Two hexokinases of the shrimp Penaeus (Litopenaeus) vannamei are differentially expressed during oxygen limited conditions.

The white shrimp Penaeus (Litopenaeus) vannamei is the most cultivated shrimp worldwide. Compared to other shrimp species, it has higher resistance to adverse conditions. During hypoxia, the shrimp reduces oxygen consumption and adjusts energy metabolism via anaerobic glycolysis, among other strategies. Hexokinase (HK) is the first enzyme of glycolysis and a key regulation point. In mammals and other vertebrates, there are several tissue-specific HK isoforms with differences in expression and enzyme activity. In contrast, crustacean HKs have been relatively little studied. We studied the P. vannamei HK isoforms during hypoxia and reoxygenation. We cloned two HK1 sequences named HK1-long (1455 bp) and HK1-short (1302 bp), and one HK2 (1344 bp). In normoxia, total HK1 expression is higher in hepatopancreas, while HK2 is higher in gills. Severe hypoxia (1 mg/L of DO) after 12 h exposure and 1 h of reoxygenation increased HK1 expression in both organs, but HK2 expression changed differentially. In hepatopancreas, HK2 expression increased in 6 and 12 h of hypoxia but diminished to normoxia levels after reoxygenation. In gills, HK2 expression decreased after 12 h of hypoxia. HK activity increased in hepatopancreas after 12 h hypoxia, opposite to gills. These results indicate that shrimp HK isoforms respond to hypoxia and reoxygenation in a tissue-specific manner. Intracellular glucose levels did not change in any case, showing the shrimp ability to maintain glucose homeostasis during hypoxia.

Integration of transcriptome, gut microbiota, and physiology reveals toxic responses of the red claw crayfish (Cherax quadricarinatus) to imidacloprid.

Imidacloprid enters the water environment through rainfall and causes harm to aquatic crustaceans. However, the potential chronic toxicity mechanism of imidacloprid in crayfish has not been comprehensively studied. In this study, red claw crayfish (Cherax quadricarinatus) were exposed to 11.76, 35.27, or 88.17 µg/L imidacloprid for 30 days, and changes in the physiology and biochemistry, gut microbiota, and transcriptome of C. quadricarinatus and the interaction between imidacloprid, gut microbiota, and genes were studied. Imidacloprid induced oxidative stress and decreased growth performance in crayfish. Imidacloprid exposure caused hepatopancreas damage and decreased serum immune enzyme activity. Hepatopancreatic and plasma acetylcholine decreased significantly in the 88.17 µg/L group. Imidacloprid reduced the diversity of the intestinal flora, increased the abundance of harmful flora, and disrupted the microbiota function. Transcriptomic analysis showed that the number of up-and-down-regulated differentially expressed genes (DEGs) increased significantly with increasing concentrations of imidacloprid. DEG enrichment analyses indicated that imidacloprid inhibits neurotransmitter transduction and immune responses and disrupts energy metabolic processes. Crayfish could alleviate imidacloprid stress by regulating antioxidant and detoxification-related genes. A high correlation was revealed between GST, HSPA1s, and HSP90 and the composition of gut microorganisms in crayfish under imidacloprid stress. This study highlights the negative effects and provides detailed sequencing data from transcriptome and gut microbiota to enhance our understanding of the molecular toxicity of imidacloprid in crustaceans.

Dietary exposure to 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) induces oxidative damage promoting cell apoptosis primarily via mitochondrial pathway in the hepatopancreas of carp, Cyprinus carpio.

