High salinity impedes efficient nitrogen removal from mariculture wastewater, which inhibits the colonization and nitrogen removal capabilities of nitrogen-removing microbes. This study aimed to isolate and characterize a salt-tolerant heterotrophic nitrification-aerobic denitrification bacterial strain. We evaluated 30 bacterial strains isolated from Portunus trituberculatus aquaculture ponds, among which Pseudomonas aeruginosa SH3 exhibited superior nitrogen removal efficiencies (99 % of NH₄⁺-N, 71 % of NO₂^--N, and 85 % fof NO₃^--N at a salinity of 30 ‰) than the other strains. Single-factor experiments demonstrated that SH3 effectively removed either NH₄⁺-N or NO₂^--N across various C/N ratios (10-20), pH levels (7-9), salinity levels (15-35 ‰), and temperatures (25-35 °C), highlighting its promising nitrogen removal capabilities under conditions suitable for mariculture. Genomic analysis showed that SH3 removes NH₄⁺-N through ammonia assimilation and nitrification and converts NO₂^--N and NO₃^--N via denitrification and assimilatory nitrate reduction. Bioaugmentation with SH3 reduced the startup period by 14 d, addressing a common challenge of prolonged startup times in a moving-bed biofilm reactor used for nitrogen removal in marine recirculating aquaculture systems. Meanwhile, bioaugmentation maintained minimal fluctuations in nitrogen levels throughout the operational period, resulting in consistently low concentrations of NO₂^--N and NH₄⁺-N, both below 1 mg/L. Therefore, strain SH3 exhibits robust nitrogen removal capabilities, demonstrating its practicality and reliability in mariculture wastewater treatment along with providing robust data support for industrial-scale applications.

Biogenic amines (BAs) play an important role in the aggressive behavior of crustaceans. In mammals and birds, 5-HT and its receptor genes (5-HTRs) are characterized as essential regulators involved in neural signaling pathways during aggressive behavior. However, only one 5-HTR transcript has been reported in crabs. In this study, the full-length cDNA of the 5-HTR1 gene, named Sp5-HTR1, was first isolated from the muscle of the mud crab Scylla paramamosain using the reverse-transcription polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends (RACE) methods. The transcript encoded a peptide of 587 amino acid residues with a molecular mass of 63.36 kDa. Western blot results indicate that the 5-HTR1 protein was expressed at the highest level in the thoracic ganglion. Furthermore, the results of quantitative real-time PCR show that the expression levels of Sp5-HTR1 in the ganglion at 0.5, 1, 2, and 4 h after 5-HT injection were significantly upregulated compared with the control group (p < 0.05). Meanwhile, the behavioral changes in 5-HT-injected crabs were analyzed with EthoVision. After 0.5 h of injection, the speed and movement distance of the crab, the duration of aggressive behavior, and the intensity of aggressiveness in the low-5-HT-concentration injection group were significantly higher than those in the saline-injection and control groups (p < 0.05). In this study, we found that the Sp5-HTR1 gene plays a role in the regulation of aggressive behavior by BAs, including 5-HT in the mud crab. The results provide reference data for the analysis of the genetic mechanism of aggressive behaviors in crabs.

