Transgenic Fish Research Articles

Growth Performance in Sangkuriang Catfish (Clarias gariepinus Var. Sangkuriang) and Transgenic Mutiara Catfish (Clarias gariepinus) using Low Protein Feed

This research was conducted at the hatchery building 4, Faculty of Fisheries and Marine Sciences, Universitas Padjadjaran. The implementation starts from November to December 2022. This experiment aims to test the growth performance of the G5 transgenic mutiara catfish by providing low protein feed. The treatment design used the completely randomized design method with four treatments (A:39% feed protein; B:31% feed protein; C:14% feed protein for transgenic catfish and D:39% feed protein for non transgenic fish as comparison) with three replications. Data were analyzed using Sigmaplot 15.0 for test parameters absolute weight gain (Wg), feed conversion ratio (FCR), protein efficiency ratio (PER), and protein retention (PR) and using the Duncan Multiple Range Test (DMRT) at a 95% confidence level. The best treatment results for feed efficiency and protein efficiency were obtained in treatments A and B with values ​​of 1.25 and 1.43 as well as 2.04 and 2.23. Transgenic mutiara catfish can grow on a low protein diet is a new finding from this study.

Evaluation of the endocrine activity of surface water samples using aquatic eleuthero-embryos - a comparison with in vitro assays.

Over the previous decade numerous new approach methodologies (NAMs) have been developed and validated for the detection of endocrine activity of individual chemicals or environmental samples. These NAMs can be largely separated into three categories, in silico tools, in vitro assays and in vivo assays using organisms or life stages not considered as laboratory animals, each with their own advantages and disadvantages. While in vitro assays provide more mechanistic information, the use of whole organisms, such as fish or amphibian embryos provides a more holistic view of the net effects of an environmental sample on hormonal activity. A panel of bioassays was used to test the endocrine activity of several samples from the Danube River at Novi Sad, Serbia. The results of the in vitro assays have been published previously. Here we present the results of the in vivo assays that were performed at the same time on the same samples. These whole organism assays are based on the use of transgenic fish and amphibian eleuthero-embryos and included the Xenopus Eleuthero-embryo Thyroid Assay (XETA), the Rapid Estrogen ACTivity In Vivo assay (REACTIV) and the Rapid Androgen Disruption Activity Reporter (RADAR) assay. Discrepancies between the different in vitro assays have previously been reported. The results of the in vivo studies also indicate discrepancies between the in vivo and in vitro data with an underestimation of the endocrine activity by the in vitro tests. Therefore, a battery of tests is advised with the initial diagnostic performed with in vivo tests to cover a wider range of modes of action and to allow the appropriate in vitro assay(s) to be selected to confirm the mode of action.

Peonidin-3-O-glucoside and Resveratrol Increase the Viability of Cultured Human hFOB Osteoblasts and Alter the Expression of Genes Associated with Apoptosis, Osteoblast Differentiation and Osteoclastogenesis.

High-throughput RNA-sequencing can determine the impact of nutrients and their combinations on gene transcription levels in osteocytes, and clarify the biological pathways associated with their impact on bone tissues. Previously, we reported that resveratrol (RES) and peonidin-3-O-glucoside (POG) increased osteoblastogenesis, as well as reduced osteoclastogenesis in transgenic teleost fish models. Here, we perform whole-genome transcriptomic profiling of osteoblasts treated with POG or RES to provide a comprehensive understanding of alterations in gene expression and the molecular mechanisms involved. Cultured human fetal osteoblastic hFOB 1.19 cells were treated with the test compounds, and then RNA was used to prepare RNA-seq libraries, that were sequenced using a NovaSeq 6000. Treatment with POG or RES increased osteoblast proliferation and reduced apoptosis. Transcriptomic profiling showed that of the 29,762 genes investigated, 3177 were differentially expressed (1481 upregulated, 1696 downregulated, FDR ≤ 0.05) in POG-treated osteoblasts. In the RES-treated osteoblasts, 2288 genes were differentially expressed (DGEs, 1068 upregulated, 1220 downregulated, FDR ≤ 0.05). Ingenuity® Pathway Analysis (IPA) of DGEs from RES or POG-treated osteoblasts revealed significant downregulation of the apoptosis, osteoarthritis and HIF1α canonical pathways, and a significant reduction in Rankl mRNA expression. The data suggest that RES and POG have both anabolic and anticlastogenic effects.

