Acute hepatopancreatic necrosis disease (AHPND) is a severe disease affecting recently stocked cultured shrimps. The disease is mainly caused by V. parahaemolyticus that harbors the pVA1 plasmid; this plasmid contains the pirA and pirB genes, which encode a delta-endotoxin. AHPND originated in China in 2009 and has since spread to several other Asian countries and recently to Latin America (2013). Many Asian strains have been sequenced, and their sequences are publicly accessible in scientific databases, but only four strains from Latin America have been reported. In this study, we analyzed nine pVA1-harboring V. parahaemolyticus sequences from strains isolated in Mexico along with the 38 previously available pVA1-harboring V. parahaemolyticus sequences and the reference strain RIMD 2210633. The studied sequences were clustered into three phylogenetic clades (Latin American, Malaysian, and Cosmopolitan) through pangenomic and phylogenomic analysis. The nucleotide sequence alignment of the pVA1 plasmids harbored by the Asian and Latin American strains confirmed that the main structural difference in the plasmid between the Asian and Latin American strains is the absence of the Tn3 transposon in the Asian strains; in addition, some deletions in the pirAB region were found in two of the Latin American strains. Our study represents the most robust and inclusive phylogenomic analysis of pVA1-harboring V. parahaemolyticus conducted to date and provides insight into the epidemiology of AHPND. In addition, this study highlights that disease diagnosis through the detection of the pirA and pirB genes is an inadequate approach due to the instability of these genes.
Biofloc technology improves water quality and promote the growth of beneficial bacteria community in shrimp culture. However, little is known about the bacteria community structure in both water and gut of cultured organisms. To address this, the current study characterised the metagenomes derived from water and shrimp intestine samples of novel Rapid BFTTM with probiotic and clearwater treatments using 16S V4 region and full length 16S sequencing. Bacteria diversity of water and intestine samples of Rapid BFTTM and probiotic treatments were similar. Based on the 16S V4 region, water samples of >20 μm biofloc had the highest abundance of amplicon sequence variant (ASV). However, based on full length 16S, no clear distinction in microbial diversity was observed between water samples and intestine samples. Proteobacteria was the most abundant taxon in all samples based on both 16S V4 and full length 16S sequences. Vibrio was among the highest genus based on 16S V4 region but only full length 16S was able to discern up to species level, with three Vibrios identified-V. harveyi, V. parahaemolyticus and V. vulnificus. Vibrio harveyi being the most abundant species in all treatments. Among water samples, biofloc water samples had the lowest abundance of all three Vibrios, with V. vulnificus was present only in bioflocs of <20 μm. Predicted functional profiles of treatments support the beneficial impacts of probiotic and biofloc inclusion into shrimp culture system. This study highlights the potential displacement of opportunistic pathogens by the usage of biofloc technology (Rapid BFTTM) in shrimp culture.
Cryptides are a subfamily of bioactive peptides that exist in all living organisms. They are latently encrypted in their parent sequences and exhibit a wide range of biological activities when decrypted via in vivo or in vitro proteases. Cationic cryptides tend to be drawn to the negatively charged membranes of microbial and cancer cells, causing cell death through various mechanisms. This makes them promising candidates for alternative antimicrobial and anti-cancer therapies, as their mechanism of action is independent of gene mutations. In the current study, we employed an in silico approach to identify novel cationic cryptides with potential antimicrobial and anti-cancer activities in atypical and systematic strategy by reanalysis of a publicly available RNA-seq dataset of Pacific white shrimp (Penaus vannamei) in response to bacterial infection. Out of 12 cryptides identified, five were selected based on their net charges and potential for cell penetration. Following chemical synthesis, the cryptides were assayed in vitro to test for their biological activities. All five cryptides demonstrated a wide range of selective activity against the tested microbial and cancer cells, their anti-biofilm activities against mature biofilms, and their ability to interact with Gram-positive and negative bacterial membranes. Our research provides a framework for a comprehensive analysis of transcriptomes in various organisms to uncover novel bioactive cationic cryptides. This represents a significant step forward in combating the crisis of multi-drug-resistant microbial and cancer cells, as these cryptides neither induce mutations nor are influenced by mutations in the cells they target.
