Automatic Shrimp Fry Counting Method Using Multi-Scale Attention Fusion.

Shrimp fry counting is an important task for biomass estimation in aquaculture. Accurate counting of the number of shrimp fry in tanks can not only assess the production of mature shrimp but also assess the density of shrimp fry in the tanks, which is very helpful for the subsequent growth status, transportation management, and yield assessment. However, traditional manual counting methods are often inefficient and prone to counting errors; a more efficient and accurate method for shrimp fry counting is urgently needed. In this paper, we first collected and labeled the images of shrimp fry in breeding tanks according to the constructed experimental environment and generated corresponding density maps using the Gaussian kernel function. Then, we proposed a multi-scale attention fusion-based shrimp fry counting network called the SFCNet. Experiments showed that our proposed SFCNet model reached the optimal performance in terms of shrimp fry counting compared to CNN-based baseline counting models, with MAEs and RMSEs of 3.96 and 4.682, respectively. This approach was able to effectively calculate the number of shrimp fry and provided a better solution for accurately calculating the number of shrimp fry.

Effects of the putative probiotics Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis on white leg shrimp, Litopenaeus vannamei, immune response, gut histology, water quality, and growth performance.

Background: A commercially significant species in the aquaculture sector globally, particularly in Egypt, is Litopenaeus vannamei. Aim: The experiment's objective was to ascertain how Sanolife PRO-F impacted the growth, water quality, immunological response, and intestinal morphometry of L. vannamei. Methods: In the current investigation, which lasted 12 weeks, Sanolife PRO-F was administered to shrimp post-larvae at diet doses of 0 (control), 1 (group one), 2 (group two), and 3 (group three) g/kg diet, respectively. Each experimental group had three repetitions. Results: In the current study, shrimp fed on probiotic-treated diets showed a considerable improvement in growth performance measures and survival rate, and the nonspecific immune response was also enhanced. Shrimp fed probiotic diets had longer and more intestinal villi overall. Shrimp fed on the G2 and G3 diets showed no appreciable differences in growth or intestinal morphology. With the G2 and G3 diet, the water had lower concentrations of nitrite and ammonia. Conclusion: The study's findings indicate that Sanolife PRO-F treatment at 2-3 g/kg feed promotes the growth of shrimp, immunological response, gut health and function, and water quality.

Effects of Lactobacillus plantarum Ep-M17 on growth, immunity and intestinal microbiota of Penaeus vannamei under Microcystin-LR stress.

Microcystins (MCs) are biologically active cyclic heptapeptide compounds released by cyanobacteria in water bodies, and MC-LR is one of the most widespread and toxic isoforms. It frequently poses a serious threat to Penaeus vannamei aquaculture. Our previous study revealed that the supplementation of Lactobacillus plantarum Ep-M17 has a probiotic effect on P. vannamei health and whether Ep-M17 can alleviate the stressful effects of MC-LR on shrimp remains unclear. Therefore, in the present work, shrimp were fed MC-LR alone or combined with Ep-M17 for six weeks, and then evaluated the effects on histology, enzyme activity, gene expression, and intestinal flora. The results showed that MC-LR stress lead to slow growth and reduced survival rates in shrimp. However, feeding Ep-M17 significantly increased both the growth rate and survival rate. Meanwhile, MC-LR stress caused severe tissue damage in the hepatopancreas and intestines of shrimp, but Ep-M17 significantly reduced the toxic effects and protected the integrity of these tissues. Additionally, Ep-M17 significantly enhanced the activities of antioxidant enzymes and digestive enzymes, and induced higher expression of immune-related genes, thereby promoting the digestive and immune responses in shrimp. Furthermore, MC-LR stress disrupted the intestinal flora in shrimp intestines, while the use of Ep-M17 significantly increased the abundance of immune- and metabolism-related bacteria and inhibited the growth of pathogenic bacteria to maintain intestinal flora balance and intestinal health. In conclusion, our results indicate that Ep-M17 can reduce the toxic effect of MC-LR on shrimp and has a positive function in the prevention and control of shrimp diseases caused by MC-LR.

Microfibers from cigarette butts can induce exoskeletal alteration in whiteleg shrimp (Penaeus vannamei).

