Microfluidic culture platforms for C. elegans bacterial interaction and digestion studies at single-organism resolution

Increasing interest in understanding fundamental biological processes, among which aging and nutrition, has led to the study of simple and powerful model organisms, as the round-worm Caenorhabditis elegans (C. elegans). The nematode, selected by Sydney Brenner , shares slightly over one-third of its genetic base code with human beings, making it an optimal model organism for many human related studies. It has been established that bacteria, besides being the principal nutrition source for these nematodes, play an active role in their aging process. In later life stage, i.e. after the larval development, the bacterial grinder of the worms loses efficiency permitting viable bacteria to reach and colonize the gut of the nematodes . Intestinal persistency of bacteria is highly related both to the strain of the hosted organism and of the host; in the first case the pathogenicity of bacteria determines colonization whereas in the second case the speed of the ingestion and digestion mechanism adopted by the host is one of the causes that can determine the occurrence of a condition of illness. Conventional methods commonly employed by biologists for C. elegans observation, widely applied in studies of interaction between worms and bacteria, food intake and processing analysis, result in some cases to be time consuming and difficult to operate. In order to solve these study related issues, such as observations of digestion related pheno-types on individual worms during the entire developmental time and reproducible food stimulation, microfluidics techniques, based on control of microliters of fluid in a laminar flow regime conditions, revealed to be useful for a consistent increase of the overall accuracy. Technical improvement in research and new analytical tools allowed single worm reversible immobilization and imaging and high throughput analysis, but gathering phenotypic information at single-cell or individual organism level, with higher throughput and better precision than conventional methods, still remains challenging. Combining the multiple technical advantages brought by microfluidics to the study of C. elegans, we focus our attention in developing new devices and protocols aiming to investigate how microbiota interact with the host organism. In particular, we intend to develop a novel microfluidic approach for performing long-term live imaging, bacterial processing and standardized food intake assays in an automated way. Worms sorting and immobilization by passive PDMS structures and accurate food concentration dispens-ing will be accomplished. We intend to obtain quantitative measurement of C. elegans food digestion, intake and development during the adult stage first and then in all stages, showing how digestion process, food intake and bacterial hosting is highly correlated with multiple characterizing phenotypes. To obtain food absorption measurement at single worm resolution, we apply an optical method of quantification using green fluorescent protein (GFP) labeled E. coli bacteria together with fluorescent microscopy techniques. Feeding behaviour and physiological digestion response under different exposure conditions will be tested in order to investigate how these factors impact on the development of the organism.