A 10-year analysis of RASFF notifications for mycotoxins in nuts. Trend in key mycotoxins and impacted countries.

The demand for tree nuts has significantly grown in recent years as epidemiological studies and clinical intervention trials demonstrated an inverse relationship between tree nut consumption and chronic diseases. However, mycotoxins are one of the main hazards responsible for increased "Rapid Alert System for Food and Feed" (RASFF) notifications and border rejections on nuts and nut products exported to the E.U. countries in the past few years. Mycotoxins are secondary metabolites that present serious threats to human and animal health. The most prevalent, toxic, and carcinogenic mycotoxins observed in human food and animal feed are the aflatoxins (AFs). This work analyzed notifications from the RASFF on nuts and nut products contaminated with mycotoxins, for a 10-year period from 2011 to 2021. A total of 4752 mycotoxin notifications were published on RASFF for food products worldwide, 63% (n = 3000) were notified in "nuts, nut products and seeds". It was observed that 95% (n = 2669) notifications were due to AFs. Over half of these notifications (52%, n = 1545) were reported for groundnuts, where 29% (n = 441) of the notifications were received for groundnuts from China alone. Border rejection was reported for 91% (n = 2560) of the nuts and nut products which received the notifications from the E.U. countries. This study proffers understanding into the major reasons for RASFF notifications on nuts and nut products exported to E.U. countries. Also, the implications of this issue with some recommendations that could reduce the incidents of notifications for tree nuts have been outlined.

Development of peptide nucleic acid-based bead array technology for Bacillus cereus detection.

Numerous novel methods to detect foodborne pathogens have been extensively developed to ensure food safety. Among the important foodborne bacteria, Bacillus cereus was identified as a pathogen of concern that causes various food illnesses, leading to interest in developing effective detection methods for this pathogen. Although a standard method based on culturing and biochemical confirmative test is available, it is time- and labor-intensive. Alternative PCR-based methods have been developed but lack high-throughput capacity and ease of use. This study, therefore, attempts to develop a robust method for B. cereus detection by leveraging the highly specific pyrrolidinyl peptide nucleic acids (PNAs) as probes for a bead array method with multiplex and high-throughput capacity. In this study, PNAs bearing prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbone with groEL, motB, and 16S rRNA sequences were covalently coupled with three sets of fluorescently barcoded beads to detect the three B. cereus genes. The developed acpcPNA-based bead array exhibited good selectivity where only signals were detectable in the presence of B. cereus, but not for other species. The sensitivity of this acpcPNA-based bead assay in detecting genomic DNA was found to be 0.038, 0.183 and 0.179 ng for groEL, motB and 16S rRNA, respectively. This performance was clearly superior to its DNA counterpart, hence confirming much stronger binding strength of acpcPNA over DNA. The robustness of the developed method was further demonstrated by testing artificially spiked milk and pickled mustard greens with minimal interference from food metrices. Hence, this proof-of-concept acpcPNA-based bead array method has been proven to serve as an effective alternative nucleic acid-based method for foodborne pathogens.

Comparative effects of different bacterial lipopolysaccharides on modulation of immune levels to improve survival of the black tiger shrimp.

