Genome-Wide Identification of Vitellogenin Gene Family and Comparative Analysis of Their Involvement in Ovarian Maturation in Exopalaemon carinicauda.

Vitellogenin (Vtg) is a precursor of yolk proteins in egg-laying vertebrates and invertebrates and plays an important role in vitellogenesis and embryonic development. However, the Vtg family remains poorly characterized in Exopalaemon carinicauda, a major commercial mariculture species found along the coasts of the Yellow and Bohai Seas. In this study, 10 Vtg genes from the genomes of E. carinicauda were identified and characterized. Phylogenetic analyses showed that the Vtg genes in crustaceans could be classified into four groups: Astacidea, Brachyra, Penaeidae, and Palaemonidae. EcVtg genes were unevenly distributed on the chromosomes of E. carinicauda, and a molecular evolutionary analysis showed that the EcVtg genes were primarily constrained by purifying selection during evolution. All putative EcVtg proteins were characterized by the presence of three conserved functional domains: a lipoprotein N-terminal domain (LPD_N), a domain of unknown function (DUF1943), and a von Willebrand factor type D domain (vWD). All EcVtg genes exhibited higher expression in the female hepatopancreas than in other tissues, and EcVtg gene expression during ovarian development suggested that the hepatopancreas is the main synthesis site in E. carinicauda. EcVtg1a, EcVtg2, and EcVtg3 play major roles in exogenous vitellogenesis, and EcVtg3 also plays a major role in endogenous vitellogenesis. Bilateral ablation of the eyestalk significantly upregulates EcVtg mRNA expression in the female hepatopancreas, indicating that the X-organ/sinus gland complex plays an important role in ovarian development, mostly by inducing Vtg synthesis. These results could improve our understanding of the function of multiple Vtg genes in crustaceans and aid future studies on the function of EcVtg genes during ovarian development in E. carinicauda.

Regulation Mechanism of Dopamine Receptor 1 in Low Temperature Response of Marsupenaeus japonicus.

Dopamine receptors (DARs) are important transmembrane receptors responsible for receiving extracellular signals in the DAR-mediated signaling pathway, and are involved in a variety of physiological functions. Herein, the D1 DAR gene from Marsupenaeus japonicus (MjDAD1) was identified and characterized. The protein encoded by MjDAD1 has the typical structure and functional domains of the G-protein coupled receptor family. MjDAD1 expression was significantly upregulated in the gills and hepatopancreas after low temperature stress. Moreover, double-stranded RNA-mediated silencing of MjDAD1 significantly changed the levels of protein kinases (PKA and PKC), second messengers (cyclic AMP (cAMP), cyclic cGMP, calmodulin, and diacyl glycerol), and G-protein effectors (adenylate cyclase and phospholipase C). Furthermore, MjDAD1 silencing increased the apoptosis rate of gill and hepatopancreas cells. Thus, following binding to their specific receptors, G-protein effectors are activated by MjDAD1, leading to DAD1-cAMP/PKA pathway-mediated regulation of caspase-dependent mitochondrial apoptosis. We suggest that MjDAD1 is indispensable for the environmental adaptation of M. japonicus.

Molecular Characterization Related to Ovary Early Development Mechanisms after Eyestalk Ablation in Exopalaemon carinicauda.

Eyestalk ablation is an effective method to promote ovarian development in crustaceans. Herein, we performed transcriptome sequencing of ovary and hepatopancreas tissues after eyestalk ablation in Exopalaemon carinicauda to identify genes related to ovarian development. Our analyses led to the identification of 97,383 unigenes and 190,757 transcripts, with an average N50 length of 1757 bp. In the ovary, four pathways related to oogenesis and three related to oocyte rapid growth were enriched. In the hepatopancreas, two vitellogenesis-associated transcripts were identified. Furthermore, short time-series expression miner (STEM) and gene ontology (GO) enrichment analyses revealed five terms related to gamete generation. In addition, two-color fluorescent in situ hybridization results suggested that dmrt1 might play a vital role in oogenesis during the early stage of ovarian development. Overall, our insights should support future studies focusing on investigating oogenesis and ovarian development in E. carinicauda.

