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The mud crab Scylla paramamosain is a key species in China due to its high nutritional value and great economic worth and has grown in popularity. Myo-inositol can modulate versatile physiological functions in aquatic animals. In the present study, S. paramamosain megalopa were given graded concentrations of myo-inositol (0, 1, 2, 4, and 8 ppm) by water immersion to explore how their metamorphosis would be affected. The results showed that supplementing with myo-inositol remarkably increased transformation and survival rate from megalopa to crablet by at least 1.16 and 1.26 times, respectively. To decipher the molecular mechanism of how myo-inositol increases metamorphosis and survival rate, we further performed transcriptome-based gene expression profiling of both megalopa and crablet treated with myo-inositol. The integrative transcriptome analyses predicted that the differentially expressed genes (DEGs) were significantly enriched in chitinase activity, structural constituent of cuticle, and chitin binding, which are associated with the decomposition and reconstruction of cuticle. qPCR results confirmed that myo-inositol mediated gene expression levels of the above cuticle-related pathways. Considering the importance of the cuticle in exoskeleton formation and molting, it can be concluded that myoinositol-induced changes in the cuticle decomposition and reconstruction might have accelerated the transformation from megalopa to crablet of S. paramamosain. Besides, numerous DEGs were significantly enriched in protein digestion and absorption, amino sugar and nucleotide sugar metabolism. It implies that myo-inositol may improve survival by regulating energy or nutrient absorption. Additionally, the accelerated metamorphosis by myo-inositol may improve survival from megalopa to crablet of S. paramamosain. Overall, this study will provide the first insights into the underlying molecular mechanisms by which myo-inositol increases metamorphosis and survival. [ABSTRACT FROM AUTHOR]

Mud crab (Scylla paramamosain) has become an important mariculture crab along the southeast coast of China due to its strong adaptability, delicious taste, and rich nutrition. Several vertebrate steroid hormones and their synthesis-related genes and receptors have been found in crustaceans, but there are few reports on their synthesis process and mechanism. 3-beta-hydroxysteroid dehydrogenase (HSD3B) is a member of the Short-chain Dehydrogenase/Reductase (SDR) family, and an indispensable protein in vertebrates' steroid hormone synthesis pathway. In this study, the SpHsd3b gene sequence was obtained from the transcriptome data of S. paramamosain , and its full-length open reading frame (ORF) was cloned. The spatial and temporal expression pattern of SpHsd3b was performed by quantitative real-time PCR (qRT-PCR). SpHsd3b dsRNA interference (RNAi) and HSD3B inhibitor (trilostane) were used to analyze the function of Sp HSD3B. The results showed that the SpHsd3b gene has an 1113 bp ORF encoding 370 amino acids with a 3β-HSD domain. Sp HSD3B has lower homology with HSD3B of vertebrates and higher homology with HSD3B of crustaceans. SpHsd3b was expressed in all examined tissues in mature crabs, and its expression was significantly higher in the testes than in the ovaries. SpHsd3b expression level was highest in the middle stage of testicular development, while its expression was higher in the early and middle stages of ovarian development. RNAi experiment and trilostane injection results showed that Sp HSD3B had regulatory effects on several genes related to gonadal development and steroid hormone synthesis. 15-day trilostane suppression could also inhibit ovarian development and progesterone level of hemolymph. According to the above results, crustaceans may have steroid hormone synthesis pathways like vertebrates, and the Hsd3b gene may be involved in the gonadal development of crabs. This study provides further insight into the function of genes involved in steroid hormone synthesis in crustaceans. • Steroid hormone synthesis pathway-related gene— Hsd3b was identified from Scylla paramamosain. • SpHsd3b knockdown can decrease the expression of gonadal development and steroid hormone synthesis-related genes. • Trilostane can inhibit ovarian development and lower progesterone level of hemolymph. • crustaceans may have steroid hormone synthesis pathways like vertebrate. [ABSTRACT FROM AUTHOR]

The mud crab Scylla paramamosain is one of the economically important aquaculture species in China. The larval development of the mud crab is characterized by two significant morphological changes, from the 5th zoea (Z5) to the megalopa (M) stage and from the M to the first juvenile crab (C1) stage. In this study, we found that methyl farnesoate (MF) could prohibit the Z5 to M metamorphosis in a concentration-dependent manner, and that a concentration of 10 μ M MF could completely prohibit the Z5 metamorphosis. Farnesoic acid (FA) could also prohibit the Z5 metamorphosis, but its effects seemed to be concentration-independent. In addition, MF could delay rather than prohibit the M to C1 metamorphosis, while FA had no effect on the M to C1 metamorphosis at all. To summarize, it is hypothesized that either absence of MF and FA, or at least very low levels of these substances, might be necessary for a successful Z5 to M metamorphosis. Résumé: Le crabe de boue Scylla paramamosain est l'une des espèces d'aquaculture économiquement importante en Chine. Le développement larvaire de ce crabe est caractérisé par deux changements morphologiques significatifs, de la 5ème zoé (Z5) au stade mégalope (M) et de M au premier stade juvénile crabe (C1). Dans cette étude nous avons trouvé que le méthyl-farnésoate (MF) pouvait inhiber la métamorphose de Z5 d'une manière dose-dépendante, et qu'une concentration de 10 μ M de MF pouvait complètement empêcher la métamorphose de la Z5. L'acide farnésoïque (FA) peut aussi inhiber la métamorphose de la Z5, mais ses effets semblent indépendants de la dose. De plus, MF peut retarder plutôt qu'inhiber la métamorphose de M à C1, tandis que FA n'a aucun effet sur la métamorphose de M à C1. En résumé, l'hypothèse est émise que, soit l'absence de MF et FA, soit au moins un faible taux de ces substances, serait nécessaire pour une métamorphose réussie de Z5 à M. [ABSTRACT FROM AUTHOR]