To prevent loss from disease, immunostimulants have been used as dietary supplements to improve immunity and survival of shrimps. Among the various types of immunostimulants, there is increasing evidence that a diet enriched with bacterial lipopolysaccharide can reduce the mortality rate of shrimp under exposure to pathogens. Here, the immunostimulatory effects of bacterial lipopolysaccharide (LPS) from various bacterial sources were explored. Bacterial LPS was extracted from a shrimp pathogen, Vibrio harveyi and its effects were compared with the commercially available LPS from the non-shrimp pathogen, Escherichia coli. Our results revealed that the LPS from V. harveyi was different in molecular size but contained similar functional groups to that from E. coli. To understand their molecular mechanisms, bacterial LPS from the two sources were applied as a supplementary diet and fed to juvenile shrimp for 4-week feeding period before tissue samples were collected for transcriptomic analysis by next generation sequencing. Gene expression profiling revealed that major immune-related genes such as pattern recognition proteins (PRPs), proteinases and proteinase inhibitors, prophenoloxidase systems (proPO system), antimicrobial peptides (AMPs), signaling transduction pathways, heat shock proteins (HSPs), oxidative stress responses, and other immune-related molecules such as mucins and peritrophins were modulated in the groups of shrimp fed with bacterial LPS from both sources, but at different levels. The results suggest that bacterial LPS could modulate shrimp immune system, and different LPS sources led to different activation of immune pathways. Additionally, metabolic-related genes were affected by LPS, suggesting that energy was required for immune stimulation. In the V. harveyi pathogen challenge trial, all shrimp groups fed with diets containing LPS from both bacterial sources showed better survival than the control group without LPS. When comparing groups fed with LPS supplemented diets, the higher concentration of LPS (8 µg/body weight) from E. coli resulted in a better survival rate than a lower concentration (4 µg/body weight). Conversely, shrimp fed with a diet containing LPS from V. harveyi showed a lower survival rate when a higher dose of LPS (8 µg/body weight) was administered than the group fed with a lower concentration of LPS (4 µg/body weight). This could be due to overstimulation of shrimp immune responses, especially by LPS derived from shrimp pathogens, resulting in a reverse effect. These results confirm that immunity in shrimp upon administration of bacterial LPS depends on the origin and dose of the LPS administered.

A rapid colorimetric lateral flow test strip for detection of live Salmonella Enteritidis using whole phage as a specific binder.

Specific antibodies are essential components of immunoassay, which can be applied for the detection of pathogens. However, producing an antibody specific to live bacterial pathogens by the classical method of immunizing animals with live pathogens can be impractical. Phage display technology is an effective alternative method to obtain antibodies with the desired specificity against selected antigenic molecules. In this study, we demonstrated the power of a microarray-based technique for obtaining specific phage-derived antibody fragments against Salmonella, an important foodborne pathogen. The selected phage-displayed antibody fragments were subsequently employed to develop a lateral flow test strip assay for the detection of live Salmonella. The test strips showed specificity to Salmonella Enteritidis without cross-reactivity to eight serovars of Salmonella or other bacteria strains. The test strip assay requires 15 min, whereas the conventional biochemical and serological confirmation test requires at least 24 h. The microarray screening technique for specific phage-based binders and the test strip method can be further applied to other foodborne pathogens.

Isolation and Characterization of Mannanase-Producing Bacteria for Potential Synbiotic Application in Shrimp Farming.

Prebiotics such as mannan-oligosaccharides (MOS) are a promising approach to improve performance and disease resistance in shrimp. To improve prebiotic utilization, we investigated the potential probiotics and their feasibility of synbiotic use in vitro. Two bacterial isolates, Man26 and Man122, were isolated from shrimp intestines and screened for mannanase, the enzyme for mannan digestion. The crude mannanase from both isolates showed optimal activities at pH 8 with optimum temperatures at 60 °C and 50 °C, respectively. The enzymes remained stable at pH 8−10 for 3 h (>70% relative activity). The thermostability range of Man26 was 20−40 °C for 20 min (>50%), while that of Man122 was 20−60 °C for 30 min (>50%). The Vmax of Man122 against locust bean gum substrate was 41.15 ± 12.33 U·mg−1, six times higher than that of Man26. The Km of Man26 and Man122 were 18.92 ± 4.36 mg·mL−1 and 34.53 ± 14.46 mg·mL−1, respectively. With the addition of crude enzymes, reducing sugars of copra meal, palm kernel cake, and soybean meal were significantly increased (p < 0.05), as well as protein release. The results suggest that Man26 and Man122 could potentially be used in animal feeds and synbiotically with copra meal to improve absorption and utilization of feedstuffs.

Differential distribution of eicosanoids and polyunsaturated fatty acids in the Penaeus monodon male reproductive tract and their effects on total sperm counts.

