (Cd) and lead (Pb) bioaccumulation in three species of oniscidean isopods - Armadillidium granulatum Brandt, Armadillidium vulgare (Latreille) and Porcellio laevis Latreille which were exposed for three weeks to a contaminated diet, and to determine the morphological and ultrastructural changes in hepatopancreas. Metal accumulation, determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), was linearly associated with the exposed concentration and was a function of the metal and the species tested. All three species accumulated lower levels of Pb than Cd. A. vulgare accumulated the largest concentration of Pb, especially at the higher doses, whereas P. laevis showed the greatest Cd accumulation, and the highest Cd concentration was lethal for all exposed species. The highest concentrations of Pb and Cd induced significant changes both in the general morphology of tubules and in the ultrastructural organization of epithelial cells in hepatopancreas. Some Pb/Cd induced alterations include: brush border disorganization; reduction of the basal labyrinth formed by the plasma membrane; condensation of some cytoplasm areas and of chromatin; rough endoplasmic reticulum and mitochondrial alterations; increase of secondary lysosomes and of type B granules in S cells. Some of the ultrastructural changes observed overlap with those induced by prolonged starvation, whereas others can be useful biomarkers of heavy metal toxicity. This study has confirmed that in terrestrial isopods, the accumulation of the different metals occurs in a species-specific manner; therefore ecological monitoring and assessment studies should consider each species individually. The research has confirmed that in the terrestrial isopods the accumulation of the different metals occurs in a species-specific way; therefore each species should first be evaluated in view of its employ in biomonitoring programs.

White feces syndrome of shrimp arises from transformation, sloughing and aggregation of hepatopancreatic microvilli into vermiform bodies superficially resembling gregarines.

Accompanying acute hepatopancreatic necrosis disease (AHPND) in cultivated Asian shrimp has been an increasing prevalence of vermiform, gregarine-like bodies within the shrimp hepatopancreas (HP) and midgut. In high quantity they result in white fecal strings and a phenomenon called white feces syndrome (WFS). Light microscopy (LM) of squash mounts and stained smears from fresh HP tissue revealed that the vermiform bodies are almost transparent with widths and diameters proportional to the HP tubule lumens in which they occur. Despite vermiform appearance, they show no cellular structure. At high magnification (LM with 40-100x objectives), they appear to consist of a thin, outer membrane enclosing a complex of thicker, inter-folded membranes. Transmission electron microscopy (TEM) revealed that the outer non-laminar membrane of the vermiform bodies bore no resemblance to a plasma membrane or to the outer layer of any known gregarine, other protozoan or metazoan. Sub-cellular organelles such as mitochondria, nuclei, endoplasmic reticulum and ribosomes were absent. The internal membranes had a tubular sub-structure and occasionally enclosed whole B-cells, sloughed from the HP tubule epithelium. These internal membranes were shown to arise from transformed microvilli that peeled away from HP tubule epithelial cells and then aggregated in the tubule lumen. Stripped of microvilli, the originating cells underwent lysis. By contrast, B-cells remained intact or were sloughed independently and whole from the tubule epithelium. When sometimes engulfed by the aggregated, transformed microvilli (ATM) they could be misinterpreted as cyst-like structures by light microscopy, contributing to gregarine-like appearance. The cause of ATM is currently unknown, but formation by loss of microvilli and subsequent cell lysis indicate that their formation is a pathological process. If sufficiently severe, they may retard shrimp growth and may predispose shrimp to opportunistic pathogens. Thus, the cause of ATM and their relationship (if any) to AHPND should be determined.

Effect of cardiac glycosides from Nerium indicum on feeding rate, digestive enzymes activity and ultrastructural alterations of hepatopancreas in Pomacea canaliculata.

Cardiac glycosides from Nerium indicum showed potent molluscicide activity against Pomacea canaliculata (GAS), but the toxicological mechanism is still far less understood. Effects of sublethal treatments of cardiac glycosides on feeding rate, digestive enzymes and ultrastructural alterations of the hepatopancreas in GAS were evaluated in this study. Exposure of GAS to sublethal concentrations of cardiac glycosides resulted in a significant reduction of feeding rate of GAS. The amylase, cellulose and protease activity were increase significantly at the end of 24 h followed by significant inhibition after 48 h of exposure while lipase activity was not affected significantly at the end of 24 h followed by a significant inhibition after 48 h of exposure during experimental period. The main ultrastructural alterations of hepatopancreas observed in snails under cardiac glycosides treatment comprised disruption of nuclear membrane, increased vesiculation and dilatation of endoplasmic reticulum, and vacuolization and swelling of mitochondrial compared to the untreated GAS. These results, for the first time, provide systematic evidences showing that cardiac glycosides seriously impairs the hepatopancreas tissues of GAS, resulting in inhibition of digestive enzymes activity and feeding rate and cause GAS death in the end.