To investigate the mechanisms of BDE-47 on hepatotoxicity in fish, this study examined the effects of dietary exposure to BDE-47 (40 and 4000 ng/g) on carp for 42 days. The results showed that BDE-47 significantly increased carp's condition factor and hepatosomatic index. Pathological results revealed unclear hepatic cord structure, hepatocytes swelling, cellular vacuolization, and inflammatory cell infiltration in the hepatopancreas of carp. Further investigation showed that ROS levels significantly increased on days 7, 14, and 42. Moreover, the activities of antioxidant enzymes SOD, GSH, CAT, and GST increased significantly from 1 to 7 days, and the transcription levels of antioxidant enzymes CAT, Cu-Zn SOD, Mn-SOD, GST, and GPX, and antioxidant pathway genes Keap1, Nrf2, and HO-1 changed significantly at multiple time-points during the 42 days. The results of apoptosis pathway genes showed that the mitochondrial pathway genes Bax, Casp3, and Casp9 were significantly upregulated and Bcl2 was significantly downregulated, while the transcription levels of FADD and PERK were significantly enhanced. These results indicate that BDE-47 induced oxidative damage in hepatopancreas, then it promoted cell apoptosis mainly through the mitochondrial pathway. This study provides a foundation for analyzing the mechanism of hepatotoxicity induced by BDE-47 on fish.

Toxicity of three microcystin variants on the histology, physiological and metabolism of hepatopancreas and intestinal microbiota of Litopenaeus vannamei.

Microcystins (MCs) are prevalent harmful contaminants within shrimp aquaculture systems, exhibiting a diverse array of variants. Gut microbiota can engage in mutual interactions with the host through the gut-liver axis. In this study, the shrimp Litopenaeus vannamei were subjected to three different variants of MCs (LR, YR, RR) at a concentration of 1 µg/L each, and elucidated the alterations in both intestinal microbiota and hepatopancreas physiological homeostasis. The results showed that all three variants of MCs prompted histological alterations in the hepatopancreas, induced elevated levels of oxidative stress biomarkers (H2O2, T-SOD, and CAT), disturbed the transcription levels of immune-related genes (Crus, ALF, and Lys), along with an increase in apoptotic genes (Casp-3 and P53). Furthermore, the metabolic profiles of the hepatopancreas were perturbed, particularly in amino acid metabolism such as "lysine degradation" and "ß-alanine metabolism"; the mTOR and FoxO signaling were also influenced, encompassing alterations in the transcription levels of related genes. Additionally, the alterations were observed in the intestinal microbiota's diversity and composition, particularly potential beneficial bacteria (Alloprevotella, Bacteroides, Collinsella, Faecalibacterium, and Prevotellaceae UCG-001), which exhibited a positive correlation with the metabolite berberine. These findings reveal that the three MCs variants can impact the health of the shrimp by interfering with the homeostasis of intestinal microbial and hepatopancreas physiology.

Evaluation of Collagenase Activity from Crab Hepatopancreas in Different Model Systems.

The effect of Kamchatka crab hepatopancreas containing three collagenolytic isoenzymes Collagenase KK and proteinases of Streptomyces lavendulae on metabolic activity and cell death were carried out on in vitro models. It was shown that changes in the protein structure under the influence of Collagenase KK occurred earlier than under the effect of bacterial proteinases. At the same time, activity of Collagenase KK was significantly higher than that of bacterial proteinases (p<0.01). Both preparations had a pronounced time- and dose-dependent effects on metabolic activity of cells. Collagenase KK had low cytotoxic effect, and cells mainly died by apoptosis. Thus, hepatopancreas collagenase has a high activity and proapoptotic effect on cells and can be used in low concentrations for enzymatic disaggregation of tissues.

Comprehensive Multi-omics Approaches Provide Insights to Summer Mortality in the Clam Meretrix petechialis.