Eicosanoids, which are oxygenated derivatives of polyunsaturated fatty acids (PUFAs), serve as signaling molecules that regulate spermatogenesis in mammals. However, their roles in crustacean sperm development remain unknown. In this study, the testis and vas deferens of the black tiger shrimp Penaeus monodon were analyzed using ultra-high performance liquid chromatography coupled with Orbitrap high resolution mass spectrometry. This led to the identification of three PUFAs and ten eicosanoids, including 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and (±)15-hydroxyeicosapentaenoic acid ((±)15-HEPE), both of which have not previously been reported in crustaceans. The comparison between wild-caught and domesticated shrimp revealed that wild-caught shrimp had higher sperm counts, higher levels of (±)8-HEPE in testes, and higher levels of prostaglandin E2 (PGE2) and prostaglandin F2α in vas deferens than domesticated shrimp. In contrast, domesticated shrimp contained higher levels of (±)12-HEPE, (±)18-HEPE, and eicosapentaenoic acid (EPA) in testes and higher levels of 15d-PGJ2, (±)12-HEPE, EPA, arachidonic acid (ARA), and docosahexaenoic acid (DHA) in vas deferens than wild-caught shrimp. To improve total sperm counts in domesticated shrimp, these broodstocks were fed with polychaetes, which contained higher levels of PUFAs than commercial feed pellets. Polychaete-fed shrimp produced higher total sperm counts and higher levels of PGE2 in vas deferens than pellet-fed shrimp. In contrast, pellet-fed shrimp contained higher levels of (±)12-HEPE, (±)18-HEPE, and EPA in testes and higher levels of (±)12-HEPE in vas deferens than polychaete-fed shrimp. These data suggest a positive correlation between high levels of PGE2 in vas deferens and high total sperm counts as well as a negative correlation between (±)12-HEPE in both shrimp testis and vas deferens and total sperm counts. Our analysis not only confirms the presence of PUFAs and eicosanoids in crustacean male reproductive organs, but also suggests that the eicosanoid biosynthesis pathway may serve as a potential target to improve sperm production in shrimp.

Shrimp genome sequence contains independent clusters of ancient and current Endogenous Viral Elements (EVE) of the parvovirus IHHNV.

BACKGROUND: Shrimp have the ability to accommodate viruses in long term, persistent infections without signs of disease. Endogenous viral elements (EVE) play a role in this process probably via production of negative-sense Piwi-interacting RNA (piRNA)-like fragments. These bind with Piwi proteins to dampen viral replication via the RNA interference (RNAi) pathway. We searched a genome sequence (GenBank record JABERT000000000) of the giant tiger shrimp (Penaeus monodon for the presence of EVE related to a shrimp parvovirus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV). RESULTS: The shrimp genome sequence contained three piRNA-like gene clusters containing scrambled IHHNV EVE. Two clusters were located distant from one another in pseudochromosome 35 (PC35). Both PC35 clusters contained multiple sequences with high homology (99%) to GenBank records DQ228358 and EU675312 that were both called "non-infectious IHHNV Type A" (IHHNV-A) when originally discovered. However, our results and those from a recent Australian P. monodon genome assembly indicate that the relevant GenBank records for IHHNV-A are sequence-assembly artifacts derived from scrambled and fragmental IHHNV-EVE. Although the EVE in the two PC35 clusters showed high homology only to IHHNV-A, the clusters were separate and distinct with respect to the arrangement (i.e., order and reading direction) and proportional content of the IHHNV-A GenBank records. We conjecture that these 2 clusters may constitute independent allele-like clusters on a pair of homologous chromosomes. The third EVE cluster was found in pseudochromosome 7 (PC7). It contained EVE with high homology (99%) only to GenBank record AF218266 with the potential to protect shrimp against current types of infectious IHHNV. One disadvantage was that some EVE in PC7 can give false positive PCR test results for infectious IHHNV. CONCLUSIONS: Our results suggested the possibility of viral-type specificity in EVE clusters. Specificity is important because whole EVE clusters for one viral type would be transmitted to offspring as collective hereditary units. This would be advantageous if one or more of the EVE within the cluster were protective against the disease caused by the cognate virus. It would also facilitate gene editing for removal of non-protective EVE clusters or for transfer of protective EVE clusters to genetically improve existing shrimp breeding stocks that might lack them.

