Fernand F. Fagutao

Increased bacterial load in shrimp hemolymph in the absence of prophenoloxidase

Invertebrates rely on their innate immune responses to protect themselves from pathogens, one of which is melanization of bacteria mediated by the activation of phenoloxidase (PO). Furthermore, invertebrate hemolymph, even that of healthy individuals, has been shown to contain bacterial species. The mechanisms that prevent these bacteria from proliferating and becoming deleterious to the host are, however, poorly understood. Here, we show that knocking down the activity of the inactive precursor of PO [prophenoloxidase (proPO)] by RNA interference resulted in a significant increase in the bacterial load of kuruma shrimp, Marsupenaeus japonicus, even in the absence of a bacterial or viral challenge. Silencing of proPO also led to a sharp increase in shrimp mortality. In addition, the hemolymph of proPO‐depleted shrimp had significantly lower hemocyte counts and PO activity than control samples. Microarray analysis after proPO silencing also showed a decrease in the expression of a few antimicrobial peptides, but no effect on the expression of the genes involved in the clotting system. Treatment with antibiotics prior to and after proPO dsRNA injection, to counteract the loss of proPO, resulted in a significant increase in shrimp survival. Our results therefore show that the absence of proPO renders the shrimp incapable of controlling bacteria present in the hemolymph, and that proPO is therefore essential for its survival.

Transglutaminase regulates immune-related genes in shrimp

Transglutaminase (TGase) is known to be involved in blood coagulation, a conserved defence mechanism among invertebrates. Gene silencing of TGase was previously shown to render shrimp susceptible to both bacterial and viral infections suggesting that TGase is an essential component of the shrimp immune system. Here, we examine the effects of the absence of TGase on the transcriptomic profile of kuruma shrimp by microarray analysis, focussing on genes that are involved in shrimp immunity. Total RNAs from shrimp haemocytes injected with dsRNA specific for TGase and control samples were isolated at 3 and 7 days p.i. and analyzed by microarray. Results revealed that TGase silencing affects the expression of genes in shrimp and caused significant down-regulation of the expressions of crustin and lysozyme. Furthermore, TGase-depleted samples were found to have lower haemocyte counts and higher total bacterial counts in their haemolymph. These results suggest that TGase is an important component of the shrimp immune response and is involved in the regulation of some immune-related genes particularly antimicrobial peptides.

Uncovering the Mechanisms of Shrimp Innate Immune Response by RNA Interference

Because of the importance of shrimp in world aquaculture, there is much interest in understanding their immune system in order to improve their resistance to pathogenic microorganisms. An effective tool in studying genes involved in the immune response in shrimp is RNA interference (RNAi). RNAi, first recognized as an antiviral response against RNA viruses, is a cellular mechanism that is triggered by double-stranded RNAs and results in the degradation of homologous genes. In this review, we describe the current studies of genes in shrimp that employed RNAi technology to elucidate or confirm their functions. We also review the potential of RNAi to elicit antiviral response in shrimp.

Effects of ergothioneine from mushrooms (Flammulina velutipes) on melanosis and lipid oxidation of kuruma shrimp (Marsupenaeus japonicus).

The antimelanosic and antioxidative properties of a hot water extract prepared from the fruiting body of the edible mushroom (Flammulina velutipes) were evaluated by dietary supplementation in Kuruma shrimp (Marsupenaeus japonicus) for possible aquaculture application. The extract contained ergothioneine (ERT) at a level of 2.05 mg/mL. A commercial standard of l-ergothioneine (l-ERT) and the mushroom extract showed inhibitory activity against mushroom polyphenoloxidase (PPO). Feeding of the extract had no adverse effects on the immune systems of the shrimp under the present experimental conditions. Supplementation of the extract in the diet significantly suppressed PPO activities in the hemolymphs of the shrimp. Expression of the prophenoloxidase (proPO) gene decreased in the hemocyte of the Kuruma shrimp fed with the mushroom extract. Consequently, development of melanosis was significantly suppressed in the supplement fed shrimp during ice storage. Lipid oxidation was also effectively controlled in the supplement fed group throughout the storage period. In vitro experiments showed that l-ERT effectively inhibited the activation of proPO in the hemocyte lysate supernatant (HLS). The transcript of the proPO gene in the hemocyte showed lower expression in the l-ERT-treated HLS. It was concluded that dietary supplementation of the mushroom extract in shrimp could be a promising approach to control post mortem development of melanosis and lipid oxidation in shrimp muscles.

