scholarly journals Gut microbiome analysis of fast- and slow-growing Rainbow Trout (Oncorhynchus mykiss)

2019 ◽  
Author(s):  
Pratima Chapagain ◽  
Brock Arivett ◽  
Beth M Cleveland ◽  
Donald M. Walker ◽  
Mohamed Salem
Keyword(s):  
Rainbow Trout ◽  
Dna Extraction ◽  
Gut Microbiome ◽  
Significant Variation ◽  
Pathogenic Bacteria ◽  
Extraction Methods ◽  
Fish Growth ◽  
Indicator Taxa ◽  
Fast Growing ◽  
Slow Growing

Abstract Background Diverse microbial communities colonizing the intestine of fish contribute to their growth, digestion, nutrition, and immune function. We hypothesized that the gut microbiome of rainbow trout could be associated with differential growth rates observed in fish breeding programs. If true, harnessing the functionality of this microbiome can improve profitability of aquaculture. To test this hypothesis, four full-sibling families were stocked in the same tank and fed an identical diet. Two fast-growing and two slow-growing fish were selected from each family. Five different extraction methods were used to obtain DNA from feces for 16S rRNA microbiome profiling. These methods were Promega-Maxwell, phenol-chloroform, MO-BIO, Qiagen-Blood, Qiagen-Stool. Methods were compared according to DNA integrity, cost, feasibility and inter-sample variation based on non-metric multidimensional scaling ordination (nMDS) clusters. Results Differences in DNA extraction methods result in significant variation in identification of bacteria that compose the gut microbiome. Promega-Maxwell had the lowest inter-sample variation and was therefore used for the subsequent analyses. The gut microbiome was different from that of the environment (feed and water). However, feed and gut shared a large portion of their microbiome suggesting significant contribution of the feed in shaping the gut microbiota. Beta diversity of the bacterial communities showed significant variation between breeding families but not between the fast- and slow-growing fish. An indicator analysis determined that cellulose, amylose degrading and amino acid fermenting bacteria (Clostridium, Leptotrichia and Peptostreptococcus) as indicator taxa of the fast-growing fish. In contrary, pathogenic bacteria (Corynebacterium and Paeniclostridium) were identified as slow-growing indicator taxa. Conclusion DNA extraction methodology should be taken into account for accurate profiling of the gut microbiome. Although the microbiome was not significantly different between the fast- and slow-growing fish groups, some bacterial taxa with functional implications were indicative of fish growth rate. Further studies are warranted to explore how bacteria are transmitted and potential usage of the indicator bacteria of fast-growing fish for development of probiotics that may improve fish health and growth.

scholarly journals Analysis of the fecal microbiota of fast- and slow-growing rainbow trout (Oncorhynchus mykiss)

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Pratima Chapagain ◽  
Brock Arivett ◽  
Beth M. Cleveland ◽  
Donald M. Walker ◽  
Mohamed Salem
Keyword(s):  
Growth Rate ◽  
Gut Microbiota ◽  
Dna Extraction ◽  
Significant Variation ◽  
Extraction Methods ◽  
Fish Growth ◽  
Indicator Taxa ◽  
Fast Growing ◽  
Dna Extraction Methods ◽  
Slow Growing

Abstract Background Diverse microbial communities colonizing the intestine of fish contribute to their growth, digestion, nutrition, and immune function. We hypothesized that fecal samples representing the gut microbiota of rainbow trout could be associated with differential growth rates observed in fish breeding programs. If true, harnessing the functionality of this microbiota can improve the profitability of aquaculture. The first objective of this study was to test this hypothesis if gut microbiota is associated with fish growth rate (body weight). Four full-sibling families were stocked in the same tank and fed an identical diet. Two fast-growing and two slow-growing fish were selected from each family for 16S rRNA microbiota profiling. Microbiota diversity varies with different DNA extraction methods. The second objective of this study was to compare the effects of five commonly used DNA extraction methods on the microbiota profiling and to determine the most appropriate extraction method for this study. These methods were Promega-Maxwell, Phenol-chloroform, MO-BIO, Qiagen-Blood/Tissue, and Qiagen-Stool. Methods were compared according to DNA integrity, cost, feasibility and inter-sample variation based on non-metric multidimensional scaling ordination (nMDS) clusters. Results Differences in DNA extraction methods resulted in significant variation in the identification of bacteria that compose the gut microbiota. Promega-Maxwell had the lowest inter-sample variation and was therefore used for the subsequent analyses. Beta diversity of the bacterial communities showed significant variation between breeding families but not between the fast- and slow-growing fish. However, an indicator analysis determined that cellulose, amylose degrading and amino acid fermenting bacteria (Clostridium, Leptotrichia, and Peptostreptococcus) are indicator taxa of the fast-growing fish. In contrary, pathogenic bacteria (Corynebacterium and Paeniclostridium) were identified as indicator taxa for the slow-growing fish. Conclusion DNA extraction methodology should be carefully considered for accurate profiling of the gut microbiota. Although the microbiota was not significantly different between the fast- and slow-growing fish groups, some bacterial taxa with functional implications were indicative of fish growth rate. Further studies are warranted to explore how bacteria are transmitted and potential usage of the indicator bacteria of fast-growing fish for development of probiotics that may improve fish health and growth.

