Utilization of a Gender-Based Sorting Machine for Crustacean Selection in Bioconcentration Studies with the Freshwater Amphipod Hyalella azteca.

Bioconcentration factors (BCFs) are determined by fish flow-through tests performed according to Organisation for Economic Co-operation and Development test guideline 305. These are time-consuming and expensive and use a large number of animals. An alternative test design using the freshwater amphipod Hyalella azteca for bioconcentration studies has been recently developed and demonstrated a high potential. For bioconcentration studies using H. azteca, male amphipods are preferred compared with female organisms. Manual sexing of male adult amphipods is, however, time-consuming and requires care and skill. A new fully automatic sorting and dispensing machine for H. azteca based on image analysis has recently been developed by the company Life Science Methods. Nevertheless, an anesthesia step is necessary prior to the automatic selection. In the present study, we show that a single-pulse of 90 min of tricaine at the concentration of 1 g/L can be used and is recommended to select H. azteca males manually or automatically using the sorting machine. In the second part, we demonstrate that the machine has the ability to select, sort, and disperse the males of a culture batch of H. azteca as efficiently as manual procedures. In the last part of the study, BCFs of two organic substances were evaluated using the H. azteca bioconcentration test (HYBIT) protocol, with an anesthetizing step and robotic selection compared with manual selection without an anesthetizing step. The different BCF values obtained were in accordance with those indicated in the literature and showed that an anesthetizing step had no effect on the BCF values. Therefore, these data validated the interest in this sorting machine for selecting males to perform bioconcentrations studies with H. azteca. Environ Toxicol Chem 2023;42:1075-1084. © 2023 SETAC.

Deep learning image recognition enables efficient genome editing in zebrafish by automated injections.

One of the most popular techniques in zebrafish research is microinjection. This is a rapid and efficient way to genetically manipulate early developing embryos, and to introduce microbes, chemical compounds, nanoparticles or tracers at larval stages. Here we demonstrate the development of a machine learning software that allows for microinjection at a trained target site in zebrafish eggs at unprecedented speed. The software is based on the open-source deep-learning library Inception v3. In a first step, the software distinguishes wells containing embryos at one-cell stage from wells to be skipped with an accuracy of 93%. A second step was developed to pinpoint the injection site. Deep learning allows to predict this location on average within 42 µm to manually annotated sites. Using a Graphics Processing Unit (GPU), both steps together take less than 100 milliseconds. We first tested our system by injecting a morpholino into the middle of the yolk and found that the automated injection efficiency is as efficient as manual injection (~ 80%). Next, we tested both CRISPR/Cas9 and DNA construct injections into the zygote and obtained a comparable efficiency to that of an experienced experimentalist. Combined with a higher throughput, this results in a higher yield. Hence, the automated injection of CRISPR/Cas9 will allow high-throughput applications to knock out and knock in relevant genes to study their mechanisms or pathways of interest in diverse areas of biomedical research.

Analysis of RNAseq datasets from a comparative infectious disease zebrafish model using GeneTiles bioinformatics.

We present a RNA deep sequencing (RNAseq) analysis of a comparison of the transcriptome responses to infection of zebrafish larvae with Staphylococcus epidermidis and Mycobacterium marinum bacteria. We show how our developed GeneTiles software can improve RNAseq analysis approaches by more confidently identifying a large set of markers upon infection with these bacteria. For analysis of RNAseq data currently, software programs such as Bowtie2 and Samtools are indispensable. However, these programs that are designed for a LINUX environment require some dedicated programming skills and have no options for visualisation of the resulting mapped sequence reads. Especially with large data sets, this makes the analysis time consuming and difficult for non-expert users. We have applied the GeneTiles software to the analysis of previously published and newly obtained RNAseq datasets of our zebrafish infection model, and we have shown the applicability of this approach also to published RNAseq datasets of other organisms by comparing our data with a published mammalian infection study. In addition, we have implemented the DEXSeq module in the GeneTiles software to identify genes, such as glucagon A, that are differentially spliced under infection conditions. In the analysis of our RNAseq data, this has led to the possibility to improve the size of data sets that could be efficiently compared without using problem-dedicated programs, leading to a quick identification of marker sets. Therefore, this approach will also be highly useful for transcriptome analyses of other organisms for which well-characterised genomes are available.

Establishment and optimization of a high throughput setup to study Staphylococcus epidermidis and Mycobacterium marinum infection as a model for drug discovery.

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Robotic injection of zebrafish embryos for high-throughput screening in disease models.

The increasing use of zebrafish larvae for biomedical research applications is resulting in versatile models for a variety of human diseases. These models exploit the optical transparency of zebrafish larvae and the availability of a large genetic tool box. Here we present detailed protocols for the robotic injection of zebrafish embryos at very high accuracy with a speed of up to 2000 embryos per hour. These protocols are benchmarked for several applications: (1) the injection of DNA for obtaining transgenic animals, (2) the injection of antisense morpholinos that can be used for gene knock-down, (3) the injection of microbes for studying infectious disease, and (4) the injection of human cancer cells as a model for tumor progression. We show examples of how the injected embryos can be screened at high-throughput level using fluorescence analysis. Our methods open up new avenues for the use of zebrafish larvae for large compound screens in the search for new medicines.

Automated microinjection of cell-polymer suspensions in 3D ECM scaffolds for high-throughput quantitative cancer invasion screens.

Cell spheroids (CS) embedded in 3D extracellular matrix (ECM) serve as in vitro mimics for multicellular structures in vivo. Such cultures, started either from spontaneous cell aggregates or single cells dispersed in a gel are time consuming, applicable to restricted cell types only, prone to high variation, and do not allow CS formation with defined spatial distribution required for high-throughput imaging. Here, we describe a method where cell-polymer suspensions are microinjected as droplets into collagen gels and CS formation occurs within hours for a broad range of cell types. We have automated this method to produce CS arrays in fixed patterns with defined x-y-z spatial coordinates in 96 well plates and applied automated imaging and image analysis algorithms. Low intra- and inter-well variation of initial CS size and CS expansion indicates excellent reproducibility. Distinct cell migration patterns, including cohesive strand-like - and individual cell migration can be visualized and manipulated. A proof-of-principle chemical screen is performed identifying compounds that affect cancer cell invasion/migration. Finally, we demonstrate applicability to freshly isolated mouse breast and human sarcoma biopsy material - indicating potential for development of personalized cancer treatment strategies.

A high-throughput screen for tuberculosis progression.

One-third of the world population is infected with Mycobacterium tuberculosis and multi-drug resistant strains are rapidly evolving. The noticeable absence of a whole organism high-throughput screening system for studying the progression of tuberculosis is fast becoming the bottleneck in tuberculosis research. We successfully developed such a system using the zebrafish Mycobacterium marinum infection model, which is a well-characterized model for tuberculosis progression with biomedical significance, mimicking hallmarks of human tuberculosis pathology. Importantly, we demonstrate the suitability of our system to directly study M. tuberculosis, showing for the first time that the human pathogen can propagate in this vertebrate model, resulting in similar early disease symptoms to those observed upon M. marinum infection. Our system is capable of screening for disease progression via robotic yolk injection of early embryos and visual flow screening of late-stage larvae. We also show that this system can reliably recapitulate the standard caudal vein injection method with a throughput level of 2,000 embryos per hour. We additionally demonstrate the possibility of studying signal transduction leading to disease progression using reverse genetics at high-throughput levels. Importantly, we use reference compounds to validate our system in the testing of molecules that prevent tuberculosis progression, making it highly suited for investigating novel anti-tuberculosis compounds in vivo.