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Salinization of freshwater ecosystems is a pressing global issue. Changes in salinity can exert severe pressure on aquatic animals and jeopardize their survival. Procambarus clarkii is a valuable freshwater aquaculture species that exhibits some degree of salinity tolerance, making it an excellent research model for freshwater aquaculture species facing salinity stress. In the present study, crayfish were exposed to acute low salt (6 ppt) and high salt (18 ppt) conditions. The organisms were continuously monitored at 6, 24, and 72 h using RNA-Seq to investigate the mechanisms of salt stress resistance. Transcriptome analysis revealed that the crayfish responded to salinity stress with numerous differentially expressed genes, and most of different expression genes was observed in high salinity group for 24h. GO and KEGG enrichment analyses indicated that metabolic pathways were the primary response pathways in crayfish under salinity stress. This suggests that crayfish may use metabolic pathways to compensate for energy loss caused by osmotic stress. Furthermore, gene expression analysis revealed the differential expression of immune and antioxidant-related pathway genes under salinity stress, implying that salinity stress induces immune disorders in crayfish. More genes related to cell proliferation, differentiation, and apoptosis, such as the Foxo, Wnt, Hippo, and Notch signaling pathways, responded to high-salinity stress. This suggests that regulating the cellular replication cycle and accelerating apoptosis may be necessary for crayfish to cope with high-salinity stress. Additionally, we identified 36 solute carrier family (SLC) genes related to ion transport, depicting possible ion exchange mechanisms in crayfish under salinity stress. These findings aimed to establish a foundation for understanding crustacean responses to salinity stress and their osmoregulatory mechanisms.

Background: Salinity is one of the main influencing factors in the culture environment and is extremely important for the survival, growth, development and reproduction of aquatic animals.Methods: In this study, a comparative transcriptome analysis (maintained for 45 days in three different salinities, 30 psu (HC group), 18 psu (MC group) and 3 psu (LC group)) was performed by high-throughput sequencing of economically cultured Penaeus monodon. P. monodon gill tissues from each treatment were collected for RNA-seq analysis to identify potential genes and pathways in response to low salinity stress.Results: A total of 64,475 unigenes were annotated in this study. There were 1,140 upregulated genes and 1,531 downregulated genes observed in the LC vs. HC group and 1,000 upregulated genes and 1,062 downregulated genes observed in the MC vs. HC group. In the LC vs. HC group, 583 DEGs significantly mapped to 37 signaling pathways, such as the NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, and PI3K-Akt signaling pathway; in the MC vs. HC group, 444 DEGs significantly mapped to 28 signaling pathways, such as the MAPK signaling pathway, Hippo signaling pathway and calcium signaling pathway. These pathways were significantly associated mainly with signal transduction, immunity and metabolism.Conclusions: These results suggest that low salinity stress may affect regulatory mechanisms such as metabolism, immunity, and signal transduction in addition to osmolarity in P. monodon. The greater the difference in salinity, the more significant the difference in genes. This study provides some guidance for understanding the low-salt domestication culture of P. monodon.

As a highly conserved transcription factor family, DMRTs (Doublesex and Mab-3-related transcription factors) are involved in sex determination and sexual differentiation in numerous animals. In the present study, we identified a DM (Doublesex/Mab-3)-domain gene in Penaeus monodon, which we named PmDMRT11E because it shared many similarities with DMRT11E homologs and has a close relationship in the phylogeny of different species. The three-dimensional structure and amino acid alignments indicated the stability and conservation of the DM domain. Real-time PCR analysis revealed that PmDMRT11E was highly and preferentially expressed in the testis of sub-adult shrimp. During embryogenesis, the expression level of PmDMRT11E was higher at the nauplius than at other periods. PmDMRT11E dsRNA injection significantly decreased the transcription factor Sox9 and increased ovarian marker Foxl2 expression, implying its important regulatory role in the mechanism of male determination. Moreover, dsDMRT11E caused abnormal sperm development, indicating that PmDMRT11E may be closely related to gonadal development and spermatogenesis.