Application of proteomics and metabolomics to assess ammonia stress response and tolerance mechanisms of juvenile ornate rock lobster Panulirus ornatus.

Ammonia is a common pollutant in the aquatic ecosystem and closed aquaculture systems. It may pose a threat to the lobster growth, reproduction and survival. However, there is lack of research of the mechanisms on the toxic effects ammonia at molecular levels. In this work, proteomics and metabolomics were integrated to analyze the proteome and metabolome responses in the ornate spiny lobster Panulirus ornatus treated with ammonia (20 mg L-1) for 48 h. A total of 199 proteins and 176 metabolites were significantly altered in P. ornatus following ammonia treatment. The responsive proteins and metabolites were predominantly involved in immune response, phase I and phase II biotransformation, carbohydrate metabolism, amino acid metabolism, and lipid metabolism. Furthermore, an increase in urea levels was observed, and amino acid metabolism was induced, indicating that the urea cycle was utilized to biotransform ammonia so as to reduce endogenous ammonia content. Ammonia exposure also affected the antioxidant system and induced cellular apoptosis. Overall, our results provide comprehensive insights into the molecular mechanisms underlying the response of P. ornatus to ammonia stress. We believe that the data reported herein should contribute to the development of novel, efficient methods for P. ornatus aquaculture.

A chromosome-level genome of the kuruma shrimp (Marsupenaeus japonicus) provides insights into its evolution and cold-resistance mechanism.

Marsupenaeus japonicus is an important marine crustacean species. However, a lack of genomic resources hinders the use of whole genome sequencing to explore their genetic basis and molecular mechanisms for genome-assisted breeding. Consequently, we determined the chromosome-level genome of M. japonicus. Here we determine the chromosome-level genome assembly for M. japonicus with a total of 665.19 Gb genomic sequencing data, yielding an approximately1.54 Gb assembly with a contig N50 size of 229.97 kb and a scaffold N50 size of 38.27 Mb. With the high-throughput chromosome conformation capture (Hi-C) technology, we anchored 18,019 contigs onto 42 pseudo-chromosomes, accounting for 99.40% of the total genome assembly. Analysis of the present M. japonicus genome revealed 24,317 protein-coding genes and a high proportion of repetitive sequences (61.56%). The high-quality genome assembly enabled the identification of genes associated with cold-stress and cold tolerance in kuruma shrimp through the comparison of eyestalk transcriptomes between the low temperature-stressed shrimp (10 °C) and normal temperature shrimp (28 °C). The genome assembly presented here could be useful in future studies to reveal the molecular mechanisms of M. japonicus in response to low temperature stress and the molecular assisted breeding of M. japonicus in low temperature.

Improved genome assembly of Chinese shrimp (Fenneropenaeus chinensis) suggests adaptation to the environment during evolution and domestication.

A high-quality reference genome is necessary to determine the molecular mechanisms underlying important biological phenomena; therefore, in the present study, a chromosome-level genome assembly of the Chinese shrimp Fenneropenaeus chinensis was performed. Muscle of a male shrimp was sequenced using PacBio platform, and assembled by Hi-C technology. The assembled F. chinensis genome was 1.47 Gb with contig N50 of 472.84 Kb, including 57.73% repetitive sequences, and was anchored to 43 pseudochromosomes, with scaffold N50 of 36.87 Mb. In total, 25,026 protein-coding genes were predicted. The genome size of F. chinensis showed significant contraction in comparison with that of other penaeid species, which is likely related to migration observed in this species. However, the F. chinensis genome included several expanded gene families related to cellular processes and metabolic processes, and the contracted gene families were associated with virus infection process. The findings signify the adaptation of F. chinensis to the selection pressure of migration and cold environment. Furthermore, the selection signature analysis identified genes associated with metabolism, phototransduction, and nervous system in cultured shrimps when compared with wild population, indicating targeted, artificial selection of growth, vision, and behavior during domestication. The construction of the genome of F. chinensis provided valuable information for the further genetic mechanism analysis of important biological processes, and will facilitate the research of genetic changes during evolution.

Characterization of p53 From the Marine Crab Portunus trituberculatus and Its Functions Under Low Salinity Conditions.