Aquatic organisms have to produce proteins or factors that help maintain a stable relationship with microbiota and prevent colonization by pathogenic microorganisms. In crustaceans and other aquatic invertebrates, relatively few of these host factors have been characterized. In this study, we show that the respiratory glycoprotein hemocyanin is a crucial host factor that modulates microbial composition and diversity in the hepatopancreas of penaeid shrimp. Diseased penaeid shrimp (Penaeus vannamei), had an empty gastrointestinal tract with atrophied hepatopancreas, expressed low hemocyanin, and high total bacterial abundance, with Vibrio as the dominant bacteria. Similarly, shrimp depleted of hemocyanin had mitochondrial depolarization, increased reactive oxygen species (ROS) levels, and dysregulation of several energy metabolism-related genes. Hemocyanin silencing together with ROS scavenger (N-acetylcysteine) treatment improved microbial diversity and decreased Vibrio dominance in the hepatopancreas. However, fecal microbiota transplantation after hemocyanin knockdown could not restore the microbial composition in the hepatopancreas. Collectively, our data provide, to our knowledge, new insight into the pivotal role of hemocyanin in modulating microbial composition in penaeid shrimp hepatopancreas via its effect on mitochondrial integrity, energy metabolism, and ROS production.
The microsporidian parasite Enterocytozoon hepatopenaei (EHP) is an emerging problem in the marine shrimp industry, primarily in Asian countries such as China, Thailand, India, Malaysia, Indonesia, and Vietnam. A screening was conducted to investigate the prevalence of EHP after a fixed period of culturing for 1 rearing cycle in 3 states of Malaysia. The screening stages covered Penaeus vannamei post larvae (PL) and after 14-30, 31-50, 51-70, and 71-90 d of culture in 1 production cycle. A total of 279 samples were amplified using a PCR assay targeting the gene encoding a spore wall protein (SWP) of EHP. The EHP infection was initially detected in the hatchery and increased to 96.6% after the shrimp were transferred to the pond. The positive EHP sequence showed 91 to 100% similarity to sequences from India, Thailand, Vietnam, Indonesia, and Latin America. EHP infection increased throughout 1 rearing cycle due to factors such as the cannibalistic feeding habits of shrimp and the presence of unknown vectors or carriers of EHP in the culture ponds. Hence, the finding from the current study will be fundamental for other studies concerning EHP.
Acute hepatopancreatic necrosis disease (AHPND) has caused severe losses in farmed populations of marine shrimp Penaeus vannamei and P. monodon. The causative agents are unique strains of the bacteria Vibrio parahaemolyticus and related Vibrio species. The disease emerged in the People's Republic of China (China) and Vietnam in 2010 and spread throughout South-East Asia; it was later reported in countries in both North and South America. The disease has had significant economic impacts on the shrimp aquaculture industry. From 2010 to 2016, combined losses from China, Malaysia, Mexico, Thailand and Vietnam due primarily to outbreaks of AHPND, including losses at the farm gate and those resulting from a drop in feed sales and exports, were estimated at over US$ 44 billion. Other economic losses include those associated with processing facilities, decreased community revenues resulting from increased unemployment, financial investments, and the costs of implementing diagnostic and control measures. The reduced employment opportunities and increases in debt burden and investment risk have had sociological impacts. The responses to the disease have led to a gradual recovery of the shrimp industry in affected countries. These response efforts have included the implementation of changes in farming systems and management, including, among others, enhanced biosecurity and the use of AHPND-free and AHPND-resistant shrimp. This situation of losses and recovery illustrates the importance of having a multi-level response plan in place to prevent, or to reduce the risk of, outbreaks of disease.