Doublesex (Dsx) is a polymorphic transcription factor of the DMRTs family, which is involved in male sex trait development and controls sexual dimorphism at different developmental stages in arthropods. However, the transcriptional regulation of the Dsx gene is largely unknown in decapods. In this study, we reported the cDNA sequence of PmDsx in Penaeus monodon, which encodes a 257 amino acid polypeptide. It shared many similarities with Dsx homologs and has a close relationship in the phylogeny of different species. We demonstrated that the expression of the male sex differentiation gene Dsx was predominantly expressed in the P. monodon testis, and that PmDsx dsRNA injection significantly decreased the expression of the insulin-like androgenic gland hormone (IAG) and male sex-determining gene while increasing the expression of the female sex-determining gene. We also identified a 5′-flanking region of PmIAG that had two potential cis-regulatory elements (CREs) for the PmDsx transcription. Further, the dual-luciferase reporter analysis and truncated mutagenesis revealed that PmDsx overexpression significantly promoted the transcriptional activity of the PmIAG promoter via a specific CRE. These results suggest that PmDsx is engaged in male reproductive development and positively regulates the transcription of the PmIAG by specifically binding upstream of the promoter of the PmIAG. It provides a theoretical basis for exploring the sexual regulation pathway and evolutionary dynamics of Dmrt family genes in P. monodon.

The black tiger shrimp, Penaeus monodon, is important in both fishery and aquaculture and is the second-most widely cultured shrimp species in the world. However, the current strains cannot meet the market needs in various cultural environments, and the genome resources for P. monodon are still lacking. Restriction-site associated DNA sequencing (RADseq) has been widely used in genetic linkage map construction and in quantitative trait loci (QTL) mapping. We constructed a high-density genetic linkage map with RADseq in a full-sib family. This map contained 6524 single nucleotide polymorphisms (SNPs) and 2208 unique loci. The total length was 3275.4 cM, and the genetic distance was estimated to be 1.1 Mb/cM. The sex trait is a dichotomous phenotype, and the same interval was detected as a QTL using QTL mapping and genome-wide association analysis. The most significant locus explained 77.4% of the phenotype variance. The sex locus was speculated to be the same in this species based on the sequence alignments in Mozambique, India, and Hawaii populations. The constructed genetic linkage map provided a valuable resource for QTL mapping, genome assembly, and genome comparison for shrimp. The demonstrated common sex locus is a step closer to locating the underlying gene.

The experiment simulated the logistical transportation of Penaeus monodon. It set four distinct gradient Vitamin C (Vc) addition levels, including 0mg/L, 111mg/L, 222mg/L, and 333mg/L, to investigate the impact of Vc on the anti-stress ability of P. monodon during transportation. And the survival rate following transportation and a 15-day interim rearing period were recorded. Moreover, the alterations in tissue structure and activity of the enzymes alkaline phosphatase (ACP), alkaline phosphatase (AKP), total superoxide dismutase (T-SOD), and total superoxide dismutase (T-AOC) were checked. The results showed that with the increase of Vc supplemental level, the survival rate of P. monodon after transportation and after 15 days of temporary rearing increased to varying degrees. ACP and AKP in the hepatopancreas increased first and then decreased. T-SOD activity in the 0mg/L group was the lowest and then decreased gradually. The activity of total antioxidant capacity (T-AOC) in the 0mg/L group was the lowest and then stabilized. The branchial tissue structure also changed. The branchial tissue blood cell disorder decreased, the diaphragm gradually narrowed, the cornered cortex gradually recovered, and the swelling decreased. In conclusion, Vc positively affects the survival rate of P. monodon after transportation and temporary cultivation and alleviates the stress of P. monodon. The amount of Vc added at about 333mg/Lin, the transportation of P. monodon could play a positive role. The experimental results provide primary data for the transportation of P. monodon. [ABSTRACT FROM AUTHOR]

In this study, three genes, PmATP synthase, PmNa+-K+-ATPase and PmToll receptor gene, were selected to study their relative expression in different tissues of Penaeus monodon. The results showed that the mRNA level of PmATP synthase gene was not significantly up-regulated under low salt stimulation (P> 0.05),while under high salinity stimulation, the transcription level of PmATP synthase mRNA reached the maximum at 16th h (P<0.05).The expression level of PmNa+-K+-ATPase mRNA increased at 4th h and 16th h, which was significantly higher than that in the beginning under low salt stimulation (P<0.05) and then decreased at 32nd h,,while under high salinity stimulation, the expression level of PmNa+-K+- ATPase mRNA reached the highest at 8th h and 32nd h. The expression level of PmToll receptor mRNA reached the highest at 4th h after low salinity stimulation (P<0.05), and then down-regulated after 16th h, while under high salinity stress, the expression level was up-regulated and reached the maximum at 8th h, which was 2.4 times of that the in the control group (P<0.05) and decreased at 16th h after stress. PmATP synthase gene was relatively high expression, while PmNa+-K+-ATPase and PmToll receptor gene expression were low, so we guess that the large amounts of ATP of the body synthesis mainly use for the growth of the body. PmNa+-K+-ATPase and PmToll receptor gene expression are relatively high, which indicate that the body osmotic pressure regulation and the body’s immune process consume a lot of energy [ABSTRACT FROM AUTHOR]

