Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849
Performing Department
Fisheries & Allied Aquaculture
Non Technical Summary
Fish are a vital source of food for humans, providing ∼16% of all high quality animal protein. Fish are the main animal protein consumed by the world's population, according to the Food and Agriculture Organization (FAO) of the United Nations. Aquaculture is currently the fastest-growing food production system worldwideand the modern aquaculture industry provides effective means for intensive fish production. However, this rapidly growing industry continues to face severe disease problems, many of which are caused by opportunistic bacterial pathogens. Despite all the information available regarding disease prevention on farmed fish, common farming practices place a lot of pressure on the animals and their symbionts that are part of their well-being.Fish serve as hosts to a wide range of microorganisms such as bacteria, yeast, virus, archaea, and protozoans.These complex commensal communities are commonly referred to as the microbiota or microbiome. Microbes inhabit many niches on and in the host including skin, gill, and the gastrointestinal tract and are well-adapted to live in intimate contact with the hosts' mucosas. Besides providing important health benefits to their host's, the microbiome also protects the host from incoming pathogens through various mechanisms, including competition for space or binding sites, competition for nutrients with bacteria with similar nutrient requirements, and by direct inhibition through the release of inhibitory molecules.Studies of bacterial abundance, diversity, host-symbionts interactions, and are essential to recognize and understand the benefits that a healthy microbiome brings to its host. This proyect aimsat filling some of the gaps in our current knowledge on the factors affecting the microbiome of fishes.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
0%
Goals / Objectives
The overall objective of this project is to characterize if (and if so, how) dysbiosis is induced using standard aquaculture practices. For most of the studies, I anticipate to use channel catfish, as it is the main aquaculture species in the US. However, there might be times in when the use of channel catfish is not ideal and we will use a model organism (zebrafish).
Project Methods
The overall layout for these experiments is as follows:Fish husbandry. Fish will be purchased from commercial suppliers, and quarantine under standard husbandry conditions until use. After the quarantine period, fish will be stocked into 20 tanks, 37 L each, at a stocking rate of 40 fish per tank and maintained as previously described [23]. They will be fed once daily to approximately 2% body weight per day with commercial catfish feed containing 32% crude protein PMB (Alabama Catfish Feed Mill, L.L.C, Uniontown, Al). Water quality will be monitored daily, and parameters were maintained at 80 ppm alkalinity, 150 ppm hardness, 27.6 ± 0.50 ?C, pH: 7.84 ± 0.15 (mean ± SD), ammonia and nitrites were kept at non-detectable levels with 12:12 h photoperiod throughout the experiment.Induction of Dysbiosis. Different factors (see specific objectives below) will be tested to see if they could induce dysbiosis or alter the gut/skin microbiomes in any substantial and permanent manner. Each treatment will consist of at least 5 replicates (= tanks). Treatments will include treated group(s) (i.e. different antibiotic concentration or vaccine route in addition to a later stressor, ie, exposure to an opportunistic pathogen), a control group (fish will not be exposed to the treatment but will be exposed to a stressor), and a system control (no treatment; no stressor).Sample collection. At different samples time (depending on the specific objective), the intestinal content of 5 fish per replica will be pooled, and DNA extractions will be carried our as previously described [24]. Briefly, DNA will be extracted using DNeasy Blood & Tissue kit (Qiagen, Valencia, CA, USA) following manufacturer's instructions (Total DNA from Animal Tissues, Spin column Protocol) with modifications including double digestion with proteinase K and pre-treatment with lysozyme for lysis of Gram-positive bacteria and RNase A treatment. DNA will be eluted with 100 µl elution buffer. DNA samples will be quantified using a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and normalized into 100 ng/µl with ddH2O.Next generation sequencing. DNA samples will be submitted to MR DNA® (Shallowater, TX, USA) for