Supplementation of ex situ produced bioflocs improves immune response against AHPND in Pacific whiteleg shrimp (Litopenaeus vannamei) postlarvae.

The emergence of Vibrio diseases, including acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio spp., had resulted in heavy losses in global shrimp production. Biofloc technology is a closed aquaculture system developed as one of the sustainable solutions to increase system resilience in the shrimp industry. In this study, biofloc was formed externally (ex situ biofloc) with probiotics Bacillus sp. strain BME and Bacillus sp. strain BCE, diatom microalgae Chaetoceros calcitrans, and a consortium of nitrifying bacteria, in the ratio of 1:1:6:6 as a starter. The study showed that the ex situ biofloc supplementation in Pacific whiteleg shrimp (L. vannamei) postlarvae culture can increase the shrimp culture performance (shrimp survival and growth), reduce Vibrio counts in the water and shrimp body, and provide stimulation of the shrimp immune response through humoral immune responses, such as pattern recognition protein (C-type lectin) and melanization process (proPO). Overall, the results indicate that the supplementation of ex situ biofloc provided protection to shrimp under Vibrio infection, regardless of the timing of addition (before, simultaneously, or after addition of Vibrio sp. strain VPA). This suggests that the ex situ biofloc can be effective as a preventive and a supportive treatment against potential AHPND infection in L. vannamei postlarvae culture. Taken together, the ability of the ex situ biofloc to modulate immune-related gene expression and resistance of L. vannamei against potentially AHPND-causing Vibrio sp. strain makes it an effective aquaculture technology for infectious disease control in shrimp production with high-density and minimal water exchange culture. KEY POINTS: • Supplementation of ex situ produced biofloc in shrimp postlarvae culture. • Ex situ biofloc reduces Vibrio counts in the water and shrimp body. • Ex situ biofloc stimulates shrimp humoral immune responses and survival.

The evolution of multiplex detection of mycotoxins using immunoassay platform technologies.

Mycotoxins present serious threats not only for public health, but also for the economy and environment. The problems become more complex and serious due to co-contamination of multiple hazardous mycotoxins in commodities and environment. To mitigate against this issue, accurate, affordable, and rapid multiplex detection methods are required. This review presents an overview of emerging rapid immuno-based multiplex methods capable of detecting mycotoxins present in agricultural products and feed ingredients published within the past five years. The scientific principles, advantages, disadvantages, and assay performance of these rapid multiplex immunoassays, including lateral flow, fluorescence polarization, chemiluminescence, surface plasmon resonance, surface enhanced Raman scattering, electrochemical sensor, and nanoarray are discussed. From the recent literature landscape, it is predicted that the future trend of the detection methods for multiple mycotoxins will rely on the advance of various sensor technologies, a variety of enhancing and reporting signals based on nanomaterials, rapid and effective sample preparation, and capacity for quantitative analysis.

Identification of novel SARS-CoV-2 RNA dependent RNA polymerase (RdRp) inhibitors: From in silico screening to experimentally validated inhibitory activity.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has posed a serious threat to global health and the economy for over two years, prompting the need for development of antiviral inhibitors. Due to its vital role in viral replication, RNA-dependent RNA polymerase (RdRp) is a promising therapeutic target. Herein, we analyzed amino acid sequence conservation of RdRp across coronaviruses. The conserved amino acids at the catalytic binding site served as the ligand-contacting residues for in silico screening to elucidate possible resistant mutation. Molecular docking was employed to screen inhibitors of SARS-CoV-2 from the ZINC ChemDiv database. The top-ranked compounds selected from GOLD docking were further investigated for binding modes at the conserved residues of RdRp, and ten compounds were selected for experimental validation. Of which, three compounds exhibited promising antiviral activity. The most promising candidate showed a half-maximal effective concentration (EC50) of 5.04 µM. Molecular dynamics simulations, binding free-energy calculation and hydrogen bond analysis were performed to elucidate the critical interactions providing a foundation for developing lead compounds effective against SARS-CoV-2.