Cigarette butts (CB) are a source of microfibers (MFs) in aquatic environments, posing a risk to the health of aquatic organisms. Research has been focused on examining the toxicity of CBs on ecological receptors, including invertebrates. More focus has been on death, growth, or movement inhibition of but less on exoskeletal effects in malacostracans. We evaluated the alteration in the carapace structure of whiteleg shrimp (Penaeus vannamei Boone, 1931) caused by MFs derived from CBs (CB-MF). Exposure to CB-MF damaged the gills, the main organs adsorbing calcium in shrimps to generate a hard carapace, disturbing calcium uptake via respiration. Rapid ecdysis caused on CB-MF exposure reduced the environmental adaptation capacity of crustaceans in the absence of normal pigments in the chromatophore of the carapace. These findings indicate that MFs released from CBs released into the aquatic environment can adversely affect exoskeletal alteration within the overall ecosystem.

Transcriptomic analysis following polystyrene nanoplastic stress in the Pacific white shrimp, Litopenaeus vannamei.

Plastics are widely produced for industrial and domestic applications due to their unique properties, and studies on the toxic effects of nanoplastics (NPs) on aquatic animals are essential. In this study, we investigated the transcriptomic patterns of Litopenaeus vannamei after NPs exposure. We found that the lysosome pathway was activated when after NPs exposure, with up-regulated DEGs, including glucocerebrosidase (GBA), hexosaminidase A (HEXA), sphingomyelin phosphodiesterase-1 (SMPD1), and solute carrier family 17 member 5 (SLC17A5). In addition, the PI3K-Akt signaling pathway was strongly affected by NPs, and the upstream genes of PI3K-Akt, including epidermal growth factor receptor (EGFR), integrin subunit beta 1 (ITGB1) and heat shock protein 90 (HSP90) were up-regulation. Other genes involved in lipogenesis, such as sterol regulatory element binding transcription factor 1 (SREBP-1c), fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD-1), were down-regulated. However, the contents of triglycerides (TG) and total cholesterol (TCH) in L. vanname hepatopancreas were reduced, which indicated that the ingestion of NPs led to the disturbance of hepatic lipid metabolism. What more, NPs treatment of L. vannamei also caused oxidative stress. In addition, NPs can damage part of the tissue structure and affect the physiological function of shrimps. The results of this study provide valuable ecotoxicological data to improve the understanding of the biological fate and effects of nanoplastics in L. vannamei.

New insights into the regulation mechanism of Pacific white shrimp (Litopenaeus vannamei) hepatopancreas under 4-nonylphenol exposure using transcriptome analysis.

4-Nonylphenol (4-NP) is one of the common endocrine-disrupting chemicals (EDCs) in estuaries and coastal zones, which can exert detrimental effects on the physiological function of aquatic organisms. However, the molecular response triggered by 4-NP remains largely unknown in Pacific white shrimp (Litopenaeus vannamei). In this study, transcriptomic analysis was performed to investigate the underlying mechanisms of 4-NP toxicity in the hepatopancreas of L. vannamei. Nine RNA-Seq libraries were generated from L. vannamei at 0 h, 24 h, and 48 h following exposure to 4-NP. Compared with 0 h vs 24 h, 962 up- and 463 down-regulated differentially expressed genes (DEGs) were identified, indicating that many genes in L. vannamei were induced to resist adverse circumstances by 4-NP exposure. In contrast, 902 up- and 1027 down-regulated DEGs were revealed in the comparison of 0 h vs 48 h, demonstrating that prolonged exposure to the stress from 4-NP resulted in more inhibited genes. To validate the accuracy of the transcriptome data, eight DEGs were selected for quantitative real-time polymerase chain reaction (qRT-PCR), which were consistent with the RNA-Seq results. Through KEGG pathway enrichment analysis, three specific pathways related to hormonal effects and endocrine function of L. vannamei were enriched significantly, including tyrosine metabolism, insect hormone biosynthesis, and melanogenesis. After 4-NP stress, genes involved in tyrosine metabolism (Tyr) and melanogenesis pathway (AC, CBP, Wnt, Frizzled, Tcf, and Ras) were induced to promote melanin pigment to help shrimp resist adverse environments. In the insect hormone biosynthesis, ALDH, CYP15A1, CYP15A1/C1, and JHE genes were activated to synthesize juvenile hormone (JH), while Spook, Phm, Sad, and CYP18A1 were induced to generate molting hormone. There is an enhanced interaction between the molting hormone and JH, with JH playing a dominant role and maintaining its "classic status quo action". Our study demonstrated that 4-NP exposure led to impairments of biological functions in L. vannamei hepatopancreas. The genes and pathways identified provide novel insights into the molecular mechanisms underlying 4-NP toxicity effects in prawns and enrich the information on the toxicity mechanism of crustaceans in response to EDCs exposure.