Scylla paramamosain is one of the commercially crucial marine crustaceans belonging to the genus Scylla, which is commonly distributed along the coasts of China. Proteomic data are scarce for the mud crab. Salinity is one of the ecological factors that affect S. paramamosain during overwintering. To understand the energy metabolism mechanism adapted to salinity, we analysed the proteomics of S. paramamosain hepatopancreas in response to two different salinities (4‰ and 25‰). A total of 1283 proteins were detected, of which 426 down‐regulated proteins and 83 up‐regulated proteins were associated with metabolism (p < 0.05). Among these proteins related to metabolic pathways (16 pathways), we identified angiotensin‐converting enzyme, trypsin, glutamate dehydrogenase, aminomethyl transferase, hexokinase, pyruvate kinase, glucose‐6‐phosphate dehydrogenase, L‐lactate dehydrogenase, superoxide dismutase, alkaline phosphatase, catalase and xanthine dehydrogenases. Additionally, real‐time PCR showed mRNA expression of differentially expressed genes correlated positively with their protein levels. Compared with 25‰ salinity group, 4‰ salinity group was involved in amino acid metabolism, carbohydrate metabolism, energy metabolism, lipid metabolism and inorganic ion metabolism by regulating related proteins, which made the metabolism of the overwintering S. paramamosain more vigorous in the low salt environment, and hepatopancreas consumed more energy to maintain life activities. These data are the first to reveal the molecular mechanism of overwintering S. paramamosain in response to different salinities at the protein level. The study provides important information for the aquaculture production of S. paramamosain during indoor overwintering. [ABSTRACT FROM AUTHOR]

The mud crab Scylla paramamosain is a major marine crab species for aquaculture in the southern coastal regions of China. This study investigated the differences in the quality of low-salt cultured and wild mud crabs and the the salinity tolerance and growth performance of their offspring. Ovarian development and fatty acid and amino acid compositions in the hepatopancreas and ovaries of mature low-salt and wild mud crabs were compared. The gonadosomatic index was significantly lower, but the hepatopancreatic index was significantly higher in the low-salt mud crab than in the wild mud crab. The contents of C18:1, C22:6n3 (DHA), DHA/EPA, and n-3/n-6 fatty acids in the ovaries and hepatopancreas of the low-salt mud crab were significantly higher than those of the wild mud crab. Except for proline in the hepatopancreas of the low-salt group, there were no significant differences in amino acids in the ovaries and hepatopancreas between the two groups. Although the survival rate of larval mud crabs in the low-salt mud crab was significantly higher than that in the wild mud crab when the salinity was 24‰, there was no significant difference in the juvenile survival between the two groups. However, the survival rate of larval and juvenile mud crabs in the low-salt group was significantly higher than in the wild group when the salinity was 5 ‰ and 3 ‰, respectively. The final body weight, average weight, weight gain, and specific weight gain rate of male and female offspring in the low-salt group were significantly higher than in the wild group. However, the weight gain and specific growth rate of male offspring in the low-salt group in September–October were significantly lower than those of male mud crabs in the wild group. This study indicates that the mud crab can be domesticated for culture and breeding at low salinity. • The fatty acid accumulation in the ovaries and hepatopancreas of Scylla paramamosain was superior at low salinity. • The offspring of Scylla paramamosain at low salinity could adapt to sudden salinity drops and had better growth performance. • Scylla paramamosain adapted to low salinity showed a stable inheritance in offspring. [ABSTRACT FROM AUTHOR]

Mud crab (Scylla paramamosain) is an important economic species in China. Vibrio parahaemolyticus infection have caused a great economic loss in mud crab farming. The mechanism involved in the immune responses of mud crab to V. parahaemolyticus is unclear. In this study, the physiological and immune response to V. parahaemolyticus infection were investigated in S. paramamosain. The results showed that V. parahaemolyticus infection decreased total hemocyte counts, led to cytological damage, and caused high mortality. Transcriptome analysis showed that 1327 differentially expressed genes (DEGs), including 809 up-regulated and 518 down-regulated ones, were obtained after V. parahaemolyticus challenge. These DEGs were mainly involved in the immune response and infectious disease. Additionally, transcriptome analysis revealed that Toll, immune deficiency (IMD), and prophenoloxidase signalling pathways played essential roles in antibacterial immunity against V. parahaemolyticus infection in mud crab. • V. parahaemolyticus led to cytological damage, and caused high mortality. • 1327 DEGs were obtained after V. parahaemolyticus challenge. • Toll, IMD, and prophenoloxidase signalling pathways played important roles in response to V. parahaemolyticus infection. [ABSTRACT FROM AUTHOR]

Mud crab (Scylla paramamosain) is one of the most economically-important marine crabs in China. However, research on mechanisms of reproductive regulation is not sufficient. Vitellogenesis-inhibiting hormone (VIH) is a member of the crustacean hyperglycemia hormones (CHH) family, which plays an essential role in the regulation of gonadal development and maturation in crustaceans, and current studies on the regulation of Vih transcription in crabs are relatively rare. Our previous studies on the transcriptional regulation of mud crab Vih (SpVih) have proved that the binding site of Oct4/Sox9 transcription factor may be the key region for positively regulating the expression of SpVih. In this study, the electrophoretic mobility shift assay (EMSA) experiment confirmed that the nuclear protein extracted from the eyestalk could bind to the key region of SpVih promoter, and these specific bindings were dependent on the presence of Oct4/Sox9 binding sites. Two specific binding complex bands were detected in the supershift group of EMSA supershift experiments by Oct4 and Sox9 antibodies, further confirming the specific recognition of these two transcription factors on the key regulatory region of SpVih. In vitro, Oct4 and Sox9 gene overexpression vectors and SpVih core promoter fragment vector were constructed and co-transfected into HEK293T cells. As a result, SpVih activity increased with the concentration of transcription factors. In vivo , when Oct4 and Sox9 dsRNA were injected into the eyestalks of mud crab, respectively, the expression level of SpVih decreased significantly after interference with Oct4 or Sox9 , and the expression level of SpVtg in the ovary and hepatopancreatic increased. Both in vitro and in vivo experiments showed that Oct4 and Sox9 had a positive regulatory effect on SpVih. The GST pull-down experiment was carried out by purified Oct4 and Sox9 proteins, and the results showed that there was an interaction between them. It was speculated that they regulated the expression of SpVih through the interaction. [ABSTRACT FROM AUTHOR]