Bivalve mass mortalities have been reported worldwide, which not only can be explained as a result of pathogen infection, but may reflect changes in environments. Although these episodes were often reported, there was limited information concerning the molecular responses to various stressors leading to summer mortality. In the present work, RNA sequencing (RNA-seq), tandem mass tagging (TMT)-based quantitative proteomics, and 16S rRNA sequencing were used to explore the natural outbreak of summer mortality in the clam Meretrix petechialis. We identified a total of 172 differentially expressed genes (DEGs) and 222 differentially expressed proteins (DEPs) in the diseased group compared to the normal group. The inconsistent expression profiles of immune DEGs/DEPs may be due to the immune dysregulation of the diseased clams. Notably, 11 solute carrier family genes were found among the top 20 down-regulated genes in the diseased group, indicating that weakened transmembrane transport ability might occur in the diseased clams. Integration analysis of transcriptomic and proteomic results showed that many metabolic processes such as "arginine and proline metabolism" and "tyrosine metabolism" were inhibited in the diseased group, suggesting metabolic inhibition. Moreover, 16S rRNA sequencing revealed that the microbial composition of clam hepatopancreas was disordered in the diseased group. The comparison of DEGs expression between the natural summer mortality event and an artificial challenge experiment involving both Vibrio infection and heat stress revealed 9/15 genes showing similar expression trends between the two conditions, suggesting that the summer mortality might be caused by a combination of high temperature and Vibrio infection. These results would deepen our understanding of summer mortality and provide candidate resistance markers for clam resistance breeding.

Cellular and transcriptomic response to pathogenic and non-pathogenic Vibrio parahaemolyticus strains causing acute hepatopancreatic necrosis disease (AHPND) in Litopenaeus vannamei.

The shrimp industry has historically been affected by viral and bacterial diseases. One of the most recent emerging diseases is Acute Hepatopancreatic Necrosis Disease (AHPND), which causes severe mortality. Despite its significance to sanitation and economics, little is known about the molecular response of shrimp to this disease. Here, we present the cellular and transcriptomic responses of Litopenaeus vannamei exposed to two Vibrio parahaemolyticus strains for 98 h, wherein one is non-pathogenic (VpN) and the other causes AHPND (VpP). Exposure to the VpN strain resulted in minor alterations in hepatopancreas morphology, including reductions in the size of R and B cells and detachments of small epithelial cells from 72 h onwards. On the other hand, exposure to the VpP strain is characterized by acute detachment of epithelial cells from the hepatopancreatic tubules and infiltration of hemocytes in the inter-tubular spaces. At the end of exposure, RNA-Seq analysis revealed functional enrichment in biological processes, such as the toll3 receptor signaling pathway, apoptotic processes, and production of molecular mediators involved in the inflammatory response of shrimp exposed to VpN treatment. The biological processes identified in the VpP treatment include superoxide anion metabolism, innate immune response, antimicrobial humoral response, and toll3 receptor signaling pathway. Furthermore, KEGG enrichment analysis revealed metabolic pathways associated with survival, cell adhesion, and reactive oxygen species, among others, for shrimp exposed to VpP. Our study proves the differential immune responses to two strains of V. parahaemolyticus, one pathogenic and the other nonpathogenic, enlarges our knowledge on the evolution of AHPND in L. vannamei, and uncovers unique perspectives on establishing genomic resources that may function as a groundwork for detecting probable molecular markers linked to the immune system in shrimp.

Acute Ammonia Causes Pathogenic Dysbiosis of Shrimp Gut Biofilms.

Acute ammonia exposure has detrimental effects on shrimp, but the underlying mechanisms remain to be fully explored. In the present study, we investigated the impact of acute ammonia exposure on the gut microbiota of the white shrimp Litopenaeus vannamei and its association with shrimp mortality. Exposure to a lethal concentration of ammonia for 48 h resulted in increased mortality in L. vannamei, with severe damage to the hepatopancreas. Ammonia exposure led to a significant decrease in gut microbial diversity, along with the loss of beneficial bacterial taxa and the proliferation of pathogenic Vibrio strains. A phenotypic analysis revealed a transition from the dominance of aerobic to facultative anaerobic strains due to ammonia exposure. A functional analysis revealed that ammonia exposure led to an enrichment of genes related to biofilm formation, host colonization, and virulence pathogenicity. A species-level analysis and experiments suggest the key role of a Vibrio harveyi strain in causing shrimp disease and specificity under distinct environments. These findings provide new information on the mechanism of shrimp disease under environmental changes.

Histopathological effects of long-term exposure to realistic concentrations of cadmium in the hepatopancreas of Sparus aurata juveniles.

