Use of Artemia as model organism to study epigenetic control of phenotypes relevant for aquaculture species

Epigenetics is the study of reversible changes in gene function that occur without modifications in the DNA sequence. Epigenetic variation that are environmentally determined, contributes to inter-individual variation in gene expression and thus to variation in development of phenotypes in an individual or even throughout the entire population. These epigenetic modifications can in some cases be inherited by subsequent generations, even when the initial external stimuli is no longer present in the environment of these organisms. Epigenetic modification occurs through different molecular mechanisms including non-coding small RNA molecules, DNA methylation or histone tail modifications. Among the environmental stressors animals (aquatic or terrestrial) can encounter, pathogen attack and heat stress are quite common. The work presented in this thesis indicated that, the organisms respond to biotic or abiotic environmental stressors with battery of adaptive phenotypes (i.e. increased resistance, different gene expression patterns and protein production). Interestingly, overall results leave no doubt on the role of epigenetic modifications, such as DNA methylation, histone H3 and H4 acetylation and H3K4Me3 on emergence of these new phenotypes

Non-mammalian model organisms in epigenetic research : an overview

Recent advances in sequencing technology and genome editing tools had an indisputably enormous impact on our understanding of complex biological pathways and their genetic and epigenetic regulation. Unlike genetics, a study of phenotype development as a result of genotypic diversity, epigenetics studies the emergence of (possibly heritable) phenotypic assortment from one DNA sequence. Epigenetic modifications (i.e., DNA methylation, histone tail modifications, noncoding RNA interference, and many others) are diverse and can bring an additional layer of complexity to phenotype development and it's inheritance. Still, today, detailed mechanisms behind the development of epigenetic marks, their interaction, and their role in transgenerational inheritance of phenotypes are not fully understood. Therefore, chromatin biology and epigenetic research have a rich history of chasing discoveries in a variety of model organisms, including yeast, worms, flies, fish, and plants. Use of these models has opened numerous new avenues for investigation in the field. In the coming future, model organisms will continue to serve as an inseparable part of studies related to interpreting complex genomic and epigenomic data, gene–protein functional relationship, various diseases pathways, aging, and many others. Use of the model organism will provide insights not only into novel genetic players but also the profound impact of epigenetics on phenotype development. Here, we present a brief overview of the most commonly used nonmammalian model organism (i.e., fruit fly, nematode worm, zebrafish, and yeast) as potential experimental systems for epigenetic studies

Recombinant DnaK orally administered protects axenic European sea bass against vibriosis

Vibrio anguillarum causes high mortality in European sea bass (Dicentrarchus labrax) larviculture and is a hindering factor for successful sustainable aquaculture of this commercially valuable species. Priming of the innate immune system through administration of immunostimulants has become an important approach to control disease outbreaks in marine fish larviculture. This study was conducted to evaluate immunostimulation by Escherichia coli HSP70 (DnaK) in axenic European sea bass larvae in order to protect the larvae against vibriosis. DnaK stimulates the immune response in crustaceans and juvenile fish against bacterial infections. The use of axenic fish larvae allows to study immunostimulation in the absence of an interfering microbial community. At 7 days post-hatching, larvae received a single dose of alginate encapsulated recombinant DnaK. Two non-treated control groups in which animals either received empty alginate microparticles (C1) or no alginante microparticles (C2 and C3) were included in the study. Eighteen hours later, all larvae, except the ones from group C3 (non-infected control) were challenged with V. anguillarum (10(5) CFU, bath infection). Mortality was daily recorded until 120 h post infection and at 18, 24, and 36 h post infection, larvae were sampled for expression of immune related genes. Results showed that V. anguillarum induced an immune response in axenic sea bass larvae but that the innate immune response was incapable to protect the larvae against deadly septicaemic disease. In addition, we showed that administration of alginate encapsulated DnaK to axenic European sea bass larvae at DAH7 resulted in a significant, DnaK dose dependent, upreglation of immune sensor, regulatory and effector genes. Significant upregulation of cxcr4, cas1 and especially of hep and dic was correlated with significant higher survival rates in V. anguillarum infected larvae. In the future recombinant DnaK might perhaps be used as a novel immunostimulant in sea bass larviculture

Probing the phenomenon of trained immunity in invertebrates during a transgenerational study, using brine shrimp Artemia as a model system