The aquatic microbial community is sensitive to environmental change; however, the impacts of those changes combined with disease outbreaks affecting S. paramamosain are unknown. Thus, from March to October, we explored the interaction between aquacultural environmental conditions and microbial composition and function in open-air aquaculture ponds containing S. paramamosain in Southern China. The microbial community structure was significantly positively correlated with microbial community function. The environment variables such as temperature and salinity during May and June changed more quickly compared with other periods, resulting changes in the structure and function of the microbial community affected S. paramamosain survivability, with higher crab mortality observed from May to June compared with other periods. These included changes in the relative abundance of Microtrichales, Synechococcales, Rhodobacterales, Chitinophagales, and SAR11_clade, and corresponding functions associated with glycolysis and/or gluconeogenesis, porphyrin and chlorophyll metabolism, photosynthetic proteins, and transcription factors. These changes could impact S. paramamosain mortality and be used to evaluate the health status of the ponds. Though the environment variables during July~October changed slowly comparing to May and June, the ponds microflora changed which benefit S. paramamosain survivability with correspondingly low S. paramamosain mortality. Therefore, rapid environmental change alters the structure and function of the aquatic microflora, increasing S. paramamosain mortality. [ABSTRACT FROM AUTHOR]

• Sp-IAG promoter has a dsx and a foxl-2 binding sites that can regulate its expression. • Cell experiment and RNAi experiment proved that dsx can enhance Sp-IAG expression. • Cell experiment and RNAi experiment showed that foxl-2 can inhibit Sp-IAG expression. Scylla paramamosain is an important cultured crab species on the southeast coast of China. However, the molecular regulation mechanism of its gonadal development still has not been thoroughly studied. Dsx (doublesex) and foxl-2 (forkhead transcription factor gene 2) are important transcription factors involved in gonadal development. So far, studies on the functions of dsx and foxl-2 in crustaceans are very limited. Insulin-like androgenic gland hormone (IAG) is an effector molecule that regulates the differentiation, development and sex maintenance of testes in crustaceans. In this study, the promoter region of Sp-IAG was predicted, and several potential binding sites of dsx and foxl-2 were found. Site-directed mutagenesis was performed on the predicted potential binding sites, and their promoter activity was analyzed. The results showed that there was a dsx and a foxl-2 binding site, respectively, that could regulate the expression of Sp-IAG. In order to verify the regulatory effect of these two transcription factors on Sp-IAG , we constructed the expression plasmids of dsx and foxl-2 and co-transfected them into HEK293T cell lines with the promoter of Sp-IAG , respectively. The results showed that dsx could significantly promote the expression of Sp-IAG , while foxl-2 could inhibit its expression substantially. Then we carried out in vivo RNA interference experiment on mud crabs. The expression of dsx and foxl-2 in crabs was interfered respectively. The results of qRT-PCR showed that the expression of Sp-IAG was significantly inhibited after interfering with dsx , while significantly increased after interfering with foxl-2 , which was consistent with the cell experiment. In conclusion, dsx and foxl-2 transcription factors play opposite roles in regulating the expression of Sp-IAG. [ABSTRACT FROM AUTHOR]

Background: Scylla paramamosain is a commercially important mud crab. The microbiota is a community that inhabits the crab intestine, and is important for physiological functional and host health. Results: Proteobacteria, Firmicutes, Bacteroidetes, Tenericutes, Spirochaetae and Fusobacteria were the dominant phyla of the 36 representative phyla. Eleven genera of the 820 representative genera were considered as core gut microbiota and were distributed in the five dominant phyla. The core genus of the Proteobacteria included Arcobacter, Photobacterium, Vibrio, Shewanella and Desulfovibrio. The other four phyla contained one or two genera. Male and female crab samples had two different core genera, (male samples: Psychrilyobacter & Lactococcus; female samples: Clostridium_sensu_stricto_11 and Candidatus_Bacilloplasma). Conclusions: This is the first time core intestinal microbiota have been identified in crab from nine coastal regions of southern China. This study provides sequencing data related to the gut microbiota of S. paramamosain, and may contribute to probiotic development for S. paramamosain aquaculture industries. [ABSTRACT FROM AUTHOR]

Mud crabs, Scylla paramamosain , are one of the most economical and nutritious crab species in China and South Asia. Inconsistent with the high development of commercial mud crab aquaculture, effective immunological methods to prevent frequently-occurring diseases have not yet been developed. Thus, high mortalities often occur throughout the different developmental stages of this species resulting in large economic losses. In recent years, numerous attempts have been made to use various advanced biological technologies to understand the innate immunity of S. paramamosain as well as to characterize specific immune components. This review summarizes these research advances regarding cellular and humoral responses of the mud crab during pathogen infection, highlighting hemocytes and gills defense, pattern recognition, immune-related signaling pathways (Toll, IMD, JAK/STAT, and prophenoloxidase (proPO) cascades), immune effectors (antimicrobial peptides), production of reactive oxygen species and the antioxidant system. Diseases affecting the development of mud crab aquaculture and potential disease control strategies are discussed. Image 1 • Hemocytes and gills immune defenses are reviewed in the mud crab Scylla paramamosain. • Knowledge of pattern recognition and immune-related signaling pathways is updated. • Humoral responses, both immune effectors and antioxidant function, are described. • Strategies are proposed for disease mitigation and control in mud crab aquaculture. [ABSTRACT FROM AUTHOR]

Abstract Mud crab (Scylla paramamosain) is an economically important crustacean species for fisheries and aquaculture industry in China and other Southeast Asian countries. In this study, the sex ratio, morphological dimorphism, and the characteristics of morphological traits between female and male S. paramamosain juveniles at three different pond-culture stages were determined. The sex ratio skewed towards female crablets as growth and development proceeded. Three growth traits exhibited significant differences between sexes when the crablets were at two months of pond-culture stage, however, the number of growth traits with significant differences increased to seven when the crablets were at three and four months of pond-culture stages. Further, significant positive correlations were detected among all the 190 trait-pairs, irrespective of the pond-culture stages of the crablets. Apart from this, the path coefficients and determination coefficients varied between sexes among the three pond-culture stages, suggesting that the growth traits have different effects on body weight (BW) of female and male crablets during different pond-culture stages. The effect of growth traits on BW in both sexes at different pond-culture stages was determined by constructing six multiple regression equations. Analysis of the width-weight relationship revealed that male crablets had positive growth allometry whereas the females had isometric growth. In addition, an obvious sexual dimorphism in cheliped size and abdomen width was observed with growth and development. A discriminant function equation was successfully constructed for sex identification based on morphological indexes, with a high accuracy of 90.4%. Highlights • The sex ratio and sexual dimorphism of Scylla paramamosain crablets were evaluated. • The morphological traits affecting body weight varied during the different developmental stages. • A sex discriminant equation based on morphological indexes was constructed, with a high accuracy of 90.4%. • The findings serve as the baseline information for prospective artificial selective breeding and farming of S. paramamosain. [ABSTRACT FROM AUTHOR]