In recent decades, cadmium has emerged as an environmental stressor in aquatic ecosystems due to its persistence and toxicity. It can enter water bodies from various natural and anthropogenic sources and, once introduced into aquatic systems, can accumulate in sediments and biota, leading to bioaccumulation and biomagnification in the food chain. For this reason, the effects of cadmium on aquatic life remain an area of ongoing research and concern. In this paper, a multidisciplinary approach was used to assess the effects of long-term exposure to an environmental concentration on the hepatopancreas of farmed juveniles of sea bream, Sparus aurata. After determining metal uptake, metallothionein production was assessed to gain insight into the organism's defence response. The effects were also assessed by histological and ultrastructural analyses. The results indicate that cadmium accumulates in the hepatopancreas at significant concentrations, inducing structural and functional damage. Despite the parallel increase in metallothioneins, fibrosis, alterations in carbohydrate distribution and endocrine disruption were also observed. These effects would decrease animal fitness although it did not translate into high mortality or reduced growth. This could depend on the fact that the animals were farmed, protected from the pressure deriving from having to search for food or escape from predators. Not to be underestimated is the return to humans, as this species is edible. Understanding the behaviour of cadmium in aquatic systems, its effects at different trophic levels and the potential risks to human health from the consumption of contaminated seafood would therefore be essential for informed environmental management and policy decisions.

Exposure to virgin and marine incubated microparticles of biodegradable and conventional polymers modulates the hepatopancreas transcriptome of Mytilus galloprovincialis.

Biodegradable polymers have been proposed as an alternative to conventional plastics to mitigate the impact of marine litter, but the research investigating their toxicity is still in its infancy. This study evaluates the potential ecotoxicological effects of both virgin and marine-incubated microparticles (MPs), at environmentally relevant concentration (0.1 mg/l), made of different biodegradable polymers (Polycaprolactone, Mater-Bi, cellulose) and conventional polymers (Polyethylene) on Mytilus galloprovincialis by using transcriptomics. This approach is increasingly being used to assess the effects of pollutants on organisms, obtaining data on numerous biological pathways simultaneously. Whole hepatopancreas de novo transcriptome sequencing was performed, individuating 972 genes differentially expressed across experimental groups compared to the control. Through the comparative transcriptomic profiling emerges that the preponderant effect is attributable to the marine incubation of MPs, especially for incubated polycaprolactone (731 DEGs). Mater-Bi and cellulose alter the smallest number of genes and biological processes in the mussel hepatopancreas. All microparticles, regardless of their polymeric composition, dysregulated innate immunity, and fatty acid metabolism biological processes. These findings highlight the necessity of considering the interactions of MPs with the environmental factors in the marine ecosystem when performing ecotoxicological evaluations. The results obtained contribute to fill current knowledge gaps regarding the potential environmental impacts of biodegradable polymers.

MicroRNA sequencing analysis reveals immune responses in hepatopancreas of Fenneropenaeus penicillatus under white spot syndrome virus infection.

White Spot Disease is one of the most harmful diseases of the red tail shrimp, which can cause devastating economic losses due to the highest mortality up to 100% within a few days. MicroRNAs (miRNAs) are large class of small noncoding RNAs with the ability to post-transcriptionally repress the translation of target mRNAs. MiRNAs are considered to have a significant role in the innate immune response of crustaceans, particularly in relation to antiviral defense mechanisms. Numerous crustacean miRNAs have been verified to be required in host immune defense against viral infection, however, till present, the miRNAs functions of F. penicillatus defense WSSV infection have not been studied yet. Here in this study, for the first time, miRNAs involved in the F. penicillatus immune defense against WSSV infection were identified using high-throughput sequencing platform. A total of 432 miRNAs were obtained including 402 conserved miRNAs and 30 novel predicted miRNAs. Comparative analysis between the WSSV-challenged group and the control group revealed differential expression of 159 microRNAs in response to WSSV infection. Among these, 48 were up-regulated and 111 were down-regulated. Ten candidate MicroRNAs associated with immune activities were randomly selected for qRT-PCR analysis, which confirming the expression profiling observed in the MicroRNA sequencing data. As a result, most differentially expressed miRNAs were down-regulated lead to increase the expression of various target genes that mediated immune reaction defense WSSV infection, including genes related to signal transduction, Complement and coagulation cascade, Phagocytosis, and Apoptosis. Furthermore, the genes expression of the key members in Toll and Imd signaling pathways and apoptotic signaling were mediated by microRNAs to activate host immune responses including apoptosis against WSSV infection. These results will help to understand molecular defense mechanism against WSSV infection in F. penicillatus and to develop an effective WSSV defensive strategy in shrimp farming.

