Effects of Shrimp Shell-Derived Chitosan on Growth, Immunity, Intestinal Morphology, and Gene Expression of Nile Tilapia (Oreochromis niloticus) Reared in a Biofloc System.

Chitosan (CH) shows great potential as an immunostimulatory feed additive in aquaculture. This study evaluates the effects of varying dietary CH levels on the growth, immunity, intestinal morphology, and antioxidant status of Nile tilapia (Oreochromis niloticus) reared in a biofloc system. Tilapia fingerlings (mean weight 13.54 ± 0.05 g) were fed diets supplemented with 0 (CH0), 5 (CH5), 10 (CH10), 20 (CH20), and 40 (CH40) mL·kg-1 of CH for 8 weeks. Parameters were assessed after 4 and 8 weeks. Their final weight was not affected by CH supplementation, but CH at 10 mL·kg-1 significantly improved weight gain (WG) and specific growth rate (SGR) compared to the control (p < 0.05) at 8 weeks. Skin mucus lysozyme and peroxidase activities were lower in the chitosan-treated groups at weeks 4 and 8. Intestinal villi length and width were enhanced by 10 and 20 mL·kg-1 CH compared to the control. However, 40 mL·kg-1 CH caused detrimental impacts on the villi and muscular layer. CH supplementation, especially 5-10 mL·kg-1, increased liver and intestinal expressions of interleukin 1 (IL-1), interleukin 8 (IL-8), LPS-binding protein (LBP), glutathione reductase (GSR), glutathione peroxidase (GPX), and glutathione S-transferase (GST-α) compared to the control group. Overall, dietary CH at 10 mL·kg-1 can effectively promote growth, intestinal morphology, innate immunity, and antioxidant capacity in Nile tilapia fingerlings reared in biofloc systems.

Zebrafish ambra1b knockout reveals a novel role for Ambra1 in primordial germ cells survival, sex differentiation and reproduction.

BACKGROUND: AMBRA1 is an intrinsically disordered protein, working as a scaffold molecule to coordinate, by protein-protein interaction, many cellular processes, including autophagy, mitophagy, apoptosis and cell cycle progression. The zebrafish genome contains two ambra1 paralogous genes (a and b), both involved in development and expressed at high levels in the gonads. Characterization of the zebrafish paralogous genes mutant lines generated by CRISPR/Cas9 approach showed that ambra1b knockout leads to an all-male population. RESULTS: We demonstrated that the silencing of the ambra1b gene determines a reduction of primordial germ cells (PGCs), a condition that, in the zebrafish, leads to the development of all-male progeny. PGC reduction was confirmed by knockdown experiments and rescued by injection of ambra1b and human AMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued by injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding region, thus suggesting that interaction with this complex is involved in PGC protection from loss. Results from zebrafish embryos injected with murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could indirectly regulate this protein through CUL4-DDB1 interaction. According to this, Ambra1+/- mice showed a reduced Stat3 expression in the ovary together with a low number of antral follicles and an increase of atretic follicles, indicating a function of Ambra1 in the ovary of mammals as well. Moreover, in agreement with the high expression of these genes in the testis and ovary, we found significant impairment of the reproductive process and pathological alterations, including tumors, mainly limited to the gonads. CONCLUSIONS: By exploiting ambra1a and ambra1b knockout zebrafish lines, we prove the sub-functionalization between the two paralogous zebrafish genes and uncover a novel function of Ambra1 in the protection from excessive PGC loss, which seems to require binding with the CUL4-DDB1 complex. Both genes seem to play a role in the regulation of reproductive physiology.

Zebrafish Mutant Lines Reveal the Interplay between nr3c1 and nr3c2 in the GC-Dependent Regulation of Gene Transcription.

Glucocorticoids mainly exert their biological functions through their cognate receptor, encoded by the nr3c1 gene. Here, we analysed the glucocorticoids mechanism of action taking advantage of the availability of different zebrafish mutant lines for their receptor. The differences in gene expression patterns between the zebrafish gr knock-out and the grs357 mutant line, in which a point mutation prevents binding of the receptor to the hormone-responsive elements, reveal an intricate network of GC-dependent transcription. Particularly, we show that Stat3 transcriptional activity mainly relies on glucocorticoid receptor GR tethering activity: several Stat3 target genes are induced upon glucocorticoid GC exposure both in wild type and in grs357/s357 larvae, but not in gr knock-out zebrafish. To understand the interplay between GC, their receptor, and the mineralocorticoid receptor, which is evolutionarily and structurally related to the GR, we generated an mr knock-out line and observed that several GC-target genes also need a functional mineralocorticoid receptor MR to be correctly transcribed. All in all, zebrafish mutants and transgenic models allow in vivo analysis of GR transcriptional activities and interactions with other transcription factors such as MR and Stat3 in an in-depth and rapid way.

