A molecular approach to identify parrotfish (Sparisoma) species during early ontogeny.

Sparisoma species (parrotfish) comprise an important functional group contributing to coral-reef resilience. The morphological diagnostic characteristics for species identification are clearly described for adult forms but not for the early stages. Consequently, many taxonomical listings of Sparisoma larvae are restricted to the genus level. The aims of this study are to determine whether the morphological and molecular identification techniques are useful to assign the species taxonomic level to Sparisoma larvae occurring in the Gulf of Mexico and whether there is a set of diagnostic features that could be used to discriminate between species in larvae of different developmental stages. Morphological assignment of Sparisoma was performed based on morphological and meristic features for 30 larvae collected in the Gulf of Mexico from late August to mid-September 2015. To corroborate and complement the morphological assignments, molecular identification was carried out using DNA sequences from regions of two mitochondrial genes, mitochondrial cytochrome oxidase I (mtDNA COI) and mitochondrial 16S rRNA (mtDNA 16S rRNA). COI and 16S gene trees for Sparisoma and related fish taxa were constructed using sequences available in the NCBI (National Center for Biotechnology Information) GenBank and BOLD (Barcode of Life Data) databases. Two morphotypes were identified based on morphology, but no diagnostic characteristics for species discrimination were found. Molecular identification, in contrast, successfully discriminated four early development stages of Sparisoma atomarium, three stages of Sparisoma radians, and two stages of Sparisoma chrysopterum and Sparisoma aurofrenatum, therefore demonstrating the successful and necessary application of molecular taxonomic approaches for species-level identifications of Sparisoma larvae.

RpuS/R Is a Novel Two-Component Signal Transduction System That Regulates the Expression of the Pyruvate Symporter MctP in Sinorhizobium fredii NGR234.

The SLC5/STAC histidine kinases comprise a recently identified family of sensor proteins in two-component signal transduction systems (TCSTS), in which the signaling domain is fused to an SLC5 solute symporter domain through a STAC domain. Only two members of this family have been characterized experimentally, the CrbS/R system that regulates acetate utilization in Vibrio and Pseudomonas, and the CbrA/B system that regulates the utilization of histidine in Pseudomonas and glucose in Azotobacter. In an attempt to expand the characterized members of this family beyond the Gammaproteobacteria, we identified two putative TCSTS in the Alphaproteobacterium Sinorhizobium fredii NGR234 whose sensor histidine kinases belong to the SLC5/STAC family. Using reverse genetics, we were able to identify the first TCSTS as a CrbS/R homolog that is also needed for growth on acetate, while the second TCSTS, RpuS/R, is a novel system required for optimal growth on pyruvate. Using RNAseq and transcriptional fusions, we determined that in S. fredii the RpuS/R system upregulates the expression of an operon coding for the pyruvate symporter MctP when pyruvate is the sole carbon source. In addition, we identified a conserved DNA sequence motif in the putative promoter region of the mctP operon that is essential for the RpuR-mediated transcriptional activation of genes under pyruvate-utilizing conditions. Finally, we show that S. fredii mutants lacking these TCSTS are affected in nodulation, producing fewer nodules than the parent strain and at a slower rate.

Transcriptomic assessment of dietary fishmeal partial replacement by soybean meal and prebiotics inclusion in the liver of juvenile Pacific yellowtail (Seriola lalandi).

BACKGROUND: Seriola lalandi is an important species for aquaculture, due to its rapid growth, adaptation to captivity and formulated diets, and high commercial value. Due to the rise in fishmeal (FM) price, efforts have been and still are made to replace it partially or entirely with vegetable meals in diets for carnivorous fish. The use of prebiotics when feeding vegetable meals has improved fish health. METHODS: Four experimental diets were assessed in juveniles, the control diet consisted of FM as the main protein source, the second diet included 2% of GroBiotic®-A (FM-P), in the third diet FM was partially replaced (25%) by soybean meal (SM25), and the fourth consisted of SM25 with 2% of GroBiotic®-A (SM25-P). Growth was evaluated and RNA-seq of the liver tissue was performed, including differential expression analysis and functional annotation to identify genes affected by the diets. RESULTS: Growth was not affected by this level of FM replacement, but it was improved by prebiotics. Annotation was achieved for 59,027 transcripts. Gene expression was affected by the factors: 225 transcripts due to FM replacement, 242 due to prebiotics inclusion, and 62 due to the interaction of factors. The SM25-P diet showed the least amount of differentially expressed genes against the control diet. CONCLUSION: The replacement of FM (25%) by soybean meal combined with prebiotics (2%) represents a good cost-benefit balance for S. lalandi juveniles since the fish growth increased and important metabolic and immune system genes in the liver were upregulated with this diet.

Quantitative real-time PCR normalization for gene expression studies in the plant pathogenic fungi Lasiodiplodia theobromae.

Lasiodiplodia theobromae is a highly virulent plant pathogen. It has been suggested that heat stress increases its virulence. The aim of this work was to evaluate, compare, and recommend normalization strategies for gene expression analysis of the fungus growing with grapevine wood under heat stress. Using RT-qPCR-derived data, reference gene stability was evaluated through geNorm, NormFinder and Bestkeeper applications. Based on the geometric mean using the ranking position obtained for each independent analysis, genes were ranked from least to most stable as follows: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), ß-tubulin (TUB) and elongation factor-1α (EF1α). Using RNAseq-derived data based on the calculated tagwise dispersion these genes were ordered by increasing stability as follows: GAPDH, ACT, TUB, and EF1α. The correlation between RNAseq and RTqPCR results was used as criteria to identify the best RT-qPCR normalization approach. The gene TUB is recommended as the best option for normalization among the commonly used reference genes, but alternative fungal reference genes are also suggested.