Differential responses of hepatopancreas transcriptome between fast and slow growth in giant freshwater prawns (Macrobrachium rosenbergii) fed a plant-based diet.

Efficient utilisation of plant-based diets in the giant freshwater prawn, Marcrobrachium rosenbergii, varies according to individual, suggesting that it might be associated with differences in physiological and metabolic responses. Therefore, we aimed to investigate the individual differences in the growth response of shrimp fed to a soybean-based diet (SBM). Two hundred shrimp were fed SBM for 90 days, and specific growth rate (SGR) was determined individually. Fast- and slow-growing shrimp (F-shrimp vs. S-shrimp), with the highest and lowest 5% SGRs, respectively, were sampled to determine haemolymph chemistry and carcass composition. The hepatopancreas of these shrimps were used for transcriptome analysis through RNA sequencing (RNA-Seq). The results showed no significant differences in haemolymph chemistry parameters. In terms of carcass proximate composition, F-shrimp exhibited higher protein composition than did S-shrimp, suggesting that F-shrimp have higher protein anabolism. Using RNA-seq and real-time reverse transcription polymerase chain reaction (qRT-PCR), the expression levels of several genes encoding physiologic and metabolic enzymes were found to be upregulated in F-shrimp compared to in S-shrimp, suggesting that these enzymes/proteins mediated the efficient use of SBM-based diets for growth promotion in shrimp. Various DEGs associated with the immune system were observed, indicating a difference in immune processes between F- and S-shrimp. The expression of several housekeeping genes was found to be upregulated in S-shrimp. Collectively, the upregulated expression of several enzymes associated with physiological and/or metabolic processes and increased protein anabolism may be attributed to the efficient use of SBM for maximal growth in shrimp.

Assessing the ecotoxicity of florfenicol exposure at environmental levels: A case study of histology, apoptosis and microbiota in hepatopancreas of Eriocheir sinensis.

The intensification of production practices in the aquaculture industry has led to the indiscriminate use of antibiotics to combat diseases and reduce costs, which has resulted in environmental pollution, posing serious threats to aquaculture sustainability and food safety. However, the toxic effect of florfenicol (FF) exposure on the hepatopancreas of crustaceans remains unclear. Herein, by employing Chinese mitten crab (Eriocheir sinensis) as subjects to investigate the toxic effects on histopathology, oxidative stress, apoptosis and microbiota of hepatopancreas under environment-relevant (0.5 and 5 µg/L), and extreme concentrations (50 µg/L) of FF. Our results revealed that the damage of hepatopancreas tissue structure caused by FF exposure in a dose-and time-dependent manner. Combined with the increased expression of apoptosis-related genes (Caspase 3, Caspase 8, p53, Bax and Bcl-2) at mRNA and protein levels, activation of catalase (CAT) and superoxide dismutase (SOD), and malondialdehyde (MDA) accumulation, FF exposure also induced oxidative stress, and apoptosis in hepatopancreas. Interestingly, 7 days exposure triggered more pronounced toxic effect in crabs than 14 days under environment-relevant FF concentration. Integrated biomarker response version 2 (IBRv2) index indicated that 14 days FF exposure under extreme concentration has serious toxicity effect on crabs. Furthermore, 14 days exposure to FF changed the diversity and composition of hepatopancreas microbiota leading remarkable increase of pathogenic microorganism Spirochaetes following exposure to 50 µg/L of FF. Taken together, our study explained potential mechanism of FF toxicity on hepatopancreas of crustaceans, and provided a reference for the concentration of FF to be used in culture of Chinese mitten crab.