Development of a Transparent Transgenic Zebrafish Cellular Phenotype Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-) to Study NF-kB Activity.

NF-κB signaling has broad effects on cell survival, tissue growth, and proliferation activities. It controls many genes that are involved in inflammation and thus is a key player in many inflammatory diseases. The elevation of NF-κB activators is associated with elevated mortality, especially in cancer and cardiovascular diseases. The zebrafish has emerged as an important model for whole-organism in vivo modeling in translational research. In vertebrates, in-vivo spatial resolution is limited due to normal opacification of skin and subdermal structure. For in vivo imaging, skin transparency by blocking the pigmentation via chemical inhibition is required and the maintenance of this transparency is vital. The Casper(roy-/-, nacre-/-) mutant of zebrafish maintains this transparency throughout its life and serves as an ideal combination of sensitivity and resolution for in vivo stem cell analyses and imaging. We developed an NF-kB:GFP/Casper transparent transgenic zebrafish cellular phenotype to study inflammatory processes in vivo. We outline the experimental setup to generate a transparent transgenic NF-kB/Casper strain of zebrafish through the cross-breeding of Casper and NF-kB transgenic adult fish and have generated F01 in the form of heterozygous progeny. The transgenic F01 progeny was further inbred to generate heterozygous progenies from F1 to F4 generations. Furthermore, it continued to successfully develop the homozygous strain Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-) in the F05 generation. This novel strain of F05 generation showed 100% homozygosity in the transgenic transparent progeny of Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-). The strain has been confirmed by generating the F06 generation of homozygous progeny and again verified and validated for its homogeneity in the F07 generation. The newly developed novel transparent transgenic strain of the NF-kB reporter line has been coined as "Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-)gmc1". We have established a newly generated phenotype of transparent transgenic zebrafish for time-lapse in vivo confocal microscopy to study the cellular phenotype and pathologies at the cellular level over time. This will allow for quantifying the changes in the NF-kB functional activities over time and allow the comparison of control and cardiac-oncology experimental therapeutics. We validated the newly developed Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-)gmc1 homozygous strain of zebrafish by studying the inflammatory response to bacterial lipopolysaccharide (LPS) exposure, tolerance, and the inhibitory role of a potential novel drug candidate against LPS-induced inflammation. The results establish the unique application of newly developed strains by identifying hit and lead drug candidates for experimental therapeutics.

Analysis of medaka GAP43 gene promoter activity in transgenic lines

We produced transgenic medaka fish lines that mimicked the expression of the GAP43 gene. Fish lines with the proximal 2-kilobase (kb) 5′-untranslated region (UTR) as the expression promoter specifically expressed enhanced green fluorescent protein (EGFP) in neural tissues, such as the brain, spinal cord, and peripheral nerves, and its expression decreased with growth, but persisted until adulthood. A functional analysis of the promoter using partially deleted UTRs revealed that functions related to neural tissue-specific promoter activity were widely distributed in the region upstream of the proximal 400-b. Furthermore, the distal half of the 2-kb UTR contributed to expression throughout the brain, while the region 400-b upstream of the proximal 600-b was strongly associated with expression in specific areas, such as the telencephalon. In addition, a region from 957 to 557b upstream of the translation initiation site was important for the long-term maintenance of promoter activity into adulthood. Among the transcription factors with recognition sequences in this region, Sp1 and CREB1 have been suggested to play important roles in the GAP43 promoter expression characteristics, such as strong expression in the telencephalon and long-term maintenance of expression.

