Genome editing in cultured fishes

With external fertilization, high fecundity, and established methods for propagation and larval rearing for cultured species, fish provide systems well suited to genome-editing procedures. While early experiments utilized zinc-finger nucleases and transcription activator-like effector nucleases (TALENs), most recent ones have used the CRISPR/Cas9 editor, and achieved rates of targeted genomic insertion well above those of classical transgenic methods, with lower frequencies of off-site integration. Genome-editing experiments with cultured fishes have focused on improving growth rate and disease resistance, achievement of reproductive confinement, and other valued traits. As reviewed here, advances in knowledge of key molecular pathways and, in some cases, favorable alterations of phenotype have been achieved. For example, loss-of-function of myostatin, a negative regulator of muscle growth, led to increased muscle mass, greater weight, and greater fillet yield in genome-edited lines of red sea bream, tiger puffer, and Nile tilapia than in their unedited counterparts. The red sea bream line become the first genome-edited animal to reach commercial production. As for all animals, wide adoption of genome-edited fishes will depend upon addressing issues of regulation, consumer acceptance, and breeding infrastructure.

Black Sea turbot sperm motility depending on the dilution of seawater

The sperm motility of fish reflects their fertilizing ability. Sperm activation in fish with external fertilization occurs in an isotonic medium (sea or freshwater). The duration of sperm motility is a compromise between the level of energy reserves possessed by the sperm and the process of osmotic damage it experiences in the activating medium. Under natural conditions, various biotic and abiotic factors can affect the characteristics of motility. At the same time, when assessing the reproductive potential of males in laboratory conditions, the dilution and methods of its activation have a significant effect on the characteristics of sperm quality (namely, speed, the proportion of motile spermatozoa, and the time of their activity). In our work, we determined the main characteristics of the motility of the Black Sea turbot sperm at a dilution of 1:10 and 1: 100 with seawater. We have shown that, the average speed and percentage of motile sperm significantly decrease with higher dilution of seawater.

Zebrafish Models in Therapeutic Research of Cardiac Conduction Disease

Malfunction in the cardiac conduction system (CCS) due to congenital anomalies or diseases can cause cardiac conduction disease (CCD), which results in disturbances in cardiac rhythm, leading to syncope and even sudden cardiac death. Insights into development of the CCS components, including pacemaker cardiomyocytes (CMs), atrioventricular node (AVN) and the ventricular conduction system (VCS), can shed light on the pathological and molecular mechanisms underlying CCD, provide approaches for generating human pluripotent stem cell (hPSC)-derived CCS cells, and thus improve therapeutic treatment for such a potentially life-threatening disorder of the heart. However, the cellular and molecular mechanisms controlling CCS development remain elusive. The zebrafish has become a valuable vertebrate model to investigate early development of CCS components because of its unique features such as external fertilization, embryonic optical transparency and the ability to survive even with severe cardiovascular defects during development. In this review, we highlight how the zebrafish has been utilized to dissect the cellular and molecular mechanisms of CCS development, and how the evolutionarily conserved developmental mechanisms discovered in zebrafish could be applied to directing the creation of hPSC-derived CCS cells, therefore providing potential therapeutic strategies that may contribute to better treatment for CCD patients.

Collagen XI Impact on Structure and Function of the Vertebrate Inner Ear in a Zebrafish Model

The ear is essential to maintaining balance and hearing; both of which can be linked to one another and significantly impact a person’s quality of life. Although aging and damage are more common reasons for hearing loss, congenital ear defects still have a considerable impact on our population. The function of the ear can be affected by structural deformities to the ear and its components which results in hearing loss. Mutations and single nucleotide polymorphisms in the gene encoding Collagen XI alpha one chain (COL11A1) protein can play a role in hearing and balance dysfunction in humans as seen in disorders such as Stickler Type 2 and Marshall Syndrome, and nonsyndromic hearing loss deafness autosomal dominant 37 (DFNA37). Due to its transparency, external fertilization, the zebrafish model system was used to create a COL11A1 zebrafish counterpart (Col11a1a) knockdown and knockout genetic model. This research highlights the importance of Col11a1a in the development and structure of the inner ear as a whole including the hair cells, kinocilia, and otolith formation. Studying the development and structural changes of the inner ear can provide insight into hearing loss and potential interventions.