Salvia miltiorrhiza polysaccharide promotes the health of crayfish (Procambarus clarkii) by promoting hemocyte phagocytosis, protecting hepatopancreas and enhancing intestinal barrier function.

Plant polysaccharides as immunomodulators are considered one of the effective measures to reduce antibiotic therapy in aquaculture. The immunomodulatory function of Salvia miltiorrhiza polysaccharides (SMP) has been demonstrated and begun to be applied in vertebrates, but its potential effect on crustaceans is unclear. In this study, crayfish (Procambarus clarkii) was fed with 0 %, 0.3 %, 0.7 %, 1.1 %, and 1.5 % SMP for 4 weeks to investigate the effects of SMP on hemocytes phagocytosis, hepatopancreatic function, and intestinal barrier function. The results revealed that hemocyte phagocytic activity was increased in all SMP groups. During the process of hemocytes phagocytic recognition and formation of phagosomes and phagolysosomes, the mRNA expression levels of mas, hem, rab3, ctsb, and lamp-1 were up-regulated mainly in the 0.3 % SMP group. During the clearance phase of phagocytosis, respiratory burst activity, ROS level, T-SOD, CAT, GST, and LZM activities were mainly increased in the 1.5 % SMP group. Hepatopancreas AKP and GOT activity were no significant change in all SMP groups. ACP activity was significantly enhanced in the 1.1 % SMP group. The GPT activity of 0.3-0.7 % SMP group was significantly decreased. The 0.7 % SMP group had the highest intestinal fold height. The highest index values of OTUs, Ace, Chao, and Shannon were in the 0.3 % SMP group. The dietary addition of 0.3 % SMP led to a tendency of increased relative abundance of Firmicutes and Bacteroidota at the phylum level, while the relative abundance of Proteobacteria at the phylum level decreased. In conclusion, dietary SMP could promote crayfish health by enhancing phagocytosis, protecting hepatopancreas and enhancing intestinal barrier function. This study contributes to the theoretical foundation for exploring the potential application of plant polysaccharides in crustaceans.

Adaptation of Glucose Metabolism to Limb Autotomy and Regeneration in the Chinese Mitten Crab.

Limb autotomy and regeneration represent distinctive responses of crustaceans to environmental stress. Glucose metabolism plays a pivotal role in energy generation for tissue development and regeneration across various species. However, the relationship between glucose metabolism and tissue regeneration in crustaceans remains elusive. Therefore, this study is aimed at analyzing the alterations of glucose metabolic profile during limb autotomy and regeneration in Eriocheir sinensis, while also evaluating the effects of carbohydrate supplementation on limb regeneration. The results demonstrated that limb autotomy triggered a metabolic profile adaption at the early stage of regeneration. Hemolymph glucose levels were elevated, and multiple glucose catabolic pathways were enhanced in the hepatopancreas. Additionally, glucose and ATP levels in the regenerative limb were upregulated, along with increased expression of glucose transporters. Furthermore, the gene expression and activity of enzymes involved in gluconeogenesis were repressed in the hepatopancreas. These findings indicate that limb regeneration triggers metabolic profile adaptations to meet the elevated energy requirements. Moreover, the study observed that supplementation with corn starch enhanced limb regeneration capacity by promoting wound healing and blastema growth. Interestingly, dietary carbohydrate addition influenced limb regeneration by stimulating gluconeogenesis rather than glycolysis in the regenerative limb. Thus, these results underscore the adaptation of glucose metabolism during limb autotomy and regeneration, highlighting its essential role in the limb regeneration process of E. sinensis.

Integrated physiological, transcriptome, and metabolome analyses of the hepatopancreas of Litopenaeus vannamei under cold stress.

