Prawn Tissues Research Articles

Effects of eyestalk ablation and seawater temperature on d-glutamate levels in the reproductive tissues of male kuruma prawn Marsupenaeus japonicus.

D-Glutamate, a novel d-amino acid found in animal tissues, exclusively exists in the male reproductive tissues of the kuruma prawn, Marsupenaeus japonicus. Herein, changes in the d-glutamate content were determined in the male reproductive tissues of M. japonicus during acclimation to breeding seawater temperatures of 18-22°C and unilateral eyestalk ablation. The d-glutamate content in the testis increased with increasing seawater temperature and with unilateral eyestalk ablation. This suggests that both stimulations induced d-glutamate synthesis in the testis. Although the d-alanine content in the testis increased after unilateral eyestalk ablation, it did not change with elevated seawater temperature. Furthermore, we determined the d-glutamate distribution in the M. japonicus spermatophore. This indicates that d-glutamate is crucial in prawn fertilization.

Microplastic Contamination in the Tissue of Giant Freshwater Prawns Macrobrachium rosenbergii, Thailand

Microplastics are everywhere and may be harmful to the biota environment. This study investigated microfiber contamination in giant freshwater prawn tissues, one of Thailand’s economic species. The prawn samples were taken from the Trang River in southern Thailand in September 2022. Three tissue sections (head, middle, and tail) of 60 prawns were studied and the samples were digested with 10 % potassium hydroxide (KOH) solution. We utilized a stereomicroscope to study the microplastics and Fourier transform infrared spectroscopy (FT-IR) to determine the types of polymer. There were 3.50 ± 0.34 microfibers in the head per 5 g weight, 2.87 ± 0.29 per 5 g weight in the middle, and 3.10 ± 2.28 per 5 g weight in the tail. Fiber appearance is obvious, the predominant color is blue, the most common size is in the range of 101-500 µm, and the polymer type found is cotton rayon polypropylene (PP). The correlation analysis of microfiber content (in the stomach and intestine), carapace length, abdomen length, stomach weight, and body weight in giant freshwater prawns was not correlated with tissue microfiber content (p > 0.01). It was found that the number of microplastics in giant freshwater prawns was not significantly different (p > 0.05) among the 3 sections of prawn tissue (head tissue, middle tissue, and tail tissue). HIGHLIGHTS Digestion of prawns with KOH 10 % Microplastic contamination was discovered in giant freshwater prawn tissues Fiber shape was most found in giant freshwater prawns The polymer type discovered is cotton rayon polypropylene GRAPHICAL ABSTRACT

Autophagy increases the survival rate of Macrobrachium rosenbergiiin after Aeromonas hydrophila infection

Autophagy is known as crucial components of crustacean innate immunity against pathogens. The dynamic changes of autophagy level during infectious disease is apparently related to disease severity, which needs to deeply understand for a health development and management of aquaculture. In this study, the role of autophagy during A. hydrophila infection was elucidated in M. rosenbergii. Adult freshwater prawn was firstly administered A. hydrophila to assess the virulence of the isolated strain. Pathological analysis of hepatopancreas and intestine tissues of moribund prawn showed significantly pathological changes, including cellular swelling and vacuolation, contraction or karyorrhexis of nuclei, dissolution and necrosis of villi, structural destruction of the striate border. A significant apoptosis and autophagy was observed in hepatopancreas tissues of the infected shrimp. Moreover, the rapamycin-pretreated groups showed high survival rate in contrast with drastic high mortality in autophagy-suppressed groups. Further analysis showed that the elevated autophagy levels restricted apoptosis signals. We conclude that autophagy plays a positive role during A. hydrophila infection and may be is a new therapeutic target for bacterial disease.

Perfluoroalkyl acids in representative edible aquatic species from the lower Yangtze River: Occurrence, distribution, sources, and health risk.