The mud crab (Scylla paramamosain) is an important economic breeding species along the southeast coast of China. As a crustacean, molting is critical for growth and reproduction. At each molting, a new external cuticle of mud crab is inherently regenerated, but along with molting the crabs are easily susceptible to pathogenic infection that might result in a low survival rate particularly at juvenile stages. To elucidate the potential response mechanism of juvenile mud crabs infected with bacteria in the molting process, integrated transcriptomic and metabolomic techniques were applied and the hormones, associated-hormone genes or immune genes modulated by challenge with Staphylococcus aureus or Vibrio alginolyticus were analyzed. The omic analysis showed that S. aureus or V. alginolyticus infection caused decreases in steroid hormones (cortisol, cortodoxone, cortisone, deoxycorticosterone) and fatty acid-derived hormones (prostaglandin A2, Δ17–6-keto prostaglandin F1α, 13,14-Dihydro-15-keto-tetranor prostaglandin F1α, prostaglandin B2, 2,3-Dinor-11β-prostaglandin F2α, thromboxane B2, and 11-Dehydro thromboxane B2). Correspondingly, transcriptomic analysis revealed that both S. aureus and V. alginolyticus infection downregulated the expression levels of fatty acid-derived hormone synthases. In addition, both bacterial infections caused upregulation in Toll, IMD, the Jak/Stat pathway, antimicrobial peptide, and the prophenoloxidase system. The study provides a new insight into the effects of bacteria on the juvenile crab molting process in vivo that lead to the modulation of both immune-associated genes and hormones. • Bacterial infection upregulated many immune genes during the molting process. • Steroid hormones and fatty acid-derived hormones were reduced during the molting process with bacterial infection. • Cortisol were significantly reduced after infection with both S. aureus and V. alginolyticus. • Many peptide hormones of mud crab were upregulated after bacterial infection. [ABSTRACT FROM AUTHOR]

The mud crab (Scylla paramamosain) is an important mariculture species, which has been successfully cultivated in inland saline-alkaline water of China. However, the study on the flavor quality of mud crab cultivated in inland saline-alkaline water has not been reported. The aim of this study was to investigate and compare the non-volatile flavor substances in the hepatopancreas and muscles of mud crabs cultured in inland saline-alkaline water (IS, 1.5 psu), coastal low salinity seawater(CS, 5 psu) and normal salinity seawater(NS, 13 psu). The results showed that there were no significant differences in total free amino acids (TAA) and essential amino acids (EAA) in muscle and hepatopancreas among the three groups. The contents of glycine (Gly) and alanine (Ala) in muscle and hepatopancreas in IS group were significantly higher than those in NS and CS groups. Taurine (Tau) content in IS-♂ group was significantly higher than that in NS-♂ group. The results of the principal component analysis showed that the similarity of amino acid composition and content in muscle and hepatopancreas of each population was related to the culture environment. To sum up, the mud crabs cultured in inland low saline-alkaline water can maintain the balance of total amino acid and essential amino acid content, the sweet flavor of edible parts was slightly enhanced, while the equivalent umami concentration was decreased. ● Inland saline-alkaline culture is a new way for the development of mud crab industry. ● Inland low saline-alkaline culture has an influence on the flavor of mud crabs. ● The contents of Gly and Ala in the muscle of saline-alkaline mud crabs are higher. ● The changes of free amino acids are related to osmoregulation. [ABSTRACT FROM AUTHOR]

Mud crab, swimming crab, and Chinese mitten crab are the most popular economic crabs cultured in China. However, little knowledge is available about the composition of amino acids, fatty acids, and heavy metals in those cultured crabs, despite the large amounts consumed.Scylla paramamosain,Portunus trituberculatus, andEriocheir sinensiswere, respectively, employed for an investigation of these three crabs. The results on the three representative species revealed that muscle, hepatopancreas, and ovary each had its own compositional pattern. The composition of total amino acids in all the crabs was well balanced. The proportion of flavor amino acid in muscles was higher than that in hepatopancreas and ovary. The composition of total fatty acids showed that two functional unsaturated fatty acids, docosahexaenoic acid (DHA, C22:6n-3) and eicosapentaenoic acid (EPA, C20:5n-3), were both abundant in the three crabs. The ratios of n-3/n-6 polyunsaturated fatty acids (PUFAs) in these crabs also showed that they were rich in n-3 PUFA. Among the seven metals explored, zinc, copper, and chromium were the most abundant. It also deserved attention that these crabs were threatened by high accumulation of cadmium in the edible hepatopancreas, which might arise from the pollution of water used for aquaculture. This data could be used as a reference baseline for monitoring the food properties of the three crabs in levels of amino acids, fatty acids, and metals. [ABSTRACT FROM PUBLISHER]

Abstract: Over past decades, the farming of mud crab Scylla paramamosain has expanded rapidly in China. In our previous experiments, the results showed that the mud crab S. paramamosain in China could be divided into northern and southern populations and these two populations had differences in the adaptive capacity to cultivation environmental conditions. In order to verify this, the present study measured and compared the mitochondrial function and metabolic enzyme activity of these two populations during seasonal acclimatization. It was shown that the mitochondrial respiration rates in the gill, hepatopancreas and muscle of the northern population were significantly higher than those of the southern population in winter, spring and summer although the differences in summer were less pronounced than in winter and spring. In winter and spring, both state 2 and 3 mitochondrial respiration rates measured using either succinate or pyruvate+malate as the substrate in the gill of the northern population were consistently significantly higher than those of the southern population. The measurement of metabolic enzyme activity during different seasons showed that for the mitochondrial cytochrome C oxidase in the gill, hepatopancreas and muscle, the differences in activity between the northern and southern populations were in general consistent with those of the mitochondrial function. For example, the mitochondrial cytochrome C oxidase activities in the gill of the northern population were significantly higher than those of the southern population in winter, spring and summer. However, activity of lactate dehydrogenase, pyruvate kinase and hexokinase in the muscle were slightly different between the northern and southern populations. Only in summer the lactate dehydrogenase and pyruvate kinase activities in the muscle of the southern population were significantly higher than those of the northern population. In summary, the results of the present study showed that compared to the southern population, the northern population had a better compensation mechanism in mitochondrial metabolite level during cooler seasons, which might indicate that the northern population adapts better to low temperature than the southern population. Therefore, the temperature for the farming of the southern population and northern population should be different. [Copyright &y& Elsevier]