Nicotine reduces social dominance and neutralizes experience-dependent effects during social conflicts in zebrafish (Danio rerio)

Nicotine, a psychoactive pollutant, binds to nicotinic acetylcholine receptors and disrupts the cholinergic modulation and reward systems of the brain, leading to attention deficit, memory loss, and addiction. However, whether nicotine affects social behaviors remains unknown. We assessed the effects of nicotine on the fighting behavior of zebrafish. Adult zebrafish treated with 5 μM nicotine were used in dyadic fighting tests with size-matched control siblings. The results indicate that nicotine treatment not only significantly reduced the likelihood of winning but also impaired the winner–loser effects (winner and loser fish did not show higher winning and losing tendencies in the second fight, respectively, after treatment.) Nicotine led to a considerable increase in c-fos-positive signals in the interpeduncular nucleus (IPN) of the brain, indicating that nicotine induces neural activity in the habenula (Hb)–IPN circuit. We used transgenic fish in which the Hb–IPN circuit was silenced to verify whether nicotine impaired the winner–loser effect through the Hb–IPN pathway. Nicotine-treated fish in which the medial part of the dorsal Hb was silenced did not have a higher winning rate, and nicotine-treated fish in which the lateral part of the dorsal Hb was silenced did not have a higher loss rate. This finding suggests that nicotine impairs the winner–loser effect by modulating the Hb–IPN circuit. Therefore, in these zebrafish, nicotine exposure impaired social dominance and neutralized experience-dependent effects in social conflicts, and it may thereby disturb the social hierarchy and population stability of such fish.

Regionalized Protein Localization Domains in the Zebrafish Hair Cell Kinocilium.

Sensory hair cells are the receptors for auditory, vestibular, and lateral line sensory organs in vertebrates. These cells are distinguished by "hair"-like projections from their apical surface collectively known as the hair bundle. Along with the staircase arrangement of the actin-filled stereocilia, the hair bundle features a single, non-motile, true cilium called the kinocilium. The kinocilium plays an important role in bundle development and the mechanics of sensory detection. To understand more about kinocilial development and structure, we performed a transcriptomic analysis of zebrafish hair cells to identify cilia-associated genes that have yet to be characterized in hair cells. In this study, we focused on three such genes-ankef1a, odf3l2a, and saxo2-because human or mouse orthologs are either associated with sensorineural hearing loss or are located near uncharacterized deafness loci. We made transgenic fish that express fluorescently tagged versions of their proteins, demonstrating their localization to the kinocilia of zebrafish hair cells. Furthermore, we found that Ankef1a, Odf3l2a, and Saxo2 exhibit distinct localization patterns along the length of the kinocilium and within the cell body. Lastly, we have reported a novel overexpression phenotype of Saxo2. Overall, these results suggest that the hair cell kinocilium in zebrafish is regionalized along its proximal-distal axis and set the groundwork to understand more about the roles of these kinocilial proteins in hair cells.

Ronidazole Is a Superior Prodrug to Metronidazole for Nitroreductase-Mediated Hepatocytes Ablation in Zebrafish Larvae.

The liver plays a very important role in physiological processes of the human body. Liver regeneration has developed into an important area of study in liver disease. The Mtz (metronidazole)/NTR (nitroreductase)-mediated cell ablation system has been widely used to study the processes and mechanisms of liver injury and regeneration. However, high concentrations and toxic side effects of Mtz severely limit the application of the Mtz/NTR system. Therefore, screening new analogs to replace Mtz has become an important means to optimize the NTR ablation system. In this study, we screened five Mtz analogs including furazolidone, ronidazole, ornidazole, nitromide, and tinidazole. We compared their toxicity on the transgenic fish line Tg(fabp10a: mCherry-NTR) and their specific ablation ability on liver cells. The results showed that Ronidazole at a lower concentration (2 mM) had the same ability to ablate liver cells comparable with that of Mtz (10 mM), almost without toxic side effects on juvenile fish. Further study found that zebrafish hepatocyte injury caused by the Ronidazole/NTR system achieved the same liver regenerative effect as the Mtz/NTR system. The above results show that Ronidazole can replace Mtz with NTR to achieve superior damage and ablation effects in zebrafish liver.