Perfluoroalkyl acid (PFAA) exposure poses a potential hazard to wildlife and humans. Food consumption is one of the main routes of PFAA exposure for the general population, with aquatic organisms being the major contributors. To evaluate the risk of coastal residents' intake of wild aquatic organisms, 14 PFAAs were detected in crucian carp and oriental river prawn from 18 sampling sites from the lower reaches of Yangtze River. The total PFAA (∑PFAA) concentrations ranged from 5.9 to 51.3ng/g wet weight (ww) in the muscle of crucian carp and river prawn, suggesting the potential risk to human and wildlife. Perfluorooctanesulfonate (PFOS), perfluorooctanoic acid (PFOA) and long-chain PFAAs (C≥10) were the main pollutants in the tissues of crucian carp and river prawn, which are known for their higher bioaccumulation capacity. The ∑PFAA concentration in all the samples showed an increasing trend from upstream to downstream and was higher in the south bank, owing to population density, prevailing winds, background pollution and industrial emission. Principal component analysis-multiple linear regression and Pearson correlation analysis showed that WWTP effluent, industrial pollution and surface runoff ware the main sources of PFAAs in the aquatic organisms and industrial pollution highest contributor, suggesting better regulation is needed to manage them. The assessment of risk to human health and wild life suggested a low risk for most residents of cities along the Yangtze River except for resident of Nantong, where frequent consumption of wild aquatic organisms may cause potential risk to human health, especially for traditional eaters and middle-aged people.

Heavy Metals and their Effects on Macroinvertebrates Present in the Ojo River, Lagos, Nigeria

Aim: To provide information regarding the presence of heavy metals in the tissues of crab (Potamon fluviatile), prawn (Macrobrachium rosenbergii), and crayfish (Metanephrops australiensis) obtained from the Ojo river of Lagos State, Nigeria.
 Study Design: Commercially sold marine crustacean samples (crab, prawn, and crayfish) obtained from the Ojo river of Lagos, Nigeria, were assessed for the presence of heavy metals, and also the potential health risks for local consumers.
 Place and Duration of Study: Ojo river, located close to Ojo local government secretariat, Ojo, Lagos State, Nigeria.
 Methodology: Live samples of macroinvertebrates such as Metanephrops australiensis, Potamon fluviatile, and Macrobrachium rosenbergii were purchased from the fishermen at the riverside in Ojo and immediately transferred to the laboratory. The samples were oven-dried and ground into a fine powder, then subjected to sample digestion and finally atomic absorption spectrophotometer (AAS), to obtain the various heavy metal concentrations present in each sample.
 Results: The result indicated variations in the metal body load among species. All the metals were below the FAO/WHO permissible limit for food consumption except for Cd in crayfish which was slightly beyond the set limit. The highest concentration examined were found in crayfish, followed by crab and prawn. Zinc and iron were of higher concentrations in the tissues of the macroinvertebrates while lead was the least concentrated metal present only in the tissues of crayfish and absent in the tissues of both crab and prawn.
 Conclusion: The human health risk evaluation for the marine organisms indicated that both the crab and the prawn samples examined were safe for consumption, while crayfish may not be considered safe for consumption. Also, the potential health risk from consuming seafood exposed to these metals should not be ignored.

Spatial Variation in Contaminant Occurrence in Marine Fishes and Prawns from Coastal Tanzania.

There are limited data on organic contaminants in marine biota from coastal Tanzania, especially on the occurrence of industrial-use contaminants such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). The present study, performed in 2018-2019 in coastal Tanzania and Zanzibar Island, aimed at assessing spatial variation in the occurrence of PCBs; brominated flame retardants (BFRs), including PBDEs; and organochlorine pesticides, including dichlorodiphenyltrichloroethane (DDT), among three locations that differ in degree of anthropogenic activity. Analyzed samples included edible tissues of marine fishes and prawns representing different trophic levels and habitats. The results indicate a mainland-island difference, with fishes and prawns collected on Zanzibar having significantly lower PCB and DDT concentrations but higher concentrations of hexachlorobenzene compared to the two mainland locations. The highest contaminant concentrations were found in fishes and prawns collected around central Dar es Salaam harbor, with median ΣPCBs ranging from 22.3 to 577 ng/g lipid weight and ΣDDTs from 22.7 to 501 ng/g lipid weight, suggesting local sources. Concentrations of PBDEs were similar among locations, suggesting more diffuse sources. None of the "newer-type" BFRs, including compounds introduced as replacements for PBDEs, were detected in the present study. Stable isotope values of carbon (δ13 C) and nitrogen (δ15 N) varied among locations, and the relationship between contaminants and δ15 N varied among locations and habitat (pelagic/demersal). Concentrations measured in the present study are below European guidelines for human consumption of fishes and prawns. However, industrial-use contaminants should be monitored in developing countries because they are contaminants of emerging concern as a result of increasing industrialization and global trade of used products and wastes. Environ Toxicol Chem 2022;41:321-333. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Diet‐tissue discrimination and turnover of δ13C and δ15N in muscle tissue of a penaeid prawn

