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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.

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.