Portunus trituberculatus, or the swimming crab, is tolerant of reduced salinity; however, the molecular mechanism of this tolerance is not clear. Cells can be damaged by hyperosmotic salinity. The protein p53, sometimes referred to as "the guardian of the genome," displays versatile and important functions under changing environmental conditions. Herein, the P. trituberculatus p53 gene (designated as Ptp53) was cloned and studied. The full-length Ptp53 cDNA comprised 1,544bp, with a 1,314bp open reading frame, which encodes a putative polypeptide of 437 amino acids. Quantitative real-time reverse transcription PCR assays revealed ubiquitous expression of Ptp53 in all tissues examined, with the gills showing the highest expression level. Extensive apoptosis was detected under low salinity conditions using terminal deoxynucleotidyl transferase nick-end-labeling staining. Oxidative stress was induced under low salinity conditions, consequently leading to apoptosis. Low salinity stress caused significant upregulation of Ptp53 mRNA and protein levels in the gills. Moreover, compared with that in the control group, the mortality of Ptp53-silenced crabs under low salinity stress was enhanced significantly. Taken together, our findings suggest that Ptp53, via regulation of apoptosis and antioxidant defense, played important functions in the low salinity stress response of the swimming crab.

Integrated physiological, transcriptome and metabolome analyses of the hepatopancreas of the female swimming crab Portunus trituberculatus under ammonia exposure.

Ammonia is a common environmental pollutant in aquatic ecosystem and is also a significant concern in closed aquaculture systems. The threat of ammonia has been increasing with rising anthropogenic activities including intensified aquaculture. In this study, we aimed to investigate ammonia toxicity and metabolism mechanisms in the hepatopancreas, a major organ for Vitellogenin (Vtg) synthesis and defending against ammonia stress, of female swimming crab Portunus trituberculatus which is an important fishery and aquaculture species, by integrating physiological, transcriptome and metabolome analyses. The results revealed that ammonia exposure (10 mg/L, an environmentally relevant concentration) resulted in a remarkable reduction in vtg expression and depression of multiple signaling pathways for reproductive regulators including methyl farnesoate, ecdysone and neuroparsin, demonstrating for the first time that ammonia impairs swimming crab female reproduction. In addition, a number of important genes and metabolites in glycolysis, the Krebs cycle, fatty acid ß-oxidation and synthesis were significantly downregulated, indicating that changes in ammonia levels lead to a general depression of energy metabolism in hepatopancreas. After ammonia exposure, an increased level of urea and a reduction of amino acid catabolism were observed in hepatopancreas, suggesting that urea cycle was utilized to biotransform ammonia, and amino acid catabolism was decreased to reduce endogenous ammonia generation. Furthermore, antioxidant systems were altered following ammonia exposure, which was accompanied by proteins and lipid oxidations, as well as cellular apoptosis. These results indicate that ammonia leads to metabolic suppression, oxidative stress and apoptosis in P. trituberculatus hepatopancreas. The findings improve the understanding for the mechanisms of ammonia toxicity and metabolism in P. trituberculatus, and provide valuable information for assessing potential ecological risk of environmental ammonia and improving aquaculture management.

The role of Nrf2 in mitigating cadmium-induced oxidative stress of Marsupenaeus japonicus.

