Transcriptome Analysis Reveals Common and Differential Response to Low Temperature Exposure Between Tolerant and Sensitive Blue Tilapia (Oreochromis aureus)

Tilapias are very important to the world's aquaculture. As befitting fish of their tropical origin, their distribution, and culture practices are highly affected by low temperatures. In this study, we used genetic and genomic methodologies to reveal pathways involved in the response and tolerance of blue tilapia (Oreochromis aureus) to low temperature stress. Cold tolerance was characterized in 66 families of blue tilapia. Fish from cold-tolerant and cold-sensitive families were sampled at 24 and 12°C, and the transcriptional responses to low-temperature exposure were measured in the gills and liver by high-throughput mRNA sequencing. Four hundred and ninety four genes displayed a similar temperature-dependent expression in both tolerant and sensitive fish and in the two tissues, representing the core molecular response to low temperature exposure. KEGG pathway analysis of these genes revealed down-regulation of focal-adhesion and other cell-extracellular matrix (ECM) interactions, and up-regulation of proteasome and various intra-cellular proteolytic activities. Differential responses between cold-tolerant and cold-sensitive fish were found with genes and pathways that were up-regulated in one group and down-regulated in the other. This reverse response was characterized by genes involved in metabolic pathways such as glycolysis/gluconeogenesis in the gills and biosynthesis of amino-acids in the liver, with low temperature down-regulation in tolerant fish and up-regulation in sensitive fish

Genetic perspective on stress response and disease resistance in aquaculture.

Despite continuous progress and improvements in aquaculture technologies and husbandry techniques, fish diseases remain a major limiting factor in the fish culture industry. The aqua- culture environment exposes fish to repeated acute stress, which leads to physiological respons- es that have suppressive effects on growth, reproduction, and immune capacity. The strong link between stress and susceptibility to diseases in farm animals has long been acknowledged, and parameters of high and low stress response are associated with disease resistance in fish. Few studies on genetic aspects of immune response to stress have been conducted. The estimated heritability of several parameters of the innate immune response is mostly moderate. Quantitative trait loci have been found for different responses to stress conditions. Gene expres- sion studies show that hundreds of genes can be involved in fishes’ physiological and immuno- logical response to stress. This general response to stress is controlled by a few major genes at the top of the pathway, which activate a cascade of reactions, having a significant effect on the overall health of the fish. Selective breeding for disease resistant fish is an attractive strategy for disease prevention and several studies have reported progress in this field. However, the bio- logical pathways of stress response and disease resistance are not well characterized, and their genetic basis and control are still poorly understood. Extensive research is still needed for a bet- ter understanding of these pathways, and this should be a collaborative effort of researchers from different fields: genetics, immunology, pathology, physiology, and endocrinology

Improving Salinity Tolerance in Tilapias: Past Experience and Future Prospects

With increasing scarcity of fresh water available for aquaculture, especially in arid regions, development of tilapias that tolerate high salinity would increase fish (and hence, animal protein) production. We review culture practices, nutrition, physiology, and genetics, and propose approaches to improving salinity tolerance in tilapias. Physiological studies of biochemical pathways underlying phenotypic differences in salt tolerance can lead to genetic studies of intra and interspecific variation. Molecular technology can lead to studies on osmoregulation-related biochemical pathways, for which the euryhaline tilapia is an attractive model. Functional genomics and proteomics are powerful tools for studying the molecular bases of environmental adaptation and metabolic connections to osmoregulatory physiology. Both provide avenues for discovering novel pathways related to osmoregulation with relevance to aquaculture. Dietary supplementation with NaCl and optimized acclimation protocols are immediate and practical ways to improve salt tolerance. Inter-specific variation in salinity tolerance may be used to select salt-tolerant species and develop salt-tolerant hybrids. In the long term, quantitative trait loci associated with, or genes involved in, saltwater tolerance may facilitate marker-assisted or gene-assisted selection for this trait in tilapia

Genomic approaches to identifyingsex-determininggenesintilapia

Variation in sex determination mechanisms among the different tilapia species has been recog- nized for over 40 years. We have looked for associations between microsatellite DNA markers and sex in families from different species and strains of tilapia. We found that at least two dif- ferent linkage groups are involved in sex determination in this group of fishes. In two species, O. niloticus and T. zillii, we found evidence for male heterogamety with a major sex-determining locus on linkage group 1 (LG1). In two other species, O. aureus (Israeli strain) and O. karongae, we found evidence for female heterogamety with a major locus for sex determination on LG3. In O. aureus (Egyptian strain) and in O. mossambicus, loci associated with sex determination were found on both LG1 and LG3, and a complex mechanism of sex determination was detected. Physical mapping by fluorescence in situ hybridization (FISH) suggests that LG3 corresponds to the largest chromosome pair, and that there is recombination suppression in the sex determina- tion region. The sex-determining region in O. niloticus has been mapped to an 11cM region between markers GM201 and UNH995 on LG1. A BAC contig containing UNH995 was identi- fied and several BACs in the contig were end- or shotgun sequenced. BLAST analysis of these sequences identified Tetraodon chromosome 5 as the homolog of tilapia LG1. Additional SNP and microsatellite markers were developed from published cichlid ESTs and the order of these markers is consistent between tilapia and Tetraodon. We have narrowed the sex-determining region to a 2.6cM interval which corresponds to a 400 kb region of Tetraodon chr5. We are com- pleting genetic and physical maps across this region in order to identify the gene(s) responsible for sex determination in this species

