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Reconstruction and annotation of transcripts, particularly for a species without reference genome, plays a critical role in gene discovery, investigation of genomic signatures, and genome annotation in the pre-genomic era. This is the first study to use Single-molecule real-time (SMRT) sequencing for reporting the full-length transcriptome of Portunus pelagicus. Overall, 16.26 Gb of raw reads were obtained, including 7,068,387 subreads, with average length of 2,300 bp and N50 length of 3,594 bp. In total, 351,870 circular consensus sequences (CCS) reads were extracted, including 255,378 full-length non-chimeric (FLNC) reads with mean length of 3,423 bp.70,407 genes were obtained after eliminating redundant sequences, and 56,557 (80.33%) genes were annotated in at least one database, 17,267 (24.52%) genes were annotated in all of the seven databases. Further, 68,797 coding sequences (CDS) were identified, including 36,848 complete CDS. A total of 1,730 unigenes were predicted to be transcription factors (TFs). Finally, 11,894 long noncoding RNA (lncRNA) transcripts were predicted by different computational approaches and 147,262 single sequence repeat (SSR)s were obtained. The transcriptome data reported herein are bound to serve as a basis for future studies on P. pelagicus.

Portunus trituberculatus (Crustacea: Decapoda: Brachyura), commonly known as the swimming crab, is of major ecological importance, as well as being important to the fisheries industry. P. trituberculatus is also an important farmed species in China due to its rapid growth rate and high economic value. Here, we report the genome sequence of the swimming crab, which was assembled at the chromosome scale, covering ~1.2 Gb, with 79.99% of the scaffold sequences assembled into 53 chromosomes. The contig and scaffold N50 values were 108.7 kb and 15.6 Mb, respectively, with 19,981 protein-coding genes and a high proportion of simple sequence repeats (49.43%). Based on comparative genomic analyses of crabs and shrimps, the C2H2 zinc finger protein family was found to be the only gene family expanded in crab genomes, and its members were mainly expressed in early embryonic development and during the flea-like larval stage, suggested it was closely related to the evolution of crabs. Combined with transcriptome and Bulked Segregant Analysis (BSA) providing insights into the genetic basis of salinity adaptation in P. trituberculatus, strong immunity and rapid growth of the species were also observed. In addition, the specific region of the Y chromosome was located for the first time in the genome of P. trituberculatus, and Dmrt1 was identified as a key sex determination gene in this region. Decoding the swimming crab genome not only provides a valuable genomic resource for further biological and evolutionary studies, but is also useful for molecular breeding of swimming crabs.

Water salinity condition is an important factor for artificial propagation of the swimming crab (Portunus trituberculatus). Low salinity (LS)-resistant strains are preferred by crab industries. Single nucleotide polymorphism (SNP), the third generation of molecular markers, can be utilized in the breeding of LS-resistant species of P. trituberculatus. Our earlier study identified 615 genes differentially expressed in low-salinity stress compared to the controls. Although thousands of SNP loci have been found, it is hard to identify a SNP marker in correlation with a desired trait. In this study, time-of-flight mass spectrometry (TOF-MS), as an efficient method to select SNPs for the tolerance of LS challenge, was utilized for SNP typing. Fifty gene segments were amplified based on comparative transcriptomics in our earlier study, a total of 18 511 bp DNA fragments were amplified, and eighty-five SNP markers were found. The frequency of the SNPs was estimated to be 0.46 per 100 base pairs of DNA sequences. The rate of the conversion mutation was 81%, while the transversion mutation was 19%. The mutation rate of the G/T (C/A), A/T and G/C was 26%, 12% and 7%, respectively. Eight SNP markers were found to significantly correlate with the adaption of low salinity. Of the eight SNP markers, three linked-SNPs were found in the cuticle proportion gene, and another three SNPs were found in three new genes, and the rest two were found in aquaporin gene and chloride channel gene. The development of these SNP markers found in our study could be primarily used for breeding LS-resistant strains of P. trituberculatus.