RationaleStable isotopes are used to study trophic and movement ecology in aquatic systems, as they provide spatially distinct, time‐integrated signatures of diet. Stable isotope ecology has been used to quantify species‐habitat relationships in many important fisheries species (e.g., penaeid prawns), with approaches that typically assume constant values for diet‐tissue discrimination and diet‐tissue steady state, but these can be highly variable. Here we provide the first report of these processes in Metapenaeus macleayi (eastern school prawn).MethodsHere we explicitly measure and model carbon (δ13C) and nitrogen (δ15N) diet‐tissue discrimination and turnover in eastern school prawn muscle tissue as a function of experimental time following a change in diet to an isotopically distinct food source.ResultsDiet‐tissue discrimination factors were 5 and 0.6‰ for δ13C and δ15N, respectively. Prawn muscle tissue reached an approximate steady state after approximately 50 and 30 days for δ13C and δ15N. Half‐lives indicated faster turnover of δ15N (~8 days) than δ13C (~14 days).ConclusionsOur diet‐tissue discrimination factors deviate from ‘typical’ values with larger values for carbon than nitrogen isotopes, but are generally similar to those measured in other crustaceans. Similarly, our estimates of isotopic turnover align with those in other penaeid species. These findings confirm muscle tissue as a reliable indicator of long‐term diet and movement patterns in eastern school prawn.

A newly identified Hippo homologue from the oriental river prawn Macrobrachium nipponense is involved in the antimicrobial immune response

The Hippo signalling pathway plays a vital role in organ size control, cell proliferation, apoptosis, and immune regulation. In this study, a Hippo homologue with three isoforms (named MnHippo-a, MnHippo-b, and MnHippo-c) was isolated and characterized for the first time from the freshwater prawn Macrobrachium nipponense. The deduced amino acid sequences of MnHippo-a (698 aa), MnHippo-b (688 aa), and MnHippo-c (656 aa) were highly similar, and they all contained an N-terminal S_TKc (serine/threonine protein kinase catalytic) domain and a C-terminal Mst1_SARAH (Sav/Rassf/Hpo) domain. MnHippo-a and MnHippo-c were derived from alternative splicing. Phylogenetic analysis was performed, and the results revealed that MnHippo was a member of the clade containing STPK4 and Hippo of Penaeus vannamei. The expression distribution showed that MnHippo was constitutively expressed in various tissues of uninfected prawns and highly expressed in the hepatopancreas and intestine. In prawns challenged with Vibrio parahaemolyticus and Staphylococcus aureus, the expression of MnHippo in haemocytes was significantly upregulated. Furthermore, in MnHippo-knockdown prawns injected with V. parahaemolyticus or S. aureus, the transcription levels of five antimicrobial peptides were downregulated. MnHippo silencing weakened the clearance of V. parahaemolyticus and S. aureus in prawns. The survival rate of the MnHippo-dsRNA group was obviously decreased from 2 to 6 days post-injection with V. parahaemolyticus or S. aureus. Hence, MnHippo might be involved in the antibacterial immune defence of M. nipponense.

