Freshwater Aquarium Microbiome

Introduction

Microbiomes, the communities of microorganisms living together in particular habitats, are vital for maintaining ecological balance. The habitat hosting the microbial communities can be anything from the gastrointestinal tract of a cow to the soils of the land (1). Of recent particular interest are those of aquatic ecosystems, which contain diverse arrays of bacterial communities. The fish themselves oftentimes possess more microbial cells than fish cells in their bodies (2). Of increasing use in identifying the microbes present in these microbiomes is 16S amplicon sequencing. The 16S rRNA gene encodes for 16S rRNA, which is an important constituent of the prokaryotic ribosome 30S subunit. It is noteworthy to point out that this gene is only found in bacteria and archaea (3). The 16S gene has a number of conserved regions, which enables the 16S gene to be recognized across many different microbes through only a few sets of primers catered to this conserved region. The variable regions in the gene are indicative to which specific microbes are products of the amplification of the microbiomes. Up until 2012, there have not been very many studies pertaining to the characterization of the microbiome pertaining to the water associated with freshwater ornamental fishes (4)...This experiment aims to obtain a report on the bacterial community composition of the freshwater fish tank housing four generations of convict cichlids and on the saltwater fish tank, located on the 3rd floor of CSB. To accomplish this, a protocol modeled after Smith et al. and Patin et al. will be established (4,5). 600 mL of aquarium water will be filtered through a 0.22 um filter unit. The concentrated microbial biomass trapped on the filter will be treated with a Puregene Qiagen kit with some modifications from the protocol. Afterwards amplification of the bacterial 16S rRNA gene will be carried out and sequencing at the University of Illinois will ensue.

Materials and Methods

Water Sampling

Sterivex Filters

1 L of water from the surface of the freshwater fish tank and saltwater fish tank were filtered through separate 0.22 um sterivex filter units via a peristaltic pump. Air was pushed through following filtration to initiate the removal of residual water contained in the filter units.

Nanopure Filtration

Due to the low concentration of DNA extracted from filtering 1 L of water through the sterivex filter unit, 2 L of water from the freshwater and saltwater fish tanks were filtered through the free 0.22 um filter membranes via a reusable filter funnel and with the aid of a vacuum source.

Spectra Field-Pro Peristaltic Pump (~$2600)

- eDNA Sampler Usage

DNA Extraction

DNA extraction from the 0.22 um sterivex filter unit followed the protocol outlined in the DNeasy Powerwater Sterivex Kit with no noteworthy deviations from this protocol (). As expected, 100 ul of product was obtained. 2 ul were tested on the nanodrop to assess the quantity and quality of the DNA obtained. The DNA extracted from the freshwater sterivex filter unit absorbed at a concentration of 3.6 ng/ul at 260 nm, which was a lower concentration of DNA than expected. Its A260/A280 ratio was 1.80, which indicates that the protein and inhibitor removal steps were successful. The recorded A260/A230 ration was very low at 0.04, which may indicate the presence of excess salt left in solution, for most of the salts used in this DNA extraction protocol absorb at 230 nm. This low ratio suggests that improvements to the overall quantity of DNA obtained may be higher than 3.6 ng/ul through purification techniques aimed to get the excess salt out.

File:PowerWater_Sterivex.pdf

Primer Construction

Forward primer design: 5'-AATGATACGGCGACCACCGAGATCTACAC-ATCGTACG-TATGGTAATT-GT-GTGCCAGCMGCCGCGGTAA-3' | 5'-adaptor-barcode-pad-linker-V4 region primer-3'

Reverse primer design: 5'-CAAGCAGGAAGACGGCATACGAGAT-AGTCAGTCAG-CC-GGACTACHVGGGTWTCTAAT-3' | 5'-adaptor-pad-linker-V4 region primer-3'

The pads serve the purpose of preventing hairpin formation and manipulating the melting point of the primers, for the adaptors need to anneal to the flow cell, a glass slide with lanes of two different types of oligo (complementary to the adaptors), at 65°C in order for sequencing to ensue (). The linkers are noncomplementary to the 16S gene so that alignment for bioinformatics processing can be carried out efficiently (). The adaptors, pads, and linkers are needed on both the forward and reverse primers because bridge amplification is utilized in Illumina sequencing platforms. During this process, one side of the adaptor sequences hybridizes to the flow cell, a complement of this original fragment is produced by a polymerase, the double strand of DNA is denatured and washed away, and then the single strand folds over and hybridizes to the second type of oligo on the flow cell (). Polymerases generate the complementary strand, and this double stranded bridge is denatured, which results in two single stranded copies tethered to the flow cell (). This process is repeated many times. After bridge amplification, the reverse strands are cleaved and cut off, which is why the barcode is only important for the forward primer.

References

2. Savage, D. C. Microbial ecology of the gastrointestinal tract. Ann Rev Microbiol. 1977. 31. 107-133.

3. Smith, K. F.; Schmidt, V.; Rosen, G. E.; Amaral-Zettler, L. Microbial Diversity and Potential Pathogens in Ornamental Fish Aquarium Water. Public Library of Science One. 2012. 7.

Page History

This page was created by Sara L Simonson in the spring of 2021