Temperature is a limiting factor in the growth of aquatic organisms and can directly affect many chemical and biological processes, including metabolic enzyme activity, aerobic respiration, and signal transduction. In this study, physiological, transcriptomic, and metabolomic analyses were performed to characterize the response of Litopenaeus vannamei to cold stress. We subjected L. vannamei to gradually decreasing temperatures (24 °C, 20 °C, 18 °C, 14 °C, and 12 °C) and studied the changes in the hepatopancreas. The results showed that extreme cold stress (12 °C) caused structural damage to the hepatopancreas of L. vannamei. However, shrimp exhibited response mechanisms to enhance cold tolerance, through regulating changes in key genes and metabolites in amino acid, lipid metabolism, and carbohydrate metabolism, including (a) increased level of methylation in cells to enhance cold tolerance; (b) increased content of critical amino acids, such as proline, alanine, glutamic acid and taurine, to ameliorate energy metabolism, protect cells from cold-induced osmotic imbalance, and promote ion transport and DNA repair; (c) accumulation of unsaturated fatty acids to improve cell membrane fluidity; and (d) regulation of the metabolic pattern shift to rely on anaerobic metabolism with a gradual decrease in aerobic metabolism and enhance glycolysis to produce enough ATP to maintain energy metabolic balance. When the temperature dropped further, cold stress impaired antioxidant and immune defense responses in shrimp. This study provides an integrated analysis of the physiology, transcriptome, and metabolome of L. vannamei in response to cold stress.

Flow cytometric analysis of hepatopancreatic cells from Armadillidium vulgare highlights terrestrial isopods as efficient environmental bioindicators in ex vivo settings.

Several studies have reported the high bioindication capacity of Isopoda (Crustacea, Oniscidea), which is related to their important ability to accumulate contaminants, usefulness in soil ecotoxicology and bioindication activities. Any change in the isopod population, diversity and life cycle can indicate relevant pollution levels. The analysis of target tissues, such as the hepatopancreas, is another emerging approach (from a cytologic/histological level) to detect contaminant accumulation from different sources. In this study, tissue disaggregation procedures were optimised in the hepatopancreas, and flow cytometry (FC) was applied to detect cell viability and several cell functions. After disaggregation, two hepatopancreatic cell types, small (S) and big (B), were still recognisable: they differed in morphology and behaviour. The analyses were conducted for the first time on isopods from sites under different conditions of ecological disturbance through cytometric re-interpretation of ecological-environmental parameters. Significant differences in cell functional parameters were found, highlighting that isopod hepatopancreatic cells can be efficiently analysed by FC and represent standardisable, early biological indicators, tracing environmental-induced stress through cytologic/histologic analyses.

Comparison of Cd, Cu, Se, and Zn Concentration in the Muscle and Hepatopancreas of Sepia pharaonis and Uroteuthis duvauceli in the North of Persian Gulf (Iran).

The objective of this research was to examine and contrast the levels of cadmium (Cd), copper (Cu), zinc (Zn), and selenium (Se) in the muscle and hepatopancreas tissues of two species, namely pharaoh cuttlefish (Sepia pharaonis) and Indian squid (Uroteuthis duvauceli), from the Persian Gulf. A total of thirty individuals of each species were gathered in January 2009 from the northern waters of the Persian Gulf. The metal concentrations were significantly higher in muscle tissue (p < 0.05) than in other tissues. S. pharaonis had higher metal concentrations than U. duvauceli. In the muscle and hepatopancreas samples of S. pharaonis, the highest mean concentrations were found to be for Zn (58.45 ± 0.96 µg/g dw) and Cu (1541.47 ± 192.15 µg/g dw), respectively. In U. duvauceli, the highest concentration of measured elements was seen for Zn in both muscle (36.52 ± 0.56 µg/g dw) and hepatopancreas (60.94 ± 2.65 µg/g dw). Se had the lowest concentration among the elements measured in both species. There was a negative and significant correlation between Cu and biometrical factors (total body length and weight) in both muscle and hepatopancreas samples of S. pharaonic and only in the muscle samples of U. duvauceli (p < 0.01, R2 = - 052; p < 0.01, R2 = - 0.055). However, there was a strong correlation between Zn and biometrical factors in hepatopancreas samples of both species. The comparison of metal concentrations with standards revealed that only Cd levels in S. pharaonis exceeded the ESFA and WHO standards, whereas other metals were below the standards.