Penaeid shrimp counteract high ammonia stress by generating and using functional peptides from hemocyanin, such as HMCs27.

Agricultural and anthropogenic activities release high ammonia levels into aquatic ecosystems, severely affecting aquatic organisms. Penaeid shrimp can survive high ammonia stress conditions, but the underlying molecular mechanisms are unknown. Here, total hemocyanin and oxyhemocyanin levels decreased in Penaeus vannamei plasma under high ammonia stress. When shrimp were subjected to high ammonia stress for 12 h, 24 hemocyanin (HMC) derived peptides were identified in shrimp plasma, among which one peptide, designated as HMCs27, was chosen for further analysis. Shrimp survival was significantly enhanced after treatment with the recombinant protein of HMCs27 (rHMCs27), followed by high ammonia stress. Transcriptome analysis of shrimp hepatopancreas after treatment with or without rHMCs27 followed by high ammonia stress revealed 973 significantly dysregulated genes, notable among which were genes involved in oxidation and metabolism, such as cytochrome C, catalase (CAT), isocitrate dehydrogenase, superoxide dismutase (SOD), trypsin, chymotrypsin, glutathione peroxidase, glutathione s-transferase (GST), and alanine aminotransferase (ALT). In addition, levels of key biochemical indicators, such as SOD, CAT, and total antioxidant capacity (T-AOC), were significantly enhanced, whereas hepatopancreas malondialdehyde levels and plasma pH, NH3, GST, and ALT levels were significantly decreased after rHMCs27 treatment followed by high ammonia stress. Moreover, high ammonia stress induced hepatopancreas tissue injury and apoptosis, but rHMCs27 treatment ameliorated these effects. Collectively, the current study revealed that in response to high ammonia stress, shrimp generate functional peptides, such as peptide HMCs27 from hemocyanin, which helps to attenuate the ammonia toxicity by enhancing the antioxidant system and the tricarboxylic acid cycle to decrease plasma NH3 levels and pH.

Endogenous enzymatic antioxidant status of whiteleg shrimp (Litopenaeus vannamei) following exposure to sublethal concentrations of silver nanoparticles.

Immunotoxicity of silver nanoparticles (AgNPs) to whiteleg shrimp (Litopenaeus vannamei) was assessed using redox-status orchestrating enzymes. To this end, the shrimp was exposed to sublethal AgNPs concentrations (0 % LC50: control; 25 % LC50: 0.97 mg/L; 50 % LC50: 1.95 mg/L; 75 % LC50: 2.92 mg/L). During the experiment, the behavior of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (GPx) was monitored, besides total antioxidant capacity (TAC) and malondialdehyde (MDA). The hepatopancreas SOD activity reduced about 63 %-76 % at.%50 LC50 and %75 LC50 AgNPs treatments, and CAT decreased in both tissues at 50 % LC50 AgNPs. TAC exhibited a U-form response in the hepatopancreas organ against stress caused by AgNPs, and hepatopancreas MDA displayed a time-dependent increase. Taken together, AgNPs triggered severe immunotoxicity through suppression of CAT, SOD, and TAC in the hepatopancreas tissue.

Thermal acclimation capacity and standard metabolism of the Pacific white shrimp Litopenaeus vannamei (Boone, 1931) at different temperature and salinity combinations.