Background: The mud crab Scylla paramamosain is an economically important species for aquaculture in China and has sexually dimorphic between females and males. Understanding sex differentiation in this species is essential for the development of monosex aquaculture. The Dmrt genes play a vital role in sex differentiation in animals., Methods and Results: In this study, two dmrt-like transcript variants, Spdmrt-like-tv1 and Spdmrt-like-v2, were cloned. SpDmrt-like-tv1 contained a DM domain, while SpDmrt-like-tv2 contained a DM and a DMA domain. Spdmrt-like-tv1 and Spdmrt-like-tv2 were both specifically expressed in testis. During testicular development, the expression level of Spdmrt-like-tv1 increased from stage I to stage II (P > 0.05) and then decreased from stage II to stage III (P < 0.05). The expression level of Spdmrt-like-tv2 in stages I and II was significantly higher than that in stage III (P < 0.05). During embryonic development, the expression level of Spdmrt-like-tv1 was higher in the mid-embryonic stage compared with the early and late stages, but the differences were not significant. Moreover, the expression level of Spdmrt-like-tv2 was stable and remained high throughout embryonic development. Furthermore, the expression level of Spdmrt-like-tv2 was significantly higher than that of Spdmrt-like-tv1. Knockdown of Spdmrt-like variants indicated that the regulative target gene of Spdmrt-like-tv1 was Spsox21, and the regulative target genes of Spdmrt-like-tv2 were Spfoxl2 and Spsox21. Combined with the results in our previously published peer-reviewed articles that the expression of Spfoxl2 in the testis was significantly higher than that in the ovary, and Spfoxl2 negatively regulated Spvtg expression. Spsox21 played a role in the development and maintenance of testis as well as in the process of neural development and regulation of body segmentation., Conclusion: Therefore, we suggest that Spdmrt-like-tv1 and Spdmrt-like-tv2 might be involved in testicular development and embryonic development, and Spdmrt-like-tv2 might play more important roles in these two developmental processes by regulating the expression of Spfoxl2 and Spsox21 due to its high expression., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The mud crab (Scylla paramamosain) is a carnivorous portunid crab, mainly distributed along the southeastern coast of China. Mitochondrial DNA analysis in a previous study indicated a high level of genetic diversity and a low level of genetic differentiation. In this study, population genetic diversity and differentiation of S. paramamosain were investigated using nine microsatellite markers. In total, 397 wild specimens from 11 locations on the southeastern coast of China were sampled and genotyped. A high level of genetic diversity was observed, with the number of alleles, and the observed and expected heterozygosity per location in the range 7.8-9.6, 0.62-0.77 and 0.66-0.76, respectively. AMOVA analysis indicated a low level of genetic differentiation among the 11 locations, despite the fact that a statistically significant fixation index (FST) value was found (FST=0.0183, P<0.05). Out of 55 pairwise location comparisons, 39 showed significant FST values (P<0.05), but all of them were lower than 0.05, except for one between Sanmen and Shantou locations. No significant deficiency of heterozygotes (inbreeding coefficient FIS=0.0007, P>0.05) was detected for all locations except Sanmen and Zhanjiang. Cluster analysis using UPGMA showed that all locations fell into one group except Sanmen. Significant association was found between genetic differentiation in terms of FST/(1-FST) and the natural logarithm of geographical distance (r2=0.1139, P=0.02), indicating that the genetic variation pattern closely resembled an isolation by distance model. This study supports the proposal of high genetic diversity and low genetic differentiation in S. paramamosain along the southeastern coast of China. [ABSTRACT FROM AUTHOR]

Abstract: In this paper, we analyzed population genetic diversity of Scylla paramamosain in Hainan Island of China based on COI gene sequence. Totally 92 individuals were collected from six localities: Haikou, Wenchang, Wanning, Sanya, Dongfang and Danzhou. A 761 bp fragment was sequenced, defining 32 different haplotypes. H4 was the most frequent haplotype, existing in all six localities, while the majority of haplotypes were rare ones, existing in only one or two individuals. Haplotype diversity (h) and nucleotide diversity (π) ranged from 0.625 to 0.914 and from 0.001 to 0.003, with an average of 0.841 and 0.002, respectively. Genetic distance ranged from 0.001 to 0.003 within localities and from 0.002 to 0.003 between localities. The AMOVA analysis indicated a low level of genetic differentiation among six localities (F ST = 0.0176, P > 0.05). Neutrality tests and mismatch analysis implied a potential population expansion event for this crab species. [Copyright &y& Elsevier]

Abstract: The climatic oscillations of the Pleistocene are believed to have greatly impacted the distribution and population dynamics of many terrestrial and marine taxa. Studies investigating the evolutionary effects of palaeo-climatic fluctuations in the East Asia Seas are scarce and the limited studies available suggest two competing evolutionary patterns: high genetic diversity and clear population structure or low genetic diversity and homogeneous population structure. To explore the general consequence of sea level oscillations in the East Asia Seas, the population demographic history and genetic structure of the mud crab, Scylla paramamosain, were investigated using specimens sampled from 11 localities along the Chinese coast. Molecular data (COI, 16S rDNA and concatenated sequences) depicted a star-like tree and suggested a recent population demographic expansion for the species. Through pairwise F ST comparisons, although shallow differentiation was found between the Beibu Bay and other sites, a genetic homogeneous population structure with high gene flow (Nem>5) was observed between most localities. Lower sea levels during the penultimate glaciation (ca. 140kya) and the sea level highstand of MIS5e (130–116kya) could be responsible for the population bottleneck and subsequent demographic expansion inferred for S. paramamosain, and the reversal of interglacial seasonal circulations and long larval development may have facilitated gene flow leading to a homogeneous population structure via planktonic larvae transport between the South China and East China Seas. These molecular, ecological and oceanographic clues indicate that the basin model of recruitment may provide the best explanation for the genetic structure and population history revealed for S. paramamosain. [Copyright &y& Elsevier]