Unlocking the potential of N-acetylcysteine: Improving hepatopancreas inflammation, antioxidant capacity and health in common carp (Cyprinus carpio) via the MAPK/NF-κB/Nrf2 signalling pathway.

N-acetylcysteine (NAC) positively contributes to enhancing animal health, regulating inflammation and reducing stress by participating in the synthesis of cysteine, glutathione, and taurine in the body. The present study aims to investigate the effects of dietary different levels of NAC on the morphology, function and physiological state of hepatopancreas in juvenile common carp (Cyprinus carpio). 450 common carps were randomly divided into 5 groups: N1 (basal diet), N2 (1.5 g/kg NAC diet), N3 (3.0 g/kg NAC diet), N4 (4.5 g/kg NAC diet) and N5 (6.0 g/kg NAC diet), and fed for 8 weeks. The results indicated that dietary 3.0-6.0 g/kg NAC reduced hepatopancreas lipid vacuoles and nuclear translocation, and inhibited apoptosis in common carp. Simultaneously, the activities of hepatopancreas alanine aminotransferase and aspartate aminotransferase progressively increased with rising dietary NAC levels. Dietary NAC enhanced the non-specific immune function of common carp, and exerted anti-inflammatory effects by inhibiting the MAPK/NF-κB signaling pathway. Additionally, dietary 3.0-6.0 g/kg NAC significantly improved the antioxidant capacity of common carp, which was associated with enhanced glutathione metabolism, clearance of ROS and the activation of Nrf2 signaling pathway. In summary, NAC has the potential to alleviate inflammation, mitigate oxidative stress and inhibit apoptosis via the MAPK/NF-κB/Nrf2 signaling pathway, thereby improving hepatopancreas function and health of common carp. The current findings provide a theoretical basis for promoting the application of NAC in aquaculture and ecological cultivation of aquatic animals.

Comparative transcriptome analysis of hepatopancreas reveals the potential mechanism of shrimp resistant to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus carrying a pathogenic plasmid (VPAHPND) is one of the main causative agents of acute hepatopancreatic necrosis disease (AHPND) in shrimp aquaculture. Knowledge about the mechanism of shrimp resistant to VPAHPND is very helpful for developing efficient strategy for breeding AHPND resistant shrimp. In order to learn the mechanism of shrimp resistant to AHPND, comparative transcriptome was applied to analyze the different expressions of genes in the hepatopancreas of shrimp from different families with different resistance to VPAHPND. Through comparative analysis on the hepatopancreas of shrimp from VPAHPND resistant family and susceptible family, we found that differentially expressed genes (DEGs) were mainly involved in immune and metabolic processes. Most of the immune-related genes among DEGs were highly expressed in the hepatopancreas of shrimp from resistant family, involved in recognition of pathogen-associated molecular patterns, phagocytosis and elimination of pathogens, maintenance of reactive oxygen species homeostasis and other immune processes etc. However, most metabolic-related genes were highly expressed in the hepatopancreas of shrimp from susceptible family, involved in metabolism of lipid, vitamin, cofactors, glucose, carbohydrate and serine. Interestingly, when we analyzed the expression of above DEGs in the shrimp after VPAHPND infection, we found that the most of identified immune-related genes remained at high expression levels in the hepatopancreas of shrimp from the VPAHPND resistant family, and most of the identified metabolic-related genes were still at high expression levels in the hepatopancreas of shrimp from the VPAHPND susceptible family. The data suggested that the differential expression of these immune-related and metabolic-related genes in hepatopancreas might contribute to the resistance variations of shrimp to VPAHPND. These results provided valuable information for understanding the resistant mechanism of shrimp to VPAHPND.