In aquatic environments, rising temperatures reduce the oxygen content of the water while increasing the oxygen demand of organisms. In intensive shrimp culture, it is of great importance to know the thermal tolerance of cultured species and their oxygen consumption since this affects the physiological condition. In this study, the thermal tolerance of Litopenaeus vannamei was determined by dynamic and static thermal methodologies at different acclimation temperatures (15, 20, 25, and 30 °C) and salinities (10, 20, and 30 ppt). The oxygen consumption rate (OCR) was also measured to determine the standard metabolic rate (SMR) of shrimp. Acclimation temperature significantly affected the thermal tolerance and SMR of Litopenaeus vannamei (P < 0.01). Salinity had a large effect on SMR (P < 0.01) but did not influence the thermal acclimation of the shrimp (P > 0.01). Litopenaeus vannamei is a species that has high thermal tolerance and can survive at extreme temperatures (CTmin-CTmax: 7.2-41.9 °C) with its large dynamic (988, 992, and 1004 °C2) and static thermal polygon areas (748, 778 and 777 °C2) developed at the above temperature and salinity combinations and resistance zone (1001, 81 and 82 °C2). The optimal temperature range of Litopenaeus vannamei is the 25-30 °C range, where a decrease in standard metabolism is determined with increasing temperature. Given the SMR and optimal temperature range, the results of this study indicate that Litopenaeus vannamei should be cultured at 25-30 °C for effective production.

The effect of air exposure and re-water on gill microstructure and molecular regulation of Pacific white shrimp Penaeus vannamei.

The Penaeus vannamei is an important shrimp species with enormous commercial and ecological values. In production process, the air exposure resistance is vital for live transportation without water. We tested the air exposure resistant ability of P. vannamei, and carried out gill histological observation and gene expression analysis. The physiology and molecular response to the air exposure stress of P. vannamei was revealed. We found that body weight could affect the air exposure tolerance. Air exposure caused epithelial cell of gill filament shrinking and tissue fluid exudation within half of hour, and triggered oxidative stress response. After retrieved to water, epithelial cell shrinking and tissue fluid exudation recovered gradually, but oxidative and antioxidant response is still going on. Organisms reduced oxidative stress by regulating levels of antioxidants and antioxidant enzymes that remove reactive oxygen species (ROS) and RNA and DNA processing to repair tissue damage, and expression of apoptosis associated-genes altered. Furthermore, the survive shrimps could live steadily more than 5 days, and their gill filament recovered to normal state, proving that the damage of air exposure is reversible. These findings could be considered in the waterless live transportation of P. vannamei.

Role of myo-inositol supplementation against toxicity of excessive dietary copper in Pacific white shrimp Litopenaeus vannamei.

Raw materials for making dried shrimp (a type of foodstuff) are mostly from farmed shrimp and preliminary findings indicated that head copper (Cu) concentrations in some commercial dried shrimp products exceeded the safe limit specified in pollution-free aquatic products (50 mg/kg), which may influence food safety. Therefore, a 63-day feeding trial was conducted to explore effects of dietary Cu concentrations on accumulation of Cu in tissues, growth performance, immune response and antioxidant status of Pacific white shrimp (Litopenaeus vannamei). Moderating effect of myo-inositol (MI, adding 200 mg/kg diet) on the adverse impacts caused by excessive dietary Cu was also investigated. 600 shrimp (initial weight: 0.89 ± 0.00 g) were divided into five groups: 37.08 mg Cu/kg diet group (control group), 62.57 mg Cu/kg diet group, 125.99 mg Cu/kg diet group, 63.41 mg Cu/kg diet group (supplemented with MI) and 119.19 mg Cu/kg diet group (supplemented with MI). The results showed that dietary Cu concentrations increased from 37.08 to over 62.57 mg/kg, hepatopancreas Cu concentrations raised from 29.04 to 233.43-263.65 mg/kg, and muscle Cu concentrations only increased from 6.22 to 6.99-8.39 mg/kg. Report to control group, excessive Cu concentration (125.99 mg/kg) didn't significantly affect growth performance, but it notably reduced whole body lipid content and immune response, induced oxidative stress and damaged the hepatopancreas structure, which was ameliorated by MI supplementation. The results suggested that consuming shrimp head and its processed products weren't recommended. Cu concentrations of commercial feeds for Pacific white shrimp should be controlled below 62.57 mg/kg. Additionally, MI supplementation mitigated the negative impacts induced by excessive dietary Cu.

Energy metabolism response of Litopenaeus vannamei to combined stress of acute cold exposure and waterless duration: Implications for physiological regulation and waterless live transport.