Immunopathogenesis of hematopoietic tissues in response to Vibrio parahaemolyticus (VPAHPND) infection in Macrobrachium rosenbergii

In crustacean, hemocytes are known as crucial components of crustaceans’ innate immunity against pathogens. Drastic hemocytes reduction during infectious disease is apparently related to disease severity and calls for a health status evaluation and aquaculture management. The molecular pathogenesis of hemocytes loss during bacterial infection was elucidated with VPAHPND challenged in M. rosenbergii. We report herein a correlation between hemocyte loss and the pathogenicity and aggressive immune response in hematopoietic tissues of moribund M. rosenbergii. In this study, adult freshwater prawn was administered an LC50 dose of VPAHPND; bacterial clearance ensued, and success was reached within 24 h. Hemocytes increased in survival, yet drastically decreased in moribund prawn. Pathological analysis of hematopoietic tissue of moribund prawn showed apparent abnormal signs, including the presence of bacteria, a small number of mitotic cells, cellular swelling, loosening of connective tissue, and karyorrhectic nuclei cells. A significant upregulation of a core apoptotic machinery gene, caspase-3, was detected in hematopoietic tissue of moribund shrimp, but not in those of Escherichia coli DH5α (non-pathogenic bacteria) and VPAHPND survival prawn. The highest level was found in the moribund group, which confirms the occurrence of apoptosis in this hematopoietic tissue. Further, our results suggest that hematopoietic tissue damage may arise from inflammation triggered by an aggressive immune response. Immune activation was indicated by the comparison of immune-related gene expression between controls, E. coli (DH5α)-infected (non-pathogenic), and VPAHPND-infected survival groups with moribund prawn. RT-PCR revealed a significant upregulation of all genes in hematopoietic tissues and hemocytes within 6–12 h and declined by 24 h. This evident related to the almost VPAHPND are clearance in survival and E. coli (DH5α) challenged group in contrast with drastic high expression was determined in moribund group. We conclude that a reduction of renewing circulating hemocytes in fatally VPAHPND-infected prawn was caused by an acute self-destructive immune response by hematopoietic cells.

Genomic and antibody-based assays for the detection of Indian strains of Macrobrachium rosenbergii nodavirus and extra small virus associated with white tail disease of Macrobrachium rosenbergii.

White tail disease (WTD) of cultured Macrobrachium rosenbergiiis caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). Since both the viruses have small single strand RNA as genetic material with short generation time, they are more prone to mutations. Hence detection methods developed for one strain may be suboptimal for the detection of isolates from the different geographical locations. In the present study two new genomic based methods (RT-PCR and dot-blot hybridization) along with one immunological method (polyclonal antibodies based detection) were developed for the detection of Indian isolates of MrNV and XSV. Among genomic based methods, RT-PCR assay developed was most sensitive. Sensitivity of detection of RT-PCR was 1fg (both MrNV and XSV) of total RNA extracted from purified viral inoculum preparation. In case of WTD positive whole tissue total RNA, the limit of detection was 10fg for both MrNV and XSV. Dot-blot hybridization had a detection limit of 10pg and 0.1ng for MrNV and XSV respectively when RNA extracted from viral inoculum preparation was used; 0.1ng and 1ng when WTD positive whole tissue total RNA was used. Polyclonal antibodies against recombinant proteins (MrNV and XSV capsid) were synthesised. Western blotting and indirect ELISA revealed that the antibodies produced to be specific and highly sensitive. Recombinant protein (antigen) of MrNV and XSV capsid were detected at the dilution of 1:8000. However in case of infected prawn tissue sample, MrNV and XSV were detected at the dilution of 1:32,000 and 1:64,000 respectively. All methods developed are field applicable.

Function of the MOB kinase activator-like 1 in the innate immune defense of the oriental river prawn (Macrobrachium nipponense)

The monopolar spindle one binder (MOB) protein, a key signal transducer of the Hippo signaling pathway, is involved in growth control and cancer. In this study, a new MOB kinase activator-like 1 of the oriental river prawns, Macrobrachium nipponense, (MnMOB1) was isolated and characterized. The open reading frame of MnMOB1 consisted of 651 nucleotides that encoded 216 amino acid residues and contained the Mob1_phocein domain. Phylogenetic analysis revealed that MnMOB1 clustered together with the MOB1 from Penaeus vannamei. The distribution of MnMOB1 expression in various tissues of normal prawn revealed that the MnMOB1 expression was highest in the hepatopancreas followed by those in the intestines, gill, heart, stomach, and hemocytes. In prawns challenged with Staphylococcus aureus and Vibrio parahaemolyticus, the expression levels of MnMOB1 in the hepatopancreas, gills, and intestine were upregulated. Furthermore, the expression levels of crustins and anti-lipopolysaccharide factors in prawn injected with S. aureus and V. parahaemolyticus and MnMOB1 knockdown were significantly decreased relative to those in the control group. These findings indicated that MnMOB1 is involved in the regulation of antimicrobial peptide expression and plays a crucial role in the innate immunity of M. nipponense.

