BackgroundMacrobrachium rosenbergii, is one of a major freshwater prawn species cultured in Southeast Asia. White tail disease (WTD), caused by Macrobrachium rosenbergii nodavirus (MrNV), is a serious problem in farm cultivation and is responsible for up to 100% mortality in the post larvae stage. Molecular data on how M. rosenbergii post-larvae launches an immune response to an infection with MrNV is not currently available. We therefore compared the whole transcriptomic sequence of M. rosenbergii post-larvae before and after MrNV infection.ResultsTranscriptome for M. rosenbergii post-larvae demonstrated high completeness (BUSCO Complete: 83.4%, fragmentation: 13%, missing:3.3%, duplication:16.2%; highest ExN50 value: 94%). The assembled transcriptome consists of 96,362 unigenes with N50 of 1308 bp. The assembled transcriptome was successfully annotated against the NCBI non-redundant arthropod database (33.75%), UniProt database (26.73%), Gene Ontology (GO) (18.98%), Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups (EggNOG) (20.88%), and Kyoto Encyclopedia of Genes and Genome pathway (KEGG) (20.46%). GO annotations included immune system process, signaling, response to stimulus, and antioxidant activity. Differential abundance analysis using EdgeR showed 2413 significantly up-regulated genes and 3125 significantly down-regulated genes during the infection of MrNV.ConclusionsThis study reported a highly complete transcriptome from the post-larvae stage of giant river prawn, M. rosenbergii. Differential abundant transcripts during MrNV infection were identified and validated by qPCR, many of these differentially abundant transcripts as key players in antiviral immunity. These include known members of the innate immune response with the largest expression change occurring in the M. rosenbergii post-larvae after MrNV infection such as antiviral protein, C-type lectin, prophenol oxidase, caspase, ADP ribosylation factors, and dicer.
Background Massive parallel sequencing technologies have enabled the elucidation of plant phylogenetic relationships from chloroplast genomes at a high pace. These include members of the family Rhamnaceae. The current Rhamnaceae phylogenetic tree is from 13 out of 24 Rhamnaceae chloroplast genomes, and only one chloroplast genome of the genus Ventilago is available. Hence, the phylogenetic relationships in Rhamnaceae remain incomplete, and more representative species are needed. Results The complete chloroplast genome of Ventilago harmandiana Pierre was outlined using a hybrid assembly of long- and short-read technologies. The accuracy and validity of the final genome were confirmed with PCR amplifications and investigation of coverage depth. Sanger sequencing was used to correct for differences in lengths and nucleotide bases between inverted repeats because of the homopolymers. The phylogenetic trees reconstructed using prevalent methods for phylogenetic inference were topologically similar. The clustering based on codon usage was congruent with the molecular phylogenetic tree. The groups of genera in each tribe were in accordance with tribal classification based on molecular markers. We resolved the phylogenetic relationships among six Hovenia species, three Rhamnus species, and two Ventilago species. Our reconstructed tree provides the most complete and reliable low-level taxonomy to date for the family Rhamnaceae. Similar to other higher plants, the RNA editing mostly resulted in converting serine to leucine. Besides, most genes were subjected to purifying selection. Annotation anomalies, including indel calling errors, unaligned open reading frames of the same gene, inconsistent prediction of intergenic regions, and misannotated genes, were identified in the published chloroplast genomes used in this study. These could be a result of the usual imperfections in computational tools, and/or existing errors in reference genomes. Importantly, these are points of concern with regards to utilizing published chloroplast genomes for comparative genomic analysis. Conclusions In summary, we successfully demonstrated the use of comprehensive genomic data, including DNA and amino acid sequences, to build a reliable and high-resolution phylogenetic tree for the family Rhamnaceae. Additionally, our study indicates that the revision of genome annotation before comparative genomic analyses is necessary to prevent the propagation of errors and complications in downstream analysis and interpretation.
Loop-mediated isothermal amplification (LAMP) is a rapid, specific, and effective DNA amplification assay. In this study, a duplex LAMP (dLAMP) assay combined with chromatographic lateral flow dipstick (LFD) was developed for the simultaneous identification of Vibrio vulnificus and V. parahaemolyticus. Each primer set that comprised F3, B3, FIP, and BIP recognized the V. vulnificus rpoS gene and the V. parahaemolyticus tlh gene. Digoxigenilated and biotinylated LAMP amplicons of V. vulnificus and V. parahaemolyticus, respectively, were hybridized with corresponded fluorescein isothiocyanate (FITC)-labeled probes for LFD detection. The optimum conditions were 90 min at 63°C. The dLAMP-LFD had high specificity to V. vulnificus and V. parahaemolyticus and showed no cross-amplification with 59 isolates of other bacteria. The detection limit of dLAMP-LFD with a pure culture of V. vulnificus was 2.2 × 10 4 CFU/mL, corresponding to 41 CFU per reaction, which was equal to that of duplex PCR (dPCR). In the case of V. parahaemolyticus, the detection limit of dLAMP-LFD with a pure culture was 2.2 × 10 3 CFU/mL, corresponding to 4.1 CFU per reaction, 100 times more sensitive than that of dPCR. In the spiked shrimp samples, the detection limit of dLAMP-LFD for V. vulnificus was 2.2 × 10 5 CFU/g, corresponding to 410 CFU per reaction, 10 times more sensitive than that of dPCR. In the case of V. parahaemolyticus, the detection limit of dLAMP-LFD in spiked shrimp samples was 2.2 × 10 4 CFU/g, corresponding to 41 CFU per reaction, 100 times more sensitive than that of dPCR. These results demonstrate that the simple dLAMP-LFD assay had high specificity and high sensitivity for the concurrent detection of V. vulnificus and V. parahaemolyticus.
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