Maintaining the homeostasis of energy metabolism is crucial for organism's stress tolerance and survival. Acute cold exposure (AC) and waterless duration (WD) represent the two predominate abiotic stressors during waterless live transport of Litopenaeus vannamei. Although previous reports have explored the physiological response of L. vannamei to combined stress AC + WD, the roles of energy metabolism response in regulation of stress tolerance remains unknown. The present study comparatively examined the variations of energy metabolism-related indicators in hemolymph (cortisol, hemocyanin, glucose and lactate), hepatopancreas and muscle tissues (levels of lactate and glycogen, activities of hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and adenosine triphosphatase (ATPase), and ATP levels). Combined stress significantly disturbed the homeostasis of energy metabolism with the increase in levels of hemocyanin, glucose and lactate, and decrease in glycogen and ATP content (P < 0.05). In addition, the activities of HK, PFK, PK, and SDH initially elevated and then decreased with the prolongation of combined stress from 3h to 9h duration, while the activity of lactate dehydrogenase (LDH) remained gradual elevation and ATPase activity decreased in a duration time dependent manner throughout the experiment. These alterations revealed that exposure to combined stress could accelerate anaerobic metabolism at initial stage and inhibit aerobic metabolism in a duration time-dependent manner, following with the reduction of energy biosynthesis and the disturbance of energy metabolism equilibrium. On the other hand, the progressive impairment on hepatopancreas tissue was observed under combined stress. In summary, the deficiency of ATP supply and histopathological injures on hepatopancreas tissue might the underlying mechanisms inducing mortality of L. vannamei during live transport.

Insights into the response mechanism of Litopenaeus vannamei exposed to cold stress during live transport combining untargeted metabolomics and biochemical assays.

Unfavorable conditions severely affect the survival quality of Litopenaeus vannamei (L. vannamei) during live transport and the molecular response mechanism needs to be clarified. In this study, metabolomics combined with conventional assay on physiological and histopathological responses were applied to deepen the understanding of L. vannamei to cold stress and provide. A solid foundation for regulation of transportation management. Physiological and biochemical analysis revealed the significant disturbance of glycolysis in hepatopancreas tissue. Furthermore, metabolomics based on the technique of ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry identified the significantly differential metabolites between acute cold exposure (AC) and normal control (NC) groups. Moreover, KEEG result indicated that the pathways of amino acid, glycerophospholipid, and nuclei acid metabolism and ABC transporters in hepatopancreas were significantly disturbed. Furthermore, histopathology and ultrastructure verified the impairment in hepatopancreas during cold stress. Overall, the concurrent use of metabolomics and biochemical assays was demonstrated to be sensitive and effective in providing new insights into the response mechanism of L. vannamei to cold stress.

Silencing of Nrf2 in Litopenaeus vannamei, decreased the antioxidant capacity, and increased apoptosis and autophagy.

Nuclear factor E2-related factor 2 (Nrf2) is a multifunctional transcription factor that plays an important role in antioxidant activities. However, its effect on antioxidant capacity in Litopenaeus vannamei, an economically important crustacean, remains unclear. In this study, the role of Nrf2 in response to oxidative stress in L. vannamei was determined by its effect on relevant gene expression and enzymatic activity. Nrf2 was cloned and analyzed. Results revealed that Nrf2 contains a 1575 bp open reading frame encoding 524 amino acids and a conserved bZIP Maf domain. The sequence similarity of Nrf2 between L. vannamei and Homarus americanus is 81%. Although the Nrf2 expression was detected in all tissues, the Nrf2 expression levels were the highest in the hepatopancreas, followed by the eyestalk and muscle. RNA interference significantly decreased the expression of antioxidant-related genes (SOD, GPX, CAT, Trx, and HO-1; p < 0.05), significantly upregulated the expression of autophagy genes (Atg3, Atg4, Atg5, Atg10, and Atg12; p < 0.05) and apoptosis genes (Caspase-3 and P53; p < 0.05). Moreover, SOD, CAT, and GPX enzyme activities decreased whereas the MDA activity increased. The histological results of the shrimp injected with dsRNA-Nrf2 showed that the hepatic tubules were irregularly arranged, the lumen was abnormal, and a few hepatic tubules were significantly enlarged compared with those of the dsRNA-EGFP group. The hepatocytes were also vacuolated. In conclusion, this study provided evidence that Nrf2 is involved in the regulation of antioxidant capacity, oxidative stress, apoptosis, and autophagy in shrimp.

Transcriptomic analysis of gills in nitrite-tolerant and -sensitive families of Litopenaeus vannamei.