Nuclear factor-erythroid 2-related factor-2 (Nrf2) is an important modulator of cellular responses against Cd in mammalian cells. However, whether such modulation is conserved in Marsupenaeus japonicas remains unknown.In our study, the shrimps were injected with dsRNA targeting Nrf2 at 4 µg g-1 body weight (b.w.) or sulforaphane (SFN) at 5 µg g-1 b.w., and then were exposed to 40 mg L-1 CdCl2 for 48 h. After Nrf2 knockdown, the Cd content increased, but decreased in the SFN group. This suggested that Nrf2 could promote Cd excretion. A terminal deoxynulceotidyl transferase nick-end-labeling (TUNEL) assay revealed that the Nrf2 knockdown increased the number of apoptotic cells in M. japonicas, while SFN decreased the number of apoptotic cells. After Nrf2 knockdown, the total antioxidant capacity (T-AOC), superoxide dismutase (Sod) activity, and related gene expression decreased significantly, while the malondialdehyde (MDA) content increased remarkably. By contrast, SFN injection alleviated the oxidative stress, as evidenced by increased T-AOC, Sod activity, sod mRNA expression and a reduced MDA content. Similarly, detoxification related enzyme activities (ethoxyresorufin O-deethylase and glutathione-S-transferase (GST)) and their corresponding gene expressions (cyp3a (cytochrome P450 family 3 subfamily A) and gst) were suppressed in the ds-Nrf2 injection group, while they were elevated in the SFN group. In addition, ds-Nrf2 activated mitochondrial apoptotic pathway, as evidenced the mRNA and protein levels of caspase-3, Bcl2 associated X protein (Bax), and p53, while SFN treatment suppressed them. These results displayed that in M. japonicus Cd-induced cellular oxidative damage probably acts via the Nrf2 pathway.

Integrated transcriptomic and metabolomic responses in the hepatopancreas of kuruma shrimp (Marsupenaeus japonicus) under cold stress.

In aquatic ecosystems, the temperature of the water is an important ecological factor that modulates aquatic organisms' metabolism, growth, development, and reproduction. In this study, the morphological, transcriptomic, and metabolomic analyses of response of Marsupeneus japonicus to acute cold stress was investigated. The results revealed that low temperature caused profound morphological damage to the hepatopancreas. Transcriptomic responses suggested that energy and primary metabolism, cytoskeleton structure, and apoptosis signaling were altered. The metabolic responses to cold stress included changes of multiple amino acids and unsaturated fatty acids. Combined transcriptomic and metabolomic data indicated that energy metabolism pathways were downregulated in the hepatopancreas under cold stress. However, M. japonicus increased ATP and unsaturated fatty acids production to ameliorate. Moreover, cold stress caused significant attenuation of macrophage apoptosis. This study provides key information to increase our understanding of low-temperature tolerance in shrimp.

Comparative accumulation and transcriptomic analysis of juvenile Marsupenaeus japonicus under cadmium or copper exposure.

Waterborne metals may be hazardous to aquatic organisms and trigger stress responses. The present study aimed to assess the effect of exposure to 100 µg/L cadmium (Cd) or copper (Cu) for 48 h on juvenile Marsupenaeus japonicus, in terms of bioaccumulation and the whole body transcriptome. The results demonstrated that Cu accumulation in M. japonicas was much higher than that of Cd. Meanwhile, transcriptome analysis identified 1802 and 2670 differentially expressed genes (DEGs) after 48 h exposure to 100 µg/L Cd and Cu, respectively. Among them, 851 DEGs responded to both metals. Cd and Cu stress shared genes were related to the cytoskeleton, immunity, antioxidation, and detoxification. Metallothionein 1 (MT1) was specifically induced in the Cd-stress response, while glycometabolism, heat shock protein 90 (HSP90), metallothionein 2 (MT2), apoptosis, and iron transport-related genes were changed specifically in response to Cu stress. In addition, real-time PCR was used to verify the expression patterns of 28 randomly selected DEGs. The sequencing and real-time PCR results were consistent. Moreover, based on the number of significantly modulated genes and their expression levels, we deduced that Cu acts as a stronger stress inducer than Cd in M. japonicus. The identified Cd and Cu stress related genes and pathways will provide new insights into the common and different molecular mechanisms underlying Cd and Cu toxicity effects in M. japonicus.

Comparative proteomic investigation of Marsupenaeus japonicus hepatopancreas challenged with Vibrio parahaemolyticus and white spot syndrome virus.