Electrochemical degradation of 17α-ethinylestradiol: Transformation products, degradation pathways and in vivo assessment of estrogenic activity

Conventional water treatment methods are not efficient in eliminating endocrine disrupting compounds (EDCs) in wastewater. Electrochemical Advanced Oxidation Processes (eAOPs) offer a promising alternative, as they electro-generate highly reactive species that oxidize EDCs. However, these processes produce a wide spectrum of transformation products (TPs) with unknown chemical and biological properties. Therefore, a comprehensive chemical and biological evaluation of these remediation technologies is necessary before they can be safely applied in real-life situations. In this study, 17α-ethinylestradiol (EE2), a persistent estrogen, was electrochemically degraded using a boron doped diamond anode with sodium sulfate (Na2SO4) and sodium chloride (NaCl) as supporting electrolytes. Ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry was used for the quantification of EE2 and the identification of TPs. Estrogenic activity was assessed using a transgenic medaka fish line. At optimal operating conditions, EE2 removal reached over 99.9% after 120 min and 2 min, using Na2SO4 and NaCl, respectively. The combined EE2 quantification and in vivo estrogenic assessment demonstrated the overall estrogenic activity was consistently reduced with the degradation of EE2, but not completely eradicated. The identification and time monitoring of TPs showed that the radical agents readily oxidized the phenolic A-ring of EE2, leading to the generation of hydroxylated and/or halogenated TPs and ring-opening products. eAOP revealed to be a promising technique for the removal of EE2 from water. However, caution should be exercised with respect to the generation of potentially toxic TPs.

Environmental microbes determine macrophage response towards saponin-induced inflammation in zebrafish larvae

The microbial consortium within an organism is crucial for its development and immune status. Alteration of the host microbiome by antibiotics or antinutritional factors may contribute to increased disease susceptibility. Here, we investigated whether exposure to different microbes could influence zebrafish larval microbiota composition and modulate their immune response towards a saponin challenge. Adult zebrafish were exposed to the antibiotic oxytetracycline (OxyT) or control tank water and their intestinal content was harvested after 30 ​h (24 ​h exposure, 6 ​h wash-out). Subsequently, zebrafish embryos were exposed to either OxyT-treated content or non-treated content from 3 to 6 days post fertilization (dpf). At 6 dpf part of the group received a saponin challenge until 8 dpf. Zebrafish larvae exposed to OxyT-treated adult gut content (3–6 dpf) showed an altered microbiota composition compared to controls. Interestingly, larvae exposed to saponin-treated OxyT-content showed fewer macrophages (as visualized by fluorescent microscopy using mpx:GFP114;mpeg:mCherry transgenic fish) in the overall fish as well as around the gut area than saponin-treated control-exposed larvae. Fewer macrophages were associated with a decreased expression of interleukin 22 (il22) in larvae exposed to saponin-treated OxyT-content compared to controls. Overall, this study shows that exposure to different microbial environments early in life might affect disease susceptibility of larval zebrafish.

Zebrafish as a model for drug induced liver injury: state of the art and beyond

Zebrafish as a preclinical drug induced liver injury (DILI) model provides multiple advantages ranging from ease of breeding and maintenance, availability of different strains and transgenic fish amenable to study liver function, and highly conserved liver structure and function with the human liver. In this review, the authors have aimed to provide an account of the metabolic enzymes that take roles in drug detoxification in both human and zebrafish in a comparative manner and exemplify several recent models in studying liver functionality. Moreover, the authors emphasize the difficulties associated with studying idiosyncratic DILI in preclinical models and propose that zebrafish could be an important complement to mice in testing functions of genes that are associated with DILI with respect to different drugs in human genome-wide association studies (GWAS) Catalog. Finally, this review highlights the state-of-the-art in the development of novel transgenic reporter strains that can be used to study degree and molecular mechanisms of hepatotoxicity caused by drugs in zebrafish. All of these will help researchers to use effectively the available resources in the zebrafish DILI models, while advocating potential leads that can be taken to provide advancements in a better understanding and treatment of DILI.

