Quantitative PCR assays to detect humpback whale (Megaptera novaeangliae), shortbelly rockfish (Sebastes jordani), and common murre (Uria aalge) in marine water samples

AbstractMonitoring aquatic species by identification of environmental DNA (eDNA) is becoming more common. In order to obtain quantitative datasets for individual species, species-specific quantitative PCR (qPCR) assays are required. Here, we present detailed methodology of qPCR assay design and testing, including in silico, in vitro, and in vivo testing, and comment on the challenges associated with assay design and performance. We use the presented methodology to design assays for three important marine organisms common in the California Current Ecosystem (CCE): humpback whale (Megaptera novaeangliae), shortbelly rockfish (Sebastes jordani), and common murre (Uria aalge). All three assays have excellent sensitivity and high efficiencies ranging from 92% to 99%. However, specificities of the assays varied from species-specific in the case of common murre to the genus-specific shortbelly rockfish assay, to the humpback whale assay which cross-amplified with other two other whale species, including one in a different family. All assays detected their associated targets in complex environmental water samples.

Download Full-text

Quantitative PCR assays to detect whales, rockfish, and common murre environmental DNA in marine water samples of the Northeastern Pacific

PLoS ONE ◽

10.1371/journal.pone.0242689 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0242689

Author(s):

Elizabeth A. Andruszkiewicz ◽

Kevan M. Yamahara ◽

Collin J. Closek ◽

Alexandria B. Boehm

Keyword(s):

Quantitative Pcr ◽

Water Samples ◽

Environmental Dna ◽

Humpback Whale ◽

Environmental Water ◽

Individual Species ◽

Common Murre ◽

Assay Design

Monitoring aquatic species by identification of environmental DNA (eDNA) is becoming more common. To obtain quantitative eDNA datasets for individual species, organism-specific quantitative PCR (qPCR) assays are required. Here, we present detailed methodology of qPCR assay design and testing, including in silico, in vitro, and in vivo testing, and comment on the challenges associated with assay design and performance. We use the presented methodology to design assays for three important marine organisms common in the California Current Ecosystem (CCE): humpback whale (Megaptera novaeangliae), shortbelly rockfish (Sebastes jordani), and common murre (Uria aalge). All three assays have excellent sensitivity and high efficiencies ranging from 92% to 99%. However, specificities of the assays varied from species-specific in the case of common murre, genus-specific for the shortbelly rockfish assay, and broadly whale-specific for the humpback whale assay, which cross-amplified with other two other whale species, including one in a different family. All assays detected their associated targets in complex environmental water samples.

Download Full-text

Gluconeogenesis in liver and kidney of common murre (Uria aalge)

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1988.254.6.r903 ◽

1988 ◽

Vol 254 (6) ◽

pp. R903-R907 ◽

Cited By ~ 3

Author(s):

G. R. Herzberg ◽

J. T. Brosnan ◽

B. Hall ◽

M. Rogerson

Keyword(s):

Amino Acids ◽

Phosphoenolpyruvate Carboxykinase ◽

Uria Aalge ◽

Kidney Tubules ◽

Major Site ◽

Common Murre ◽

Liver And Kidney ◽

Gluconeogenic Substrates ◽

Better Than

Phosphoenolpyruvate carboxykinase (PEPCK) in murre liver occurs in both cytoplasmic and mitochondrial forms. During a 3-day fast, hepatic PEPCK increases from 9.1 U/g with 19% cytosolic to 12.2 U/g with 35% cytosolic. The increase in activity is due almost entirely to increased cytosolic activity. PEPCK in murre kidney was present only in the mitochondrial compartment. Gluconeogenesis in vitro was determined in both hepatocytes and kidney tubules isolated from 3-day-fasted murres. In hepatocytes, lactate was the best substrate, but both pyruvate and alanine were good gluconeogenic substrates. This observation is consistent with the existence of a cytosolic form of PEPCK. In the kidney, glycerol was the best substrate but was only slightly better than lactate. Alanine and pyruvate were not as effective as gluconeogenic precursors, presumably because of the lack of cytosolic PEPCK. We propose that the major site of gluconeogenesis from amino acids in the murre is the liver, since this is a much larger organ than the kidney and has a cytosolic form of PEPCK necessary for gluconeogenesis from oxidized substrates.