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

Salt-alkali tolerance is one of the important breeding traits of

The crustacean hyperglycemic hormone (CHH) gene of Portunus trituberculatus (Pt-CHH) consists of four exons and three introns spanning 3849 bp in size and generating two mature mRNA, Pt-CHH1, and Pt-CHH2. The primary gene transcript produces a cDNA encoding for the putative Pt-CHH2 from exons 1, 2, 3, and 4 and an alternative transcript encodes for a putative Pt-CHH1 peptide from exons 1, 2, and 4. A promoter fragment of about 3 kb was obtained by genomic walking. The tissue-specific expression pattern is examined by reverse transcriptase chain reaction, and the results show that Pt-CHH1 is detected in the eyestalk, brain, muscle, and blood. However, Pt-CHH2 is detected in the ganglia thoracalis and gill. The results indicate that the expression of Pt-CHH2 in the gill might suggest a potential role in osmoregulation. The Pt-CHH transcript level in the gill increases when the crab is exposed to low salinity. The injection of dsRNA for Pt-CHH causes a significant reduction in Pt-CHH2 transcript level and the activity of Na(+)/K(+)-ATPase, and carbonic anhydrase (CA) show a serious decrease. In conclusion, this study provides molecular evidence to support the osmoregulatory function of Pt-CHH2.

We analysed the metabolic rate of large individuals (LIs) and small individuals (SIs) (based on body weight) of 80‐day‐old specimens of Portunus trituberculatus injected with 40 mg/kg florfenicol (FLR). The results showed that FLR contents in the gills and hepatopancreas were higher in the SIs than in the LIs. The activities of erythromycin N‐demethylase and 7‐ethoxyresorufin‐O‐deethylase related to cytochrome oxidase P450 (CYP) were induced after FLR injection and showed a size‐dependent pattern; however, no significant difference in aminopyrine N‐demethylase activity was found between the LIs and SIs. The activity of the phase II enzyme glutathione S‐transferase (GST) increased in the hepatopancreas after FLR injection, and it was higher in the SIs than the LIs. Moreover, induction of CYP2, CYP3, GST, ABCB and ABCG was observed in the first 24 hr after FLR injection. FLR caused more biomolecular damage in the SIs than the LIs, displaying an obvious body weight‐ and time‐dependent pattern. In summary, the SIs exhibited faster FLR uptake and slower depuration in this species, and FLR caused more biomolecular damage to the SIs.

Portunus trituberculatus is one of the main mariculture crabs in China and has obvious sexual dimorphism. The development of a genetic sex identification method is key to clarifying the mechanisms of sex determination. Based on an insertion-deletion (inDel) marker closely linked to the sex of P. trituberculatus, we successfully established a sex identification method with the advantages of high efficiency, low cost and low false-positive rates using conventional PCR and agarose gel electrophoresis. Based on the method, the sex ratio of seven full-sib families at different developmental stages were analyzed, including zoea, megalopa and II juvenile crab. Our results showed that the sex ratio of P. trituberculatus in early development stages approximated the theoretical sex ratio (1:1) of the XY sex determination system, although there were three families with sex ratio deviations from 1:1 at the juvenile stage. This study is important for detailing the mechanisms of sex determination, and is also conducive to the development of sex-control technologies in this species.

The aim of the present study was to evaluate the possible effects of inbreeding on the carbohydrate metabolism of Portunus trituberculatus juveniles from different inbred families by measuring the activities of carbohydrate metabolism-related enzymes and glycogen content. The results show that inbreeding weakens the activities of hexokinase in the hepatopancreas and muscle; pyruvate kinase, muscles and serum; and lactate dehydrogenase, gills, but enhances the activity of succinate dehydrogenase in the muscles and gills. The real-time PCR results indicated that the relative mRNA expression levels of the metabolism-related genes decreased as the inbreeding generations increased. The results illustrate that inbreeding gradually reduces carbohydrate metabolism in P. trituberculatus.

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.

The swimming crab, Portunus trituberculatus, is an important farmed species in China, and it has been studied extensively, which requires more information on its genetic background. To date, information on the proteomics of P. trituberculatus is scarce. In this study, we used isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics to investigate growth regulation in P. trituberculatus. Total proteins were isolated from five tissues (eyestalk, gill, heart, hepatopancreas and muscle). Equal quantities of protein from each tissue were pooled at the proteome level using the iTRAQ method. A total of 961 proteins were identified using liquid chromatography coupled with tandem mass spectrometry and de novo sequencing data. Using a 1.2-fold change in expression as a physiologically significant benchmark, 30 differentially expressed proteins (DEPs) were found to undergo differential expression related to the growth of P. trituberculatus, and most of the growth-related proteins, including those involved in metabolism, immune responses, DNA duplication and protein synthesis, were upregulated, indicating that conservation of energy is an important strategy to cope with growth. There was a high consistency between the expression levels determined using iTRAQ and mRNA, highlighting the high reproducibility of our proteomic approach and its great value in elucidating the molecular mechanisms of P. trituberculatus.