PCR amplification and Next- Generation Sequencing using Illumina MiSeq platform targeting the 16S rRNA gene V4 variable region. Universal bacterial primers 515 F (5'-GTGCCAGCMGCCGCGGTAA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') with a barcode on the forward primer will be used to generate a 300 bp amplicon. The HotStarTaq Plus Master Mix Kit (Qiagen, USA) will be used under the following PCR conditions: an initial denaturation for 3 minutes at 94 °C followed by 28 cycles of denaturing at 94 °C for 30 s, annealing at 53 °C for 40 s, extension at 72 °C for 1 min and a final elongation step for 5 min at 72 °C. The PCR products will be run through a 2% agarose gel to verify successful amplification and relative band intensity of the target DNA. Multiple samples will be pooled together and purified using calibrated Ampure XP beads to prepare the Illumina DNA library. Samples will be sequenced as paired-end reads on the Illumina MiSeq platform following the manufacturer's instructions. Resulting sequencing will be processed using a proprietary pipeline (MR DNA, Shallowater, TX, USA). Sequencing data will be joined, and all barcodes, ambiguous base calls, and sequences <150 bp will be removed. Denoising of sequences will be performed, and operational taxonomic units (OTUs) were generated. Cut-offs for OTU assignment will be defined at a 97% similarity (<3% sequence variation) in concurrence with the prokaryotic species concept [25]. Taxonomic classifications will be obtained using BLASTn against the Green Genes database [26].Data analysis. Rarefaction curves, observed OTUs, shared OTUs, ACE, CHAO1, good coverage and Shannon Evenn index (SEI) will be calculated using Mothur v.1.33.3 [27] after standardization of each sample type to the sample yielding the least number of total sequences. One-way Anova will be used followed by Tuckey's test for all-pairwise comparison to determine differences in diversity index between the groups. The PCoA (Principal coordinate analysis) using the variables treatment, time, and groups will be performed using optimized position to visualize the similarities or dissimilarities of the samples. OTU and Genera tables including all samples will be loaded into PRIMER (Primer E Ltd, Plymouth, UK) for clustering using group average and ANOSIM analysis (group, treatments, sampling time). To accompany the results of the ANOSIM, a PERMANOVA statistical test will be used to analyze the diversity across groups sampled. In addition, PERMDISP test, will be run under the null hypothesis of no differences of within-group multivariate dispersion across sampling time points, to give insight on within- and between- group dispersion to enable more accurate interpretation of the PERMANOVA and ANOSIM results [28]. PERMANOVA, ANOSIM, and PERMIDISP test will be conducted using 9,999 permutations of data, following the recommendation of Clarke and Gorley [29]. A Pairwise test will be done when statistical tests identified significant effects within main test (P≤ 0.05) to determine the difference between and within groups across sampling events. Similarity percentage (SIMPER) analysis on a Bray-Curtis similarity matrix assembled from a Genus level OTU table will be conducted to determine specific taxonomical differences between communities. The relative abundance at phylum and genus level comparison was using One-way ANOVA.Specific objectives include:Effect of medicated feed on the gut microbiome of fishes. Commercial, FDA-approved, antibiotics will be tested including Aquaflor (Florfenicol). Different doses and withdraw periods will be compared to determine the permanent effect (if any) of using medicated feed in channel catfish.Effect of vaccination on the, gill, skin and gut microbiome of fishes. Autogenous vaccines against columnaris disease, enteric septicemia of catfish, and Aeromonas hydrophila will be administered to the fish by immersion, by IP injection, and orally. Different doses and adjuvants will be compared.Effect of mechanical injury and stress on the gut microbiome of fishes: understanding the brain-gut nexus. Fish are subjected to mechanical injury during seining and transport. It has been shown in mammalian models that hormones can regulate the microbial community composition in the gut and that gut microbes in fact can modulate brain development and behavior by producing neurotransmitor precursors. Stress will be induced using feed depravation, crowding, etc... while seine-induced mechanical injuries will be mimic by small skin abrasions using scalpels.