Nanoplastics (NPs) are an abundant, long-lasting, and widespread type of environmental pollution that is of increasing concern because of the serious threats they might pose to ecosystems and species. Identifying the ecological effects of plastic pollution requires understanding the effects of NPs on aquatic organisms. Here, we used the Pacific white shrimp (Litopenaeus vannamei) as a model species to investigate whether ingestion of polystyrene NPs affects gut microbes and leads to metabolic changes in L. vannamei. The abundance of Proteobacteria increased and that of Bacteroidota decreased after NPs treatment. Specifically, Vibrio spp., photobacterium spp., Xanthomarina spp., and Acinetobacter spp. increased in abundance, whereas Sulfitobacter spp. and Pseudoalteromonas spp. decreased. Histological observations showed that L. vannamei exposed to NP displayed a significantly lower intestinal fold height and damaged intestinal structures compared with the control group. Exposure to NPs also stimulated alkaline phosphatase, lysozyme, and acid phosphatase activity, resulting in an immune response in L. vannamei. In addition, the content of triglycerides, total cholesterol, and glucose were significantly altered after NP exposure. These results provided significant ecotoxicological data that can be used to better understand the biological fate and effects of NPs in L. vannamei.
Global shrimp aquaculture farmers have suffered major economic losses due to disease outbreaks. A notable shrimp disease is Acute Hepatopancreatic Necrosis Disease (AHPND), which is caused by a new strain of Vibrio parahaemolyticus bacteria (VpAHPND) that mainly inhabits the shrimp gut and damages the hepatopancreas. Fewer studies have investigated whether this disease will affect shrimp muscle functioning or cause any muscle damage. We challenged Penaeus monodon shrimp with VpAHPND bacteria using an immersion method. Expression of Dystrophin gene, an important regulatory gene for maintenance of muscle integrity, was quantified from muscle samples using qRT-PCR. Additional verification was conducted by determining calcium concentration and bta-miR-4286 and dre-miR-107b miRNAs expression. P. monodon dystrophin gene demonstrated the highest expression level during AHPND infection when muscle calcium concentration was detected at its lowest level at 6 h post-infection (hpi). The highest muscle calcium concentration, determined at 36 hpi, was supported by higher bta-miR-4286 miRNA expression and lower dre-miR-107b miRNA expression in VpAHPND-infected samples compared to uninfected samples at the same time point. We deduced an interactive relationship between dystrophin gene expression, calcium concentration, and miRNA expression in P. monodon muscle tissues triggered by the invading VpAHPND bacterium.
Hepatopancreatic microsporidiosis in cultivated Litopenaeus vannamei and Penaeus monodon is caused by the newly emerged pathogen Enterocytozoon hepatopenaei (EHP). It has been detected in shrimp cultured in China, Vietnam and Thailand and is suspected to have occurred in Malaysia and Indonesia and to be associated with severely retarded growth. Due to retarded shrimp growth being reported at farms in the major grow-out states of Tamilnadu, Andhra Pradesh and Odisha in India, shrimp were sampled from a total of 235 affected ponds between March 2014 and April 2015 to identify the presence of EHP. PCR and histology detected a high prevalence of EHP in both P. monodon and L. vannamei, and infection was confirmed by in situ hybridization using an EHP-specific DNA probe. Histology revealed basophilic inclusions in hepatopancreas tubule epithelial cells in which EHP was observed at various developmental stages ranging from plasmodia to mature spores. The sequence of a region of the small subunit rDNA gene amplified by PCR was found to be identical to EHP sequences deposited in GenBank. Bioassays confirmed that EHP infection could be transmitted orally to healthy shrimp. Histology also identified bacterial co-infections in EHP-infected shrimp sampled from slow-growth ponds with low-level mortality. The data confirm that hepatopancreatic microsporidiosis caused by EHP is prevalent in shrimp being cultivated in India. EHP infection control measures thus need to be implemented urgently to limit impacts of slowed shrimp growth.
Parapenaeopsis sculptilis (Heller, 1862) locally referred to as ?udang kulit keras? in Malaysia has profound biological, ecological, aquacultural and conservational significance. The reproductive biology of this important penaeid from the coastal waters of Perak, Peninsular Malaysia, was studied during the period between February 2012 to January 2013. Females outnumbered males with a sex ratio of M: F= 1:3 (P < 0.05). Four maturity stages of female gonads viz., immature, maturing, mature and spent stages were distinguished. The first stage of sexual maturity was attained at a length of 9.3 cm, and female P. sculptilis showed a peak gonadosomatic index during the month of April, August and October, indicating that P. sculptilis potentially breeds throughout the year. The findings of this study would greatly contribute towards the understanding of gonadal maturation, spawning season and breeding biology, which could be important for the effective population management of this prawn species.
