Metagenomic analysis between free-living and cultured Epinephelus fuscoguttatus under different environmental conditions in Indonesian waters.

In this study, we analyzed and compared feces of free-living and cultivated fish species, Epinephelus fuscoguttatus under different environmental conditions in Indonesian waters. Metagenome analysis was performed using Illumina MiSeq sequencing of the whole metagenomic DNA isolated from fish feces samples. The analysis covered both prokaryotic and eukaryotic DNA. Feces samples from mariculture fish revealed a highly stable distribution of several orders of bacteria when compared to samples from free-living fish, which were highly diverse and dominated by Vibrionales, Pseudomonales, Rhizobiales and non-classifiable Alphaproteobacteria. The eukaryotic content of the samples was dominated by residues of the host and nine additional fish species that formed a portion of the diet. Investigations on functional annotations for predominant bacterial taxa, using Gene Ontology enrichment, revealed a number of functions related to DNA metabolic processes, especially DNA repair, as well as antibiotic response in the free-living fish species.

Microbiome analysis and detection of pathogenic bacteria of Penaeus monodon from Jakarta Bay and Bali.

Penaeus monodon, the Asian black tiger shrimp is one of the most widely consumed marine crustaceans worldwide. In this study, we examine and compare the fecal microbiota of P. monodon from highly polluted waters around Jakarta Bay, with those of less polluted waters of Bali. Using next generation sequencing techniques, we identified potential bacterial pathogens and common viral diseases of shrimp. Proteobacteria (96.08%) was found to be the most predominant phylum, followed by Bacteriodetes (2.32%), Fusobacteria (0.96%), and Firmicutes (0.53%). On the order level, Vibrionales (66.20%) and Pseudoaltermonadales (24.81%) were detected as predominant taxa. qPCR profiling was used as a confirmatory step and further revealed Vibrio alginolyticus and Photobacterium damselae as two potential pathogenic species present in most of the samples. In addition, viral diseases for shrimp were discovered among the samples, WSSV in Jakarta free-living samples, YHV in Bali free-living samples and IHHNV in both.

Microbial Diversity and Parasitic Load in Tropical Fish of Different Environmental Conditions.

In this study we analysed fecal bacterial communities and parasites of three important Indonesian fish species, Epinephelus fuscoguttatus, Epinephelus sexfasciatus and Atule mate. We then compared the biodiversity of bacterial communities and parasites of these three fish species collected in highly polluted Jakarta Bay with those collected in less polluted Indonesian areas of Cilacap (E. sexfasciatus, A. mate) and Thousand Islands (E. fuscoguttatus). In addition, E. fuscoguttatus from net cages in an open water mariculture facility was compared with free living E. fuscoguttatus from its surroundings. Both core and shared microbiomes were investigated. Our results reveal that, while the core microbiomes of all three fish species were composed of fairly the same classes of bacteria, the proportions of these bacterial classes strongly varied. The microbial composition of phylogenetically distant fish species, i.e. A. mate and E. sexfasciatus from Jakarta Bay and Cilacap were more closely related than the microbial composition of more phylogentically closer species, i.e. E. fuscoguttatus, E. sexfasciatus from Jakarta Bay, Cilacap and Thousand Islands. In addition, we detected a weak negative correlation between the load of selected bacterial pathogens, i.e. Vibrio sp. and Photobacterium sp. and the number of endoparasites. In the case of Flavobacterium sp. the opposite was observed, i.e. a weak positive correlation. Of the three recorded pathogenic bacterial genera, Vibrio sp. was commonly found in E. fuscoguttatus from mariculture, and lessly in the vicinity of the net cages and rarely in the fishes from the heavily polluted waters from Jakarta Bay. Flavobacterium sp. showed higher counts in mariculture fish and Photobacteria sp. was the most prominent in fish inside and close to the net cages.

Population structure of the Indonesian giant tiger shrimp Penaeus monodon: a window into evolutionary similarities between paralogous mitochondrial DNA sequences and their genomes.

Here we used both microsatellites and mtCR (mitochondrial DNA control region) sequences as genetic markers to examine the genetic diversity and population structure of Penaeus monodon shrimp from six Indonesian regions. The microsatellite data showed that shrimp from the Indian and the Pacific Ocean were genetically distinct from each other. It has been reported previously that P. monodon mtCR sequences from the Indo-Pacific group into two major paralogous clades of unclear origin. Here we show that the population structure inferred from mtCR sequences matches the microsatellite-based population structure for one of these clades. This is consistent with the notion that this mtCR clade shares evolutionary history with nuclear DNA and may thus represent nuclear mitochondrial pseudogenes (Numts).