Functional Analysis of C-type Lysozyme in Penaeid Shrimp

Background: Lysozyme is ubiquitous in many organisms and functions primarily in bacterial lysis. Results: The absence of lysozyme in shrimp resulted in the increase of bacteria in the hemolymph leading to mortality. Conclusion: Lysozyme is essential to shrimp survival particularly in regulating bacterial communities in the hemolymph. Significance: A clear understanding of the immune effectors in shrimp, such as AMPs, is vital in ensuring resistance against bacterial pathogens. Lysozyme is an enzyme that cleaves the β-1,4-glycosidic linkages between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, leading to bacterial lysis. Recently, lysozyme has been found to have anti-HIV and anti-cancer properties in mammals. However, most functional analyses were done in vitro using purified or recombinant lysozyme protein. Here, we used RNA interference to silence c-type lysozyme expression in penaeid shrimp, Marsupenaeus japonicus, to analyze the function of lysozyme in vivo. Silencing of lysozyme expression by dsRNA lysozyme (dsLYZ) led to 100% mortality without any artificial bacterial infection in 5 days. Lysozyme deficiency caused the number of hemocytes in hemolymph to decrease from 1.3 × 107 to 2.3 × 106 cells/ml and caused the number of bacteria to increase from 78 to 764 colony-forming units/ml. Suppression of bacterial growth using oxytetracycline and kanamycin showed improvement in mortality, suggesting that shrimp mortality post- dsLYZ injection can be attributed to bacterial growth in the shrimp hemolymph. The majority of the bacteria, identified by 16 S rRNA analysis, were Gram-negative species such as Vibrio and Pseudomonas. Furthermore, PKH26 staining showed that the dsLYZ-injected shrimp were unable to eliminate non pathogenic Escherichia coli or Staphylococcus aureus in 24 h. These data suggest that c-type lysozyme in shrimp serves to regulate the growth of bacterial communities, particularly Gram-negative bacteria, in the hemolymph.

Characterization of crustin antimicrobial proteins from Japanese spiny lobster Panulirus japonicus.

Crustin antimicrobial proteins (PJC1-4) were identified from a phyllosoma library of Japanese spiny lobster, Panulirus japonicus. The deduced amino acid sequences of PJC1-4 contained open reading frames of 130, 139, 124 and 150 amino acid residues, respectively. These proteins contained a glycine-rich region at the N-terminus and 12 conserved cysteine residues containing a single whey acidic protein (WAP) domain at the C-terminus. A phylogenetic tree and sequences alignment analyses revealed that PJC1-4 are more closely related to shrimp crustins than to other lobster crustins. Transcripts of PJC1, 3 and 4 were detected in heart, nerves, intestine, hemocytes, gills and hepatopancreas, while transcripts of PJC2 were detected only in nerves.

Hyper-expansion of large DNA segments in the genome of kuruma shrimp, Marsupenaeus japonicus