​Effect of Phosphorus and Sulphur Application on Growth, Yield Attributes, Nutrient Uptake, Quality and Economics of Blackgram (Vigna mungo L.): A Review

Phosphorus and sulphur is a key element involved in various functions in growth and metabolism of blackgram. It is frequently a major limiting nutrient for plant growth in most Indian soils. Black gram is the major source of protein for the vegetarian diet and important pulse crop both in terms of area and production. Pulses have been in focus in recent times due to the continuous upswing in their prices. The low productivity of blackgram is due to intensive farming practices and use of exhaustive and high yield cultivars and imbalanced external fertilization. Therefore proper fertilization (site specific and balanced fertilization) is needed to increase the production and productivity of blackgram. Blackgram is very much responsive to phosphorus and sulphur application. In crop plants, the nutrient interactions are generally measured in terms of growth response, yield response and changes in concentration and uptake of nutrients. Studies have indicated synergistic effect between phosphorus and sulphur but their relationship depends on their rate of application and crop species etc. A better understanding of nutrient interaction is helpful in maximizing fertilizer use efficiency and net profit. The most of the results revealed that different levels of P and S had a significant effect on growth attributes, yield attributes, grain and haulm yield and quality prarameters of blackgram. The conjoint application up to 60 kg P2O5 ha-1 + 40 kg S ha-1 were found to be superior and synergistic in improving the growth and yield attributes of blackgram. The present paper is a critical review from research findings of eminent scientists on sulphur and phosphorus effect over black gram.

External fertilization is orchestrated by a pH-regulated soluble adenylyl cyclase controlling sperm motility and chemotaxis

The reaction of CO2 with H2O to form HCO3- and H+ is one of the most important chemical equilibria in cells. In mammalian sperm, a soluble adenylyl cyclase (sAC) serves as cellular HCO3- sensor that conveys the equilibrium state via cAMP synthesis to cAMP-signaling molecules. The function of sAC and cAMP in non-mammalian sperm is largely unknown. Here, we identify sAC orthologs in sea urchin and salmon sperm that, surprisingly, are activated by alkaline pH rather than HCO3-. Two amino-acid residues required for HCO3- binding of mammalian sAC are lacking in pH-regulated sAC. Orthologs identified in ten other phyla are also lacking either one of these key residues, suggesting that pH control is widespread among non-mammalian metazoan. The pH-sensitive sAC controls several functions of sperm from external fertilizers. Upon spawning, alkalization triggers cAMP synthesis and, thereby, activates motility of quiescent sperm. Egg-derived chemoattractants also alkalize sperm and elevate cAMP, which then-modulates pacemaker HCN channels to trigger a chemotactic Ca2+ response. Finally, the sAC and the voltage- and cAMP-activated Na+/H+ exchanger sNHE mutually control each other. A picture of evolutionary significance is emerging: motility and sensory signaling of sperm from both internal and external fertilizers rely on cAMP, yet, their sAC is regulated by HCO3- or pHi, respectively. Acidification of aquatic habitats due to climate change may adversely affect pH-sensing by sAC and thereby sexual reproduction in the sea.

DNA methylation and histone modifications are essential for regulation of stem cell formation and differentiation in zebrafish development

The complex processes necessary for embryogenesis require a gene regulatory network that is complex and systematic. Gene expression regulates development and organogenesis, but this process is altered and fine-tuned by epigenetic regulators that facilitate changes in the chromatin landscape. Epigenetic regulation of embryogenesis adjusts the chromatin structure by modifying both DNA through methylation and nucleosomes through posttranslational modifications of histone tails. The zebrafish is a well-characterized model organism that is a quintessential tool for studying developmental biology. With external fertilization, low cost and high fecundity, the zebrafish are an efficient tool for studying early developmental stages. Genetic manipulation can be performed in vivo resulting in quick identification of gene function. Large-scale genome analyses including RNA sequencing, chromatin immunoprecipitation and chromatin structure all are feasible in the zebrafish. In this review, we highlight the key events in zebrafish development where epigenetic regulation plays a critical role from the early stem cell stages through differentiation and organogenesis.

