A Neurexin2aa deficiency results in axon pathfinding defects and increased anxiety in zebrafish.

Neurexins are presynaptic transmembrane proteins that control synapse activity and are risk factors for autism spectrum disorder. Zebrafish, a popular model for behavioral studies, has six neurexin genes, but their functions in embryogenesis and behavior remain largely unknown. We have previously reported that nrxn2a is aberrantly spliced and specifically dysregulated in motor neurons (MNs) in models of spinal muscular atrophy. In this study, we generated nrxn2aa-/- mutants by CRISPR/Cas9 to understand nrxn2aa function at the zebrafish neuromuscular junction (NMJ) and to determine the effects of its deficiency on adult behavior. Homozygous mutant embryos derived from heterozygous parents did not show obvious defects in axon outgrowth or synaptogenesis of MNs. In contrast, maternal-zygotic (MZ) nrxn2aa-/- mutants displayed extensively branched axons and defective MNs, suggesting a cell-autonomous role for maternally provided nrxn2aa in MN development. Analysis of the NMJs revealed enlarged choice points in MNs of mutant larvae and reduced co-localization of pre- and post-synaptic terminals, indicating impaired synapse formation. Severe early NMJ defects partially recovered in late embryos when mutant transcripts became strongly upregulated. Ultimately, however, the induced defects resulted in muscular atrophy symptoms in adult MZ mutants. Zygotic homozygous mutants developed normally but displayed increased anxiety at adult stages. Together, our data demonstrate an essential role for maternal nrxn2aa in NMJ synapse establishment, while zygotic nrxn2aa expression appears dispensable for synapse maintenance. The viable nrxn2aa-/- mutant furthermore serves as a novel model to study how an increase in anxiety-like behaviors impacts other deficits.

Feed Restriction Modulates Growth, Gut Morphology and Gene Expression in Zebrafish.

A reduction in daily caloric or nutrient intake has been observed to promote health benefits in mammals and other vertebrates. Feed Restriction (FR), whereby the overall food intake of the organism is reduced, has been explored as a method to improve metabolic and immune health, as well as to optimize productivity in farming. However, less is known regarding the molecular and physiological consequences of FR. Using the model organism, Danio rerio, we investigated the impact of a short-term (month-long) FR on growth, gut morphology and gene expression. Our data suggest that FR has minimal effects on the average growth rates, but it may affect weight and size heterogeneity in a sex-dependent manner. In the gut, we observed a significant reduction in gut circumference and generally lower mucosal heights, whereas other parameters remained unchanged. Gene Ontology (GO), EuKaryotic Orthologous Groups (KOG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified numerous metabolic, reproductive, and immune response pathways that were affected by FR. These results broaden our understanding of FR and contribute towards growing knowledge of its effects on vertebrate health.

Chromosomal-Level Assembly of the Asian Seabass Genome Using Long Sequence Reads and Multi-layered Scaffolding.

We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species' native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics.

Correction: Chromosomal-Level Assembly of the Asian Seabass Genome Using Long Sequence Reads and Multi-layered Scaffolding.

[This corrects the article DOI: 10.1371/journal.pgen.1005954.].

Proteomics Analysis of Early Developmental Stages of Zebrafish Embryos.

Zebrafish is a well-recognized organism for investigating vertebrate development and human diseases. However, the data on zebrafish proteome are scarce, particularly during embryogenesis. This is mostly due to the overwhelming abundance of egg yolk proteins, which tend to mask the detectable presence of less abundant proteins. We developed an efficient procedure to reduce the amount of yolk in zebrafish early embryos to improve the Liquid chromatography-tandem mass spectrometry (LC-MS)-based shotgun proteomics analysis. We demonstrated that the deyolking procedure resulted in a greater number of proteins being identified. This protocol resulted in approximately 2-fold increase in the number of proteins identified in deyolked samples at cleavage stages, and the number of identified proteins increased greatly by 3-4 times compared to non-deyolked samples in both oblong and bud stages. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed a high number of functional proteins differentially accumulated in the deyolked versus non-deyolked samples. The most prominent enrichments after the deyolking procedure included processes, functions, and components related to cellular organization, cell cycle, control of replication and translation, and mitochondrial functions. This deyolking procedure improves both qualitative and quantitative proteome analyses and provides an innovative tool in molecular embryogenesis of polylecithal animals, such as fish, amphibians, reptiles, or birds.

Cyberlindnera jadinii yeast as a functional protein source: Modulation of immunoregulatory pathways in the intestinal proteome of zebrafish (Danio rerio).

Yeasts contain bioactive components that can enhance fish immune robustness and disease resistance. Our study focused on analyzing intestinal immunoregulatory pathways in zebrafish (Danio rerio) using iTRAQ and 2D LC-MS/MS to quantify intestinal proteins. Zebrafish were fed either control diet (C) or diet supplemented with autolyzed Cyberlindnera jadinii (ACJ). KEGG analysis revealed that ACJ yeast diet induced increased abundance of proteins related to arginine and proline metabolism, phagosome, C-lectin receptor signaling, ribosome and PPAR signaling pathways, which can modulate and enhance innate immune responses. ACJ yeast diet also showed decreased abundance of proteins associated with inflammatory pathways, including apoptosis, necroptosis and ferroptosis. These findings indicate boosted innate immune response and control of inflammation-related pathways in zebrafish intestine. Our findings in the well annotated proteome of zebrafish enabled a detailed investigation of intestinal responses and provide insight into health-beneficial effects of yeast species C. jadinii, which is relevant for aquaculture species.

Comparative Nutritional and Histological Analysis of Malabar Red Snapper (Lutjanus malabaricus) and Asian Seabass (Lates calcarifer).

