Cataloging and Analysis of Microbial Communities in the Zebrafish Colony at the Krasnow Institute for Advanced Study

Zebrafish (Danio rerio) are important model organisms for studying developmental biology. The Krasnow Institute for Advanced Study (KIAF) at GMU houses a colony of zebrafish in their animal facility. The research presented here is part of a greater review of care protocols in order to reduce mortality rates in juvenile fish. Several samples of microbial biofilm were harvested from the aquarium system housing the fish and cultured in petri dishes at 28℃. The goal was to check for any pathogenic organisms, but none were found. Instead, investigators discovered a high diversity of microfauna that organize themselves into three distinct communities based on water conditions and quality. Type I communities were observed in conditions that zebrafish thrive in, Type II communities were observed in conditions that zebrafish deteriorate in, and Type III communities are neutral to water quality and instead react to ambient light levels. Based on these findings, zebrafish researchers at the KIAF can use microbial analysis to monitor water quality and better diagnose issues with the fish by cataloging the presence of Type I and II communities. Investigators hope that the application of these culture techniques will improve survivorship in both adult and young zebrafish at the lab.

Hello, my name is Tom Lopez and I’m currently a junior biology student at George Mason University. Today I will be presenting the cataloging and analysis of microbial communities in the Zebrafish Colony located in the Krasnow Institute of Advanced Study.

I originally applied for the OSCAR URSP Program with my mentor Dr. Valerie Olmo with an idea for a conservation biology project about an endangered and understudied species of fish. While I set up my own research project, Dr. Olmo invited me to work with the Zebrafish Colony. The researchers there were struggling to raise young fish, so she asked me to help review their care strategies. I tested different factors while raising several clutches of eggs, including water quality, feeding regimes, and disinfection protocols. While investigating the high mortality rate of young fish, I began to look at the microbial organisms that inhabited the aquarium system. I initially wanted to see if there were any pathogens or other harmful organisms which may be affecting the fish. I did not end up finding anything dangerous, but I did discover a much wider variety of biodiversity than I expected. This presentation will explore these findings.

Aquariums support many different types of microhabitats, oftentimes unintentionally. The constant flow of nutrients in the water supports several communities of microorganisms, which form biofilms across surfaces. I sought to identify exactly what organisms comprise these biofilms.

To do this, I collected several samples by scraping the biofilm off the surface, pipetting it into a petri dish, and incubating the dishes at 28 degrees centigrade. After leaving the contents of the dish to settle for 24 hours, I looked at them under a dissecting microscope. Immediately, I saw a huge diversity of life. I began recording them one by one in an effort to identify them. Among the species found living in the system were:

Tetrahymena pyriformis, a single-celled detritivore
Two kinds of rotifers, one in the class Bdelloidea and the other in the genus Euchlanis
An unidentified Vorticella species, which are sedentary filter feeders
Peranema, which are euglenids
Gastrotrichs, which are benthic worm-like animals
Non-parasitic nematodes

As I collected and analyzed more samples, I began to notice that the organisms divided themselves into three distinct communities, which I called Type 1, 2, and 3 for this project. Each community is associated with a different aquatic environment.

A Type 1 community is one which is dominated by an unidentified fungi. This organism grows in a mycelium-like pattern, branching across a surface like a web. This mycelium substrate hosts a variety of species, primarily Tetrahymena, Vorticella, gastrotrichs and rotifers. All of these organisms have been observed attaching to and living within the mycelium matrix, suggesting that the fungi provides some sort of critical factor for this ecosystem to develop.

A Type 2 community is one which is dominated by a bacterial biofilm. This film spreads like a sludge across a surface around uneaten food. This community hosts a lower diversity of organisms, but a much higher density. The primary organism in this ecosystem by a large margin is Peranema. Unlike their more well-known Euglena cousins, Peranema cannot photosynthesize, instead feeding on the bacteria which grow in the biofilm. Type 2 communities are visible with the naked eye, appearing as a brown slime dotted with several white masses. These masses are actually piles of Peranema, which congregate into these areas to feed and breed.

Type 1 and Type 2 communities are mutually exclusive, requiring different water conditions and actively repelling each other, as can be seen here. Type 1 communities have been observed in conditions with faster water flow, low nutrients from leftover food and low ammonia waste from the fish. By contrast, Type 2 communities have been observed in low-flow conditions with high nutrients and very high levels of ammonia waste. This distinction allows the communities to be used as secondary indicators by researchers to monitor aquatic health, since zebrafish thrive in the conditions preferred by Type 1 and deteriorate in the conditions preferred by Type 2.

Type 3 is an outlier community associated with high flow and high light. Only two have been observed in the system, but they were the two largest microbial communities documented. Type 3 communities are dominated by diatoms- tiny, photosynthetic ciliates which form silica shells around themselves. Samples from these communities were difficult to culture since the diatoms often died when kept in a dark incubator; however, I was able to identify the glass shells they left behind. The diatom colonies form a brown-red film which acts as a matrix for larger organisms to inhabit, primarily rotifers and a small unidentified plankton too small to record. They also seem to be associated with webs of thin red strands; these structures have yet to be identified, but are speculated to be another kind of fungi.

These are the findings I have compiled by the end of the semester. In regards to zebrafish husbandry in the Krasnow Institute, the ecology of these microorganisms can be used to improve care practices and better provide for the offspring. However, more work must still be done. There are several organisms which have not yet been identified, and the significance of the association with Type 1 and 2 communities and water quality has yet to be quantitatively determined. With further investigation of microbial analysis, researchers can use the health of all organisms in an aquarium system to better provide for the needs of the fish they want to raise. I will continue to move forward with this investigation, and I am excited to contribute to the husbandry of the model organisms upon which so much research relies. Thank you for watching!