The invertebrate's innate immune system was reported to show some form of adaptive features, termed trained immunity. However, the memory characteristics of innate immune system and the mechanisms behind such phenomena remain unclear. Using the invertebrate model Artemia, we verified the possibility or impossibility of trained immunity, examining the presence or absence of enduring memory against homologous and heterologous antigens (Vibrio spp.) during a transgenerational study. We also determined the mechanisms behind such phenomenon. Our results showed the occurrence of memory and partial discrimination in Artemia's immune system, as manifested by increased resistance, for three successive generations, of the progenies of Vibrio-exposed ancestors towards a homologous bacterial strain, rather than to a heterologous strain. This increased resistance phenotype was associated with elevated levels of hsp70 and hmgb1 signaling molecules and alteration in the expression of key innate immunity-related genes. Our results also showed stochastic pattern in the acetylation and methylation levels of H4 and H3K4me3 histones, respectively, in the progenies whose ancestors were challenged. Overall results suggest that innate immune responses in invertebrates have the capacity to be trained, and epigenetic reprogramming of (selected) innate immune effectors is likely to have central place in the mechanisms leading to trained immunity

Determining the efficacy of ginger Zingiber officinale as a potential nutraceutical agent for boosting growth performance and health status of Labeo rohita reared in a semi-intensive culture system

A 120-day feeding trial was conducted in a pilot field setting to study the nutraceutical properties of ginger powder (GP), focusing on the growth performance and health status of Indian major carp L. rohita reared under a semi-intensive culture system. L. rohita fingerlings (average weight: 20.5 g) were divided into five groups and fed a diet with no GP supplementation (control), or a diet supplemented with GP at 5 g (GP5), 10 g (GP10), 15 g (GP15), and 20 g (GP20) per kg of feed. The study was carried out in outdoor tanks (20 m(2)) following a complete randomized design with three replicates for each experimental group. Dietary supplementation of GP at 15 g.kg(-1) (GP15) of feed caused a significant increase in the growth performances of the fish. Results also showed that feeding of GP15 diet led to a significant improvement in the health status of fish as indicated by a marked change in the tested haematological indices (i.e., higher RBC, WBC, Hb, and Ht values), oxidative status (increased SOD and decreased LPO levels), biochemical parameters (increased HDL, decreased cholesterol, and triglycerides levels), and activities of the liver enzymes (decreased AST and ALT). Overall results suggested that dietary supplementation of GP could positively influence the growth and health status of L. rohita fingerlings, and hence could be an important natural nutraceutical for sustainable farming of carp

Phloroglucinol treatment induces transgenerational epigenetic inherited resistance against Vibrio infections and thermal stress in a brine shrimp (Artemia franciscana) model

Emerging, infectious diseases in shrimp like acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus and mortality caused by other Vibrio species such as Vibrio harveyi are worldwide related to huge economic losses in industrial shrimp production. As a strategy to prevent disease outbreaks, a plant-based phenolic compound could be used as a biocontrol agent. Here, using the brine shrimp (Artemia franciscana) as a model system, we showed that phloroglucinol treatment of the parental animals at early life stages resulted in transgenerational inherited increased resistance in their progeny against biotic stress, i.e., bacteria (V. parahaemolyticus AHPND strain and V. harveyi) and abiotic stress, i.e., lethal heat shock. Increased resistance was recorded in three subsequent generations. Innate immune-related gene expression profiles and potential epigenetic mechanisms were studied to discover the underlying protective mechanisms. Our results showed that phloroglucinol treatment of the brine shrimp parents significantly (P < 0.05) enhanced the expression of a core set of innate immune genes (DSCAM, proPO, PXN, HSP90, HSP70, and LGBP) in subsequent generations. We also demonstrated that epigenetic mechanisms such as DNA methylation, m6A RNA methylation, and histone acetylation and methylation (active chromatin marker i.e., H3K4Me3, H3K4me1, H3K27me1, H3 hyperacetylation, H3K14ac and repression marker, i.e., H3K27me3, H4 hypoacetylation) might play a role in regulation of gene expression leading toward the observed transgenerational inheritance of the resistant brine shrimp progenies. To our knowledge, this is the first report on transgenerational inheritance of a compound-induced robust protected phenotype in brine shrimp, particularly protected against AHPND caused by V. parahaemolyticus and vibriosis caused by V. harveyi. Results showed that epigenetic reprogramming is likely to play a role in the underlying mechanism

