In order to investigate the effects of multi-frequency ultrasound-assisted immersion freezing (MUIF) on the meat quality of Macrobrachium rosenbergii, tail meat was subjected to different MUIF treatments respectively, namely 20 + 40 kHz (MUIF-20 + 40), 20 + 60 kHz (MUIF-20 + 60), 40 + 60 kHz (MUIF-40 + 60) and 20 + 40 + 60 kHz (MUIF-20 + 40 + 60), and the immersion freezing (IF) as control. Results showed that average diameter of ice crystals was 28 μm in IF, and that was only 8 μm in MUIF-20 + 40 + 60. When compared to IF, MUIF alleviated oxidative deterioration of lipids and proteins, but only at higher ultrasound frequency (MUIF-40 + 60; MUIF-20 + 40 + 60). Carbonyl content of MUIF-20 + 40 + 60 was only 40% of that in IF. Similarly, protein denaturation was inhibited in MUIF (except for MUIF-20 + 40). Transmission electron microscopy showed greater distortion of the ultrastructural components in IF, MUIF-40 + 60, and MUIF-20 + 40 + 60, suggested by bended Z-line. In conclusion, MUIF can be an effective strategy to mitigate mechanical damage and protein deterioration in the meat of Macrobrachium rosenbergii.

Decapod iridescent virus 1 (DIV1) is a lethal virus that has a significant influence on the shrimp and prawn culture industries. The mechanism through which infected prawns respond to the DIV1 virus is currently unknown. Here, we examined in detail the clinical signs, histopathology, and humoral, cellular, and immune-related gene responses after a sub-lethal dose of DIV1 during the acute infection period of 0-120 h post infection (hpi). Interestingly, at the end of the experiment, DIV1-infected prawns had black lesions on several external regions. The DIV1-infected prawns also exhibited few karyopyknotic nuclei in the gills and intestine tissues and exhibited increasing immunological responses, as revealed by significant increases in all examined parameters, including total hemocytes, phagocytosis, lysozyme, and overall bactericidal activity, from 6 to 48 hpi. In addition, between 72 and 120 hpi, all immune response activities of DIV1-infected prawn were impaired compared with those of normal prawns, indicating negative impacts on immunological parameters. A viral load analysis of various tissues by qPCR indicated that hemocytes were the dominant initial viral target tissues, followed by the gills and hepatopancreas. An expression analysis of crucial immune-related genes by qRT‒PCR revealed various expression patterns in response to DIV1 infection; in particular, fold changes in the relative expression of anti-lipopolysaccharide factors (ALFs), prophenoloxidase (proPO), lipopolysaccharide and β-1,3-glucan binding protein (LGBP) were observed. Additionally, five common chemicals, calcium hypochlorite [Ca(OCl)2] at 16.25-130 ppm, hydrogen peroxide (H2O2) at 8.75-70 ppm, povidone iodine (PVP-I) at 3-24 ppm, benzalkonium chloride (BKC) at 20-160 ppm, and formalin at 25-200 ppm, had a significant effect on the killing of DIV1 particles in vitro within 24 h after exposure. These data will be helpful for determining the health status and immune defense mechanisms of giant river prawns during DIV1 infection periods. The study performed the first application of very common disinfectants, and the obtained information will be useful for implementing effective strategies to prevent and control DIV1 infection in both hatchery and grow-out ponds.

The endoplasmic reticulum (ER) is an organelle important for several functions of cellular physiology. This study identified the giant river prawn\'s glucose-regulated protein 78 (Mr-grp78), which is important for ER stress mechanisms. Nucleotide and amino acid analyses of Mr-grp78, as compared with other species, revealed the highest similarity scores with the grp78 genes of crustaceans. An expression analysis by quantitative RT-PCR indicated that Mr-grp78 was expressed in all tissues and presented its highest expression in the ovary (57.64 ± 2.39-fold), followed by the gills (42.25 ± 1.12), hindgut (37.15 ± 2.47), thoracic ganglia (28.55 ± 2.45) and hemocytes (28.45 ± 2.26). Expression analysis of Mr-grp78 mRNA levels under Aeromonas hydrophila induction and heat/cold-shock exposure was conducted in the gills, hepatopancreas and hemocytes. The expression levels of Mr-grp78 in these tissues were highly upregulated 12 h after bacterial infection. In contrast, under heat- and cold-shock conditions, the expression of Mr-grp78 was significantly suppressed in the gills at 24-96 h and in the hepatopancreas at 12 h (p < 0.05). A functional analysis via Mr-grp78 gene knockdown showed that Mr-grp78 transcription in the gills, hepatopancreas and muscle strongly decreased from 6 to 96 h. Furthermore, the silencing of this gene effectively increased the sensitivity of the tested prawns to heat- and pathogenic-bacterium-induced stress. The results of this study clearly demonstrate the significant functional roles of Mr-grp78 in response to both temperature and pathogen treatments.