Anesthesia serves as an effective method to mitigate the stress response in aquatic animals during aquaculture and product transportation. In this study, we assessed the anesthetic efficacy of clove oil, tricaine methane-sulfonate (MS-222), ethanol, and magnesium chloride by anesthesia duration, recovery time, 24-hour survival rate, and the behavior of mud crabs (Scylla paramamosain). Additionally, the optimal anesthetic concentration for varying body weights of mud crabs was also investigated. The results revealed that clove oil emerged as the optimal anesthetic for mud crabs, with a 24-hour survival rate surpassing those observed in MS-222 and magnesium chloride treatments. Ethanol caused amputation and hyperactivity in mud crabs. Regression analyses between the optimal anesthetic concentration of clove oil and the weight categories of 0.03–27.50 g and 27.50–399.73 g for mud crabs yielded the following equations: y = 0.0036 x³ − 0.1629 x² + 1.7314 x + 4.085 (R² = 0.7115) and y = 0.0437 x + 2.9461 (R² = 0.9549). Clove oil exhibited no significant impact on serum cortisol, glucose, lactate content, aspartate aminotransferase (AST), alanine aminotransferase (ALT) activities, or superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels in mud crabs across different treatment groups. Anesthesia induced by clove oil in mud crabs resulted in an increase in inhibitory neurotransmitters such as glycine. However, the recovery from anesthesia was associated with elevated levels of the excitatory neurotransmitters L-aspartic acid and glutamate. In conclusion, clove oil proves to be a safe and optimal anesthetic agent for mud crabs, exerting no physiological stress on the species.

With the development of the aquatic products processing industry, 6–8 million tons of shrimp and crab shell waste are produced globally annually, but, due to the lack of high-value conversion technology, crab shells are often discarded in large quantities as a by-product of processing. Pseudomonas-based microbial cell factories are capable of biosynthesis of high-value products using a wide range of substrates; however, there is currently no reliable fermentation model for producing high-value chemicals using crab shell waste by Pseudomonas strains. In this study, we first explored the culture conditions of shell fermentation using KT2440 through single-factor and orthogonal experiments, and the optimized fermentation parameters obtained are given as follows: a temperature of 30 °C, fermentation time of 42 h, substrate solid–liquid ratio of 7%, and rotational speed of 200 rpm. After optimization, the maximum cell growth was increased by 64.39% from 350.67 × 10⁸ CFU/mL to 576.44 × 10⁸ CFU/mL. Combined with engineering modification, two engineered strains, KT_+IV and KT_+lasBT, expressing exogenous proteases, were obtained, and the maximum growth was increased from 316.44 × 10⁸ CFU/mL to 1268.44 × 10⁸ CFU/mL and 616.89 × 10⁸ CFU/mL, which were 300.84% and 94.94% higher, respectively. In addition, the engineered strain KT_+NtrcT-D55E, which regulates nitrogen metabolism, was obtained, and the accumulation of intracellular polyhydroxy fatty acid esters (PHA) was increased from 20.00 mg/L to 78.58 mg/L, which was a significant increase of 292.93% relative to the control group. This study provides a theoretical basis and technical support for the high-value utilization of shrimp and crab shell resources and the development of environmentally friendly bioproducts.

With the development of the aquatic products processing industry, 6-8 million tons of shrimp and crab shell waste are produced globally annually, but due to the lack of high-value conversion technology, crab shells are often discarded in large quantities as a by-product of processing. Pseudomonas-based microbial cell factories are capable of biosynthesis of high-value products using a wide range of substrates; however, there is currently no reliable fermentation model for producing high-value chemicals using crab shell waste by Pseudomonas strains. In this study, we first explored the culture conditions of shell fermentation using KT2440 through single-factor and orthogonal experiments, and the optimized fermentation parameters obtained were: with the temperature of 30°C, fermentation time of 42 h, substrate solid-liquid ratio of 7%, and rotational speed of 200 rpm. After optimization, the maximum cell growth was increased by 64.39%, from 350.67 × 10 8 CFU/mL to 576.44 × 10 8 CFU/mL. Combined with engineering modification, two engineered strains KT+IV and KT+lasBT expressing exogenous proteases were obtained, with the maximum growth increasing by 300.84% and 94.94%, respectively. Additionally, an engineered strain KT+NtrcT-D55E regulating nitrogen metabolism was obtained, significantly increasing the accumulation of intracellular polyhydroxyalkanoates (PHA) by 292.93%, relative to the control group. This study provides a theoretical basis and technical support for the high-value utilization of shrimp and crab shell resources and the development of environmentally friendly bioproducts.