Vibrio alginolyticus is a virulent pathogen that affects crab aquacultures. In the present study, the immune responses and antioxidant status of big and small (based on body weight and size) 80-, 100- and 120-day-old specimens of Portunus trituberculatus, challenged for 72 h with Vibrio alginolyticus, were studied. The total hemocyte count (THC), and phagocytic, prophenoloxidase and phenoloxidase activities, of the big individuals (BIs) were higher than those of the small individuals (SIs) (P

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. The primary objective of this study was to describe the salinity adaptation mechanism in P. trituberculatus. 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). 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. 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.

Chitinases play an important role in many biological processes in crustaceans, including molting, digestion, and immunity. In order to further explore the immune defense mechanism of chitinase in Portunus trituberculatus, the PtCht-1 gene was cloned by RACE (rapid-amplification of cDNA ends). This cDNA with a full length of 1910 bp, and an ORF (open reading frame) 1749 bp, coded for 582 amino acid residues and was classified into P. trituberculatus chitinase GH18-group4. It had the typical structural characteristics of GH18 chitinase family. Real-time PCR was used to analyze the expression of PtCht-1 in different tissues, molting stages, after pathogen infection, and low salinity (11‰). PtCht-1 was expressed in all tissues, with the highest expression in the hepatopancreas. In the hepatopancreas of different molting stages, the expression level decreased successively during post-molt stages (A/B), pre-molt stage (D) and inter-molt stage (C). Under normal circumstances, after artificial infection with WSSV and Vibrio parahaemolyticus, the expression of PtCht-1 in hepatopancreas reached the maximum at 48 h, and in hemolymph at 72 h and 24 h, respectively. Overall PtCht-1 expression was up-regulated compared with the control group. Low salinity stress significantly inhibited the expression of PtCht-1, up to 42 folds. Under low salinity stress, the time when WSSV infection reached the peak was markedly delayed by at least 24 h. The results of this study indicate that PtCht-1, as an immune factor, is likely involved in pathogen defense of P. trituberculatus, the immune function of which may be inhibited to some extent after low salinity stress.

The purpose of this study was to investigate the effects of inbreeding on the immune responses and antioxidant status of Portunus trituberculatus juveniles. Results showed that inbreeding affected the total haemocyte count, and phagocytic, pro-phenoloxidase (propo), phenoloxidase and antibacterial activities decreased after the seventh generation. Antioxidant status showed a similar pattern: total antioxidant capacity, superoxide dismutase (SOD) activity and GSH/GSSG in the cell-free haemolymph and hepatopancreas decreased, while catalase activity in the cell-free haemolymph increased (P

MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate gene expression in organisms. To understand the underlying mechanisms behind the molecular response of the crab to low salt-stress, high-throughput Illumina/Solexa deep sequencing technology was used to investigate the expression profiles of miRNAs under low salinity challenged. Two mixed RNA pool libraries of gill tissues from low salinity challenged (LC) and the control groups (NC) were sequenced on the Illumina platform. A total of 6,166,057 and 7,032,973 high-quality reads were obtained from the NC and LC libraries, respectively. Sixty-seven miRNAs consisting of 16 known and 51 novel ones were identified, among which, 12 miRNAs were differentially expressed in LC compared to NC. Thirty-four of the target genes predicted were differentially expressed in the opposite direction to the miRNAs, which were involved in crucial processes related to osmoregulation by gene ontology (GO) functional enrichment analysis, such as anion transport processes (GO:0006820) and chitin metabolic process (GO:0006030). These results provide a basis for further investigation of the miRNA-modulating networks in osmoregulation of Portunus trituberculatus.