Anti-lipopolysaccharide factor (ALF) is an immune-related protein that is crucially involved in immune defense mechanisms against invading pathogens in crustaceans. In the current study, three different ALFs of giant river prawn (Mr-ALF3, Mr-ALF8 and Mr-ALF9) were discovered. Based on sequence analysis, Mr-ALF3 and Mr-ALF9 were identified as new members of ALFs in crustaceans (groups F and G, respectively). Structurally, each newly identified Mr-ALF contained three α-helices packed against a four-stranded β-sheet bearing the LPS-binding motif, which usually binds to the cell wall components of bacteria. Tissue expression analysis using quantitative real-time RT-PCR (qRT-PCR) demonstrated that Mr-ALF3 was expressed in most tissues, and the highest expression was in the heart and hemocytes. The Mr-ALF8 gene was highly expressed in the heart, hemocytes, midgut, hepatopacreas and hindgut, respectively, while the Mr-ALF9 gene was modestly expressed in the heart and hemocytes, respectively. The transcriptional responses of the Mr-ALFs to Aeromonas hydrophila and hot/cold temperatures were investigated by qRT-PCR in the gills, hepatopancreas and hemocytes. We found that all Mr-ALFs were clearly suppressed in all tested tissues when the experimental prawns were exposed to extreme temperatures (25 and 35 °C). Moreover, the expression levels of these genes were significantly induced in all examined tissues by 2 different concentrations of A. hydrophila (1 × 106 and 1 × 109 CFU/ml), particularly 12 and 96 h after the injection. Finally, binding activity analysis of LPS-motif peptides of each Mr-ALF revealed that the LPS peptide of Mr-ALF3 exhibited the strongest adhesion to two pathogenic Gram-negative bacteria, A. hydrophila and Vibrio harveyi, and the non-pathogenic Gram-positive Bacillus megaterium. The results also showed that the Mr-ALF8 and Mr-ALF9 peptides had mild antimicrobial effects against similar tested bacteria. Based on information obtained in this study, novel ALF genes were clearly identified. Analyses of their responses under pathogenic and temperature stresses demonstrated the binding and antimicrobial activities of these ALFs and the consequent physiological effects, indicating their crucial functional roles in the prawn immune system.

The giant river prawn (Macrobrachium rosenbergii) is one of the most farmed freshwater crustaceans in the world. Its global production has been stalling in the past decade due to the inconsistent quality of broodstock and hatchery-produced seeds. A better understanding of the role of nutrition in maturation diets will help overcome some of the production challenges. Arachidonic acid (20:4 n-6, ARA) is a fatty acid precursor of signaling molecules important for crustacean reproduction, prostaglandins E and F of the series II (PGE2 and PGF2α), and is often lacking in maturation diets of shrimp and prawns. We examined the effects of ARA in a combination of different fish oil (FO) and soybean oil (SO) blends on females\' reproductive performance and larval quality. Adult females (15.22 ± 0.13 g and 11.12 ± 0.09 cm) were fed six isonitrogenous and isolipidic diets containing one of two different base compositions (A or B), supplemented with one of three levels of Mortierella alpine-derived ARA (containing 40% active ARA): 0, 1 or 2% by ingredient weight. The two base diets differed in the percentages of (FO and SO with diet A containing 2% SO and 2% FO and diet B containing 2.5% SO and 1.5% FO, resulting in differences in proportional contents of dietary linoleic acid (18:2n-6, LOA) and docosahexaenoic acid (22:6n-3, DHA)). After the eight-week experiment, prawns fed diet B with 1 and 2% ARA supplement (B1 and B2) exhibited the highest gonadosomatic index (GSI), hepatosomatic index (HSI), egg clutch weight, fecundity, hatching rate, number of larvae, and reproductive effort compared to those fed other diets (p ≤ 0.05). Larvae from these two dietary treatments also had higher tolerance to low salinity (2 ppt). The maturation period was not significantly different among most treatments (p ≥ 0.05). ARA supplementation, regardless of the base diet, significantly improved GSI, HSI, egg clutch weight and fecundity. However, the diets with an enhanced ARA and LOA (B1 and B2) resulted in the best reproductive performance, egg hatchability and larval tolerance to low salinity. These dietary treatments also allow for effective accumulation of ARA and an n-3 lcPUFA, DHA in eggs and larvae.

The giant river prawn, Macrobrachium cf. rosenbergii, is one of the most cultivated freshwater prawns in the world and has been introduced into more than 40 countries. In some countries, this prawn is considered an invasive species that requires close monitoring. Recent changes in the taxonomy of this species (separation of M. rosenbergii and M. dacqueti) require a re-evaluation of introduced taxa. In this work, molecular analyses were used to determine which of these two species was introduced into Brazil and to establish the geographic origin of the introduced populations that have invaded Amazonian coastal waters. The species introduced into Brazil was M. dacqueti through two introduction events involving prawns originating from Vietnam and either Bangladesh or Thailand. These origins differ from historical reports of the introductions and underline the need to confirm the origin of other exotic populations around the world. The invading populations in Amazonia require monitoring not only because the biodiversity of this region may be affected by the introduction, but also because admixture of different native haplotypes can increase the genetic variability and the likelihood of persistence of the invading species in new habitats.