Metabolite profiles of brown planthopper-susceptible and resistant rice (Oryza sativa) varieties associated with infestation and mechanical stimuli.

Understanding brown planthopper (BPH) resistance mechanism will expedite selective breeding of better BPH resistant lines of rice (Oryza sativa). Metabolic responses during BPH infestation derived from wound stress imposed by insect feeding, comparing with mechanical piercing will provide an insight into resistance mechanism in rice. Therefore, this study aimed to compare the metabolic responses of needle piercing treatment and BPH feeding treatment in BPH-susceptible (KD) and BPH-resistant (RH) varieties at four different time points (0, 6, 24 and 96 h) using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Phenotypes of RH were not different among the treatments, whereas KD exhibited hopperburn symptom at 96 h post-BPH infestation. Principal component and cluster analyses revealed that metabolite profiles between KD and RH were different in response to both insect and mechanical stimuli. Metabolite profiles of RH under BPH and mechanical treatments at 24 and 96 h were different from the untreated, whereas metabolite profiles of KD after BPH infestation at 24 and 96 h were distinct from needle piercing and no treatment, suggesting that the resistant variety has an ability to adapt and defend both mechanical and insect stimuli. Metabolomics result showed that BPH infestation perturbed purine salvage biosynthesis (e.g., inosine, hypoxanthine) in both varieties, amino acid biosynthesis (e.g., phenylalanine, tryptophan) in KD, while the infestation perturbed lysine metabolism (pipecolic acid) and phenylpropanoid pathway (2-anisic acid) only in RH. BPH and mechanical stimuli perturbed phenylamide only in RH, but not in KD. These findings revealed that different rice varieties utilize different metabolites in response to insect and mechanical stimuli, resulting in different degrees of resistance.

Comparative Analysis of PacBio and Oxford Nanopore Sequencing Technologies for Transcriptomic Landscape Identification of Penaeus monodon.

With the advantages that long-read sequencing platforms such as Pacific Biosciences (Menlo Park, CA, USA) (PacBio) and Oxford Nanopore Technologies (Oxford, UK) (ONT) can offer, various research fields such as genomics and transcriptomics can exploit their benefits. Selecting an appropriate sequencing platform is undoubtedly crucial for the success of the research outcome, thus there is a need to compare these long-read sequencing platforms and evaluate them for specific research questions. This study aims to compare the performance of PacBio and ONT platforms for transcriptomic analysis by utilizing transcriptome data from three different tissues (hepatopancreas, intestine, and gonads) of the juvenile black tiger shrimp, Penaeus monodon. We compared three important features: (i) main characteristics of the sequencing libraries and their alignment with the reference genome, (ii) transcript assembly features and isoform identification, and (iii) correlation of the quantification of gene expression levels for both platforms. Our analyses suggest that read-length bias and differences in sequencing throughput are highly influential factors when using long reads in transcriptome studies. These comparisons can provide a guideline when designing a transcriptome study utilizing these two long-read sequencing technologies.

Development of a microarray lateral flow strip test using a luminescent organic compound for multiplex detection of five mycotoxins.