Nitrite stress is a major environmental factor that limits aquatic animal growth, reproduction and survival. Even so, some shrimps still can withstand somewhat high concentrations of nitrite environment. However, few studies have been conducted about the tolerance molecular mechanism of Litopenaeus vannamei in the high concentration nitrite. To identify the genes and pathways involved in the regulation of nitrite tolerance, we performed comparative transcriptomic analysis in the L. vannamei nitrite-tolerant (NT) and nitrite-sensitive (NS) families, and untreated shrimps were used as the control group. After 24 h of nitrite exposure (NaNO2, 112.5 mg/L), a total of 1521 and 868 differentially expressed genes (DEGs) were obtained from NT compared with NS and control group, respectively. Functional enrichment analysis revealed that most of these DEGs were involved in immune defense, energy metabolism processes and endoplasmic reticulum (ER) stress. During nitrite stress, energy metabolism in NT was significantly enhanced by activating the related genes expression of oxidative phosphorylation (OXPHOS) pathway and tricarboxylic acid (TCA) cycle. Meanwhile, some DEGs involved in innate immunity- related genes and pathways, and ER stress responses also were highly expressed in NT. Therefore, we speculate that accelerated energy metabolism, higher expression of immunity and ER related genes might be the important adaptive strategies for NT in relative to NS under nitrite stress. These results will provide new insights on the potential tolerant molecular mechanisms and the breeding of new varieties of nitrite tolerant L. vannamei.

In vitro fertilization and hybridization potential of the Indian white shrimp (Penaeus indicus).

Managing the reproduction of shrimp farmed in captivity is essential for selective breeding and interspecies hybridization. Procedures have not been developed for conducting in vitro fertilization in penaeid shrimp. In this study, the feasibility of in vitro fertilization (IVF) of the pre-ovulatory oocyte of Penaeus indicus was examined. Additionally, the processes of fertilization and possibility of hybridizing P. indicus with P. monodon with utilization of IVF procedures was also evaluated. The IVF was conducted by dissecting ovaries with fully developed follicles, and mixing these (~0.1 million oocytes) with 1 mL of a sperm suspension. Evaluations for fertilization and embryonic development occurred every l5 min. The eggs collected from the ovaries had the capacity for activation and fertilization. The hatching rate was 5.5 ± 1.1% of the total number of eggs fertilized, and 8.2 ± 4.8% of the nauplii developed to the post-larval stage. Results from the scanning electron micrograph evaluations provided detailed information about the changes occurring as a result of IVF in P. indicus. Interspecific hybridization of P. indicus with P. monodon did not result in hatching of embryos from the eggs, although 1% of eggs were fertilized. A reliable and inexpensive IVF procedure was developed, therefore, IVF could be an efficacious procedure for facilitation of intraspecific cross production in a penaeid breeding program. Furthermore, it was determined that IVF could be a possible procedure for interspecies hybridization between closely related species to circumvent prezygotic barriers in reproductive processes.

Combined stress of acute cold exposure and waterless duration at low temperature induces mortality of shrimp Litopenaeus vannamei through injuring antioxidative and immunological response in hepatopancreas tissue.

High mortality is a frequent occurrence during live transport of shrimp species and the biochemical mechanism remains unknown. This study aimed to explore the influence of combined stress of acute cold exposure (AC) and waterless duration (WD) on survivability and biochemical response of shrimp L. vannamei during live transport. The shrimps in NC and AC groups remained the total survivability throughout the experiment while the shrimps exposed to AC + WD stress exhibited significantly higher mortality since 6h afterwards (P < 0.05) and the median survival time was calculated at 10.46 h. Moreover, the typical combined stress points at AC + WD3h, AC + WD6h and AC + WD9h were assigned for exploring the immunological and antioxidative responses. For immunity response, the total hemocyte counts (THC) decreased with the prolongation of duration time and the activities of non-specific immunity enzymes such as phenol oxidase (PO), acid phosphatase (ACP), alkaline phosphatase (AKP), aspartate aminotransferase (AST) and alanine transaminase (ALT) were significantly elevated in AC + WD9h groups (P < 0.05). Moreover, compared with that in NC group, the significant accumulation of reactive oxygen species (ROS) was observed in AC group and then reduced in combined stress groups (P < 0.05), with the highest level of malonaldehyde (MDA) in AC and AC + WD3h groups. Overall, the significant elevation of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and total antioxidant capacity (T-AOC) was detected in AC + WD9h group (P < 0.05). Furthermore, the accumulative pathological impairment on hepatopancreas tissue revealed the cytoskeleton degradation. In addition, correlation analyses visualized the correlation between oxidative stress and biochemical response. This study not only deepens our understanding on the biochemical mechanism of shrimp mortality induced by combined stress, but also provides a potential strategy for improving the management of L. vannamei during live transport.