Expression of the toxin-antitoxin genes yefM(Lrh), yoeB(Lrh) in human Lactobacillus rhamnosus isolates.

Lactobacilli are important microorganisms in various activities, for example, diary products, meat ripening, bread and pickles, but, moreover, are associated directly with human skin and cavities (e.g., mouth, gut, or vagina). Some of them are used as probiotics. Therefore, the molecular biological investigation of these bacteria is important. Earlier we described several toxin antitoxin systems (type II) in lactobacilli. Here, we describe the structure and transcriptional regulation of genes, encoding TA system YefM-YoeB(Lrh) in three strains of Lactobacillus rhamnosus comparing stationary and exponential growth phases, the influence of stress factors and mRNA stability. The same TA system is responding to physiological and stress conditions differently in related strains. Using primer extension and RLM-RACE methods we determined three transcription start sites of RNAs in the operon. The promoter region of the operon is preceded by a conserved BOX element occurring at multiple positions in the genomes of L. rhamnosus strains. Downstream of and partially overlapping with the 3' end of the yoeB(Lrh) toxin gene, a divergently transcribed unexpected RNA was detected.

Paralogous mitochondrial control region in the giant tiger shrimp, Penaeus monodon (F.) affects population genetics inference: A cautionary tale.

The mitochondrial control region (mtCR) is a widely used genetic marker for phylogenetic, phylogeographic and population genetic inference. The analysis of mtCR in 115 Indonesian specimens of the giant tiger shrimp, Penaeus monodon, revealed 26 individuals yielding a second - apparently paralogous - sequence in addition to the putatively authentic mitochondrial haplotype. The paralogous haplotypes fell into two major haplogroups that are highly diverged with respect to the authentic mitochondrial haplotypes (average pairwise sequence divergence of 12.5% and 5.0%, respectively). A comparison with published mtCR sequences of P. monodon showed that the paralogous contaminant sequences were inadvertently included in a series of recent population genetic studies, leading to seriously compromised conclusions about genetic diversity and differentiation. The prevalence of the paralogous haplotypes throughout the sampled Indo-Pacific populations is highly skewed: From African and Indian individuals only paralogs have been sequenced, while they are completely absent from Australian individuals. This suggests that geographically unequally distributed allelic variants at binding sites of the primer pair ordinarily used to amplify mtCR in P. monodon suppressed the amplification of authentic mtCR in a wide range of samples.

Fast short-fragment PCR for rapid and sensitive detection of shrimp viruses.

This article describes a fast short-fragment PCR method for the detection of white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and monodon baculovirus (MBV). Fast two-temperature (95 degrees C denaturation and 60 degrees C annealing/extension) PCRs were performed in 5-10 microl volume samples in miniaturized microplates using a fast Peltier thermal cycler. 40 cycles were completed in 25-30 min. Rapid high-resolution agarose gel electrophoresis of 70-150 bp PCR fragments was performed in 10 min. High sensitivity of PCR product detection (50-100 pg) was obtained using ultra sensitive dyes such as GelStar and a gel documentation system equipped with a blue-light transilluminator. This novel method is faster and more sensitive than its TaqMan real-time PCR counterparts.

Two-laser, large-field hyperspectral microarray scanner for the analysis of multicolor microarrays.

We describe the development and operation of a two-laser, large-field hyperspectral scanner for analysis of multicolor genotyping microarrays. In contrast to confocal microarray scanners, in which wavelength selectivity is obtained by positioning band-pass filters in front of a photomultiplier detector, hyperspectral microarray scanners collect the complete visible emission spectrum from the labeled microarrays. Hyperspectral scanning permits discrimination of multiple spectrally overlapping fluorescent labels with minimal use of optical filters, thus offering important advantages over standard filter-based multicolor microarray scanners. The scanner uses two-sided oblique line illumination of microarrays. Two lasers are used for the excitation of dyes in the visible and near-infrared spectral regions. The hyperspectral scanner was evaluated with commercially available two-color calibration slides and with in-house-printed four-color microarrays containing dyes with spectral properties similar to their commercial genotyping array counterparts.