While lateral flow immunoassay (LFIA) is a simple technique that offers a rapid, robust, user friendly, and point-of-care test, its capacity for multiplex detection is rather limited. This study therefore combined the multiplexity of microarray technique and the simple and rapid characteristics of LFIA to enable simultaneous and quantitative detection of five mycotoxins, namely aflatoxin B1 (AFB1), deoxynivalenol (DON), fumonisin B1 (FUMB1), T-2 toxin (T-2), and zearalenone (ZON). In addition, we have synthesized a novel extra-large Stokes shift and strong fluorescence organic compound to be used as a reporter molecule which can be detected under UV light without light filter requirement. Many parameters including microarray spotting buffer, blocking buffer, and concentrations of mycotoxin antibodies were optimized for the microarray LFIA (µLFIA) construction. With the optimal conditions, the µLFIA could accurately and quantitatively detect multiple mycotoxins at the same time. The limits of detection of AFB1, DON, FUMB1, T-2, and ZON were 1.3, 0.5, 0.4, 0.4, and 0.9 ppb, respectively. The recoveries of these five mycotoxins were 70.7%-119.5% and 80.4%-124.8% for intra-assay and inter-assay, respectively. Combining the advantages of the novel reporter molecule and the multiplex capability of µLFIA test, this system could simultaneously detect multiple mycotoxins in one sample with high specificity and high sensitivity. Moreover, this system presents a promising affordable point-of-care platform to detect other analytes as well.

A chromosome-level assembly of the black tiger shrimp (Penaeus monodon) genome facilitates the identification of growth-associated genes.

To salvage marine ecosystems from fishery overexploitation, sustainable and efficient aquaculture must be emphasized. The knowledge obtained from available genome sequence of marine organisms has accelerated marine aquaculture in many cases. The black tiger shrimp (Penaeus monodon) is one of the most prominent cultured penaeid shrimps (Crustacean) with an average annual global production of half a million tons in the last decade. However, its currently available genome assemblies lack the contiguity and completeness required for accurate genome annotation due to the highly repetitive nature of the genome and technical difficulty in extracting high-quality, high-molecular weight DNA. Here, we report the first chromosome-level whole-genome assembly of P. monodon. The combination of long-read Pacific Biosciences (PacBio) and long-range Chicago and Hi-C technologies enabled a successful assembly of this first high-quality genome sequence. The final assembly covered 2.39 Gb (92.3% of the estimated genome size) and contained 44 pseudomolecules, corresponding to the haploid chromosome number. Repetitive elements occupied a substantial portion of the assembly (62.5%), the highest of the figures reported among crustacean species. The availability of this high-quality genome assembly enabled the identification of genes associated with rapid growth in the black tiger shrimp through the comparison of hepatopancreas transcriptome of slow-growing and fast-growing shrimps. The results highlighted several growth-associated genes. Our high-quality genome assembly provides an invaluable resource for genetic improvement and breeding penaeid shrimp in aquaculture. The availability of P. monodon genome enables analyses of ecological impact, environment adaptation and evolution, as well as the role of the genome to protect the ecological resources by promoting sustainable shrimp farming.

Optimization of high molecular weight DNA extraction methods in shrimp for a long-read sequencing platform.

Marine organisms are important to global food security as they are the largest source of animal proteins feeding mankind. Genomics-assisted aquaculture can increase yield while preserving the environment to ensure sufficient and sustainable production for global food security. However, only few high-quality genome sequences of marine organisms, especially shellfish, are available to the public partly because of the difficulty in the sequence assembly due to the complex nature of their genomes. A key step for a successful genome sequencing is the preparation of high-quality high molecular weight (HMW) genomic DNA. This study evaluated the effectiveness of five DNA extraction protocols (CTAB, Genomic-tip, Mollusc DNA, TIANamp Marine Animals DNA, and Sbeadex livestock kits) in obtaining shrimp HMW DNA for a long-read sequencing platform. DNA samples were assessed for quality and quantity using a Qubit fluorometer, NanoDrop spectrophotometer and pulsed-field gel electrophoresis. Among the five extraction methods examined without further optimization, the Genomic-tip kit yielded genomic DNA with the highest quality. However, further modifications of these established protocols might yield even better DNA quality and quantity. To further investigate whether the obtained genomic DNA could be used in a long-read sequencing application, DNA samples from the top three extraction methods (CTAB method, Genomic-tip and Mollusc DNA kits) were used for Pacific Biosciences (PacBio) library construction and sequencing. Genomic DNA obtained from Genomic-tip and Mollusc DNA kits allowed successful library construction, while the DNA obtained from the CTAB method did not. Genomic DNA isolated using the Genomic-tip kit yielded a higher number of long reads (N50 of 14.57 Kb) than those obtained from Mollusc DNA kits (N50 of 9.74 Kb). Thus, this study identified an effective extraction method for high-quality HMW genomic DNA of shrimp that can be applied to other marine organisms for a long-read sequencing platform.