• The full-length cDNAs of alp and acp of Scylla paramamosain were identified. • Expression of Spalp and Spacp were the highest levels in the hepatopancreas. • The core promoter region of the 5′-flanking region of Spalp and Spacp were confirmed. • Potential transcription factor binding sites were found in the 5′-flanking regions. • This study is the first one to analyze the activity of Spalp and Spacp promoters. In crustaceans, innate immunity serves as the frontline of defense against microbes. Alkaline phosphatases (ALPs) and acid phosphatases (ACPs) are essential enzymes that play a significant role in crustaceans' immune defenses. However, the function and transcriptional regulation of the alp and acp genes in the Scylla paramamosain , an important aquaculture species in China, have not been elucidated. In this study, the full-length cDNAs of Spalp and Spacp were identified, which consist of 2,718 bp and 3,768 bp, encoding 579 and 452 amino acids, respectively. Multiple sequence alignment and phylogenetic analysis showed that these two genes were conserved among different species and shared high homology with crustaceans. The mRNA expression of Spalp and Spacp were examined in eight tested tissues, with the highest levels in the hepatopancreas. The 5′-flanking regions of Spalp and Spacp were cloned and sequenced. The core promoter region of the Spalp and Spacp was −39 bp∼+8 bp and −39 bp∼+10 bp, respectively. Potential binding sequences for SOX-2, c-fos, SP1, NF-κB, GATA-1, YY1, and AP-1 transcription factors were found in the 5′-flanking regions of Spalp and Spacp. The NF-κB binding site located between −1,223 bp and −972 bp in Spalp while SP1 and AP-1 binding sites located between −1,249 bp and −514 bp in Spacp. Mutation analysis confirmed that NF-κB negatively regulated the expression of Spalp gene, and SP1 and AP-1 positively regulated Spacp gene expression. These results provide us with essential information to elucidate the function of the Spalp and Spacp in S. paramamosain. This study is the first one to analyze the activity of Spalp and Spacp promoters. [ABSTRACT FROM AUTHOR]

The article discusses a study of the cell structure and seasonal changes of the androgenic gland (AG) of the mud crab Scylla paramamosain acquired from local fish markets in Hong Kong, China. The chemicals and techniques used in dissecting microscopy and histological observations are cited. A description of the cells in different parts of the AG is offered. The ultrastructure of AG cells in mature crabs is comparable to that of type II AG cells. Crabs demonstrated differences in morphology and histology of the AG including gland size and cell boundary.

To evaluate the potential of stock enhancement for decapod crustaceans, we analyzed stock enhancement programs for 4 species: the penaeid prawns (Penaeus japonicus) in Japan and (Penaeus chinensis) China, the European lobster (Homarus gammarus) in Norway, and the mud crab (Scylla paramamosain) in Japan, for which stocking effectiveness has been intensively examined. For each species, we summarized the life cycle, fishery, and stocking programs and then comparatively analyzed the recapture rates, the yield from the released individuals (YPR), and the economic efficiency. Scylla paramamosain had some advantages in stock enhancement, including restricted living areas such as bays and estuaries, a large body size, and high values of the ratio of price per landed individual to the cost per released juvenile. These characteristics promised higher values of YPR and economic efficiency, even at lower recapture rates. Therefore, hatchery releases affect areas with small stocks of S. paramamosain. This scenario might also be inferred for H. gammarus, which had the highest YPR and does not generally migrate from the release site, although the economic efficiency was low due to the high cost of juvenile production. On the other hand, fishery productions of the penaeid prawns, which had a lower YPR, could not be augmented without a huge number of released juveniles and the maintenance of nursery habitats. The restocking is a more cost-effective measure for sustainable fishery production than only a sea ranching (put and take) operation. Therefore, hatchery releases for decapod crustaceans should be applied to rebuild wild stocks (restocking) with appropriate fishing regulations and habitat restoration. In the future, selection of target species for stock enhancement programs should take into account the necessity for restocking and their life history characteristics. [ABSTRACT FROM AUTHOR]

Mud crab (Scylla paramamosain) is an economically important cultured species in China. Hypoxia is a major environmental stressor during mud crab culture. In the present study, we investigated the oxidative stress and transcriptome changes in the gills of mud crab after intermediate hypoxia stress with dissolved oxygen (DO) 3.0 ± 0.2 mg/L (named as "DO3") and acute hypoxia stress with DO 1.0 ± 0.2 mg/L (named as "DO1") for 0, 3, 6, 12 and 24 h. The superoxide dismutase (SOD) activity of DO1 increased significantly at 3, 6 and 24 h after hypoxia stress, while SOD activity of DO3 increased significantly at 6 and 24 h. The total antioxidant capacity (T-AOC) increased significantly at 6, 12 and 24 h after hypoxia stress. The malondialdehyde (MDA) concentration of DO1 increased significantly at 6, 12 and 24 h after hypoxia stress, while MDA concentration of DO3 only increased significantly at 6 h. The lactate dehydrogenase (LDH) activity of DO1 increased significantly at 3, 6, 12 and 24 h after hypoxia stress, while LDH activity of DO3 increased significantly at 12 and 24 h. Transcriptomic analysis was conducted at 24 h of gill tissues after hypoxia stress. A total of 1052 differentially expressed genes (DEGs) were obtained, including 394 DEGs between DO1 and DO3, 481 DEGs between DO1 and control group, 177 DEGs between DO3 and control group. DEGs were enriched in the pathways related to metabolism, immune functions, ion transport, and signal transduction. Transcriptional analysis showed that glycolysis and tricarboxylic acid cycle genes were the key factors in regulating the adaptation of mud crab to hypoxia stress. [Display omitted] • First transcriptome of Scylla paramamosain gills response to hypoxia stress • A total of 1052 DEGs were obtained, and 394, 481 and 177 DEGs were gained respectively from DO1&DO3, DO1&CK and DO3&CK. • Numerous DEGs were involved in multiple metabolic-relevant pathways. [ABSTRACT FROM AUTHOR]

