The development of automated microscopy platforms has enabled large-scale observation of biological processes, thereby complementing genome scale biochemical techniques. However, commercially available systems are restricted either by fixed-field-of-views, leading to potential omission of features of interest, or by low-resolution data of whole objects lacking cellular detail. This limits the efficiency of high-content screening assays, especially when large complex objects are used as in whole-organism screening. Here we demonstrate a toolset for automated intelligent high-content screening of whole zebrafish embryos at cellular resolution on a standard wide-field screening microscope. Using custom-developed algorithms, predefined regions of interest-such as the brain-are automatically detected. The regions of interest are subsequently imaged automatically at high magnification, enabling rapid capture of cellular resolution data. We utilize this approach for acquiring 3-D datasets of embryonic brains of transgenic zebrafish. Moreover, we report the development of a mold design for accurate orientation of zebrafish embryos for dorsal imaging, thereby facilitating standardized imaging of internal organs and cellular structures. The toolset is flexible and can be readily applied for the imaging of different specimens in various applications.

Several in vitro studies have suggested that silica nanoparticles (NPs) might induce adverse effects in gut cells. Here, we used the human colon cancer epithelial cell line HCT116 to study the potential cytotoxic effects of ingested silica NPs in the presence or absence of serum. Furthermore, we evaluated different physico-chemical parameters important for the assessment of nanoparticle safety, including primary particle size (12, 70, 200, and 500 nm) and surface modification (–NH₂ and –COOH). Silica NPs triggered cytotoxicity, as evidenced by reduced metabolism and enhanced membrane leakage. Automated microscopy revealed that the silica NPs promoted apoptosis and necrosis proportional to the administered specific surface area dose. Cytotoxicity of silica NPs was suppressed by increasing amount of serum and surface modification. Furthermore, inhibition of caspases partially prevented silica NP-induced cytotoxicity. In order to investigate the role of specific cell death pathways in more detail, we used isogenic derivatives of HCT116 cells which lack the pro-apoptotic proteins p53 or BAX. In contrast to the anticancer drug cisplatin, silica NPs induced cell death independent of the p53–BAX axis. In conclusion, silica NPs initiated cell death in colon cancer cells dependent on the specific surface area and presence of serum. Further studies in vivo are warranted to address potential cytotoxic actions in the gut epithelium. The unintended toxicity of silica NPs as observed here could also be beneficial. As loss of p53 in colon cancer cells contributes to resistance against anticancer drugs, and thus to reoccurrence of colon cancer, targeted delivery of silica NPs could be envisioned to also deplete p53 deficient tumor cells.

The use of natural products in agriculture as pesticides has been strongly advocated. However, it is necessary to assess their toxicity to ensure their safe use. In the present study, mammalian cell lines and fish models of the zebrafish (Danio rerio) and medaka (Oryzias latipes) have been used to investigate the toxic effects of ten natural products which have potential applications as biopesticides. The fungal metabolites cavoxin, epi-epoformin, papyracillic acid, seiridin and sphaeropsidone, together with the plant compounds inuloxins A and C and ungeremine, showed no toxic effects in mammalian cells and zebrafish embryos. Conversely, cyclopaldic and α-costic acids, produced by Seiridium cupressi and Dittrichia viscosa, respectively, caused significant mortality in zebrafish and medaka embryos as a result of yolk coagulation. However, both compounds showed little effect in zebrafish or mammalian cell lines in culture, thus highlighting the importance of the fish embryotoxicity test in the assessment of environmental impact. Given the embryotoxicity of α-costic acid and cyclopaldic acid, their use as biopesticides is not recommended. Further ecotoxicological studies are needed to evaluate the potential applications of the other compounds.

Touch response of zebrafish larvae refers to the behaviors after the mechanical stimuli that varies under different conditions (different drugs), and statistical analysis of the behaviors in the organism level is becoming more attractive in drug screening. Collecting repeatable and reliable experimental data on the alive animals in a high-throughput manner is of importance and challenging. Thus, we for the first time introduce a multi-larvae experimentation platform for the fully automated data acquisition to screen the touch-response behaviors of zebrafish in the high-throughput manner. A multi-larvae touching strategy is designed for this platform, which provides a new alternative method to generate touch-response experimental data in a higher efficiency than in the single-larva case (in average 3.58 videos collected per well instead of only one, with 33.00 s duration on each larva instead of 55.95 s). As well, for the proof of the concept, the unknown parameters used for the strategy are decided by experiments. This platform will help in the high-throughput screening of the drugs in the whole animal (organism) level.

A touch-evoked response of zebrafish larvae provides information on the mechanism of the gene functional expressions. Recently, an automated system has been developed for precise and repeated touch-response experimentation with minor human intervention. To quantify the collected data, we propose a fully automated multi-larvae touch-response behavior inspection pipeline based on larva tracking and segmentation. Experimental data with different treatments is analyzed by using the proposed inspection platform for demonstration, and the result proves that this platform can generate comparable touch-response behavior inspection readouts efficiently and automatically. The initial results were published in 31. Workshop Computational Intelligence, and this paper summarizes and extends the main work of the respective article.

Background Oryzias latipes (Medaka) has been established as a vertebrate genetic model for over a century, and has recently been rediscovered outside its native Japan. The power of new sequencing methods now makes it possible to reinvigorate Medaka genetics, in particular by establishing a near-isogenic panel derived from a single wild population. Results Here we characterise the genomes of wild Medaka catches obtained from a single Southern Japanese population in Kiyosu as a precursor for the establishment of a near isogenic panel of wild lines. The population is free of significant detrimental population structure, and has advantageous linkage disequilibrium properties suitable for establishment of the proposed panel. Analysis of morphometric traits in five representative inbred strains suggests phenotypic mapping will be feasible in the panel. In addition high throughput genome sequencing of these Medaka strains confirms their evolutionary relationships on lines of geographic separation and provides further evidence that there has been little significant interbreeding between the Southern and Northern Medaka population since the Southern/Northern population split. The sequence data suggest that the Southern Japanese Medaka existed as a larger older population which went through a relatively recent bottleneck around 10,000 years ago. In addition we detect patterns of recent positive selection in the Southern population. Conclusions These data indicate that the genetic structure of the Kiyosu Medaka samples are suitable for the establishment of a vertebrate near isogenic panel and therefore inbreeding of 200 lines based on this population has commenced. Progress of this project can be tracked at http://www.ebi.ac.uk/birney-srv/medaka-ref-panel