BackgroundHigher crustaceans (class Malacostraca) represent the most species-rich and morphologically diverse group of non-insect arthropods and many of its members are commercially important. Although the crustacean DNA sequence information is growing exponentially, little is known about the genome organization of Malacostraca. Here, we constructed a bacterial artificial chromosome (BAC) library and performed BAC-end sequencing to provide genomic information for kuruma shrimp (Marsupenaeus japonicus), one of the most widely cultured species among crustaceans, and found the presence of a redundant sequence in the BAC library. We examined the BAC clone that includes the redundant sequence to further analyze its length, copy number and location in the kuruma shrimp genome.ResultsMj024A04 BAC clone, which includes one redundant sequence, contained 27 putative genes and seemed to display a normal genomic DNA structure. Notably, of the putative genes, 3 genes encode homologous proteins to the inhibitor of apoptosis protein and 7 genes encode homologous proteins to white spot syndrome virus, a virulent pathogen known to affect crustaceans. Colony hybridization and PCR analysis of 381 BAC clones showed that almost half of the BAC clones maintain DNA segments whose sequences are homologous to the representative BAC clone Mj024A04. The Mj024A04 partial sequence was detected multiple times in the kuruma shrimp nuclear genome with a calculated copy number of at least 100. Microsatellites based BAC genotyping clearly showed that Mj024A04 homologous sequences were cloned from at least 48 different chromosomal loci. The absence of micro-syntenic relationships with the available genomic sequences of Daphnia and Drosophila suggests the uniqueness of these fragments in kuruma shrimp from current arthropod genome sequences.ConclusionsOur results demonstrate that hyper-expansion of large DNA segments took place in the kuruma shrimp genome. Although we analyzed only a part of the duplicated DNA segments, our result suggested that it is difficult to analyze the shrimp genome following normal analytical methodology. Hence, it is necessary to avoid repetitive sequence (such as segmental duplications) when studying the other unique structures in the shrimp genome.

Edible Mushroom (Flammulina velutipes) Extract Inhibits Melanosis in Kuruma Shrimp (Marsupenaeus japonicus)

This study compared the potential of an aqueous extract of an edible mushroom (Flammulina velutipes) to prevent melanosis in cultured Kuruma shrimp (Marsupenaeus japonicus) with other antimelanosic compounds in vivo. The mushroom extract contained 9.1 mg/mL ergothioneine (ESH). Immersion of live full-grown shrimp in a 0.5% w/v solution of mushroom extract significantly reduced PPO activity in shrimp hemolymph. In addition, expression of the prophenoloxidase (proPO) gene decreased in hemocytes, suggesting that the extract blocked the activation of the proPO cascade. Consequently, the development of melanosis in the treated shrimp was significantly suppressed during ice storage. Treatment with a 0.05% w/v solution of sodium ascorbate and 4-hexyl-1,3-benzenediol had the same effect. In vitro experiments showed that ESH effectively inhibited PPO activity and activation of the proPO cascade in hemocyte lysate supernatant. This study suggests that in vivo application of F. velutipes mushroom extract is an effective natural alternative to synthetic antimelanosic agents to inhibit postmortem melanosis in shrimp. Practical Application: The extract of an edible mushroom (F. velutipes) containing ergothioneine can be a promising natural alternative to synthetic antimelanosic agents used to prevent postharvest melanosis in shrimp and other crustaceans. Furthermore, utilization of the mushroom trimmings could also help address the growing concerns on the disposal of such agricultural wastes and instead use it into a novel purpose as a source of antimelanosic and antioxidants for food and industrial application.

Differential gene expression in black tiger shrimp, Penaeus monodon, following administration of oxytetracycline and oxolinic acid

The intensification of shrimp farming systems has led to the spreading of a variety of bacterial and viral diseases that continue to plague the shrimp industry worldwide. Efforts to combat these pathogenic organisms include the use of immunostimulants, probiotics, vaccines and antibiotics. Although a few studies have already reported on the effects of various stimuli on shrimp, the effect of antibiotics, particularly on the changes in the shrimp transcriptomic profile have yet to be reported. Here we show that injecting shrimp with oxytetracycline and oxolinic acid alters the expression of genes in the black tiger shrimp, Penaeus monodon, lymphoid organ. These antibiotics, especially oxylinic acid, down-regulated the expression of a few immune-related genes, most notably penaeidin, proPO, clotting protein, profilin and whey acidic protein.