This study aimed to use isobaric tags (IBTs) to investigate the immune response of the hepatopancreas of Marsupenaeus japonicas infected with Vibrio parahaemolyticus or white spot syndrome virus (WSSV). Liquid chromatography-tandem mass spectrometry and protein sequencing identified 1005 proteins. Among them, 109 proteins were upregulated and 94 were downregulated after V. parahaemolyticus infection. After WSSV infection, 130 proteins were identified as differentially abundant, including 88 that were upregulated and 42 were downregulated. Fifty-four proteins were identified as differentially abundant after both V. parahaemolyticus and WSSV infection. A number of proteins related to cytoskeletal processes, including actin and myosin, and apoptosis-related proteins were upregulated in shrimp after V. parahaemolyticus and WSSV infection, indicating that phagocytosis and apoptosis may be involved in the response to in V. parahaemolyticus or WSSV infection. Quantitative real-time PCR was carried out to verify the reliability of the proteomic data. These data provide a basis to characterize the immunity-related processes of shrimp in response to infection with WSSV or V. parahaemolyticus.

Bioaccumulation and physiological responses in juvenile Marsupenaeus japonicus exposed to cadmium.

The heavy metal cadmium readily accumulates in organisms, causing damage. In this study, juvenile marine shrimp Marsupenaeus japonicus were exposed to cadmium (Cd2+; 5, 50 and 500 µg L-1). Cd accumulation and antioxidant-related indices were determined, and damage to biomolecules was assessed, after 24, 48 and 96 h. Cd bioaccumulation in M. japonicus increased with exposure time and concentration, which reached the highest value at 96 h. The data showed that 5, 50 and 500 µg L-1 Cd increased glutathione (GSH) content and the activities of superoxide dismutase (SOD) and glutathione S-transferase (GST) in a Cd-dose-dependent manner, but 5 and 50 µg L-1 Cd had no effect on caspase-3 activity. The expression levels of SOD, GST, heat shock protein 70 (HSP70), metallothionein (MT), p53 and caspase-3 genes were rapidly increased after 50 and 500 µg L-1 Cd exposure, and remained at a significantly higher level than in the control after 96 h of exposure. After exposure to 5, 50 and 500 µg L-1 Cd, F-value (the ratio between double-stranded DNA and total DNA) remained high at 24 h, however, as the exposure time increased, the F-value decreased in a dose-dependent manner. An increase in malondialdehyde content was also observed following exposure to 50 and 500 µg L-1 Cd. Our data suggest that Cd induces oxidative stress, molecular damage and apoptosis in juvenile M. japonicus in a concentration- and time-dependent manner.

Transcriptomic analysis provides insight into the mechanism of salinity adjustment in swimming crab Portunus trituberculatus.

BACKGROUND: Low salinity is one of the main factors limiting the distribution and survival of marine species. As a euryhaline species, the swimming crab (Portunus trituberculatus) is adaptive to relatively low salinity. However, the mechanisms underlying salinity stress responses in P. trituberculatus is not very clear. OBJECTIVES: The primary objective of this study was to describe the salinity adaptation mechanism in P. trituberculatus. METHODS: The crabs were exposed to low salinity stress, and gill tissue was sampled at 0, 12, 36, 48 and 72 h and subjected to high throughput sequencing. Subsequently, we tested the accuracy and quality of the sequencing results, and then carried out GO and KEGG bioinformatics on the differentially expressed genes (DEG). RESULTS: Each sample yielded more than 1.1 Gb of clean data and 23 million clean reads. The process was divided into early (0-12 h), middle (12-48 h), and late phase (48-72 h). A total of 1971 (1373 up-regulated, 598 down-regulated), 1212 (364 up-regulated, 848 down-regulated), and 555 (187 up-regulated, 368 down-regulated) DEGs with annotations were identified during the three stages, respectively. DEGs were mainly associated with lipid metabolism energy metabolism, and signal transduction from the three stages, respectively. CONCLUSION: A substantial number of genes were modified by salinity stress, along with a few important salinity acclimation pathways. This work provides valuable information on the salinity adaptation mechanism in P. trituberculatus. In addition, the comprehensive transcript sequences reported in this study provide a rich resource for identification of novel genes in this and other crab species.

Comparative transcriptomic analysis of Marsupenaeus japonicus hepatopancreas in response to Vibrio parahaemolyticus and white spot syndrome virus.