Rearing White Grouper (Epinephelus aeneus) in Low Salinity Water: Effects of Dietary Salt Supplementation

The white grouper (Epinephelus aeneus) is native to the Mediterranean Sea and the eastern Atlantic Ocean. It is an important species in fisheries of this region, having high market value. Our project of white grouper domestication in brackish water systems aims at developing culture methods for local conditions. The study evaluates the potential of culturing white grouper in low salinity water and the effect of an NaCl-enriched diet on this species. The results of a three-month culture period, conducted at a commercial farm, demonstrate that white grouper can grow in 3 ppt salinity and that 3% dietary salt supplementation can significantly improve its growth. However, the overall growth performance in low salinity was below expected for this species and further research is needed to develop culture practices for white grouper

Fertility Problems In The Second Generation Of A Four-Species Tilapia Cross

Reproductive problems were encountered in attempting to produce the second generation of a four-way cross of tilapia from interspecific F1 hybrids. The cross (Oreochromis mossambicus x O. aureus) x (Sarotherodon galilaeus x O. niloticus) successfully bred for one generation, how- ever, not even a single batch of progeny was obtained in the subsequent generation. It was con- cluded that the complex genetic structure of this cross caused the fertility problem. Any similar breeding programs that are based on multi-species crosses should take into consideration that reproductive problems may occur

Sex-linked markers and microsatellite locus duplication in the cichlid species Oreochromis tanganicae

Cichlid species of the genus Oreochromis vary in their genetic sex-determination systems. In this study, we used microsatellite DNA markers to characterize the sex-determination system in Oreochromis tanganicae. Markers on linkage group 3 were associated with phenotypic sex, with an inheritance pattern typical of a female heterogametic species (WZ–ZZ). Further, locus duplication was observed for two separate microsatellite markers on the sex chromosome. These results further advance our understanding of the rapidly evolving sex-determination systems among these closely related tilapia species

Removal of evolutionarily conserved functional MYC domains in a tilapia cell line using a vector-based CRISPR/Cas9 system.

MYC transcription factors have critical roles in facilitating a variety of cellular functions that have been highly conserved among species during evolution. However, despite circumstantial evidence for an involvement of MYC in animal osmoregulation, mechanistic links between MYC function and osmoregulation are missing. Mozambique tilapia (Oreochromis mossambicus) represents an excellent model system to study these links because it is highly euryhaline and highly tolerant to osmotic (salinity) stress at both the whole organism and cellular levels of biological organization. Here, we utilize an O. mossambicus brain cell line and an optimized vector-based CRISPR/Cas9 system to functionally disrupt MYC in the tilapia genome and to establish causal links between MYC and cell functions, including cellular osmoregulation. A cell isolation and dilution strategy yielded polyclonal myca (a gene encoding MYC) knockout (ko) cell pools with low genetic variability and high gene editing efficiencies (as high as 98.2%). Subsequent isolation and dilution of cells from these pools produced a myca ko cell line harboring a 1-bp deletion that caused a frameshift mutation. This frameshift functionally inactivated the transcriptional regulatory and DNA-binding domains predicted by bioinformatics and structural analyses. Both the polyclonal and monoclonal myca ko cell lines were viable, propagated well in standard medium, and differed from wild-type cells in morphology. As such, they represent a new tool for causally linking myca to cellular osmoregulation and other cell functions

Removal of evolutionarily conserved functional MYC domains in a tilapia cell line using a vector-based CRISPR/Cas9 system

Abstract MYC transcription factors have critical roles in facilitating a variety of cellular functions that have been highly conserved among species during evolution. However, despite circumstantial evidence for an involvement of MYC in animal osmoregulation, mechanistic links between MYC function and osmoregulation are missing. Mozambique tilapia (Oreochromis mossambicus) represents an excellent model system to study these links because it is highly euryhaline and highly tolerant to osmotic (salinity) stress at both the whole organism and cellular levels of biological organization. Here, we utilize an O. mossambicus brain cell line and an optimized vector-based CRISPR/Cas9 system to functionally disrupt MYC in the tilapia genome and to establish causal links between MYC and cell functions, including cellular osmoregulation. A cell isolation and dilution strategy yielded polyclonal myca (a gene encoding MYC) knockout (ko) cell pools with low genetic variability and high gene editing efficiencies (as high as 98.2%). Subsequent isolation and dilution of cells from these pools produced a myca ko cell line harboring a 1-bp deletion that caused a frameshift mutation. This frameshift functionally inactivated the transcriptional regulatory and DNA-binding domains predicted by bioinformatics and structural analyses. Both the polyclonal and monoclonal myca ko cell lines were viable, propagated well in standard medium, and differed from wild-type cells in morphology. As such, they represent a new tool for causally linking myca to cellular osmoregulation and other cell functions