Role of Intestinal Helminth Parasite of Macrobrachium vollenhovenii-Cucullanus sp. in Host Metal Accumulation in Epe Axis of the Lekki Lagoon, Lagos, Nigeria.

This paper assesses the role of intestinal helminth parasite of Macrobrachium vollenhovenii on host metal accumulation in Lekki lagoon, Lagos, Nigeria. Lead, zinc, copper, chromium and iron concentrations were determined in water, sediment, intestinal parasite, intestine of infected and non-infected prawns. Intestinal nematode parasite Cucullanus sp. was identified. Pb and Cr were not detected in the water and sediment. The sediment had higher concentrations of the metals than the water medium, and exceeded the WHO limits for Zn, Cu and Fe. Cucullanus sp. accumulated Zn 19 times, Cr several thousand times more and Fe 11 times more than the host intestine (wet wt). This parasite could have accumulated these metals from its host, reducing the concentration of these metals in the tissues of the infected prawns compared to non-infected ones. The infected and non-infected prawns accumulated Zn and Fe several times more than the water medium. The parasite influenced metal accumulation in the host tissue and pose ecological risk.

A Kruppel-like factor from Macrobrachium rosenbergii (MrKLF) involved in innate immunity against pathogen infection

Kruppel-like factors (KLFs) belong to a family of zinc finger-containing transcription factors that are widely present in eukaryotes. In the present study, a novel KLF from the giant river prawn Macrobrachium rosenbergii (designated as MrKLF) was successfully cloned and characterized. The full-length cDNA of MrKLF was 1799 bp with an open reading frame of 1332 bp that encodes a putative protein of 444 amino acids, including three conserved ZnF_C2H2 domains at the C-terminus. Multiple alignment analysis showed that MrKLF and other crustacean KLFs shared high similarity. Quantitative real-time PCR analysis revealed that MrKLF mRNA was found in different tissues of prawns and detected in the gills, hepatopancreas, and intestines. After the challenge with Vibrio parahaemolyticus and Aeromonas hydrophila, different expression patterns of MrKLF in the gills, intestines, and hepatopancreas were observed. RNA interference analysis indicated that MrKLF was involved in regulating the expression of four antimicrobial peptides, namely, Crustin (Crus) 2, Crus8, anti-lipopolysaccharide factor (ALF) 1, and ALF3. These results help promote research on M. rosenbergii innate immunity.

Employing DNA binding dye to improve detection of Enterocytozoon hepatopenaei in real-time LAMP

Enterocytozoon hepatopenaei (EHP) is a pathogen in the pancreatic tissue of prawn, as reported in recent years. Enterosporidiosis caused by EHP in Penaeus genus is spreading widely, which seriously threatens the sustainable development of shrimp aquaculture in the world. Empolying the DNA binding dye of SYTO-16, a real-time quantitative loop-mediated isothermal amplification (LAMP) method has been established herein to detect EHP. The primer sequences used in the LAMP reaction were according to the SSU rRNA gene. The LAMP assay has reached a sensitivity of 101 copies/µL and no cross-reaction with other aquatic pathogens. Compared with normal PCR, the efficiency of the LAMP technique is more sensitive, which has a shorter detection time. The use of fluorescent nucleic acid dye (SYTO-16) reveals a more satisfactory performance relative to calcein. The quantitative LAMP assay can be considered as a valid tool for rapid detection of microsporidian in prawns. Our study provides a scientific basis to follow the effect of the pathogen infection on growth of cultured penaeid shrimp.

