A TaqMan real-time PCR based assay targeting plaice (Pleuronectes platessa L.) DNA to detect predation by the brown shrimp (Crangon crangon L.) and the shore crab (Carcinus maenas L.)—Assay development and validation

Albaina, Aitor; Fox, Clive; Taylor, Natasha; Hunter, Ewan; Maillard, Marjorie; Taylor, Martin I.

doi:10.1016/j.jembe.2010.06.029

Cited by 33 publications

(39 citation statements)

References 64 publications

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“…This TaqMan probe, which targets a section of the mitochondrial cytochrome-β gene, is highly species-specific and does not cross-react against DNA from a wide range of marine organisms (Albaina et al 2010). Because the BIONESS samples had been rapidly sorted and frozen at sea (due to concerns about postmortem degradation of DNA in predator guts), it was necessary to check their identifications before processing them for molecular analysis.…”

Section: Taqman Analysis Of Stomach Samples and Bioness Samplesmentioning

confidence: 99%

“…TaqMan assays were run on a 7900HT Fast RealTime PCR System (Applied Biosystems) following Albaina et al (2010). Reactions were run in Optical 96-well reaction plates using Optical Adhesive Covers (Applied Biosystems).…”

Section: Taqman Analysis Of Stomach Samples and Bioness Samplesmentioning

confidence: 99%

“…Plates were run under real-time conditions on a single dye layer with 2 no template controls (NTCs) and 2 positive controls (DNA extracted from adult plaice muscle tissue) per 96-well plate. The assay was run using the standard protocol including an initial step of 2 min at 50°C and denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing at 60°C for 60 s. Because PCR inhibitors are often co-extracted from crustacea stomachs, samples returning negative TaqMan results were rerun following dilution and addition of BSA the following guidelines in Albaina et al (2010).…”

Section: Taqman Analysis Of Stomach Samples and Bioness Samplesmentioning

confidence: 99%

“…A positive ΔRn threshold of 0.02 was set for the assay and threshold cycle (Ct) values were computed (Albaina et al 2010); all amplification curves were also checked visually to remove occasional false positive/negative signals.…”

Section: Taqman Analysis Of Stomach Samples and Bioness Samplesmentioning

confidence: 99%

“…Given the extreme sensitivity of these methods, stringent efforts are also needed to control cross-contamination during field sampling (Symondson 2002, Passmore et al 2006. In previous papers we described the development of a TaqMan probe for plaice DNA, demonstrated its specificity and described the effects of digestion by crustacean and teleost predators on detection efficiency (Albaina et al 2010, Hunter et al 2012). Here we describe the first application of this method to study predation of plaice eggs (and larvae) in the field.…”

Section: Introductionmentioning

confidence: 99%

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Identification of marine fish egg predators using molecular probes

Fox¹,

Taylor²,

Kooij³

et al. 2012

Mar. Ecol. Prog. Ser.

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Mortality during the egg and larval stages is thought to play a major role in de termining year-class strength of many marine fish. Predation of eggs and larvae is normally considered to be a major factor but the full suite of predators responsible has rarely been identified. Potential predators on a patch of plaice Pleuronectes platessa eggs located in the eastern Irish Sea were mapped using acoustics and sampled by trawl and a plankton multi-net. Gut contents of 3373 fish, crustacea and cephalopods sampled in the area were screened using a plaice-specific TaqMan DNA probe. Herring Clupea harengus and sprat Sprattus sprattus dominated trawl catches and showed high positive TaqMan responses (77 and 75% of individuals tested respectively). Locations of clupeid schools also broadly corresponded with the distribution of fish eggs in the plankton. Whiting Merlangius merlangus were also reasonably abundant in trawl hauls and 86% of their stomachs tested positive for plaice DNA. Species showing lower levels of positive TaqMan response included mackerel Scomber scombrus, poor cod Trisopterus minutus, squid Loligo spp., dogfish Scyliorhinus canicula and weever Trachinus vipera. Nonre activity of all negative controls precluded the occurrence of cross-contamination, and positive reactions from demersal species, such as dogfish and weever, may have resulted from secondary predation. No benthic macro-crustaceans tested positive. Samples of planktonic organisms yielded 13% positive TaqMan reactions, mainly from clupeoid or sandeel (Ammodytidae) larvae, but also included some Malacostraca and Amphipoda. Use of the molecular approach allowed rapid screening of a large number of potential predators of plaice eggs, and the results provide a more holistic description of the predator community than has previously been achieved.