Protein-protein interactions (PPIs) are essential for understanding cell physiology in normal and pathological conditions, as they might involve in all cellular processes. PPIs have been widely used to elucidate the pathobiology of human and plant diseases. Therefore, they can also be used to unveil the pathobiology of infectious diseases in shrimp, which is one of the high-risk factors influencing the success or failure of shrimp production. PPI network analysis, specifically host-pathogen PPI (HP-PPI), provides insights into the molecular interactions between the shrimp and pathogens. This review quantitatively analyzed the research trends within this field through bibliometric analysis using specific keywords, countries, authors, organizations, journals, and documents. This analysis has screened 206 records from the Scopus database for determining eligibility, resulting in 179 papers that were retrieved for bibliometric analysis. The analysis revealed that China and Thailand were the driving forces behind this specific field of research and frequently collaborated with the United States. Aquaculture and Diseases of Aquatic Organisms were the prominent sources for publications in this field. The main keywords identified included "white spot syndrome virus," "WSSV," and "shrimp." We discovered that studies on HP-PPI are currently quite scarce. As a result, we further discussed the significance of HP-PPI by highlighting various approaches that have been previously adopted. These findings not only emphasize the importance of HP-PPI but also pave the way for future researchers to explore the pathogenesis of infectious diseases in shrimp. By doing so, preventative measures and enhanced treatment strategies can be identified.
Shrimp aquaculture contributes significantly to global economic growth, and the whiteleg shrimp, Penaeus vannamei, is a leading species in this industry. However, Vibrio parahaemolyticus infection poses a major challenge in ensuring the success of P. vannamei aquaculture. Despite its significance in this industry, the biological knowledge of its pathogenesis remains unclear. Hence, this study was conducted to identify the interaction sites and binding affinity between several immune-related proteins of P. vannamei with V. parahaemolyticus proteins associated with virulence factors. Potential interaction sites and the binding affinity between host and pathogen proteins were identified using molecular docking and dynamics (MD) simulation. The P. vannamei-V. parahaemolyticus protein-protein interaction of Complex 1 (Ferritin-HrpE/YscL family type III secretion apparatus protein), Complex 2 (Protein kinase domain-containing protein-Chemotaxis CheY protein), and Complex 3 (GPCR-Chemotaxis CheY protein) was found to interact with -4319.76, -5271.39, and -4725.57 of the docked score and the formation of intermolecular bonds at several interacting residues. The docked scores of Complex 1, Complex 2, and Complex 3 were validated using MD simulation analysis, which revealed these complexes greatly contribute to the interactions between P. vannamei and V. parahaemolyticus proteins, with binding free energies of -22.50 kJ/mol, -30.20 kJ/mol, and -26.27 kJ/mol, respectively. This finding illustrates the capability of computational approaches to search for molecular binding sites between host and pathogen, which could increase the knowledge of Vibrio spp. infection on shrimps, which then can be used to assist in the development of effective treatment.
The practicality of biometrics of seafood cannot be overemphasized, particularly for competent authorities of the shrimp industry. However, there is a paucity of relevant literature on the relationship between biometric and physicochemical indices of freshly harvested shrimp. This work therefore investigated the relationship between biometric (standard length (SL), total weight (TW) and condition factor (CF)) and physicochemical (moisture content, pH, titratable acidity, water activity, water retention index, colour values and fracturability) characteristics of freshly harvested Pacific white shrimp (Litopenaeus vannamei) obtained from three different farms. The relationships between these parameters were determined using correlation and regression analyses.
Since 1994, white spot syndrome virus (WSSV) has been detected in cultured shrimp Penaeus monodon in Peninsular Malaysia. The gross signs, target organs and histo-cytopathology for the viral infection were studied and it was found to infect most organs and tissues including oocytes, but not hepatopancreatocytes and epithelial cells of the midgut, which were regarded as refractory tissues. Based on a time-sequence of ultrastructural cytopathology, 4 cytopathic profiles and 6 phases of viral morphogenesis were described. The virions were elliptical to short rods with trilamilar envelopes that measured 305 +/- 30 x 127 +/- 11 nm. Viral nucleosomes were often present singly in infected nuclei and were associated with the early stages of viral replication. The structure of WSSV pathognomonic white, cuticular lesions was examined at the microscopic and ultrastructural levels and the mechanism of their formation appeared to be related to the disruption of exudate transfer from epithelial cells to the cuticle via cuticular pore canals.