Knockout of the Glucocorticoid Receptor Impairs Reproduction in Female Zebrafish.

The pleiotropic effects of glucocorticoids in metabolic, developmental, immune and stress response processes have been extensively investigated; conversely, their roles in reproduction are still less documented. It is well known that stress or long-lasting therapies can cause a strong increase in these hormones, negatively affecting reproduction. Moreover, the need of glucocorticoid (GC) homeostatic levels is highlighted by the reduced fertility reported in the zebrafish glucocorticoid receptor mutant (nr3c1ia30/ia30) line (hereafter named gr-/-). Starting from such evidence, in this study, we have investigated the role of glucocorticoid receptor (Gr) in the reproduction of female zebrafish. Key signals orchestrating the reproductive process at the brain, liver, and ovarian levels were analyzed using a multidisciplinary approach. An impairment of the kiss-GnRH system was observed at the central level in (gr-/-) mutants as compared to wild-type (wt) females while, in the liver, vitellogenin (vtg) mRNA transcription was not affected. Changes were instead observed in the ovary, particularly in maturing and fully grown follicles (classes III and IV), as documented by the mRNA levels of signals involved in oocyte maturation and ovulation. Follicles isolated from gr-/- females displayed a decreased level of signals involved in the acquisition of competence and maturation, causing a reduction in ovulation with respect to wt females. Fourier transform infrared imaging (FTIRI) analysis of gr-/- follicle cytoplasm showed major changes in macromolecule abundance and distribution with a clear alteration of oocyte composition. Finally, differences in the molecular structure of the zona radiata layer of gr-/- follicles are likely to contribute to the reduced fertilization rate observed in mutants.

Zebrafish xenograft as a tool for the study of colorectal cancer: a review.

Colorectal cancer (CRC) is the second leading cause of cancer-related death, mostly due to metastatic disease and the fact that many patients already show signs of metastasis at the time of first diagnosis. Current CRC therapies negatively impact patients' quality of life and have little to no effect on combating the tumor once the dissemination has started. Danio rerio (zebrafish) is a popular animal model utilized in cancer research. One of its main advantages is the ease of xenograft transplantation due to the fact that zebrafish larvae lack the adaptative immune system, guaranteeing the impossibility of rejection. In this review, we have presented the many works that choose zebrafish xenograft as a tool for the study of CRC, highlighting the methods used as well as the promising new therapeutic molecules that have been identified due to this animal model.

Quantity discrimination in newly hatched zebrafish suggests hardwired numerical abilities.

An intriguing hypothesis to explain the ubiquity of numerical abilities is that all vertebrates are born with hardwired neuronal networks for processing numbers. To date, only studies on human foetuses have clearly supported this hypothesis. Zebrafish hatch 48-72 h after fertilisation with an embryonic nervous system, providing a unique opportunity for investigating this hypothesis. Here, we demonstrated that zebrafish larvae exposed to vertical bars at birth acquired an attraction for bar stimuli and we developed a numerical discrimination task based on this preference. When tested with a series of discriminations of increasing difficulty (1vs.4, 1vs.3, 1vs.2, and 2vs.4 bars), zebrafish larvae reliably selected the greater numerosity. The preference was significant when stimuli were matched for surface area, luminance, density, and convex hull, thereby suggesting a true capacity to process numerical information. Converging results from two phylogenetically distant species suggests that numerical abilities might be a hallmark feature of vertebrates' brains.

DOPAL initiates αSynuclein-dependent impaired proteostasis and degeneration of neuronal projections in Parkinson's disease.

Dopamine dyshomeostasis has been acknowledged among the determinants of nigrostriatal neuron degeneration in Parkinson's disease (PD). Several studies in experimental models and postmortem PD patients underlined increasing levels of the dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is highly reactive towards proteins. DOPAL has been shown to covalently modify the presynaptic protein αSynuclein (αSyn), whose misfolding and aggregation represent a major trait of PD pathology, triggering αSyn oligomerization in dopaminergic neurons. Here, we demonstrated that DOPAL elicits αSyn accumulation and hampers αSyn clearance in primary neurons. DOPAL-induced αSyn buildup lessens neuronal resilience, compromises synaptic integrity, and overwhelms protein quality control pathways in neurites. The progressive decline of neuronal homeostasis further leads to dopaminergic neuron loss and motor impairment, as showed in in vivo models. Finally, we developed a specific antibody which detected increased DOPAL-modified αSyn in human striatal tissues from idiopathic PD patients, corroborating the translational relevance of αSyn-DOPAL interplay in PD neurodegeneration.