Sequence and expression analysis of HSP70 family genes in Artemia franciscana

Thus far, only one gene from the heat shock protein 70 (HSP70) family has been identified in Artemia franciscana. Here, we used the draft Artemia transcriptome database to search for other genes in the HSP70 family. Four novel HSP70 genes were identified and designated heat shock cognate 70 (HSC70), heat shock 70 kDa cognate 5 (HSC70-5), lmmunoglobulin heavy-chain binding protein (BIP), and hypoxia up-regulated protein I (HYOUI). For each of these genes, we obtained nucleotide and deduced amino acid sequences, and reconstructed a phylogenetic tree. Expression analysis revealed that in the juvenile state, the transcription of HSP70 and HSC70 was significantly (P 0.05) induction. Gene expression analysis demonstrated that not all members of the HSP70 family are involved in the response to heat stress and selection and that especially altered expression of HSC70 plays a role in a population selected for increased thermotolerance

Probing the protective mechanism of poly-β-hydroxybutyrate against vibriosis by using gnotobiotic Artemia franciscana and Vibrio campbellii as host-pathogen model

The compound poly-beta-hydroxybutyrate (PHB), a polymer of the short chain fatty acid beta-hydroxybutyrate, was shown to protect experimental animals against a variety of bacterial diseases, (including vibriosis in farmed aquatic animals), albeit through undefined mechanisms. Here we aimed at unraveling the underlying mechanism behind the protective effect of PHB against bacterial disease using gnotobiotically-cultured brine shrimp Artemia franciscana and pathogenic Vibrio campbellii as host-pathogen model. The gnotobiotic model system is crucial for such studies because it eliminates any possible microbial interference (naturally present in any type of aquatic environment) in these mechanistic studies and furthermore facilitates the interpretation of the results in terms of a cause effect relationship. We showed clear evidences indicating that PHB conferred protection to Artemia host against V. campbellii by a mechanism of inducing heat shock protein (Hsp) 70. Additionally, our results also showed that this salutary effect of PHB was associated with the generation of protective innate immune responses, especially the prophenoloxidase and transglutaminase immune systems - phenomena possibly mediated by PHB-induced Hsp70. From overall results, we conclude that PHB induces Hsp70 and this induced Hsp70 might contribute in part to the protection of Artemia against pathogenic V. campbellii

The 9H-Fluoren Vinyl Ether Derivative SAM461 Inhibits Bacterial Luciferase Activity and Protects Artemia franciscana From Luminescent Vibriosis

Vibrio campbellii is a major pathogen in aquaculture. It is a causative agent of the so-called “luminescent vibriosis,” a life-threatening condition caused by bioluminescent Vibrio spp. that often involves mass mortality of farmed shrimps. The emergence of multidrug resistant Vibrio strains raises a concern and poses a challenge for the treatment of this infection in the coming years. Inhibition of bacterial cell-to-cell communication or quorum sensing (QS) has been proposed as an alternative to antibiotic therapies. Aiming to identify novel QS disruptors, the 9H-fluroen-9yl vinyl ether derivative SAM461 was found to thwart V. campbellii bioluminescence, a QS-regulated phenotype. Phenotypic and gene expression analyses revealed, however, that the mode of action of SAM461 was unrelated to QS inhibition. Further evaluation with purified Vibrio fischeri and NanoLuc luciferases revealed enzymatic inhibition at micromolar concentrations. In silico analysis by molecular docking suggested binding of SAM461 in the active site cavities of both luciferase enzymes. Subsequent in vivo testing of SAM461 with gnotobiotic Artemia franciscana nauplii demonstrated naupliar protection against V. campbellii infection at low micromolar concentrations. Taken together, these findings suggest that suppression of luciferase activity could constitute a novel paradigm in the development of alternative anti-infective chemotherapies against luminescent vibriosis, and pave the ground for the chemical synthesis and biological characterization of derivatives with promising antimicrobial prospects

The genome of the extremophile Artemia provides insight into strategies to cope with extreme environments

