Automated, high-throughput, motility analysis in Caenorhabditis elegans and parasitic nematodes: Applications in the search for new anthelmintics

The scale of the damage worldwide to human health, animal health and agricultural crops resulting from parasitic nematodes, together with the paucity of treatments and the threat of developing resistance to the limited set of widely-deployed chemical tools, underlines the urgent need to develop novel drugs and chemicals to control nematode parasites. Robust chemical screens which can be automated are a key part of that discovery process. Hitherto, the successful automation of nematode behaviours has been a bottleneck in the chemical discovery process. As the measurement of nematode motility can provide a direct scalar readout of the activity of the neuromuscular system and an indirect measure of the health of the animal, this omission is acute. Motility offers a useful assay for high-throughput, phenotypic drug/chemical screening and several recent developments have helped realise, at least in part, the potential of nematode-based drug screening. Here we review the challenges encountered in automating nematode motility and some important developments in the application of machine vision, statistical imaging and tracking approaches which enable the automated characterisation of nematode movement. Such developments facilitate automated screening for new drugs and chemicals aimed at controlling human and animal nematode parasites (anthelmintics) and plant nematode parasites (nematicides)

High-throughput phenotyping of multicellular organisms: finding the link between genotype and phenotype

High-throughput phenotyping approaches (phenomics) are being combined with genome-wide genetic screens to identify alterations in phenotype that result from gene inactivation. Here we highlight promising technologies for 'phenome-scale' analyses in multicellular organisms

Tools for Behavioral Phenotyping of C. elegans

Animal behavior is critical to survival and provides a window into how the brain makes decisions and integrates sensory information. A simple model organism that allows researchers to more precisely interrogate the relationships between behavior and the brain is the nematode C. elegans. However, current phenotyping tools have technical limitations that make observing, intervening in, and quantifying behavior in diverse settings difficult. In this thesis, I develop enabling technological systems to resolve these challenges. To address scaling issues in observation and intervention in long-term behavior, I develop a platform for long-term continuous imaging, online behavior quantification, and online behavior-conditional intervention. I show that this tool is easy to build and use and can operate in an automated fashion for days at a time. I then use this platform to understand the consequences of quiescence deprivation to C. elegans health. To quantify complex animal postures, and plant and stem cell aggregate morphology, I develop an app to enable fast, versatile and quantitative annotation and demonstrate that it is both ~ 130-fold faster and in some cases less error-prone than state-of-the-art computational methods. This app is agnostic to image content and allows freehand annotation of curves and other complex and non-uniform shapes while also providing an automated way to distribute annotation tasks. This tool may be used to generate ground truth sets for testing or creating automated algorithms. Finally, I quantify C. elegans behavior using quantitative machine-learning analysis and map the worm’s behavioral repertoire across multiple physical environments that more closely mimic C. elegans’ natural environment. From this analysis, I identified subtle behaviors that are not easily distinguishable by eye and built a tool that allows others to explore our video dataset and behaviors in a facile way. I also use this analysis to examine the richness of C. elegans behavior across selected environments and find that behavior diversity is not uniform across environments. This has important implications for choice of media for behavioral phenotyping, as it suggests that the appropriate media choice may increase our ability to distinguish behavioral phenotypes in C. elegans. Together, these tools enable novel behavior experiments at a larger scale and with more nuanced phenotyping compared to currently available tools.Ph.D

Deep Learning for Microfluidic-Assisted <i>Caenorhabditis elegans</i> Multi-Parameter Identification Using YOLOv7

The Caenorhabditis elegans (C. elegans) is an ideal model organism for studying human diseases and genetics due to its transparency and suitability for optical imaging. However, manually sorting a large population of C. elegans for experiments is tedious and inefficient. The microfluidic-assisted C. elegans sorting chip is considered a promising platform to address this issue due to its automation and ease of operation. Nevertheless, automated C. elegans sorting with multiple parameters requires efficient identification technology due to the different research demands for worm phenotypes. To improve the efficiency and accuracy of multi-parameter sorting, we developed a deep learning model using You Only Look Once (YOLO)v7 to detect and recognize C. elegans automatically. We used a dataset of 3931 annotated worms in microfluidic chips from various studies. Our model showed higher precision in automated C. elegans identification than YOLOv5 and Faster R-CNN, achieving a mean average precision (mAP) at a 0.5 intersection over a union ([email protected]) threshold of 99.56%. Additionally, our model demonstrated good generalization ability, achieving an [email protected] of 94.21% on an external validation set. Our model can efficiently and accurately identify and calculate multiple phenotypes of worms, including size, movement speed, and fluorescence. The multi-parameter identification model can improve sorting efficiency and potentially promote the development of automated and integrated microfluidic platforms

Novel technologies for high-throughput and high-content studies on zebrafish larvae

The zebrafish larva is an ideal candidate for in vivo high-throughput screening: it is a small vertebrate, it is optically transparent, possesses complex organs, and is easy to culture. In addition, genetic mutants and models of human diseases are widely available. Despite these attractive features there are no tools capable of screening at sufficient throughput and resolution to fully exploit the zebrafish. Here, I present a collection of technologies that enable high-throughput studies on zebrafish larvae at cellular resolution.Engineering and Applied Science