Monitoring Nrf2/ARE Pathway Activity with a New Zebrafish Reporter System

Among multiple cytoprotective mechanisms, eukaryotic cells exhibit a complex transcriptional program relying on the Nrf2 transcription factor, which is generally recruited upon biological stressors including oxidative-stress-based cellular insults. The relevance of this master regulator has remarkably emerged in recent years in several research fields such as cancer, inflammatory disorders and age-related neurological diseases. Here, we document the generation and characterization of a novel Nrf2/ARE pathway biosensor fish which exhibits a dynamic spatiotemporal expression profile during the early developmental stages. The transgenic line is responsive to known Nrf2 pathway modulators but also to Edaravone, which direct activity on the Nrf2 pathway has never been documented in a live transgenic fish model. We also show that the reporter is faithfully activated during fin regeneration, and its degree of expression is slightly affected in a glucocerebrosidase (Gba1) morphant zebrafish model. Therefore, this novel transgenic fish may represent a valuable tool to be exploited for the characterization of zebrafish models of human diseases, as well as for primary high-throughput drug screening.

INVASIVE POTENTIAL AND REGULATION EXPERIENCE OF USING TRANSGENIC ORGANISMS IN AQUACULTURE BY WAY OF EXAMPLE OF ATLANTIC SALMON

The article considers a 30-year history of the creation, state regulation and production of AquAdvantage transgenic salmon genetically modified (GM) with the growth hormone gene. The arguments of opponents and proponents of the concept of ecological safety of the use of transgenic fish in aquaculture are critically analyzed. It has been generalized that the adaptability of transgenic fish is lower than that of its wild relatives. On this basis, it is assumed that in the event of an accidental ingestion of GM fish from aquaculture to natural populations, transgenes are under the negative influence of natural selection. Strict security measures for the maintenance of GM fish, however, should not be weakened, since there are still unexplained cases of the survival of fluorescent transgenic aquarium fish in freshwater ecosystems.

Gene Editing of the Follicle-Stimulating Hormone Gene to Sterilize Channel Catfish, Ictalurus punctatus, Using a Modified Transcription Activator-like Effector Nuclease Technology with Electroporation

Follicle-stimulating hormone (fsh) plays an important role in sexual maturation in catfish. Knocking out the fsh gene in the fish zygote should suppress the reproduction of channel catfish (Ictalurus punctatus). In this study, transcription activator-like effector nuclease (TALEN) plasmids targeting the fsh gene were electroporated into fertilized eggs with the standard double electroporation technique. Targeted fsh cleavage efficiency was 63.2% in P1fsh-knockout catfish. Ten of fifteen (66.7%) control pairs spawned, and their eggs had 32.3-74.3% average hatch rates in 2016 and 2017. Without hormone therapy, the spawning rates of P1 mutants ranged from 33.3 to 40.0%, with an average egg hatching rate of 0.75%. After confirmation of the low fertility of P1 mutants in 2016, human chorionic gonadotropin (HCG) hormone therapy improved the spawning rates by 80% for female mutants and 88.9% for male mutants, and the mean hatch rate was 35.0% for F1 embryos, similar to that of the controls (p > 0.05). Polymerase chain reaction (PCR) identification showed no potential TALEN plasmid integration into the P1 channel catfish genome. Neither the P1 nor the F1 mutant fish showed any noticeable changes in in body weight, survival rate, and hatching rate when the reproductive gene was knocked out. F1 families had a mean inheritance rate of 50.3%. The results brought us one step closer to allowing implementation of certain genetic techniques to aquaculture and fisheries management, while essentially eliminating the potential environment risk posed by transgenic, hybrid, and exotic fish as well as domestic fish.

Insights into the control and consequences of breathing adjustments in fishes-from larvae to adults.