Glucocorticoid receptor activities in the zebrafish model: a review.

Glucocorticoids (GCs) are steroid hormones that contribute to the regulation of many physiological processes, such as inflammation, metabolism and stress response, mainly through binding to their cognate receptor, GR, which works as a ligand-activated transcription factor. Due to their pleiotropy and the common medical use of these steroids to treat patients affected by different pathologies, the investigation of their mechanisms of action is extremely important in biology and clinical research. The evolutionary conservation of GC physiological function, biosynthesis pathways, as well as the sequence and structure of the GC nuclear receptors has stimulated, in the last 20 years, the use of zebrafish (a teleost of Cyprinidae family) as a reliable model organism to investigate this topic. In this review, we wanted to collect many of the most significant findings obtained by the the scientific community using zebrafish to study GCs and their receptors. The paper begins by describing the experiments with transient knockdown of zebrafish gr to gain insights, mainly during development, and continues with the discoveries provided by the generation of transgenic reporter lines. Finally, we discuss how the generation of mutant lines for either gr or the enzymes involved in GC synthesis has significantly advanced our knowledge on GC biology.

Measuring recognition memory in zebrafish larvae: issues and limitations.

Recognition memory is the capacity to recognize previously encountered objects, events or places. This ability is crucial for many fitness-related activities, and it appears very early in the development of several species. In the laboratory, recognition memory is most often investigated using the novel object recognition test (NORt), which exploits the tendency of most vertebrates to explore novel objects over familiar ones. Despite that the use of larval zebrafish is rapidly increasing in research on brain, cognition and neuropathologies, it is unknown whether larvae possess recognition memory and whether the NORt can be used to assess it. Here, we tested a NOR procedure in zebrafish larvae of 7-, 14- and 21-days post-fertilization (dpf) to investigate when recognition memory first appears during ontogeny. Overall, we found that larvae explored a novel stimulus longer than a familiar one. This response was fully significant only for 14-dpf larvae. A control experiment evidenced that larvae become neophobic at 21-dpf, which may explain the poor performance at this age. The preference for the novel stimulus was also affected by the type of stimulus, being significant with tri-dimensional objects varying in shape and bi-dimensional geometrical figures but not with objects differing in colour. Further analyses suggest that lack of effect for objects with different colours was due to spontaneous preference for one colour. This study highlights the presence of recognition memory in zebrafish larvae but also revealed non-cognitive factors that may hinder the application of NORt paradigms in the early developmental stages of zebrafish.

Zebrafish ambra1a and ambra1b Silencing Affect Heart Development.

In zebrafish, two paralogous genes, activating molecule in beclin-1 (BECN1)-regulated autophagy ambra1a and ambra1b, both required for the autophagic process and during development, encode the protein AMBRA1, a positive regulator of early steps of autophagosome formation. As transcripts for both genes are expressed during embryogenesis in the heart region, in this work, we investigated the effects of ambra1a and ambra1b knockdown on heart development by means of morpholino oligonucleotides (MOs). Silencing of the two proteins by MOs directed against the ATG translation initiation codon affects cardiac morphogenesis, resulting in a small, string-like heart with pericardial edema, whereas treatment with splice-blocking MOs does not lead to overt cardiac phenotypes, thus revealing the relevance of maternally supplied ambra1 transcripts for heart development. Co-injection of both ATG-MOs determines a more severe cardiac phenotype, with prominent pericardial edema. Whole-mount in situ hybridization (WMISH) for myosin light chain 7 (myl7), as well as ambra1 ATG-MO microinjection in zebrafish transgenic line expressing green fluorescent protein in the heart, revealed defects with the heart jogging process followed by imperfect cardiac looping. Moreover, WMISH of homeodomain transcription factor 2 isoform c (pitx2c) transcripts showed both bilateral and reversed pitx2c expression in morphants. The morphants' cardiac phenotypes were effectively rescued by co-injection of MOs with human AMBRA1 (hAMBRA1) messenger RNA (mRNA), pointing at the conservation of Ambra1 functions during evolution. Co-injections of ambra1 ATG-MOs with a hAMBRA1 mRNA mutated in the protein phosphatase 2a (PP2A) binding sites (hAMBRA1PXP) were not able to rescue the cardiac phenotypes, at the difference from wild-type hAMBRA1 mRNA, and treatment of zebrafish embryos with the specific PP2A inhibitor cantharidin resulted in similar developmental cardiac defects. These results suggest a critical role for AMBRA1 in vertebrate heart development, likely involving the binding site for the PP2A phosphatase.