Traditional broodstock diets, being susceptible to virus attacks or carrying the virus to parent shrimps, bring a huge bio-security risk to shrimp breeding. This study was carried out to determine the feasibility of Pheretima tschiliensis as a replacement of traditional bait to accelerate the maturity of parent shrimps of Penaeus monodon, which was verified by the dynamics of vitellogenin gene expression in hepatopancreas and ovary of P. monodon. The parent shrimps fed on four different diets including commercial parent shrimps feed (Diet A), Pheretima tschiliensis combination bait (Diet B), Pheretima tschiliensis (Diet C) and Nereis succinea combination bait (Diet D), and the effect of each diet on the maturity of parent shrimps was evaluated by testing the gonad weight increase rate, eggs amount, average spawning time and levels of VTG mRNA expression. The results showed that P. tschiliensis, as a closely related terrestrial species of N. succinea, had the best effect on maturation of parent shrimps. The highest gonad weight increase rate of 1845.28% and the largest consecutive spawning rate with average value of 70.66% were both in groups fed on P. tschiliensis, with no significant difference from the groups fed on N. succinea combination bait. The dynamics of VTG mRNA level were observed in hepatopancreas and ovary of P. monodon with the trend of increasing firstly and then decreasing, which reflected reproductive performance of the parent shrimps and was suggested to be an indicator of reproductive performance for selective breeding. As a result, P. tschiliensis is a suitable substitute of N. succinea based on its effect on the reproductive traits and VTG mRNA expression level in parent shrimps of P. monodon. [ABSTRACT FROM AUTHOR]

This study examined the growth response of black tiger shrimp, Penaeus monodon, reared in indoor cement ponds fed at variable daily feeding frequencies. Five hundred healthy shrimp (1.26±0.37 g initial weight) were stocked in 12 cement ponds (5.5x2.5x1.5m). Three ponds in each group were assigned to four different feeding frequency i.e., two, three, four and six times/day designated as FF1, FF2, FF3 and FF4, respectively. Artificial diet (40% fishmeal protein) was used to feed at eight percent of total shrimp body weight per day for a total period of 56 days. The shrimp length and weight were measured fortnightly and the feeding rate was adjusted according to the new weight. At the end of the trial, all shrimp remained healthy and active with the survival rate of 70-85%. The specific growth rate (SGR) were the highest in groups FF3 (4.21±0.53) and FF4 (4.23±1.04), which were significantly higher than those in FF1 (3.82±0.49) and FF2 (4.02±1.03) groups. Whole body proximate composition (protein, ash, moisture and lipid) remained similar in all the treatment groups. It could be concluded that the black tiger shrimp, P. monodon, would be grown in indoor cement ponds effectively by feeding four times day-1 to achieve significantly better growth. [ABSTRACT FROM AUTHOR]

The fertilized eggs of "Nanhai 2" Penaeus monodon bred by our research group were incubated at the same temperature (30°C), different salinity (20, 25, 30, 35, 40), different pH (7.0, 7.5, 8.0, 8.5, 9.0) and different alkalinity (2.0 mmol/L, 2.5 mmol/L, 3.0 mmol/L, 3.5 mmol/L, 4.0 mmol/L) to explore the effects of salinity, pH and alkalinity on hatching rate of fertilized eggs of P. monodon. The results showed that the hatching rate of fertilization rate of P. monodon was closely related to salinity, and the best hatching rate was obtained when the seawater salinity was 30 with the average hatching rate was 82.60%. The hatching rate was very low when the salinity was as low as 20 or as high as 40, which was significantly lower than that of other treatments (P<0.05). The hatching effect of the fertilized eggs of P. monodon was closely related to the pH value of seawater, and the slightly alkaline seawater was conducive to the normal development of the fertilized eggs. Among them, the hatching effect of the seawater pH value of 8.0 was the best, and the average hatching rate of the fertilized eggs was 80.62%. Too low or too high pH value of the seawater was not conducive to the development of the embryo, and the hatching rate of the fertilized eggs decreased in varying degrees. There was no significant correlation between the hatching effect of fertilized eggs and the change of seawater alkalinity. The average hatching rate of fertilized eggs ranged from 78.65% to 83.12% in the alkalinity range of 2.0-4.0 mmol/L. [ABSTRACT FROM AUTHOR]