Evaluation of DNA extraction methods and dilution treatment for detection and quantification of Acanthamoeba in water and biofilm by real-time PCR

2010 ◽  
Vol 62 (9) ◽  
pp. 2141-2149 ◽  
Author(s):  
Ching-Wen Chang ◽  
Ying-Chieh Wu
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Pathogenic Bacteria ◽  
Acanthamoeba Castellanii ◽  
Extraction Methods ◽  
Cell Number ◽  
Pcr Analysis ◽  
Human Pathogens ◽  
Dna Extraction Methods

Acanthamoeba, human pathogens and natural hosts of pathogenic bacteria, may be accurately detected and quantified by real-time PCR if Acanthamoeba DNA are properly extracted and PCR inhibitors are effectively eliminated. However, the optimization of DNA extraction methods has not been reported for Acanthamoeba. This study compared the effectiveness of two DNA extraction/purification methods (FastDNA® Spin Kit for soil and Wizard® SV genomic DNA Purification System) by using trophozoites and cysts of Acanthamoeba castellanii and water and biofilm samples of cooling towers. DNA of A. castellanii extracted with the FastDNA® Kit and quantified by TaqMan PCR resulted in a lower variation (CV of Ct < 3%), greater linearity (R2=0.99), and higher slopes (1.177–1.187 log fg DNA/log cell number) as compared to that by the Wizard® Kit. For field testing, the number of Acanthamoeba-positive samples and the Acanthamoeba DNA quantity were both greater with the FastDNA® Kit than with the Wizard® Kit (P=0.016 and <0.0001, respectively). Beneficial effects with dilutions of extracted DNA were also revealed with the FastDNA® Kit (P=0.0003). In conclusion, DNA extraction by the FastDNA® Kit coupled with dilution of extracted DNA and PCR analysis are recommended for detecting and quantifying environmental Acanthamoeba.

scholarly journals A comparison of different DNA extraction methods and molecular techniques for the detection and identification of foodborne pathogens

2021 ◽  
Vol 7 (3) ◽  
pp. 304-319
Author(s):  
Spyridon Andreas Papatheodorou ◽  
◽  
Panagiotis Halvatsiotis ◽  
Dimitra Houhoula ◽  
Keyword(s):  
Dna Extraction ◽  
Foodborne Pathogens ◽  
Extraction Method ◽  
Pathogenic Bacteria ◽  
Low Cost ◽  
Isoamyl Alcohol ◽  
Extraction Methods ◽  
Molecular Techniques ◽  
Bacterial Dna ◽  
Dna Extraction Method

<abstract> <p>Foodborne infections continue to plague Europe. Food safety monitoring is in crisis as the existing techniques for detecting pathogens do not keep up with the global rising of food production and consumption. Thus, the development of innovative techniques for detecting and identifying pathogenic bacteria has become critical. The aim of the present study was firstly to develop an innovative simple and low cost method of extracting bacterial DNA from contaminated food and water samples with <italic>Salmonella enteric</italic> subsp. <italic>enteric</italic> serovar Typhimurium and <italic>Listeria monocytogenes</italic> and its comparison with two commercial DNA extraction kits (Qiagen, Macherey-Nagel). Finally, pathogens' detection using two molecular techniques (PCR-electrophoresis, LAMP), in order to evaluate the best combination of DNA extraction and identification based on their sensitivity, cost, rapidity and simplicity. Considering the above criteria, among them, best was proved an in-house bacterial DNA extraction method, based on the chloroform-isoamyl alcohol protocol, with certain modifications. This technique showed statistically similar results in terms of sensitivity, compared to the commercial kits, while at the same time maintained high rapidity and much lower cost. Lastly, between the molecular techniques, LAMP was found more promising considering its simplicity, high rapidity and sensitivity. Conclusively, the in-house DNA extraction method along with the LAMP technique, was proven to be the best among the presented combinations.</p> </abstract>

Behavior and recruitment success in fish larvae: variation with growth rate and the batch effect

2005 ◽  
Vol 62 (6) ◽  
pp. 1337-1349 ◽  
Author(s):  
Lee A Fuiman ◽  
James H Cowan, Jr. ◽  
Michael E Smith ◽  
Jonathan P O'Neal
Keyword(s):  
Growth Rate ◽  
Fish Larvae ◽  
Fish Growth ◽  
Predatory Fish ◽  
Behavioral Performance ◽  
Treatment Groups ◽  
Fast Growing ◽  
Predation Mortality ◽  
Survival Skills ◽  
Slow Growing