Vibrio parahaemolyticus and white spot syndrome virus (WSSV) are pathogens that cause epidemics in kuruma shrimp (Marsupenaeus japonicus) during aquaculture, resulting in severe economic losses to local farmers. To characterise the mechanisms of the molecular responses to V. parahaemolyticus and WSSV infection in M. japonicus, the transcriptome of hepatopancreas was sequenced using next-generation sequencing after infection. A total of 29,180 unigenes were assembled, with an average length of 1,151 bp (N50 = 1,951 bp). After BLASTX searching against the Nr database (E-value cut-off = 10-5), 15,176 assembled unigenes remained, with 3,039 and 1,803 differentially expressed transcripts identified in the V. parahaemolyticus- and WSSV-infected groups, respectively. Of these, 1466 transcripts were up-regulated and 1573 were down-regulated in V. parahaemolyticus-infected shrimps, and 970 transcripts were up-regulated and 833 were down-regulated in the WSSV-infected shrimps. Additionally, 761 transcripts were differentially expressed in both V. parahaemolyticus- and WSSV-infected shrimps. Several known immune-related genes including caspase 4, integrin, crustin, ubiquitin-conjugating enzyme E2, C-type lectin, and α2-macroglobulin were among the differentially expressed transcripts. These results provide valuable information for characterising the immune mechanisms of the shrimp responses of to V. parahaemolyticus andWSSV infection.

Characterization of three caspases and their pathogen-induced expression pattern in Portunus trituberculatus.

Caspases are a family of proteases involved in many important biological processes including apoptosis and inflammation. In this study, we analyzed the expression patterns and effects on immune response in various tissues of the edible crab Portunus trituberculatus. PtCas 2, PtCas 3 and PtCas 4 share overall sequence identities of 55.88%-74.86%, 8.47%-46.54% and 20.11%-50.87%, respectively, with their other crustacean species. PtCas 2, PtCas 3 and PtCas 4 have the same caspase domain and catalytic site found in known caspases. The expression levels of the three caspases differed between tissues. Following bacterial and viral infection, the expression levels of the three caspases reached a maximum level at 24 h post-infection (hpi) in case of bacteria, whereas it was 48 hpi in virus. Moreover, the WSSV, Vibrio alginolyticus or V. parahaemolyticus induced the activities of PtCas 2-4 in a time-dependent manner. These results indicate an involvement of caspases in bacterial and viral induced immune response and demonstrate for the first time that PtCas 2, PtCas 3 and PtCas 4 are essential for optimal response to bacterial and virus infection in crabs.

Effects of florfenicol on the antioxidant status, detoxification system and biomolecule damage in the swimming crab (Portunus trituberculatus).

Florfenicol (FLR) is the most commonly used antibacterial agent in aquaculture because of its wide spectrum of activity and few side-effects. We characterized the toxicokinetics of FLR in the swimming crab (Portunus trituberculatus) after intravenous (IV) dosing (20, 40 and 80mg/kg). The results showed that FLR significantly suppressed the antioxidant system of the hepatopancreas. FLR induced transcriptional expression of phase I and phase II detoxification genes (CYP3 and GST, respectively) in a dose- and clearance time-dependent manner and altered the expression of their corresponding enzymes (erythromycin N-demethylase and glutathione S-transferase, respectively). Moreover, FLR induced the transcription of ATP-binding cassette (ABC) transporter subfamily B (ABCB) and subfamily G (ABCG), although ABCG transcription was not induced by FLR at 20mg/kg. Additionally, higher FLR doses caused significant biomolecule damage during the first 48h after delivery. This study will provide an improved understanding of the exact mechanism underlying toxicity in aquatic organisms.

Effects of ammonia-N stress on metabolic and immune function via the neuroendocrine system in Litopenaeus vannamei.