Multi-omics analysis to examine microbiota, host gene expression and metabolites in the intestine of black tiger shrimp (Penaeus monodon) with different growth performance.

Understanding the correlation between shrimp growth and their intestinal bacteria would be necessary to optimize animal's growth performance. Here, we compared the bacterial profiles along with the shrimp's gene expression responses and metabolites in the intestines between the Top and the Bottom weight groups. Black tiger shrimp (Penaeus monodon) were collected from the same population and rearing environments. The two weight groups, the Top-weight group with an average weight of 36.82 ± 0.41 g and the Bottom-weight group with an average weight of 17.80 ± 11.81 g, were selected. Intestines were aseptically collected and subjected to microbiota, transcriptomic and metabolomic profile analyses. The weighted-principal coordinates analysis (PCoA) based on UniFrac distances showed similar bacterial profiles between the two groups, suggesting similar relative composition of the overall bacterial community structures. This observed similarity was likely due to the fact that shrimp were from the same genetic background and reared under the same habitat and diets. On the other hand, the unweighted-distance matrix revealed that the bacterial profiles associated in intestines of the Top-weight group were clustered distinctly from those of the Bottom-weight shrimp, suggesting that some unique non-dominant bacterial genera were found associated with either group. The key bacterial members associated to the Top-weight shrimp were mostly from Firmicutes (Brevibacillus and Fusibacter) and Bacteroidetes (Spongiimonas), both of which were found in significantly higher abundance than those of the Bottom-weight shrimp. Transcriptomic profile of shrimp intestines found significant upregulation of genes mostly involved in nutrient metabolisms and energy storage in the Top-weight shrimp. In addition to significantly expressed metabolic-related genes, the Bottom-weight shrimp also showed significant upregulation of stress and immune-related genes, suggesting that these pathways might contribute to different degrees of shrimp growth performance. A non-targeted metabolome analysis from shrimp intestines revealed different metabolic responsive patterns, in which the Top-weight shrimp contained significantly higher levels of short chain fatty acids, lipids and organic compounds than the Bottom-weight shrimp. The identified metabolites included those that were known to be produced by intestinal bacteria such as butyric acid, 4-indolecarbaldehyde and L-3-phenyllactic acid as well as those produced by shrimp such as acyl-carnitines and lysophosphatidylcholine. The functions of these metabolites were related to nutrient absorption and metabolisms. Our findings provide the first report utilizing multi-omics integration approach to investigate microbiota, metabolic and transcriptomics profiles of the host shrimp and their potential roles and relationship to shrimp growth performance.

Double antibody pairs sandwich-ELISA (DAPS-ELISA) detects Acidovorax citrulli serotypes with broad coverage.