Housing, Husbandry and Welfare of a “Classic” Fish Model, the Paradise Fish (Macropodus opercularis)

Thanks to its small size, external fertilization and fecundity, over the past four decades, zebrafish (Danio rerio) has become the dominant fish model species in biological and biomedical research. Multiple lines of evidence, however, suggest that the reliance on only a handful of genetic model organisms is problematic, as their unique evolutionary histories makes them less than ideal to study biological questions unrelated to their historically contingent adaptations. Therefore, a need has emerged to develop novel model species, better suited for studying particular problems. The paradise fish (Macropodus opercularis) has a much more complex behavioral repertoire than zebrafish and has been a favored model animal in ethological research during the last decades of the previous century. We believe that with currently available, easily adaptable genetic toolkits, this species could be easily developed into a popular model of behavioral genetics. Despite its earlier popularity, however, the description of a detailed housing and husbandry protocol for this species is still missing from scientific literature. We present here a detailed description of how to raise and breed paradise fish successfully under laboratory conditions, and also discuss some of the challenges we faced while creating a stable breeding population for this species in our facility.

Zebrafish Models of Autosomal Dominant Ataxias

Hereditary dominant ataxias are a heterogeneous group of neurodegenerative conditions causing cerebellar dysfunction and characterized by progressive motor incoordination. Despite many efforts put into the study of these diseases, there are no effective treatments yet. Zebrafish models are widely used to characterize neuronal disorders due to its conserved vertebrate genetics that easily support genetic edition and their optic transparency that allows observing the intact CNS and its connections. In addition, its small size and external fertilization help to develop high throughput assays of candidate drugs. Here, we discuss the contributions of zebrafish models to the study of dominant ataxias defining phenotypes, genetic function, behavior and possible treatments. In addition, we review the zebrafish models created for X-linked repeat expansion diseases X-fragile/fragile-X tremor ataxia. Most of the models reviewed here presented neuronal damage and locomotor deficits. However, there is a generalized lack of zebrafish adult heterozygous models and there are no knock-in zebrafish models available for these diseases. The models created for dominant ataxias helped to elucidate gene function and mechanisms that cause neuronal damage. In the future, the application of new genetic edition techniques would help to develop more accurate zebrafish models of dominant ataxias.

Onset of sexual maturity of sexually propagated and wild Favitesabdita colonies in northwestern Philippines

Scleractinian corals are modular colonial organisms and are the main framework builders of coral reefs. Most corals reproduce by broadcast spawning with external fertilization and these processes are essential to replenish reef coral populations. Despite decades of research, many aspects of coral reproductive biology remain poorly studied. For example, two important reproductive life history traits, colony size and age at the onset of sexual maturity, are mostly unknown for many reef-building corals. In this study, wild colonies of different size classes and colonies of a known age (i.e. colonies sexually cultured and reared from larvae to adults) of the massive scleractinian Favitesabdita were examined for the presence or absence of mature oocytes to determine size and age at the onset of sexual reproduction. Fecundity at the onset of reproductive maturity across size classes of wild colonies was also determined. Surveyed and sampled colonies were grouped into three size classes based on maximum colony diameter (A = 0.1-4.0 cm, B = 4.1-8.0 cm, and C = >8.1 cm). For both wild and sexually propagated colonies, the smallest colonies containing gametes were 1.8 cm in diameter, suggesting that this is the minimum colony size at onset of sexual maturity. Colonies of size class A had lower mean oocyte counts per polyp (44 ± 6.08; mean ± SE) compared to colonies of classes B and C (469 ± 62.41, 278 ± 57.15, respectively). However, mean oocyte geometric mean diameter of size class A colonies was greater (340.38 ± 7.68 µm) than classes B and C (283.96 ± 6.94 µm, 317.57 ± 9.18 µm, respectively). Results of this study bring in to question the widely applied operational definition of coral juveniles being colonies ≤4.0 cm diameter and suggest that even quite small colonies may play a role in contributing to the natural larval pool on reefs than previously thought.