Adjustments of ventilation in fishes to regulate the volume of water flowing over the gills are critically important responses to match branchial gas transfer with metabolic needs and to defend homeostasis during environmental fluctuations in O2 and/or CO2 levels. In this focused review, we discuss the control and consequences of ventilatory adjustments in fish, briefly summarizing ventilatory responses to hypoxia and hypercapnia before describing the current state of knowledge of the chemoreceptor cells and molecular mechanisms involved in sensing O2 and CO2. We emphasize, where possible, insights gained from studies on early developmental stages. In particular, zebrafish (Danio rerio) larvae have emerged as an important model for investigating the molecular mechanisms of O2 and CO2 chemosensing as well as the central integration of chemosensory information. Their value stems, in part, from their amenability to genetic manipulation, which enables the creation of loss-of-function mutants, optogenetic manipulation, and the production of transgenic fish with specific genes linked to fluorescent reporters or biosensors.

Infertility control of transgenic fluorescent zebrafish with targeted mutagenesis of the dnd1 gene by CRISPR/Cas9 genome editing.

Transgenic technology and selective breeding have great potential for the genetic breeding in both edible fish and ornamental fish. The development of infertility control technologies in transgenic fish and farmed fish is the critical issue to prevent the gene flow with wild relatives. In this study, we report the genome editing of the dead end (dnd1) gene in the zebrafish model, using the CRISPR/Cas9 technology to achieve a loss-of-function mutation in both wild-type zebrafish and transgenic fluorescent zebrafish to develop complete infertility control technology of farmed fish and transgenic fish. We effectively performed targeted mutagenesis in the dnd1 gene of zebrafish with a single gRNA, which resulted in a small deletion (-7 bp) or insertion (+41 bp) in exon 2, leading to a null mutation. Heterozygotes and homozygotes of dnd1-knockout zebrafish were both selected by genotyping in the and generations. Based on a comparison of histological sections of the gonads between wild-type, heterozygous, and homozygous dnd1 zebrafish mutants, the dnd1 homozygous mutation (aa) resulted in the loss of germ cells. Still, there was no difference between the wild-type (AA) and dnd1 heterozygous (Aa) zebrafish. The homozygous dnd1 mutants of adult zebrafish and transgenic fluorescent zebrafish became all male, which had normal courtship behavior to induce wild-type female zebrafish spawning. However, they both had no sperm to fertilize the spawned eggs from wild-type females. Thus, all the unfertilized eggs died within 10h. The targeted mutagenesis of the dnd1 gene using the CRISPR/Cas9 technology is stably heritable by crossing of fertile heterozygous mutants to obtain sterile homozygous mutants. It can be applied in the infertility control of transgenic fluorescent fish and genetically improved farmed fish by selective breeding to promote ecologically responsible aquaculture.

CRISPR/Cas9-mediated precision integration of fat-1 and fat-2 from Caenorhabditis elegans at long repeated sequence in channel catfish (Ictalurus punctatus) and the impact on n-3 fatty acid level

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are essential for the development and health of human and animals. However, n-3 PUFAs in human diets, aquaculture and livestock feed are mainly from oily marine fishes. To improve n-3 fatty acid levels in channel catfish (Ictalurus punctatus), we generated transgenic channel catfish carrying codon optimized Caenorhabditis elegans fat-1 gene encoding an n-3 fatty acid desaturase and fat-2 gene encoding the delta12 fatty acid desaturase using CRISPR/Cas9 technology. We used a special cocktail design of donor plasmid, utilized mixed HDR, MMEJ and NHEJ repair pathways, and targeted long repeated DNA target sequence (LRS) to improve knock-in (KI) efficiency. Our results showed that two donor DNAs precisely inserted into at least four genomic sites. Detectable on-target KI efficiency of fat-1 was 9.1% (55/605), three times higher than that of fat-2, which might due to the smaller size of the donor fat-1. Sanger sequencing results suggested that HDR was the dominant repair pathway using this method, leading to the highest proportion of on-target KI events. Reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) demonstrated that the fat-1 and fat-2 transgenes had strong expression, and also greatly improved fads6, elovl2 and elovl5 gene expression in liver and muscle tissues of transgenic channel catfish. Fatty acid analysis showed the fat-1-only transgenic channel catfish had the lower levels of 14:0, 16:0, 18:2n-6, total n-6, and the higher levels of DHA, total n-3 and n-3/n-6 ratio than other genotypes including the fat-2-only, co-integration of fat-1 and fat-2, and wild-type control. In particular, DHA, total n-3 and n-3/n-6 ratio were significantly increased by 37%, 19.8% and 25.2%, respectively, in the fat-1-only transgenic fish than that in the wild-type control (P < 0.05). Our fat-1 and fat-2 KI channel catfish hold promise for improving fish nutritional value, reducing fish oil requirement in aquaculture feed, and serving as an alternative source of n-3 PUFAs.