Portunus trituberculatus is an important farmed crustacean in China. Growth traits are important economic traits that are controlled by many genes. The aim of this study was to screen growth-related genes using Illumina paired-end sequencing technology, and to identify growth-related SNPs in P. trituberculatus by association analysis. A total of 36,599,712 and 29,247,160 qualified Illumina read pairs for small size (SG) and large size crabs (LG) were obtained, giving rise to total clean bases of 7.14 and 5.72 G, respectively. A gene expression comparison showed that a total of 117 unigenes were differentially expressed between SG and LG, including 49 up-regulated and 68 down-regulated unigenes in SG and LG, respectively. GO functional enrichment analysis results showed up-regulated genes in LG were involved in three biological processes. These were cell communication, ovarian follicle cell development and cell division. Down-regulated genes in LG were involved in five biological processes, which were chitin metabolism, carbohydrate metabolism, ATP hydrolysis coupled proton transport, the steroid hormone mediated signaling pathway and fatty acid biosynthesis. Association analysis identified one growth-associated SNP, which was located in hemocyanin (comp58070-R31). This research has provided information on key genes, SNPs and biological processes related to growth, which will increase the efficiency of selective breeding programs for this important species.

Calreticulin (CRT) is a highly conserved and multifunctional endoplasmic reticulum (ER) chaperone protein and plays important roles in salinity stress response. Portunus trituberculatus is a commercially important fishery species, and water salinity conditions influence its commercial farming significantly. In order to research the function of calreticulin under salinity stress, the full-length cDNA sequence of calreticulin from P. trituberculatus (PtCRT) was firstly cloned and characterized. The complete cDNA sequence of PtCRT is 1676 bp with 1218 bp open reading frame (ORF), encoding a polypeptide of 405 amino acids. Multiple sequence alignments showed that the deduced acid amino sequences of PtCRT shared the highest homology to CRT of Fenneropenaeus chinensis (89 %). Fluorescent quantitative real-time PCR analysis indicated that PtCRT was expressed in all detected tissues and showed the highest expression level in hepatopancreas. In addition, salinity challenge significantly influenced the expression level of PtCRT in gill. Six single nucleotide polymorphisms (SNPs) were detected in cDNA sequence of PtCRT, and one SNP was associated with the salt tolerant trait. All results indicated that PtCRT plays an important role in mediating the salinity adaption of P. trituberculatus.

Na+/K+-ATPases are membrane-associated enzymes responsible for the active transport of Na+ and K+ ions across cell membranes, generating chemical and electrical gradients. These enzymes’ α-subunit provides catalytic function, binding and hydrolyzing ATP, and itself becoming phosphorylated during the transport cycle. In this study, Na+/K+-ATPase α-subunit cDNA was cloned from gill tissue of the swimming crab Portunus trituberculatus by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end methods. Analysis of the nucleotide sequence revealed that the cDNA had a full-length of 3 833 base pairs (bp), with an open reading frame of 3 120 bp, 5′ untranslated region (UTR) of 317 bp, and 3′ UTR of 396 bp. The sequence encoded a 1 039 amino acid protein with a predicted molecular weight of 115.57 kDa and with estimated pI of 5.21. It was predicted here to possess all expected features of Na+/K+-ATPase members, including eight transmembrane domains, putative ATP-binding site, and phosphorylation site. Comparison of amino acid sequences showed that the P. trituberculatus α-subunit possessed an overall identity of 75%–99% to that of other organisms. Phylogenetic analysis revealed that this α-subunit was in the same category as those of crustaceans. Quantitative real-time RT-PCR analysis indicated that this α-subunit’s transcript were most highly expressed in gill and lowest in muscle. RT-PCR analysis also revealed that α-subunit expression in crab gill decreased after 2 and 6 h, but increased after 12, 24, 48, and 72 h. In addition, α-subunit expression in hepatopancreas of crab decreased after 2-72 h. These facts indicated that the crab’s Na+/K+-ATPase α-subunit was potentially involved in the observed acute response to low salinity stress.