Accumulation kinetic trends of cesium and cadmium in the Penaeus monodon were studied using Cs-134 and Cd-109 as a tracer. The objective of this study was to quantify the uptake and loss/depuration kinetic of these two radionuclides in the Penaeus monodon. Uptake and loss/depuration kinetic of these two radionuclides in the Penaeus monodon were varied widely, displayed a simple double kinetic model of linear and exponential trend with time unless modified by moulting at the stage in the mount cycle. Therefore, the variation of Cs-134 and Cd-109 bio-concentration factor could be concluded considerably influence by moulting cycle, environmental and biological condition as well as physico-chemical that direct effects on their uptake and loss/depuration kinetic.
The exciting discovery of the semiconducting-like properties of deoxyribonucleic acid (DNA) and its potential applications in molecular genetics and diagnostics in recent times has resulted in a paradigm shift in biophysics research. Recent studies in our laboratory provide a platform towards detecting charge transfer mechanism and understanding the electronic properties of DNA based on the sequence-specific electronic response, which can be applied as an alternative to identify or detect DNA. In this study, we demonstrate a novel method for identification of DNA from different shrimp viruses and bacteria using electronic properties of DNA obtained from both negative and positive bias regions in current-voltage (I-V) profiles. Characteristic electronic properties were calculated and used for quantification and further understanding in the identification process. Aquaculture in shrimp industry is a fast-growing food sector throughout the world. However, shrimp culture in many Asian countries faced a huge economic loss due to disease outbreaks. Scientists have been using specific established methods for detecting shrimp infection, but those methods do have their significant drawbacks due to many inherent factors. As such, we believe that this simple, rapid, sensitive and cost-effective tool can be used for detection and identification of DNA from different shrimp viruses and bacteria.
Laem-Singh virus (LSNV) is a positive-sense single-stranded RNA (ssRNA) virus that was recently identified in Penaeus monodon shrimp in Thailand displaying signs of slow growth syndrome. A total of 326 shrimp collected between 1998 and 2007 from countries in the Indo-Pacific region were tested by RT-PCR for evidence of LSNV infection. The samples comprised batches of whole postlarvae, and lymphoid organ, gill, muscle or pleopod tissue of juvenile, subadult and adult shrimp. LSNV was not detected in 96 P. monodon, P. japonicus or P. merguiensis from Australia or 16 P. monodon from Fiji, Philippines, Sri Lanka and Mozambique. There was no evidence of LSNV infection in 73 healthy juvenile P. vannamei collected during 2006 from ponds at 9 locations in Thailand. However, LNSV was detected in each of 6 healthy P. monodon tested from Malaysia and Indonesia, 2 of 6 healthy P. monodon tested from Vietnam and 39 of 40 P. monodon collected from slow-growth ponds in Thailand. A survey of 81 P. monodon collected in 2007 from Andhra Pradesh, India, indicated 56.8% prevalence of LSNV infection but no clear association with disease or slow growth. Phylogenetic analysis of PCR amplicons obtained from samples from India, Vietnam, Malaysia and Thailand indicated that nucleotide sequence variation was very low (>98% identity) and there was no clustering of viruses according to site of isolation or the health status of the shrimp. The data suggests that LSNV exists as a single genetic lineage and occurs commonly in healthy P. monodon in parts of Asia.