BACKGROUND : Brine shrimp Artemia have an unequalled ability to endure extreme salinity and complete anoxia. This study aims to elucidate its strategies to cope with these stressors. RESULTS AND DISCUSSION : Here, we present the genome of an inbred A. franciscana Kellogg, 1906. We identified 21,828 genes of which, under high salinity, 674 genes and under anoxia, 900 genes were differentially expressed (42%, respectively 30% were annotated). Under high salinity, relevant stress genes and pathways included several Heat Shock Protein and Leaf Embryogenesis Abundant genes, as well as the trehalose metabolism. In addition, based on differential gene expression analysis, it can be hypothesized that a high oxidative stress response and endocytosis/exocytosis are potential salt management strategies, in addition to the expression of major facilitator superfamily genes responsible for transmembrane ion transport. Under anoxia, genes involved in mitochondrial function, mTOR signalling and autophagy were differentially expressed. Both high salt and anoxia enhanced degradation of erroneous proteins and protein chaperoning. Compared with other branchiopod genomes, Artemia had 0.03% contracted and 6% expanded orthogroups, in which 14% of the genes were differentially expressed under high salinity or anoxia. One phospholipase D gene family, shown to be important in plant stress response, was uniquely present in both extremophiles Artemia and the tardigrade Hypsibius dujardini, yet not differentially expressed under the described experimental conditions. CONCLUSIONS : A relatively complete genome of Artemia was assembled, annotated and analysed, facilitating research on its extremophile features, and providing a reference sequence for crustacean research.Additional file 1. Assembly characteristics of all assembled crustacean genomes. Characteristics listed are: species, whether the species genome is annotated yes or no, N50 of the fragments with the highest assembly hierarchy, number of fragments with the highest assembly hierarchy in the assembly, haploid genome size, assembly size, completeness of the assembly (=haploid GS/assembly size), taxonomic lineage (NCBI taxonomy), reference for the genome paper.Additional file 2. Evolution of Artemia assembly quality metrics throughout the assembly steps. Evolution of the scaffold N50, the number of fragments and the genome completeness (assembly size/ genome size) in the subsequent Artemia assembly stagesAdditional file 3 BUSCO analysis results for the A. franciscana genome assembly and annotation.Additional file 4. BLAST results for mitochondrial genes in the Artemia genome. Listed: Query accession and gene name, presence of a (significant) BLAST hit in the Artemia proteome with the highest bit score, E-value and bit score of the hit, scaffold length of the scaffold on which the hit lies, percentage of mitochondrial genes on this scaffold.Additional file 5. Taxonomic groups of alien genomes identified in the Artemia genome.Additional file 6 Expanded or contracted Artemia orthogroups compared to other Branchiopoda. Listed: Orthogroup ID, number of genes in this orthogroup in A. franciscana, D. pulex, L. arcticus, and E. texana, expanded or contracted status of the orthogroup in Artemia compared to D. pulex, L. arcticus and E. texana, conservation in Branchiopoda (whether this orthogroup contains genes for each branchiopod), comma-separated IPR description of Artemia genes in this orthogroup, Artemia genes in this orthogroup.Additional file 7 GO enrichment of Artemia compared to other Branchiopoda. Listed: GO ID, name and category, false discovery rate (FDR) and P value of the Fisher’s exact test enrichment analysis in Blast2GO, number of Artemia genes from expanded/contracted orthogroups in this GO ID, number of whole Artemia genome genes in this GO category, number of Artemia genes from expanded/contracted orthogroups in this GO ID without GO annotation. The Fisher’s Exact Test is sensitive in the direction of the test: the genes that are present in the test-set and also in the reference genome set will be deleted from the reference, but not from the test set, resulting in zero sequences in the reference set and values above zero in the test set. Significantly enriched GOs (FDR ≤ 0.05, biological process) of Artemia genes in expanded or contracted orthogroups compared to Branchiopoda are given.Additional file 8 Expanded or contracted Artemia and H. dujardini orthogroups compared to other Arthropoda. Listed: Orthogroup ID, number of genes in this orthogroup in A. franciscana and in the other arthropod species, expanded or contracted status of the orthogroup in Artemia compared to the other arthropod species, comma-separated IPR description of Artemia genes in this orthogroup, H. dujardini genes in this orthogroup, Artemia genes in this orthogroup.Additional file 9. STAR mapping statistics for differential expression analysis in Artemia. Listed: sample name, total number of reads for this sample, percentage of uniquely mapped reads, absolute number of uniquely mapped reads, percentage of multi mapped reads, absolute number of multi mapped reads.Additional file 10. Summarization statistics for differential expression analysis in Artemia. Listed: sample name, total counts, percentage of counts assigned to a gene annotation, absolute