This present study aimed to investigate the effects of inbreeding on growth traits and genetic diversity in the swimming crab, Portunus trituberculatus. To this end, full-sibling inbred groups were established at four levels of inbreeding coefficient (F values of 0.25, 0.375, 0.5, and 0.5937). The growth traits of the control group (F ≈ 0) differed significantly from each of the inbred groups at three ages (80, 100 and 120 days old). Significant inbreeding depression was found in all inbred groups (P

Cold temperature can lead to large-scale mortality of overwinter cultured swimming crab Portunus trituberculatus ; however, little is currently known about the physiology of this species under low temperature. The present study was carried out to investigate the effects of cold exposure on physiology of P. trituberculatus . Activities of antioxidant enzymes (SOD and CAT), oxidative damage to lipid and protein (indexed by contents of malondialdehyde (MDA) and protein carbonyl (PC), respectively), and expression of heat shock protein genes ( hsp70 , hsp90- 1 and hsp90-2 ) and a marker of apoptosis, caspase-3 , in muscle and hepatopancreas tissues of the crabs acclimated at the temperatures of 3, 6, 9, 12, 15 and 22 °C (control) for four weeks were measured. SOD and CAT activities in the crabs increased significantly at 9, 12 and 15 °C, but decreased markedly at 3 and 6 °C, compared with those in control crabs. Accumulation of MDA and PC in both muscle and hepatopancreas tissues was observed when the crabs were acclimated at 3 and 6 °C. Abundance of hsp70 , hsp90-1 and hsp90-2 mRNA increased with decreasing temperature, reached the maximum levels at 6 °C and then decreased sharply at 3 °C. Caspase-3 expression, in coincidence with MDA and PC contents, increased significantly at 3 and 6 °C. These results suggested that at low temperatures antioxidant defense system and heat shock proteins in swimming crabs are enhanced to prevent cellular damage and can effectively protect cells against apoptosis; however, cold stress beyond a critical level results in depression of the protective mechanisms, which in turn leads to cellular components damage, and elevated levels of apoptotic cell death. Based on the results of this study, overwintering P. trituberculatus should be cultured at temperatures above 6 °C.

The swimming crab, Portunus trituberculatus, is widely distributed throughout the coastal waters of Asian-Pacific nations and is an important economic species in this region. The aquaculture of swimming crabs has been plagued by problems associated with low growth rates, poor flesh quality, and weak disease resistance. To overcome these problems, selective breeding programs have been suggested as a means of genetically improving these traits in stock populations. In this study, we evaluated the genetic differentiation of 3 different geographical populations (Zhoushan: S; Laizhou Bay: L; and Haizhou Bay: H) using 8 polymorphic microsatellite loci. Nine strains of first filial generation were obtained, with 3 geographically populations as parental stock. We assessed the growth and survival rates of the F1 generation to identify new strains or breeds showing improvements in these economically important traits. Our results indicated that pairwise FST among populations was significantly higher than 0 (P = 0.0000) for every population pair, ranging from 0.0810 to 0.1083 for 3 different geographical populations. We observed significant heterosis for the growth and viability (survival) traits, although some strains (crossbred combinations) showed evidence of hybrid weakness in some growth measurements. One particular strain ("SL") outperformed other combinations, displaying the greatest extent of heterosis over the growth and viability (survival) traits. These results indicate that hybridization may be used to increase the performance of P. trituberculatus in aquaculture.

The swimming crab (Portunus trituberculatus) is an economically important species in Asian aquaculture. Implementing growth-related traits of P. trituberculatus into genetic breeding programmes is an ongoing effort. We used a previously published genetic linkage map of P. trituberculatus, containing 55 simple sequence repeat (SSR) markers and 172 amplified fragment length polymorphism (AFLP) techniques to map quantitative trait loci (QTL) for growth-related traits in a single full sibling F2 family. Ten growth-related traits were measured for QTL mapping. Composite interval mapping identified 9 QTL on the female map and 16 on the male map. Individual QTL with additive effects explained 11–38% of the phenotypic variance for various traits using the female parent's map, and from 1% to 21% using the male parent's map. Two QTL explaining a large percentage of variation in body weight were detected on chromosome 17 on the female map, and on chromosome 16 on the male map, and contributed 38% and 18% of the phenotypic variance respectively. This is the first study to report the detection and positioning of major QTL affecting growth in a true crab species (Brachyura). The mapping of growth-related QTL in this study raises the possibility of improving the growth of P. trituberculatus through marker-assisted selection.

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.

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.