To explore the effect of concentrated dephenolic cottonseed protein (CDCP) partly instead of fish meal on the non-specific immune of Penaeus monodon, we designed a single factor test to study the four types of non-specific immunity of P. monodon hepatopancreas after acute salinity stress. A total number of 5,400 P. monodon were selected with an initial body weight of 1.76±0.15g and randomly divide into 6 groups (3 replicates per group, 900 tails per replicate). Shrimp were feed with two protein level diets (Diet A and Diet B). After 8 weeks of feeding, P. monodon were randomly selected with an average weight of 14.71±1.06g in each feed group, and subjected to acute salinity stress of 10ppt and 20ppt after temporary rearing, and the heat shock protein 70 (HSP70), Na+-K+-ATPase, cortisol and the content of alkaline phosphatase (ALP) were measured. The results of the study showed that when the CDCP partially replaced fish meal of P. monodon diet, the hepatopancreas HSP70, and the contents of Na+-K+-ATPase and cortisol were significantly higher than those of the control group. In the acute salinity change, there was no significant difference in the survival rate of P. monodon at 48h under the two salinity stresses, and the four immune indexes showed different trends. The expression level of Cortisol was more prominent than that of HSP70, which could better reflect the stress degree of P. monodon. Most of the indicators showed a trend of rising and then falling and then rising compared with the original level, and the changes of the four indicators of hepatopancreas in the Diet B group were significantly higher than those in the Diet A group at most time point of the two salinities. [ABSTRACT FROM AUTHOR]

To explore the effect of microecological preparations on the growth and stress resistance of Litopenaeus vannamei larvae, different amounts of Bacillus and Lactobacillus bacteria were added to the L. vannamei aquaculture water during seeding. Seven groups were established and the microecological preparations were added every 5 d. Bacillus concentrations in the Y1, Y2 and Y3 groups were 10 ppm, 15 ppm and 20 ppm, respectively, and the Lactobacillus concentrations in the R1, R2 and R3 groups were 20 ppm, 25 ppm and 30 ppm. The control (C) group had no microecological preparation added. Results showed that the Y2 larvae achieved the heaviest body weights, which were significantly higher than those of all other groups (P < 0.05), except for Y3 and R3 (P > 0.05). The body weights of the Y2 group were 27.18%, 11.97%, 27.18% and 23.58% higher than the weights of the C, Y1, R1 and R2 groups, respectively. Furthermore, except for that of the R2 group (P > 0.05), the survival rate of the R3 group was significantly higher than that of the C (34.86%), Y1 (21.49%), Y2 (17.60%), Y3 (15.75%) and R1 (15.75%) groups (P < 0.05). Microecological preparation addition also improved the stress resistance of L. vannamei larvae. Results showed that under stress conditions (60 min in fresh water and a sudden temperature change of 5°C), the survival rate in the two treatment groups was markedly higher than that in the C group (14.46% and 9.65%, respectively; P < 0.05). These results demonstrated that microecological preparations significantly enhanced the growth, survival and stress resistance of L. vannamei larvae. [ABSTRACT FROM AUTHOR]

The impacts of temperature on oxygen and energy metabolism in the hepatopancreas of the black tiger shrimp (Penaeus monodon) were investigated via the effects of thermal stress on superoxide anion (O2-) production, superoxide dismutase (SOD) activity, glutathione (GSH) and adenosine triphosphate (ATP) concentration, as well as nitric oxide synthase (NOS) activity and nitric oxide (NO) concentration catalyzed by NOS. Based on biochemical analysis of the above, results showed that O2- production could be induced significantly after cold stress at 15°C and heat stress above 30°C. SOD activity showed a similar changing profile as the concentration of O2- after thermal stress between 15°C and 30°C, and GSH concentration increased significantly under both high and low temperature. The NOS activity and concentration of NO catalyzed by NOS increased significantly after heat stress. The ATP concentration increased significantly after both low and high temperature stress, but returned to the control level after 8 h of recovery. Thus, low and high temperature stress could lead to oxygen metabolism disorder in the hepatopancreas of P. monodon, which might induce antioxidant enzyme responses. Our findings suggest that SOD and GSH might play different roles in the response of the shrimp to low and high temperature, whereas NO might play an important role in the induction of many signaling pathways in response to thermal stress. Additional ATP was also produced after cold and heat stress, suggesting that more energy was required to cope with temperature extremes. Our findings indicate that temperature stress led to oxygen metabolism disorder, possibly due to the temperature being beyond the oxygen- and capacitylimitation of thermal tolerance of P. monodon. Oxygen metabolism disruption might also impact energy metabolism and other physiological activities in P. monodon. [ABSTRACT FROM AUTHOR]