Past, present and future prospect on microinjection gene transfer in aquaculture

Microinjection is arguably the most direct and reliable gene transfer method to date. Since the development of the first transgenic fish in 1984, microinjection continues to be the most commonly used gene transfer method in aquaculture. Microinjection is used to inject cells, nucleic acid, gene constructs, recombinant DNA, and morpholino oligonucleotides into fish cells, embryos, and larvae. Despite the development of various gene transfer methods, microinjection is preferable. Continuous improvement in micromanipulation tools and micro capillary further develop microinjection method. This review describes the past and present use of microinjection gene transfer methods for genetic manipulation and genetic improvement in fisheries and aquaculture, especially in fish transgenesis. Furthermore, we discuss the future prospects of microinjection method in tandem with recombinant DNA technology and CRISPR-Cas9 and its application in the aquaculture industry.

Color assortative shoaling in zebrafish, Danio rerio (Cypriniformes: Cyprinidae)

Zebrafish, Danio rerio, form shoals consisting of loosely aggregated individuals swimming in different directions. Shoal-mate composition in fish can be influenced by body size, shape, coloration, and pattern. The effect of transgenic body coloration on shoal-mate composition in wildtype D. rerio was examined. A test arena was divided into three equal sections with a wildtype zebrafish in the center compartment. For control trials, a wildtype shoal of seven individuals was placed in both side compartments. For experimental trials, one of the wildtype shoals was replaced with seven green transgenic fish. The number of visits made to each side and the time spent on each side were recorded. Control fish showed no significant difference (NSD) in the average number of visits to the left versus right side and NSD in the average time spent with either shoal. Experimental fish exhibited NSD between the average number of visits to the transgenic versus the wildtype shoal, but they spent significantly more time shoaling with the wildtype. Wildtype test fish consistently visited each side, which would enable assessment of shoal composition, but spent more time shoaling with phenotypically similar individuals, as predicted by the oddity effect. Previous studies show no significant preference by wildtype D. rerio for wildtype versus red transgenic conspecifics and indicate a more prominent role of striping pattern. This generates further questions about the influence of body coloration on shoal-mate preference. Future research could examine the effect of other transgenic colors on shoal-mate preference.

GTP-Binding Protein 1-Like (GTPBP1l) Regulates Vascular Patterning during Zebrafish Development.

Genetic regulation of vascular patterning is not fully understood. Here, we report a novel gene, gtpbp1l (GTP-binding protein 1-like), that regulates vascular development in zebrafish. Amino acid sequence comparison and a phylogenetic study showed that gtpbp1l is conserved in vertebrates. Gtpbp1l mRNA is expressed in the vasculature during embryogenesis. Knockdown of gtpbp1l by morpholino impairs the patterning of the intersegmental vessel (ISV) and caudal vein plexus (CVP), indicating the role of gtpbp1l in vasculature. Further apoptosis assays and transgenic fish tests suggested that vascular defects in gtpbp1l morphants are not due to cell death but are likely caused by the impairment of migration and proliferation. Moreover, the altered expression of vessel markers is consistent with the vascular defects in gtpbp1l morphants. Finally, we revealed that gtpbp1l is regulated by VEGF/notch and BMP signaling. Collectively, these findings showed that gtpbp1l plays a critical role in vascular patterning during zebrafish development.