Download Full-text

Field Detection ofSchistosoma japonicumCercariae in Environmental Water Samples by Quantitative PCR

Applied and Environmental Microbiology ◽

10.1128/aem.01561-10 ◽

2011 ◽

Vol 77 (6) ◽

pp. 2192-2195 ◽

Cited By ~ 21

Author(s):

Caitlin Worrell ◽

Ning Xiao ◽

Jorge E. Vidal ◽

Lin Chen ◽

Bo Zhong ◽

...

Keyword(s):

Quantitative Pcr ◽

Surface Waters ◽

Water Samples ◽

Sampling Methods ◽

Environmental Water ◽

Quantitative Detection ◽

Mouse Bioassay ◽

The Novel ◽

Water Sampling ◽

Species Specific

ABSTRACTA species-specific quantitative PCR (qPCR) assay was combined with two novel water-sampling methods and compared with the mouse bioassay for the quantitative detection ofS. japonicumin surface waters. The novel methods were capable of capturing cercariae and, with subsequent analysis through qPCR, detecting the presence of a minimum of 1 cercaria.

Download Full-text

The efficacy of antimicrobial agents is decreased in a polymicrobial environment

10.1101/2021.07.20.453069 ◽

2021 ◽

Author(s):

Thomas James O'Brien ◽

Wendy Figueroa ◽

Martin Welch

Keyword(s):

Antimicrobial Agents ◽

Individual Species ◽

Nucleotide Polymorphisms ◽

Loss Of Function ◽

Airway Infections ◽

Genes Encoding ◽

Pilus Biogenesis ◽

The Individual ◽

Species Specific

The airways of people with cystic fibrosis (CF) often harbour diverse polymicrobial communities. These airway infections can be impossible to resolve though antibiotic intervention, even though isolates of the individual species present are susceptible to the treatment when tested in vitro. This suggests that susceptibility to antimicrobial agents may be altered in the presence of other microbial species. In this work, we investigate how polymicrobial cultures of key CF-associated species respond to challenge with species-specific antimicrobial agents; colistin (targets Pseudomonas aeruginosa), fusidic acid (targets Staphylococcus aureus) and fluconazole (targets Candida albicans). We found that, compared with growth in axenic cultures, the target organism was protected (sometimes by several orders of magnitude) from the effect(s) of the antimicrobial agent when grown in a polymicrobial culture. This decreased antimicrobial efficacy in polymicrobial cultures was found to have both phenotypic and inherited components. Whole genome sequencing of the colistin-resistant P. aeruginosa isolates revealed single nucleotide polymorphisms and indels in genes encoding lipopolysaccharide (LPS) biosynthesis or pilus biogenesis. Colistin resistance associated with loss-of-function mutations in the LPS biosynthetic gene, wzy, could be complemented by expression of the wild-type wzy gene in trans. Our findings indicate that the polymicrobial nature of the CF airways is likely to have a significant impact on the clinical response to antimicrobial therapy.

Download Full-text

Development and validation of a quantitative qPCR assay for detecting Natterjack toad (Epidalea calamita) eDNA samples

Conservation Genetics Resources ◽

10.1007/s12686-021-01199-3 ◽

2021 ◽

Author(s):

Marina Reyne ◽

Amanda M. Naaum ◽

Ferdia Marnell ◽

Neil Reid ◽

Sarah J. Helyar

Keyword(s):

Water Samples ◽

Rapid Assessment ◽

Pond Water ◽

Qpcr Assay ◽

Amphibian Species ◽

Tissue Samples ◽

Species Specific ◽

Epidalea Calamita

AbstractThe Natterjack toad (Epidalea calamita) is the rarest amphibian species in Ireland, regionally Red-Listed as Endangered. We applied an eDNA approach to detect species presence in breeding pond water samples. We developed a species-specific qPCR assay targeting the cytochrome c oxidase subunit I (COI). The assay was tested in silico, in vitro (DNA extracted from tissue) and in vivo (DNA extracted from water samples). Water samples were collected from five ponds with known Natterjack toad presence or absence to validate the sensitivity and specificity of the assay. The assay was shown to be highly specific to the Natterjack toad and tested positive only against toad tissue samples and eDNA samples from ponds with known species presence. We believe this method can be used for rapid assessment of species occurrence.

Download Full-text