Raising axenic Nile tilapia Oreochromis niloticus and subsequent exposure to gnotobiotic and normal environmental conditions

During development fish larvae interact with numerous (opportunistic) bacteria in the culture medium. Maternal bacteria are present on the egg which together with microbiota in the rearing water and food colonize the gut during development. Either stable and healthy intestinal microbiota develop and the animals prosper or the intestinal microbiota become instable and the animals suffer infections and diseases. With thousands of different species potentially colonizing a fish gut, understanding (fish)-host microbe interactions is difficult. Raising animals under germ-free axenic conditions and exposing them to known microbiota provides a model allowing to study host microbe interactions in fish under controlled conditions with respect to identity and complexity of microbial colonizers. With fish, the common method to grow axenic larvae is to collect fertilized eggs, first disinfect them and then incubate them in a cocktail of disinfectants and antibiotics. Purpose build axenic incubation chambers set up in a UV-cabinet were used to culture axenic tilapia swim-up fry. In total 400 fertilized eggs were divided among 4 axenic incubation chambers, where they were raised in an antibiotic-antifungal medium. After 4 days incubation under axenic conditions, in two chambers the probiotic bacterial strain Bacilus subtilis was introduced into the rearing water (> 106 CFU per ml). After the introduction 2 chambers continued to be maintained under axenic conditions and 2 chambers under gnotobiotic conditions until the yolksack was fully adsorbed on day 9-10 post-hatching. Then the larvae were transferred to normal (non-axenic) rearing tanks with matured culture water and grown for another 14 days while fed sterilized (gamma irradiated) feed. To test for bacterial contamination the rearing medium in each chamber was sampled daily, incubated in liquid broth and then plated on agar growth medium. At different moments in time microbial DNA was extracted from guts and water and submitted to 454 pyrosequencing of PCR-amplified 16S rRNA to monitor gut colonization. Preliminary data show a successful axenic environment and gnotobiotic conditions (based on culture dependent techniques). Mortality was 11% in incubation chamber one and 13% in chamber 2 when switching from axenic to gnotobiotic conditions. When switching from either gnotobiotic or axenic conditions to matured water no swim-up fry died. Data on the imprinting effect of early gut exposure to gnotobiotic conditions, after pretreatment with antibiotics, will be reported in detail during the conference.