Acidovorax citrulli, a seedborne bacterium and quarantine pest, causes the devastating bacterial fruit blotch disease in cucurbit plants. Immunological assays such as ELISA are widely used in routine field inspections for this bacterium. However, to the best of our knowledge, none of the currently available monoclonal antibodies (MAbs) can detect all common A. citrulli strains. We therefore aimed to produce a panel of MAbs and to develop an ELISA-based method capable of detecting all A. citrulli strains. We used a high-throughput bead array technique to screen and characterize A. citrulli-specific MAbs produced from hybridoma clones. The hybridoma library was simultaneously screened against five A. citrulli strains (PSA, KK9, SQA, SQB and P) and the closely related bacterium, Delftia acidovorans. Three MAbs exhibiting different binding patterns to A. citrulli were used to develop an ELISA-based method called "double antibody pairs sandwich ELISA" (DAPS-ELISA). DAPS-ELISA employing mixtures of MAbs was able to specifically detect all 16 A. citrulli strains tested without cross-reactivity with other bacteria. By contrast, our previously developed MAb capture-sandwich ELISA (MC-sELISA) and a commercial test kit detected only 15 and 14 of 16 strains, respectively. The sensitivity of the DAPS-ELISA ranged from 5×105 to 1×106 CFU/mL, while those of the MC-sELISA and the commercial test kit ranged from 5×104 to 1×107 CFU/mL and 5×104 to 5×105 CFU/mL, respectively. DAPS-ELISA thus represents an alternative method enabling rapid, accurate, and inexpensive detection of all A. citrulli strains. The method can be applied to seed testing prior to planting as well as to routine field inspections.

Bacterial analysis in the early developmental stages of the black tiger shrimp (Penaeus monodon).

Microbial colonization is an essential process in the early life of animal hosts-a crucial phase that could help influence and determine their health status at the later stages. The establishment of bacterial community in a host has been comprehensively studied in many animal models; however, knowledge on bacterial community associated with the early life stages of Penaeus monodon (the black tiger shrimp) is still limited. Here, we examined the bacterial community structures in four life stages (nauplius, zoea, mysis and postlarva) of two black tiger shrimp families using 16S rRNA amplicon sequencing by a next-generation sequencing. Although the bacterial profiles exhibited different patterns in each developmental stage, Bacteroidetes, Proteobacteria, Actinobacteria and Planctomycetes were identified as common bacterial phyla associated with shrimp. Interestingly, the bacterial diversity became relatively stable once shrimp developed to postlarvae (5-day-old and 15-day-old postlarval stages), suggesting an establishment of the bacterial community in matured shrimp. To our knowledge, this is the first report on bacteria establishment and assembly in early developmental stages of P. monodon. Our findings showed that the bacterial compositions could be shaped by different host developmental stages where the interplay of various host-associated factors, such as physiology, immune status and required diets, could have a strong influence.

Author Correction: Transcriptome analyses reveal the synergistic effects of feeding and eyestalk ablation on ovarian maturation in black tiger shrimp.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transcriptome analyses reveal the synergistic effects of feeding and eyestalk ablation on ovarian maturation in black tiger shrimp.

Unilateral eyestalk ablation in the female black tiger shrimp Penaeus monodon is commonly employed to induce ovarian maturation. However, the importance of complementing this practice with the provision of live feed supplement (such as polychaetes) has not been emphasized in previous studies. Indeed, it has been less emphasized that female broodstock must be fed with live feeds such as polychaetes for this practice to be effective. While the effects of eyestalk ablation have been thoroughly studied in various aspects, the synergistic effects of feeding with live feeds and the ablation have never been elucidated at a transcriptome-wide level. With recent advances in the next-generation sequencing platforms, it is now possible to investigate the effects of eyestalk ablation and live feeds at the transcriptomic levels. This study employed both short-read Illumina RNA sequencing and long-read Pacific Biosciences (PacBio) isoform sequencing (Iso-seq) to generate the first high-quality ovarian reference transcriptome in P. monodon. This novel assembly allowed us to dissect the effects of feeds and eyestalk ablation and reveal their synergistic effects at the transcriptomic level through the regulation of important genes involved in fatty acid regulation, energy production, and hormone-mediated oocyte maturation pathways. The synergistic effects between the polychaete feeding and the eyestalk ablation in the process of ovarian maturation in black tiger shrimp suggest that without having proper nutrients from the polychaetes, female broodstock might not be ready to develop its ovary. However, even with proper nutrients, the eyestalk ablation is still necessary to perhaps manipulate the female endocrine of the black tiger shrimp. These findings shed the light on molecular mechanisms and key molecular pathways that lead to successful ovarian maturation.