Acoustic particle motion detection in the snapping shrimp (Alpheus richardsoni).

Many crustaceans produce sounds that might be used in communication. However, little is known about sound detection in crustaceans, hindering our understanding of crustacean acoustic communication. Sound detection has been determined only for a few species, and for many species, it is unclear how sound is perceived: as particle motion or sound pressure. Snapping shrimp are amongst the loudest and most pervasive marine sound sources. They produce snaps during interactions with conspecifics, and they also interact with soniferous heterospecifics. If they can hear, then sound could facilitate key behavioral interactions. We measured the auditory sensitivity of the snapping shrimp, Alpheus richardsoni, using auditory evoked potentials in response to a shaker table that generated only particle motion and an underwater speaker that generated both particle motion and sound pressure. Auditory detection was most sensitive between 80 and 100 Hz, and auditory evoked potentials were detected up to 1500 Hz. Snapping shrimp responded to both the shaker table and the underwater speaker, demonstrating that they detect acoustic particle motion. Crushing the statocyst reduced or eliminated hearing sensitivity. We conclude that snapping shrimp detect acoustic particle motion using the statocyst, they might detect conspecifics and heterospecifics, and hearing could facilitate key behavioral interactions.

A Novel Approach of Transducing Recombinant Baculovirus into Primary Lymphoid Cells of Penaeus monodon for Developing Continuous Cell Line.

Cell line development from shrimp is not a novel venture as researchers across the globe have been trying to have crustacean cell lines over 30 years. The reason for not attaining a crustacean or precisely a shrimp cell line is believed to be the replicative senescence and the inability to maintain telomere length in vitro. Moreover, spontaneous in vitro transformations do not happen in shrimp cells. Oncogenic induction in primary cell culture is one of the ways to attain in vitro transformation by way of disrupting the mechanisms which involve cellular senescence. In this context, a recombinant baculovirus with shrimp viral promoter IHHNV-P2 was used for the transduction aimed at immortalization. An oncogene, H-ras, was successfully amplified and cloned in to the baculoviral vector, downstream to shrimp viral promoter IHHNV-P2 and upstream to GFP. Recombinant baculovirus with H-ras was generated and used for transduction into shrimp lymphoid cells during early dividing stage. Accordingly, fibroblast-like primary cell culture got developed, and H-ras and GFP expression could be confirmed. The study suggests that the simple method of incubating recombinant baculovirus with minced tissue enables in vitro transduction during early dividing stage of the cells, and the transduction efficiency gets enhanced by adding 5 mM sodium butyrate to the culture medium.

Potential roles for microRNAs in facilitating physiological adaptation to low-temperature stress in Penaeus vannamei.

Water temperature is one of the most common physiological stressors in aquaculture. Previous studies demonstrate that organisms require miRNA activity for survival in various unfavourable environmental conditions. However, the detailed role of miRNA in response to low-temperature stress is still unclear. This study was conducted to construct a comprehensive miRNA dataset for the Penaeus vannamei after low-temperature stress. A total of 329 known miRNAs and 60 putative novel miRNAs were identified. Among them, 17 miRNAs were identified with the most significant differences, and they were found to be involved in stimulation or stress processes. The main enriched target pathways of the 17 miRNAs were the Hippo signalling pathway, autophagy, apoptosis and MAPK signalling. In addition, all the 17 miRNAs identified were up-regulated, suggesting that miRNA by inhibiting the expression of target genes constitutes an effective strategy for Penaeus vannamei to cope with low-temperature stress. The 35-putative target of the 17 miRNAs was related to apoptosis and autophagy-related proteins, such as Pxt, DRAM2, cytochrome c, ATG2B, JNK, ATG4 and API5. The analysis of miRNA expression profiles contributes to the understanding of the molecular mechanisms of low-temperature tolerance in Penaeus vannamei. This study's findings enrich current miRNA resources and offer the possibility to validate the involvement of 17 miRNAs in the response of shrimp to low-temperature stress.