Global high demand for Pacific white shrimp Penaeus vannamei has led to intensified cultivation and a wide range of disease problems, including bacterial diseases due to vibrios. Three presumptive luminescent Vibrio harveyi strains (Vh5, Vh8 and Vh10) were isolated from the hepatopancreas (Vh5) and haemolymph (Vh8 and Vh10) of diseased growout Pacific white shrimp from a farm in Setiu, Terengganu, Malaysia, using Vibrio harveyi agar (VHA) differential medium. All three strains were identified as V. harveyi by biochemical characteristics. 16S rRNA gene-based phylogenetic analyses by neighbour-joining, maximum likelihood and maximum parsimony methods showed all three strains in the V. harveyi cluster. All three strains were β-haemolytic and positive for motility, biofilm formation and extracellular products (caseinase, gelatinase, lipase, DNase, amylase and chitinase). Vh10 was subjected to pathogenicity test in Pacific white shrimp by immersion challenge and determined to have a LC50 of 6.0 × 108 CFU mL-1 after 168 h of exposure. Antibiotic susceptibility tests showed that all strains were resistant to oxytetracycline (OXT30), oleandomycin (OL15), amoxicillin (AML25), ampicillin (AMP10) and colistin sulphate (CT25) but sensitive to doxycycline (DO30), flumequine (UB30), oxolinic acid (OA2), chloramphenicol (C30), florfenicol (FFC30), nitrofurantoin (F5) and fosfomycin (FOS50). Each strain was also resistant to a slightly different combination of eight other antibiotics, with an overall multiple antibiotic resistance (MAR) index of 0.40, suggesting prior history of heavy exposure to the antibiotics. Vh10 infection resulted in pale or discoloured hepatopancreas, empty guts, reddening, necrosis and luminescence of uropods, as well as melanised lesions in tail muscle. Histopathological examination showed necrosis of intertubular connective tissue and tubule, sloughing of epithelial cells in hepatopancreatic tubule, haemocytic infiltration, massive vacuolation and loss of hepatopancreatic tubule structure.
Although invertebrates' innate immunity relies on several immune-like molecules, the diversity of these molecules and their immune response mechanisms are not well understood. Here, we show that Penaeus vannamei hemocyanin (PvHMC) undergoes specific deacetylation under Vibrio parahaemolyticus and LPS challenge. In vitro deacetylation of PvHMC increases its binding capacity with LPS and antibacterial activity against Gram-negative bacteria. Lysine residues K481 and K484 on the Ig-like domain of PvHMC are the main acetylation sites modulated by the acetyltransferase TIP60 and deacetylase HDAC3. Deacetylation of PvHMC on K481 and K484 allows PvHMC to form a positively charged binding pocket that interacts directly with LPS, whereas acetylation abrogates the positive charge to decrease PvHMC-LPS attraction. Besides, V. parahaemolyticus and LPS challenge increases the expression of Pvhdac3 to induce PvHMC deacetylation. This work indicates that, during bacterial infections, deacetylation of hemocyanin is crucial for binding with LPS to clear Gram-negative bacteria in crustaceans.
Inadequate gonadal maturation and poor spawning performance increasingly threaten the sustainability of shrimp aquaculture. Unraveling the mechanisms regulating ovarian development and maturation hence is critical to address industry challenges. Vitellogenin (Vtg), a precursor of yolk protein found in the hepatopancreas and ovary of shrimp, plays a key role in facilitating shrimp's oocyte maturation and embryonic development after oviposition. This study found that FpVtg was specifically expressed in F. penicillatus hepatopancreas and ovary. FpVtg was localized predominantly in the oocyte cytoplasm and distributed uniformly in the hepatopancreas tissue. Silencing FpVtg led to apoptosis in both hepatopancreas and ovary tissues. Furthermore, FpVtg depletion upregulated the expression of ovarian peritrophin 1, ovarian peritrophin 2, serine proteinase inhibitor 6, and juvenile hormone esterase-like carboxylesterase 1, while downregulated that of vitellogenin, delta-9 desaturase, and insulin-like receptor. KEGG pathway analysis implicated such as PI3K-AKT signaling, RNA transport, ECM-receptor interaction, hippo signaling, oocyte meiosis, and apoptosis were enriched and involved in ovarian development. These findings have provided insights into the FpVtg's reproductive role and the associated regulatory genes and pathways in F. penicillatus. This knowledge can contribute to establishing strategies to improve the breeding and aquaculture production of F. penicillatus by elucidating its vitellogenesis regulation in redtail prawn and other penaeid species. Further characterization of the implicated pathways and genes will clarify the intricacies underlying ovarian maturation.