Project description:Introduction: A major economic impediment to the aquaculture of marine finfish in land-based recirculating systems is maintaining the system salinity at a range for optimal growth. Florida Pompano (Trachinotus carolinus) are warm water, euryhaline, marine finfish shown to be a suitable candidate for low-salinity culture. Due to its popularity among sport and commercial fishers, high market value, and ability to readily consume pelleted feeds, the Florida Pompano has become a renewed target for commercialized aquaculture. Although the Florida Pompano has been successfully grown at various salinities, its optimal growth salinity has not yet been established. Studies have shown culturing at different salinities has an effect on digestion rates, feed utilization, and lipid biosynthesis. Identifying the optimal salinity for growth would result in decreased energy expenditure on maintaining homeostasis, since osmoregulation is a highly demanding process. This would allow for energy to be spent on the efficient use of nutrients. Methods: Our study was designed to determine how Florida Pompano larviculture at various salinities affects fish health with transcriptomics (RNA-seq) and fatty acid analysis. RNA-seq was used to identify genes actively transcribed and expressed at the time of sampling. After hatching in a salinity of ~ 30 ppt, larvae were reared in 345 L tanks at one of three salinities (10, 20, 30 ppt) in triplicate. Larvae samples for RNA-seq and fatty acid analysis were collected every three days until weaning. Samples for fatty acid composition were analyzed using a gas chromatography-mass spectrometry (GC/MS). RNA was extracted from homogenized whole-body samples using the Qiagen RNeasy Mini kit. Total RNA was sequenced on the Illumina HiSeq 4000 System. Raw sequences were filtered for low-quality sequences, aligned to the S. dumerili reference genome (NCBI Genome: 12614), and quantified. Each sampling day was compared across salinities to establish the differentially expressed genes (DEGs) between salinities. Results & Discussion: The numbers of DEGS in pooled samples was minimal (less than 15 total in each comparison) and the number of DEGS at each dph was variable. There were no significant differences between pooled samples or at each dph according to PERMANOVA analysis, but each dph was statistically different on pairwise analysis. Gene set enrichment analysis revealed that there was a downregulation of immune related genes in lower salinities versus higher salinities suggesting larvae may be dealing with less stress at lower salinities. Most osmoregulation, fatty acid, and immune related genes tested were not significantly different across salinities. This is likely be cause the Florida pompano is euryhaline and has a tolerance to the salinities selected. There was an increase in the expression levels of several immune related genes that may indicate an increase exposure to pathogens at later days post hatch likely related to feeding schedule. There was also an increase in fatty acid biosynthesis genes that corresponded with levels of fatty acids indicating a possible synthesis of essential fatty acids from precursors. Conclusions: From a transcriptomics perspective, the larvae did not show a negative response to lower salinities in comparison to higher salinities, which in fact showed upregulation of immune related genes. Further exploration of Florida pompano’s ability to biosynthesize essential fatty acids is necessary. This study will help enable the optimization of Florida Pompano larviculture using information on the optimal salinity and fatty acid content for growth and facilitate more cost-effective rearing methods.

Project description:Algae of the genus Dunaliella are known to be able to grow in a wide range of salinities. To investigate which genes may be important for growth under extremely low or high salinities, a survey of the Dunaliella sp. transcriptome was performed by microarray analysis. For the cDNA microarray 778 expressed sequence tags (ESTs) were generated from a cDNA library of Dunaliella sp. The cDNA microarray was used to compare the transcription profile of cells that were acclimated to extreme saline conditions (0.08 M and 4.5 M NaCl) with the transcription profile of cells acclimated to an intermediate salinity at 1.5 M NaCl. The comparative microarray analysis indicated that 211 out of the 778 ESTs differed in their expression levels more than two-fold in cells grown at extreme different salinity when compared to cells grown at intermediate salinity (1.5M NaCl). The sequences of these 211 cDNAs were classified into 169 non-redundant gene clusters. Of these genes, the expression of 36 genes was increased and 69 genes were suppressed in cells grown at low salinity. The expression of 49 genes was increased and 85 genes were suppressed in cells grown at high salinity. One-hundred thirty four genes have been revealed to show up- and down-regulation by high salinity (4.5M NaCl). Of the high salinity-regulated genes 52% were also influenced by low salinity (0.08M NaCl), revealing a dense overlap between low and high salt responses. Only 35 genes are regulated independently by low salt condition. Strong relationships, however, were observed in the expression of these extreme salinity-responsive genes based on Venn diagram analysis, which found 70 genes that responded to these two salinity conditions. Refined expression analysis using gene-specific primers and real-time PCR for selected transcripts was in agreement with microarray results for most genes tested. This study provides the first large-scale survey of the transcriptome in the microalga Dunaliella sp. and it provides a platform for further functional investigation of differentially expressed genes in Dunaliella. Keywords: Low salt(0.08M NaCl) or high salt(4.5M NaCl)-acclimated cells

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