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Section: Taqman Analysis Of Stomach Samples and Bioness Samplesmentioning

confidence: 99%

Section: Taqman Analysis Of Stomach Samples and Bioness Samplesmentioning

confidence: 99%

Section: Taqman Analysis Of Stomach Samples and Bioness Samplesmentioning

confidence: 99%

Section: Taqman Analysis Of Stomach Samples and Bioness Samplesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Identification of marine fish egg predators using molecular probes

Fox¹,

Taylor²,

Kooij³

et al. 2012

Mar. Ecol. Prog. Ser.

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Evaluation of an automated protocol for efficient and reliable DNA extraction of dietary samples

Wallinger

Staudacher

Sint

et al. 2017

Ecology and Evolution

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Molecular techniques have become an important tool to empirically assess feeding interactions. The increased usage of next‐generation sequencing approaches has stressed the need of fast DNA extraction that does not compromise DNA quality. Dietary samples here pose a particular challenge, as these demand high‐quality DNA extraction procedures for obtaining the minute quantities of short‐fragmented food DNA. Automatic high‐throughput procedures significantly decrease time and costs and allow for standardization of extracting total DNA. However, these approaches have not yet been evaluated for dietary samples. We tested the efficiency of an automatic DNA extraction platform and a traditional CTAB protocol, employing a variety of dietary samples including invertebrate whole‐body extracts as well as invertebrate and vertebrate gut content samples and feces. Extraction efficacy was quantified using the proportions of successful PCR amplifications of both total and prey DNA, and cost was estimated in terms of time and material expense. For extraction of total DNA, the automated platform performed better for both invertebrate and vertebrate samples. This was also true for prey detection in vertebrate samples. For the dietary analysis in invertebrates, there is still room for improvement when using the high‐throughput system for optimal DNA yields. Overall, the automated DNA extraction system turned out as a promising alternative to labor‐intensive, low‐throughput manual extraction methods such as CTAB. It is opening up the opportunity for an extensive use of this cost‐efficient and innovative methodology at low contamination risk also in trophic ecology.

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A real‐time PCR assay to detect predation by spiny dogfish on Atlantic cod in the western North Atlantic Ocean

Pitchford

Smith

McBride

2020

Ecology and Evolution

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Conventional observations show spiny dogfish ( Squalus acanthius Linnaeus) rarely eat Atlantic cod ( Gadus morhua Linnaeus; 0.02% of stomachs) in the northwestern Atlantic Ocean. Critics express concern that digestion may limit species‐level prey identification, and with recovery from overfishing, dogfish populations may be suppressing cod by competition or predation. This study applied a real‐time PCR TaqMan assay to identify cod in dogfish stomachs collected by cooperating fishing boats during normal trawling operations (May 2014–May 2015; Gulf of Maine, Georges Bank). Conventional methods observed 51 different prey taxa and nearly 1,600 individual prey items, but no cod were observed. Cod DNA was detected in 31 (10.5%) of the dogfish stomachs, with a higher percentage of these from the homogenate of amorphous, well‐digested prey and stomach fluids (20 stomachs or 65%) than from discrete animal tissues (11 stomachs or 35%). Re‐examination of photographs of these 11 tissue samples revealed one whole, partially digested fish that could be recognized in hindsight as cod. Cod DNA was observed in dogfish stomachs year round: in January (1 of 1 trip), February (1 of 1), May (1 of 3), June (0 of 1), July (3 of 4), August (1 of 2), and October (3 of 3). Although these data suggest higher interaction rates between dogfish and cod than previously observed, addressing the population consequences of this predator–prey relationship requires a robust sampling design, estimates of digestion rates by dogfish to account for complete degradation of DNA sequences, and consideration for dogfish scavenging during fishing operations.

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A TaqMan real-time PCR based assay targeting plaice (Pleuronectes platessa L.) DNA to detect predation by the brown shrimp (Crangon crangon L.) and the shore crab (Carcinus maenas L.)—Assay development and validation

Cited by 33 publications

References 64 publications

Identification of marine fish egg predators using molecular probes

Identification of marine fish egg predators using molecular probes

Evaluation of an automated protocol for efficient and reliable DNA extraction of dietary samples

A real‐time PCR assay to detect predation by spiny dogfish on Atlantic cod in the western North Atlantic Ocean

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