Comparative account of DNA extraction protocols in some freshwater prawns of Genus Macrobrachium (Bate, 1868) (Family Palaemonidae) from Jammu waters for PCR based applications

Genetic variations among prawns act as an important tool to characterize and differentiate between the species. Molecular and phylogenetic analysis of shrimps and prawns like any other organism rely on high yields of pure and better quality genomic DNA. In this regard isolation of DNA is the first and basic step. In spite of the availability of many protocols of DNA extraction from animal tissues, it is difficult to ascertain that which one would provide desired results for prawn tissue. In the present study, three different techniques of DNA isolation i.e., salting out, phenol-chloroform and Qiagen DNA extraction kit were performed and compared for their yield. Cephalothoracic tissue and muscle tissue of pleopods were used for isolation. Tissue samples from fresh specimens as well as from alcohol preserved specimens were employed for extraction. The quantity (µg/ml) and quality of isolated DNA were determined by UV spectrophotometry and agarose gel electrophoresis. Results showed that Phenol-chloroform method with slight modifications obtained higher yield of genomic DNA as compared to other methods. The present work also revealed that among fresh specimens cephalotoracic tissue yielded high concentration DNA than muscle tissue. However, among alcohol preserved specimens, the concentration of DNA was higher in muscle tissue of pleopods. The high quality DNA was then subjected to randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) analysis. The DNAs produced clear, sharp and reproducible PCR (Polymerse chain reaction) product pattern.

2-Transmembrane C-type lectin from oriental river prawn Macrobrachium nipponense participates in antibacterial immune response

As a type of pattern-recognition proteins (PRRs), C-type lectins (CTLs) perform important functions in non-self recognition and clearance of pathogens in innate immunity. In this study, a unique 2-transmembrane CTL (designated as Mn-2TM-cLec) with a single carbohydrate recognition domain (CRD) was isolated from Macrobrachium nipponense. The full-length cDNA of Mn-2TM-cLec consisted of 3265 bp with an 837 bp open reading frame encoding a protein with 278 amino acids. Mn-2TM-cLec was ubiquitously distributed in various tissues of normal prawn, particularly in the hemocytes, hepatopancreas, and gills. The expression of Mn-2TM-cLec was significantly up-regulated in the gills and hepatopancreas after the prawns were challenged with Staphylococcus aureus and Vibrio parahaemolyticus. RNA interference knock-down of Mn-2TM-cLec gene decreased the transcription levels of three antimicrobial peptides (anti-lipopolysaccharide factor (ALF) 1, ALF2, and Crustin (Crus) 1) after V. parahaemolyticus infection. The recombinant CRD of Mn-2TM-cLec could bind lipopolysaccharide, peptidoglycans, and diverse bacterial strains and agglutinate S. aureus and V. parahaemolyticus in a Ca2+-dependent manner. In addition, the rCRD enhanced the clearance of V. parahaemolyticus injected in prawns. In summary, Mn-2TM-cLec might act as a PRR to participate in the prawn immune defense against pathogens through its antimicrobial activity.

Distribution of abalone egg-laying hormone-like peptide in the central nervous system and reproductive tract of the male mud crab, Scylla olivacea

The mud crab, Scylla olivacea, is a high value economic marine animal in Thailand. However, collection of these crabs from natural habitat for local consumption and export has caused rapid population decline. Hence, aquaculture of this species is required and to this measure understanding of endocrine control of their reproduction must be understood. Egg laying hormone (ELH) is a neuropeptide synthesized by the bag cells (neurons) in the abdominal ganglia of Aplysia gastropods. It plays a critical role in controlling egg production and laying in gastropods, and its possible homolog (ELH-like peptide) was reported in the neural and ovarian tissues of prawns and recently in female reproductive tract of the blue swimming crab, Portunus pelagicus. In this study, we have studied the histology of the male reproductive tract in Scylla olivacea which are comprised of anterior testis, posterior testis, early proximal spermatic duct (ePSD), proximal spermatic duct (PSD), middle spermatic duct (MSD) and distal spermatic duct (DSD), by immunohistochemistry, detected an abalone ELH- immunoreactivity (aELH-ir) in epithelium of ducts in posterior testis and epithelium of all parts of spermatic duct. Furthermore, we could detect aELH-ir in neurons of cluster 9, 11, olfactory neuropil (ON) in the brain and in the small neurons located between the third and the fourth thoracic neuropils (T3-T4) and between the fourth and the fifth thoracic neuropils (T4-T5) of thoracic ganglia. Thus, the presence of aELH in male S. olivacea was designated the role of female egg laying behavior in the male mud crab.