The purpose of this study was to evaluate the immunological responses, such as phenoloxidase (PO), antibacterial, and bacteriolytic activities, and metabolic variables, such as oxyhemocyanin, lactate, and glucose levels, of Litopenaeus vannamei exposed to ambient ammonia-N at 0, 2.5, 5, 7.5, and 10 mg/L for 0, 3, 6, 12, 24, and 48 h, and determine the effects of the eyestalk hormone on the metabolic and immune functions of unilateral eyestalk-ablated L. vannamei exposed to ambient ammonia-N at 10 mg/L. The actual concentrations of the control and test solutions were 0.04, 2.77, 6.01, 8.30, and 11.36 mg/L for ammonia-N and 0.01, 0.15, 0.32, 0.44, and 0.60 mg/L for NH3-N (unionized ammonia). The results showed a significant decrease in the PO, antibacterial, and bacteriolytic activities in the plasma as well as a significant increase in the glucose and lactate levels and decreased oxyhemocyanin levels in the hemolymph of L. vannamei exposed to elevated ammonia-N levels. These findings indicated that L. vannamei exposed to ammonia-N might demonstrate weakened metabolic and immunological responses. Moreover, eyestalk removal caused a dramatic decrease in PO, antibacterial, and bacteriolytic activities, which indicated that the eyestalk hormone in L. vannamei exhibited a higher immune response due to the induction of protective mechanisms against ammonia-N stress. Eyestalk removal also caused a dramatic decrease in glucose and lactate levels, suggesting that the eyestalk hormone is involved in glucose metabolism to meet the energy requirements under ammonia-N stress conditions.

Immune response and antioxidant status of Portunus trituberculatus inoculated with pathogens.

The white spot syndrome virus (WSSV), Vibrio parahaemolyticus and V. alginolyticus are serious epidemic pathogen affecting the cultured Portunus trituberculatus and resulted in severe economic losses to local farmers. The immune and antioxidant systems are believed to be closely involved in host responses to pathogens in aerobic animals, including crustaceans. In order to explore such host-pathogen interactions in the early stage of infection in P. trituberculatus, the mRNA transcript levels of six key immune-related genes (proPO, α2M, crustin, lysozyme, NOS, and NOX) and three key antioxidant-related genes (CuZnSOD, CAT, and GPx) and their corresponding enzymatic activity were investigated in response to challenge with the three pathogens. A decrease in the expression of the proPO, crustin, and lysozyme was observed, which may reflect the immunosuppressive mechanism of the pathogens against the host. Moreover, an increase was observed in the α2M expression with time, which indicated that the pathogens could affect proteinase cascade reactions associated with the proPO system by disturbing the balance between serine proteinases and their inhibitors. Moreover, WSSV, V. parahaemolyticus and V. alginolyticus induced to increase the transcription and enzyme activities of NOS and NOX. Additionally, significant variations in the expression of the anitioxidant-related genes CuZnSOD, CAT, and GPx and their enzyme activities implied that these enzymes played a critical role in the immune response against the pathogens. The present findings indicate that the immune parameters analyzed here may be markers of the physiological status of this species after bacterial or viral infections.

Toxic responses of swimming crab (Portunus trituberculatus) larvae exposed to environmentally realistic concentrations of oxytetracycline.

Oxytetracycline (OTC) is the most commonly used antibiotics for bacterial treatment in crustacean farming in China, and because of their intensive use, the potential harmful effects on aquatic organisms are of great concern. The aim of this study was to investigate the effects of oxytetracycline (OTC) on the antioxidant system, detoxification progress, and biomolecule damage in Portunus trituberculatus larvae. In this study, larvae that belonged to four zoeal stages were exposed to four different concentrations of OTC (0, 0.3, 3, and 30 µg/L) for 3 days. The results showed that the exposure to OTC significantly suppressed the antioxidant system of, especially, zoea I (Z1) and zoea II (Z2) larvae. OTC inhibited the transcriptional expression of phase I (CYP2 and CYP3) and phase II detoxification genes (GST) in a dose-dependent manner and altered the expressions of their corresponding enzymes, namely, aminopyrine N-demethylase, erythromycin N-demethylase, and glutathione S-transferase. Moreover, 0.3 µg/L OTC activated the transcription of ATP-binding cassette (ABC) transporter subfamily B (ABCB) and subfamily G (ABCG) in the Z1 and Z2 larvae, while 3 and 30 µg/L OTC suppressed all of them. Additionally, malondialdehyde content exhibited a dose- and zoea-effect relationship to some extent, but no significant differences were observed in the F values of the Z3 and Z4 larvae, except for the 30 µg/L OTC treatment. Thus, the Z3 and Z4 larvae were less sensitive to OTC exposure than the Z1 and Z2 larvae.