Scylla paramamosain is an economically important cultured crab species in China. Cyclins and cyclin-dependent kinases (CDKs) play important roles in regulations of cell cycle and ovarian development. MiRNAs can negatively regulate gene expression at the post-transcriptional level through base-complementary pairing with the 3'-untranslated region (3-UTR) of the target gene. In this study, bioinformatics prediction showed that miR-9c and miR-263a identified from our group's gonad miRNAome of S. paramamosain may bind to the 3' UTR region of cyclin A , cyclin B , cyclin E , cyclin H , CDK1, and CDK2. Furthermore, the results of double luciferase reporter gene assay showed that the luciferase activities of HEK293T cells co-transfected with miR-9c mimics/miR-9c inhibitor and the 3'-UTR plasmid vectors of the five genes (cyclin A , cyclin B , cyclin H , CDK1, and CDK2) were significantly decreased/increased compared with those in the NC (negative control) and BC (blank control) groups. The results in miR-263a were similar to miR-9c, but all of the six genes could be regulated by miR-263a. In in vivo experiments, agomiR-9c (miR-9c enhancer) injection resulted in decreases of cyclin A and CDK1 expression level, and reverse effects were observed by injecting antagomiR-9c. AgomiR-263a decreased the expression of cyclin A , cyclin B , cyclin H , CDK1, and CDK2, but antagomiR-263a increased their expression. Both the in vitro and in vivo experiments confirmed functions of miR-9c and miR-263a in cell cycle progress of ovarian development by expression regulation of cyclin A , cyclin B , cyclin E , cyclin H , CDK1, and CDK2. The findings provide new insights into the reproductive regulation mechanism in mud crab and further enrich the knowledge of cell cycle and ovarian development regulation in invertebrates. [Display omitted] • The regulation of miR-9c and miR-263a on cyclins and CDKs was confirmed in Scylla paramamosain. • MiR-263a can inhibit the expression of cyclin A , cyclin B , cyclin E , cyclin H , CDK1 , and CDK2. • MiR-9c only inhibit the expression of cyclin A and CDK1. • MiR-263a may play a more essential role than miR-9c in regulating cell cycle genes in S. paramamosain. • The change of vtg expression level manifests that cyclins and CDKs can regulate ovarian development. [ABSTRACT FROM AUTHOR]

In May 2017, there was a massive mortality of mud crab Scylla paramamosain in a farm of Sanmen, Zhejiang Province, China. The bacterial isolates from the hepatopancreas and muscle of the diseased S. paramamosain showed the same phenotypic traits and biochemical profiles, and were identified as Photobacterium damselae subsp. damselae in accordance with the biochemical characteristics and molecular identification with the 16S rRNA and ureC genes. The morphological observation suggested that P. damselae subsp. damselae QX175062 strain was a rod-shaped bacterium with singlet flagellum. Bacterial infection tests were conducted by intramuscular injection of the QX175062 isolate into healthy mud crabs, and the results indicated that the isolate was lethal to S. paramamosain with the LD 50 value of 3.63 × 101 CFU/g (body weight). The challenged mud crabs showed similar pathological signs as those that were naturally infected. Histopathological analysis of mud crabs injected with QX175062 isolate showed agglutination and necrosis of hepatopancreatic tubules in hepatopancreas; ruptured or disordered tissue and fiber dissolution in muscle. Antibiogram study revealed that QX175062 isolate was mainly sensitive to tetracyclines, aminoglycosides, macrolides and quinolones, but resistant to β-lactam antibiotic drugs. This first case report underlines the involvement of P. damselae subsp. damselae in the mud crab aquaculture with disease outbreak and emphasizes the need of developing rapid diagnostic methods and prevention measures to reduce the economic loss caused by P. damselae subsp. damselae infection in mud crab aquaculture. • Photobacterium damselae subsp. damselae (PDD) was firstly isolated from mud crab cultured in China. • PDD was identified by biochemical and phylogenetic analyses. • The pathogenicity of isolated PDD to mud crab was confirmed by challenge experiments. • Histopathological analysis of mud crabs injected with PDD showed pathological changes in hepatopancreas and muscle. • The isolated PDD strains were highly sensitive to aminoglycosides, macrolides, tetracyclines and quinolones antibiotics. [ABSTRACT FROM AUTHOR]

Mud crab (Scylla paramamosain) is an economically important marine crustacean species commonly distributed in China and Indo-west Pacific countries. However, the disease outbreaks caused by Vibrio parahemolyticus have resulted in high mortality and disastrous economic losses in the mud crab culture industry. Immune priming has shown the capacity of protecting invertebrates against pathogens in the second exposure, with profound implications for the prevention of infectious diseases. In this study, the survival and immune responses of mud crabs received primary exposure to formalin-killed V. parahemolyticus (immune priming, IP) and secondary challenge with live counterparts (lethal challenge, LC) were investigated. The priming dose of 2 × 105 cells·g−1 of inactivated V. parahemolyticus showed the highest survival rate (75.7%). There was no significant difference among the primed groups that received lethal challenge at different time points (P >.05). The cumulative survival of the primed (FVP) group (73.5%) was significantly higher than that of the control (SA) and naïve groups after the second challenge with live V. parahemolyticus. The efficiency of pathogen clearance, total hemocyte count, and phagocytic rate were significantly increased in FVP group compared to SA group after the IP and LC treatments. By contrast, no significant changes in antibacterial activity, phagocytic index and phenoloxidase activity (excepting at 6 hpi) were found in the comparison between FVP and SA groups. These results suggested the occurrence of priming phenomenon in mud crab, primary immunization with inactivated V. parahemolyticus can improve survival following infection and enhance cellular immune responses, the particularly phagocytic rate that contributes to the elevated phagocytosis. This finding provides a novel direction in protecting mud crab against vibriosis. • Priming with inactivated V. parahemolyticus improved the survival of mud crab after infection. • Immune priming accelerated the bacterial clearance in the hemolymph of mud crab. • Increased phagocytic rate of hemocytes was responsible for the elevated phagocytic activity. • The priming-induced proliferation of phagocytic cells may contribute to the enhanced phagocytosis. [ABSTRACT FROM AUTHOR]

The mud crab, Scylla paramamosain , is an economically-important crab in China. Air exposure is an important environmental stressor during mud crab culture and transportation. Adaptive mechanisms responding to air exposure in mud crabs are still poorly understood. In this study, mud crabs were exposed to air for 120 h. Air exposure decreased total hemocyte counts, led to cytological damage, and caused high mortality. Transcriptomic analysis was conducted at 0, 6 and 96 h after air exposure. A total of 3530 differentially expressed genes (DEGs) were identified. DEGs were mainly involved in the oxidative stress response, metabolism, cellular processes, signal transduction, and immune functions. Transcriptomic analysis also revealed that genes of glycolysis and of the tricarboxylic acid cycle were key factors in regulating the mud crab adaptation to air exposure. Unlabelled Image • Air exposure induced oxidative stress, and led to cytological damage. • Some genes involved in response to air exposure were found. • Glycolysis and tricarboxylic acid cycle related pathways associated with air exposure stress were altered. [ABSTRACT FROM AUTHOR]