The epg5 knockout zebrafish line: a model to study Vici syndrome.

The EPG5 protein is a RAB7A effector involved in fusion specificity between autophagosomes and late endosomes or lysosomes during macroautophagy/autophagy. Mutations in the human EPG5 gene cause a rare and severe multisystem disorder called Vici syndrome. In this work, we show that zebrafish epg5-/- mutants from both heterozygous and incrossed homozygous matings are viable and can develop to the age of sexual maturity without conspicuous defects in external appearance. In agreement with the dysfunctional autophagy of Vici syndrome, western blot revealed higher levels of the Lc3-II autophagy marker in epg5-/- mutants with respect to wild type controls. Moreover, starvation elicited higher accumulation of Lc3-II in epg5-/- than in wild type larvae, together with a significant reduction of skeletal muscle birefringence. Accordingly, muscle ultrastructural analysis revealed accumulation of degradation-defective autolysosomes in starved epg5-/- mutants. By aging, epg5-/- mutants showed impaired motility and muscle thinning, together with accumulation of non-degradative autophagic vacuoles. Furthermore, epg5-/- adults displayed morphological alterations in gonads and heart. These findings point at the zebrafish epg5 mutant as a valuable model for EPG5-related disorders, thus providing a new tool for dissecting the contribution of EPG5 on the onset and progression of Vici syndrome as well as for the screening of autophagy-stimulating drugs. Abbreviations: ATG: autophagy related; cDNA: complementary DNA; DIG: digoxigenin; dpf: days post-fertilization; EGFP: enhanced green fluorescent protein; EPG: ectopic P granules; GFP: green fluorescent protein; hpf: hours post-fertilization; IL1B: interleukin 1 beta; Lc3-II: lipidated Lc3; mpf: months post-fertilization; mRNA: messenger RNA; NMD: nonsense-mediated mRNA decay; PCR: polymerase chain reaction; qPCR: real time-polymerase chain reaction; RAB7A/RAB7: RAB7a, member RAS oncogene family; RACE: rapid amplification of cDNA ends; RFP: red fluorescent protein; RT-PCR: reverse transcriptase-polymerase chain reaction; SEM: standard error of the mean; sgRNA: guide RNA; UTR: untranslated region; WMISH: whole mount in situ hybridization; WT: wild type.

Zebrafish ambra1b knockout reveals a novel role for Ambra1 in primordial germ cells survival, sex differentiation and reproduction

BACKGROUND: AMBRA1 is an intrinsically disordered protein, working as a scaffold molecule to coordinate, by protein-protein interaction, many cellular processes, including autophagy, mitophagy, apoptosis and cell cycle progression. The zebrafish genome contains two ambra1 paralogous genes (a and b), both involved in development and expressed at high levels in the gonads. Characterization of the zebrafish paralogous genes mutant lines generated by CRISPR/Cas9 approach showed that ambra1b knockout leads to an all-male population. RESULTS: We demonstrated that the silencing of the ambra1b gene determines a reduction of primordial germ cells (PGCs), a condition that, in the zebrafish, leads to the development of all-male progeny. PGC reduction was confirmed by knockdown experiments and rescued by injection of ambra1b and human AMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued by injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding region, thus suggesting that interaction with this complex is involved in PGC protection from loss. Results from zebrafish embryos injected with murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could indirectly regulate this protein through CUL4-DDB1 interaction. According to this, Ambra1+/- mice showed a reduced Stat3 expression in the ovary together with a low number of antral follicles and an increase of atretic follicles, indicating a function of Ambra1 in the ovary of mammals as well. Moreover, in agreement with the high expression of these genes in the testis and ovary, we found significant impairment of the reproductive process and pathological alterations, including tumors, mainly limited to the gonads. CONCLUSIONS: By exploiting ambra1a and ambra1b knockout zebrafish lines, we prove the sub-functionalization between the two paralogous zebrafish genes and uncover a novel function of Ambra1 in the protection from excessive PGC loss, which seems to require binding with the CUL4-DDB1 complex. Both genes seem to play a role in the regulation of reproductive physiology.