Predation-mortality risk for red drum (Sciaenops ocellatus) larvae does not appear to be related to their growth rate, but important differences in behavioral performance occur between batches of larvae. This conclusion is based upon field-enclosure and laboratory experiments that assessed the degree to which predation-mortality rates and behavioral survival skills vary with growth rate. In field enclosures, populations composed of 15 fast-growing larvae and 15 slow-growing larvae of a comparable size were exposed to a predatory fish. Growth rate did not affect predation rate. In the laboratory we measured 11 survival skills on 100 larvae of a common size from 10 batches of eggs. For each batch, behavioral performance of fast-growing larvae was compared with that of slow-growing larvae. Growth rate did not affect performance in 10 of the 11 survival skills, but behavioral performance varied among treatment groups (growth rate × batch), with higher performance in most survival skills for some treatment groups and consistently poorer performance for other groups. This coordinated pattern of behavioral performance forecasts differential survival among batches. The variation among batches may be related to timing of spawning within the reproductive season of this serially spawning species.

scholarly journals Direct PCR offers a fast and reliable alternative to conventional DNA isolation methods for animal gut microbiomes

2017 ◽  
Author(s):  
Elin Videvall ◽  
Maria Strandh ◽  
Anel Engelbrecht ◽  
Schalk Cloete ◽  
Charlie K. Cornwallis
Keyword(s):  
Dna Extraction ◽  
Gut Microbiome ◽  
Comprehensive Evaluation ◽  
454 Sequencing ◽  
Illumina Miseq ◽  
Extraction Methods ◽  
Direct Pcr ◽  
Faecal Samples ◽  
Dna Extraction Methods ◽  
Microbiome Data

AbstractThe gut microbiome of animals is emerging as an important factor influencing ecological and evolutionary processes. A major bottleneck in obtaining microbiome data from large numbers of samples is the time-consuming laboratory procedures, specifically the isolation of DNA and generation of amplicon libraries. Recently, direct PCR kits have been developed that circumvent conventional DNA extraction steps, thereby streamlining the laboratory process by reducing preparation time and costs. However, the reliability and efficacy of the direct PCR method for measuring host microbiomes has not yet been investigated other than in humans with 454-sequencing. Here, we conduct a comprehensive evaluation of the microbial communities obtained with direct PCR and the widely used MoBio PowerSoil DNA extraction kit in five distinct gut sample types (ileum – caecum – colon – faeces – cloaca) from 20 juvenile ostriches, using 16S rRNA Illumina MiSeq sequencing. We found that direct PCR was highly comparable over a range of measures to the DNA extraction method in caecal, colon, and faecal samples. However, the two methods recovered significantly different microbiomes in cloacal, and especially ileal samples. We also sequenced 100 replicate sample pairs to evaluate repeatability during both extraction and PCR stages, and found that both methods were highly consistent for caecal, colon, and faecal samples (rs > 0.7), but had low repeatability for cloacal (rs = 0.39) and ileal (rs = −0.24) samples. This study indicates that direct PCR provides a fast, cheap, and reliable alternative to conventional DNA extraction methods for retrieving 16S data, which will aid future gut microbiome studies of animals.

scholarly journals Identification and Characterization of Novel CircRNAs Involved in Muscle Growth of Blunt Snout Bream (Megalobrama amblycephala)

2021 ◽  
Vol 22 (18) ◽  
pp. 10056
Author(s):  
Lifang Liu ◽  
Yulong Chen ◽  
Jinghan Diao ◽  
Lifei Luo ◽  
Zexia Gao
Keyword(s):  
Muscle Development ◽  
Muscle Growth ◽  
Adherens Junction ◽  
Fish Muscle ◽  
Fish Growth ◽  
Megalobrama Amblycephala ◽  
Circular Rnas ◽  
Blunt Snout Bream ◽  
Fast Growing ◽  
Slow Growing

Circular RNAs (circRNAs), a novel class of endogenous RNAs, have been recognized to play important roles in the growth of animals. However, the regulatory mechanism of circRNAs on fish muscle growth is still unclear. In this study, we performed whole transcriptome analysis of skeletal muscles from two populations with different growth rates (fast-growing and slow-growing) of blunt snout bream (Megalobrama amblycephala), an important fish species for aquaculture. The selected circRNAs were validated by qPCR and Sanger sequencing. Pairs of circRNA–miRNA–mRNA networks were constructed with the predicted differentially expressed (DE) pairs, which revealed regulatory roles in muscle myogenesis and hypertrophy. As a result, a total of 445 circRNAs were identified, including 42 DE circRNAs between fast-growing (FG) and slow-growing (SG) groups. Many of these DE circRNAs were related with aminoglycan biosynthetic and metabolic processes, cytokinetic processes, and the adherens junction pathway. The functional prediction results showed that novel_circ_0001608 and novel_circ_0002886, competing to bind with dre-miR-153b-5p and dre-miR-124-6-5p, might act as competing endogenous RNAs (ceRNAs) to control MamblycephalaGene14755 (pik3r1) and MamblycephalaGene10444 (apip) level, respectively, thus playing an important regulatory role in muscle growth. Overall, these results will not only help us to further understand the novel RNA transcripts in M. amblycephala, but also provide new clues to investigate the potential mechanism of circRNAs regulating fish growth and muscle development.

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