In this study, a Gram-negative bacterium was isolated from diseased Scylla paramamosain and tentatively named strain QX17. The bacterial isolate was identified as Vibrio parahaemolyticus based on morphological and biochemical characteristics and molecular identification with the 16S rRNA and HSP60 genes. In the challenge experiment, S. paramamosain injected intramuscularly with the V. parahaemolyticus isolate developed pathological signs similar to the naturally diseased mud crabs. The infection experiment also showed that the median lethal dosage (LD50) for QX17 was 4.79 × 102 CFU g-1 (crab weight). Histopathological analysis of the diseased mud crabs infected with V. parahaemolyticus showed deformation and basement membrane rupture of hepatopancreatic tubules in the hepatopancreas, and disordered and broken muscle fiber in the muscle. Antimicrobial susceptibility tests revealed that QX17 was highly sensitive to most of the tested aminoglycosides, tetracyclines, and quinolones. To the best of our knowledge, this is the first study reporting isolation and antibiotic sensitivities of V. parahaemolyticus from cultured mud crabs. The discovery of V. parahaemolyticus in cultured mud crabs not only adds to the growing list of emerging pathogens in crab aquaculture in China, but also highlights the necessity of developing early detection strategies and appropriate interventions to reduce the damage caused by V. parahaemolyticus in mud crab aquaculture.

The mud crab Scylla paramamosain (Decapoda:Portunidae) is a commercially important crustacean species in China, which is broadly distributed along the southeastern coastal regions of China. In previous studies, the population from Beibu Bay showed distinct genetic lineage. Here, we report the complete mitochondrial genome sequence of S. paramamosain from Beibu Bay. The mitogenome has 15,816 base pairs and made up of total of 37 genes (13 protein-coding, 22 transfer RNAs and 2 ribosomal RNAs), and a putative control region. The complete mitogenome in the Beibu Bay population was shorter than that in the Shenzhen population, because the non-coding region between tRNA-Glu and tRNA-His was shorter in the former, and there were 59 mutations sites between these two populations. This study adds a distinct mitogenomes of S. paramamosain and will provide useful genetic information for future genetic variation identification and genetic diversity evaluation of this economic valuable crab. [ABSTRACT FROM AUTHOR]

Background: Scylla paramamosain is one of the commercially crucial marine crustaceans belonging to the genus Scylla, which is commonly distributed along the coasts of China, Vietnam, and Japan. Genomic and transcriptomic data are scarce for the mud crab. Light intensity is one of the ecological factors that affect S. paramamosain during indoor overwintering. To understand the energy metabolism mechanism adapted to light intensity, we analyzed the transcriptome of S. paramamosain hepatopancreas in response to different light intensities (0, 1.43, 40.31 μmol·m - 2 ·s - 1 )., Results: A total of 5052 differentially expressed genes were identified in low light group (LL group, 3104 genes were up-regulated and 1948 genes were down-regulated). A total of 7403 differentially expressed genes were identified in high light group (HL group, 5262 genes were up-regulated and 2141 genes were down-regulated). S. paramamosain adapts to different light intensity environments through the regulation of amino acids, fatty acids, carbon and energy metabolism. Different light intensities had a strong impact on the energy generation of S. paramamosain by influencing oxygen consumption rate, aerobic respiration, glycolysis/gluconeogenesis pathway, the citrate cycle (TCA cycle) and fatty acid degradation., Conclusion: Low light is more conducive to the survival of S. paramamosain, which needs to produce and consume relatively less energy to sustain physiological activities. In contrast, S. paramamosain produced more energy to adapt to the pressure of high light intensities. The findings of the study add to the knowledge of regulatory mechanisms related to S. paramamosain metabolism under different light intensities.

Scylla paramamosain (Crustacea) is a commercially important euryhaline species distributed along the coast of southern China and other Indo-Pacific countries. However, a sudden variation in salinity will cause injury or even death to S. paramamosain. In this paper, we simulated a sudden decrease in salinity due to heavy precipitation in crab ponds. Comparison of gill microstructures of individuals in the control group and decreased salinity group showed gills became shorter and thicker, while the top of the filaments became swollen and then returned to normal after 120 h. A total of 3962 proteins were identified by proteomic sequencing of gills after 120 h under conditions of decreased salinity. 845 proteins were differentially expressed: 371 up-regulated and 474 down-regulated. Of the enriched KEGG pathways, 20 were up-regulated and 14 were down-regulation (P < 0.05). Among the significantly enriched up-regulated pathways, six were associated with amino acid metabolism and three were associated with Na + -K + -ATPase enzymatic activities. Pathways associated with redox metabolism and energy metabolism were identified. These results showed that in response to a decrease in salinity, S. paramamosain could adapt to the environment after 120 h. Molecular mechanism of this adaptation involved amino acid metabolism and Na + -K + -ATPase ion transport. Meanwhile, energy metabolism and redox metabolism were critical to the adaptation to a sudden decrease in salinity. This study, for the first time at the protein level, revealed the molecular mechanisms underlying salinity adaption of S. paramamosain and provides theoretical guidelines for the cultivation of S. paramamosain and other marine crustaceans., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Mud crab dicistrovirus-1 (MCDV-1) was isolated from the mud crab (Scylla paramamosain), resulting in mass mortality and widespread economic loss in China. In this study, a detection method for MCDV-1 using loop-mediated isothermal amplification was developed. Two pairs of primers targeting the VP2 gene were designed. These primers were the outer primers F3 and B3, and the inner primers FIP and BIP. Optimal amplification was carried out using 0.2 μmol/L F3/B3, 1.6 μmol/L FIP/BIP, 6 mmol/L Mg(2+), 0.8 mmol/L dNTPs, and 0.8 mol/L betaine, and completed in 1h at 62°C. The products demonstrated a ladder pattern on agarose gel electrophoresis and could also be detected visually according to turbidity, or by adding SYBR Green I and observing a color change from orange to green. The proposed method could specifically amplify MCDV-1 gene fragments. Sensitivity assay revealed that six copies of the viral genome could be detected by this method, which was 1000-fold more sensitive than that of conventional PCR using constructed plasmid as amplification template. At clinical sample level, sensitivity of LAMP was 100-fold higher than that of conventional PCR., (Copyright © 2014 Elsevier B.V. All rights reserved.)