This study offers a comprehensive morpho-histological analysis of the gastrointestinal tract (GIT) of the Malabar red snapper. A comparison of its GIT morphology with that of the Asian seabass reveals similarities and differences between the two species. Additionally, the moisture content, crude protein, and ash in the fillets of Malabar red snapper and Asian seabass were slightly different, with Malabar red snapper exhibiting higher levels of essential fatty acids. Furthermore, higher levels of the polyunsaturated fatty acid (PUFA)/saturated fatty acid (SFA) ratio and docosahexaenoic acid (DHA)/eicosapentaenoic acid (EPA) ratio, and a lower omega-6/omega-3 ratio, were observed in Malabar red snapper compared to Asian seabass. The Malabar red snapper's esophagus featured protective mechanisms such as simple columnar epithelial cells, mucous-secreting glands, and goblet cells that were predominantly stained for acid and neutral mucosubstances. Furthermore, its stomach, with mucus cells that were weakly stained for acid mucosubstances, exhibited distinct regions with varying glandular densities, with the pyloric region featuring few glands. The pyloric caeca of the fish were composed of five finger-like structures and few goblet cells. Several goblet cells gradually increased from the anterior to the posterior region of the intestine. These findings provide useful insights for the aquaculture sector, focusing on Malabar red snapper.

Transcriptomic Analysis of Gill and Kidney from Asian Seabass (Lates calcarifer) Acclimated to Different Salinities Reveals Pathways Involved with Euryhalinity.

Asian seabass (or commonly known as barramundi), Lates calcarifer, is a bony euryhaline teleost from the Family Latidae, inhabiting nearshore, estuarine, and marine connected freshwaters throughout the tropical Indo-West Pacific region. The species is catadromous, whereby adults spawn in salinities between 28 and 34 ppt at the mouth of estuaries, with resultant juveniles usually moving into brackish and freshwater systems to mature, before returning to the sea to spawn again as adults. The species lives in both marine and freshwater habitats and can move quickly between the two; thus, the species' ability to tolerate changes in salinity makes it a good candidate for studying the salinity acclimation response in teleosts. In this study, the transcriptome of two major osmoregulatory organs (gills and kidneys) of young juvenile Asian seabass reared in freshwater and seawater were compared. The euryhaline nature of Asian seabass was found to be highly pliable and the moldability of the trait was further confirmed by histological analyses of gills and kidneys. Differences in major expression pathways were observed, with differentially expressed genes including those related to osmoregulation, tissue/organ morphogenesis, and cell volume regulation as central to the osmo-adaptive response. Additionally, genes coding for mucins were upregulated specifically under saline conditions, whereas several genes important for growth and development, as well as circadian entrainment were specifically enriched in fish reared in freshwater. Routing of the circadian rhythm mediated by salinity changes could be the initial step in salinity acclimation and possibly migration in euryhaline fish species such as the Asian seabass.

B Chromosomes of the Asian Seabass (Lates calcarifer) Contribute to Genome Variations at the Level of Individuals and Populations.

The Asian seabass (Lates calcarifer) is a bony fish from the Latidae family, which is widely distributed in the tropical Indo-West Pacific region. The karyotype of the Asian seabass contains 24 pairs of A chromosomes and a variable number of AT- and GC-rich B chromosomes (Bchrs or Bs). Dot-like shaped and nucleolus-associated AT-rich Bs were microdissected and sequenced earlier. Here we analyzed DNA fragments from Bs to determine their repeat and gene contents using the Asian seabass genome as a reference. Fragments of 75 genes, including an 18S rRNA gene, were found in the Bs; repeats represented 2% of the Bchr assembly. The 18S rDNA of the standard genome and Bs were similar and enriched with fragments of transposable elements. A higher nuclei DNA content in the male gonad and somatic tissue, compared to the female gonad, was demonstrated by flow cytometry. This variation in DNA content could be associated with the intra-individual variation in the number of Bs. A comparison between the copy number variation among the B-related fragments from whole genome resequencing data of Asian seabass individuals identified similar profiles between those from the South-East Asian/Philippines and Indian region but not the Australian ones. Our results suggest that Bs might cause variations in the genome among the individuals and populations of Asian seabass. A personalized copy number approach for segmental duplication detection offers a suitable tool for population-level analysis across specimens with low coverage genome sequencing.

Morpho-histological characterisation of the alimentary canal of an important food fish, Asian seabass (Lates calcarifer).

Asian seabass (Lates calcarifer) is a food fish of increasing aquaculture importance. In order to improve our understanding on the digestive system and feeding of this species, morphological and histological features of the gut were studied. Morphologically, the Asian seabass gut is defined by a short and muscular esophagus, well-developed stomach and comparatively short intestine. Mucous secreting goblet cells reactive to PAS (Periodic Acid Schiff) and AB (Alcian Blue) stain were present throughout the esophagus. The stomach was sac-like and could be distinguished into the cardiac, fundic and pyloric regions. Gastric glands and mucus cells were predominately present in the cardiac and fundic regions. Five finger-like pyloric caeca were present between the stomach and intestine. The intestine was a short, tubular structure with no morphological differences between the various regions. Histologically, the intestinal regions were similar, the main difference being in the number of goblet cells that increased from anterior to posterior intestine, with 114 ± 9, 153 ± 7 and 317 ± 21 goblet cells in the anterior, mid and posterior regions, respectively. The intestinal epithelium stained positively for PAS, but the staining was stronger for acidic glycoproteins. The rectum was similar to intestine, except for increased goblet cell numbers (anterior rectum: 529 ± 26; posterior rectum: 745 ± 29). Gut morpho-histology did not respond to salinity changes, however, there was a significant reduction of mucosal height, goblet cell numbers and muscularis thickness upon food deprivation.