Involvement of tetraspanin 8 in the innate immune response of the giant prawn, Macrobrachium rosenbergii

The tetraspanins, representing a conserved superfamily of four-span membrane proteins, are highly involved in viral and bacterial infections. Thus far, the function of the tetraspanins in crustaceans remains largely unknown. In this study, we report the cloning and expression analysis of a tetraspanin 8 from the giant freshwater prawn, Macrobrachium rosenbergii (named as MrTspan8). MrTspan8 contains a 720-bp open reading frame encoding a 239-amino acids protein, which exhibits four transmembrane domains and two extracellular loops that are typical for tetraspanins. MrTspan8 was found to be widely expressed in a variety of prawn tissues including heart, gill, muscle, gut, and hepatopancreas. Additionally, MrTspan8 expression was significantly increased in the hepatopancreas and gill of the prawns challenged by the bacterial pathogen Aeromonas hydrophila. Moreover, we show that pre-incubation of the peptides from the large extracellular loop of MrTSPAN8 protein reduced the cell death caused by A. hydrophila infection in prawn tissue, suggesting that MrTSPAN8 could be a mediator for bacterial infection to prawn.

Molecular isolation and characterization of a spätzle gene from Macrobrachium rosenbergii

Spätzle protein is an extracellular ligand of Toll receptor in Toll signaling pathway involved in the embryonic dorsoventral patterning and in the innate immunity. In this study, a spätzle gene of freshwater prawn, Macrobrachium rosenbergii (MrSpz) was isolated and characterized. The open reading frame of MrSpz consisted of 747 nucleotides encoding 248 amino acid residues containing a signal peptide and C-terminal spätzle activated domain. MrSpz shared high similarity to spätzle of Fenneropenaeus chinensis (FcSpz) at 92% identity and Marsupenaeus japonicus (MjSpz) at 83% identity. Phylogenetic analysis was performed and the results revealed that MrSpz was a member of the clade containing LvSpz3 of Litopenaeus vannamei, FcSpz and Penaeus monodon spätzle protein. The expression distribution at transcriptional level in various tissues of normal prawn revealed that the MrSpz was detected in gills, heart and hepatopancreas while no expression was observed in hemocyte, muscle and stomach. In the Aeromonas caviae challenged prawn, the expression level of MrSpz in hemocyte was increased gradually at 6, 12 and 24 h post-injection. Furthermore, in MrSpz knocked down prawn injected with Aeromonas caviae, the mortality rate were higher than that of non-related dsRNA group and control group. These results suggest that MrSpz protein may play a key role in the innate immunity of M. rosenbergii, especially in response to Gram-negative bacteria A. caviae invasion.

Integrated metabolomic and transcriptomic analysis of brain energy metabolism in the male Oriental river prawn (Macrobrachium nipponense) in response to hypoxia and reoxygenation

Hypoxia is as an endocrine disruptor, and, in crustaceans, the energy metabolic consequences of hypoxia in the brain tissue are still poorly understood. We combined gas chromatography-mass spectrometry (GC-MS)-based metabolomic analysis and high-throughput RNA sequencing to evaluate the metabolic effects and subjacent regulatory pathways in the brain tissue of the male Oriental river prawn (Macrobrachium nipponense) in response to hypoxia and reoxygenation. We recorded LC50 and heartbeats per minute of male M. nipponense juveniles. Hypoxia resulted in the generation of reactive oxygen species in the brain cells and alterations in gene expression and metabolite concentrations in the prawn brain tissue in a time-dependent manner. The transcriptomic analyses revealed specific changes in the expression of genes associated with metabolism pathways, which was consistent with the changes in energy metabolism indicated by the GC-MS metabolomic analysis. Quantitative real-time polymerase chain reaction and western blot confirmed the transcriptional induction of these genes because of hypoxia. The lactate levels increased significantly during hypoxia and decreased to normal after reoxygenation; this is consistent with a shift towards anaerobic metabolism, which may cause metabolic abnormalities in the brain tissue of M. nipponense. Overall, these results are consistent with metabolic disruption in the brain of M. nipponense exposed to hypoxia and will help in understanding how crustacean brain tissue adapts and responds to hypoxia and reoxygenation.