counts assigned to a gene annotation. * notice that this amount can be more than the sum of uniquely mapped + multi-mapped in the mapping statistics since multimapped reads are considered.Additional file 11 Differentially expressed genes under high salinity (p < 0.05). Listed: functional annotation of the differentially expressed gene, gene ID in the genome annotation and on the ORCAE platform, p value, average log fold change of gene expression under high salinity, gene regulation of the differentially expressed gene (up or down), InterPro description of the gene family to which the gene belongs.Additional file 12 Differentially expressed genes under anoxia (p < 0.05). Listed: functional annotation of the differentially expressed gene, gene ID in the genome annotation and on the ORCAE platform, p value, log fold change of gene expression under anoxia, gene regulation of the differentially expressed gene (up or down), InterPro description of the gene family to which the gene belongs.Additional file 13 GO enrichment in Artemia under high salinity. Significantly Enriched GOs (FDR ≤ 0.05) of Artemia genes differentially expressed under high salinity. Listed: GO ID, name and category, false discovery rate (FDR) and P value of the Fisher’s exact test enrichment analysis in Blast2GO, number of DEG under high salinity in this GO category, number of whole Artemia genome genes in this GO category, number of DEG under high salinity without GO annotation. The Fisher’s Exact Test is sensitive in the direction of the test: the genes that are present in the test-set and also in the reference genome set will be deleted from the reference, but not from the test set, resulting in zero sequences in the reference set and values above zero in the test set.Additional file 14 Pathway enrichment in Artemia under high salinity. Significantly enriched (Fisher’s exact test corrected for multiple testing, FDR ≤ 0.05) pathways of Artemia genes differentially expressed under high salinity. Listed in first worksheet (STRING annotation): gene number, ORCAE gene ID, STRING Daphnia pulex gene ID, BLAST identity and bit score, gene name and gene annotation. Listed in second worksheet (STRING pathway enrichment): KEGG Daphnia pulex pathway name, pathway description, number of DEG under high salinity in this pathway, number of genes in the D. pulex genome that belong to this pathway, enrichment FDR, matching D. pulex gene IDs, matching gene names in pathways shown in figures and additional files, matching D. pulex gene ID labels.Additional file 15. Consolidation of DEG analysis, GO enrichment and pathway enrichment in Artemia under high salinity.Additional file 16. The enriched Carbon metabolism pathway in Artemia under high salinity. Up- and downregulated genes are indicated on the KEGG map dpx01200.Additional file 17. GO enrichment in Artemia under anoxia. Significantly enriched GOs (FDR ≤ 0.05) of Artemia genes differentially expressed under anoxia. Listed: GO ID, name and category, false discovery rate (FDR) and P value of the Fisher’s exact test enrichment analysis in Blast2GO, number of DEG under anoxia in this GO ID, number of whole Artemia genome genes in this GO ID, number of DEG under anoxia without GO annotation. The Fisher’s Exact Test is sensitive in the direction of the test: the genes that are present in the test set and also in the reference genome set will be deleted from the reference, but not from the test set, resulting in zero sequences in the reference set and values above zero in the test set.Additional file 18 Pathway enrichment in Artemia under anoxia. Significantly enriched (Fisher’s exact test corrected for multiple testing, FDR ≤ 0.05) pathways of Artemia genes differentially expressed under anoxia. Listed in first worksheet (STRING annotation): gene number, ORCAE gene ID, STRING Daphnia pulex gene ID, BLAST identity and bit score, gene name and gene annotation. Listed in second worksheet (STRING pathway enrichment): KEGG Daphnia pulex pathway name, pathway description, number of DEG under anoxia in this pathway, number of genes in the D. pulex genome that belong to this pathway, enrichment FDR, matching D. pulex gene IDs, matching gene names in pathways shown in figures and additional files, matching D. pulex gene ID labels.Additional file 19. Consolidation of DEG analysis, GO enrichment and pathway enrichment in Artemia under anoxia.Additional file 20. The enriched N-glycan biosynthesis pathway in Artemia under anoxia. Up- and downregulated genes are indicated on the KEGG map dpx00510.Additional file 21. The enriched Basal transcription factors pathway in Artemia under anoxia. Up- and downregulated genes are indicated on the KEGG map dpx03022.Additional file 22. Augustus custom training files for Artemia. Includes probabilities, parameters and weights used for Augustus training for annotation of the Artemia genome.Additional file 23. EuGene custom parameter file for Artemia. Includes parameters used for EuGene training for annotation of the Artemia genome.Additional file 24. Sequence GC-content profiles for all samples used for differential expression analysis.The Flemish Government Special Research Fund and the Laboratory of Aquaculture & Artemia Reference Center.http://www.biomedcentral.com/bmcgenomicsam2022BiochemistryGeneticsMicrobiology and Plant Patholog