qPCR assays for Alexandrium fundyense and A. ostenfeldii (Dinophyceae) identified from Alaskan waters and a review of species-specific Alexandrium molecular assays

Vandersea, Mark W.; Kibler, Steven R.; Sant, Scott B. Van; Tester, Patricia A.; Sullivan, Kate; Eckert, Ginny L.; Cammarata, Charlayna A.; Reece, K. S.; Scott, Gail P.; Place, Allen R.; Holderied, Kris; Hondolero, Dominic; Litaker, R. Wayne

doi:10.2216/16-41.1

Cited by 33 publications

(24 citation statements)

References 90 publications

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“…Quantitative real-time PCR assays targeting ''harmful'' algal species Alexandrium monilatum, Margalefidinium polykrikoides (previously Cochlodinium), Karlodinium veneficum, Luciella spp., Pfiesteria piscicida, Pseudopfiesteria shumwayae, and Prorocentrum minimum were executed for each of the DNA samples as previously described (VA DEQ 2014, Vandersea et al 2017. For control and standard curve DNA, stocks of each dinoflagellate species were grown, enumerated, and extracted following standard methods (VA DEQ 2014).…”

Section: Microscopic and Quantitative Real-time Polymerase Chain Reacmentioning

confidence: 99%

Tracking Triploid Mortalities of Eastern Oysters Crassostrea virginica in the Virginia Portion of the Chesapeake Bay

Guévélou

Carnegie

Small

et al. 2019

Journal of Shellfish Research

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Since 2012, aquacultured eastern oysters Crassostrea virginica have been reported by oyster farmers to display mortality approaching 30%, and in some cases 85%, in areas of the lower Chesapeake Bay, VA. Based on accounts from industry, this mortality has typically affected 1-y-old oysters between May and early July, and has tended to occur in triploid oysters, which represent the vast bulk of production in the area. During this period, samples submitted for pathology have not revealed the presence of major pathogens as a cause. In 2015, to gain deeper insight into this mortality and determine whether specific sites, ploidy condition, or genetic lines were affected, oyster seed commercially produced in early 2014 were obtained from four lines, one diploid (2N DEBY) and three triploid (3N DEBY, 3N hANA, and 3N Northern). These lines were deployed in July 2014 at aquaculture farms at five Chesapeake Bay locations: Locklies Creek and Milford Haven on the western shore, and Pungoteague Creek, Nassawadox Creek, and Cherrystone Creek on the Eastern Shore. During this study, mortality was observed to peak in June at most sites, reaching a mean mortality across all tested lines of 17.0% and a cumulative mortality for the study period of 32.0% at Nassawadox Creek, the site most severely affected by mortality that followed the expected early summer mortality pattern. Interval mortality at all sites decreased to under 5% after June, but cumulative levels for the study period reached from 8.8% to 18.6% even at the sites least affected by mortality. This represents a high level of mortality given the documented absence of material involvement by major oyster pathogens such as Hapolosporidium nelsoni and Perkinsus marinus. Infiltration of gill tissues by hemocytes, observed in up to 33% of individuals at Nassawadox Creek coincident with the increase in mortality, was the only pathology observed. Harmful algal blooms were not associated with the mortality, nor were abnormal temperatures or salinities. There was no clear relationship of mortality to oyster genetic heritage, although there was variability in susceptibility among oyster lines and interactions between lines and specific sites. At some locations and in comparison with diploids, triploid oysters appeared to be more susceptible to mortality. Mortality in triploids was coincident with the timing of peak gametogenic development in diploids. Given the lack of involvement by major pathogens and the possible association of mortality with oyster gametogenesis, future work should seek to better understand the suite of environmental stressors potentially impacting cultured oysters in these systems and their interactions with the physiology and energetics of these animals.

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Section: Microscopic and Quantitative Real-time Polymerase Chain Reacmentioning

confidence: 99%

Tracking Triploid Mortalities of Eastern Oysters Crassostrea virginica in the Virginia Portion of the Chesapeake Bay

Guévélou

Carnegie

Small

et al. 2019

Journal of Shellfish Research

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“…The internal transcribed spacer (ITS) region, located between the small (18S) and large (28S) subunits of the rRNA gene, is a useful locus for eDNA-based studies of marine eukaryotes. For example, previous studies have targeted the ITS region of harmful microalgae (Andree et al 2011, Vandersea et al 2017, fungi (Op De Beeck et al 2014), parasites (Reece et al 2008) and bivalves (Insua et al 2003, Vierna et al 2010, Lazoski et al 2011, Salvi et al 2014. Wang et al (2006) found that the ITS region was a reliable genetic marker for species identification among 12 commercially harvested bivalves.…”

Section: Introductionmentioning

confidence: 99%

Developing an eDNA toolkit to quantify broadcast spawning events of the sea scallop Placopecten magellanicus: moving beyond fertilization assays

Bayer¹,

Countway²,

Wahle³

2019

Mar. Ecol. Prog. Ser.

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Broadcast spawners release their gametes into the water column for 'chance' fertilization events. However, detection of such events in near real-time is extremely difficult, but needed to improve fisheries and conservation management practices. It is practically impossible to distinguish the gametes of many species by microscopy; therefore, DNA-based techniques are preferable to detect and quantify gametes from field-collected plankton samples. We developed a quantitative PCR (qPCR) approach to detect and quantify broadcast spawning events in marine environmental DNA (eDNA) samples. We applied this approach to a commercially valuable broadcast spawning bivalve species, the sea scallop Placopecten magellanicus. Our approach includes (1) sequencing the internal transcribed spacer (ITS) region, (2) developing a novel species-specific probe and primer set, (3) testing the probe and primer set on a dilution series of sea scallop sperm to quantify the relationship between gamete abundance and DNA copy number, and (4) conducting dockside field tests of our method on plankton samples adjacent to naturally spawning sea scallops. Quantitative PCR revealed a clear relationship between DNA copy number and P. magellanicus sperm cell abundance, indicating that this method is reliable for detecting sperm release by male scallops during spawning events. Plankton samples collected during the scallop spawning season revealed spikes of scallop eDNA in both the < 20 µm (sperm) and > 20 µm (possible eggs) particle size-fractions. This method holds great potential to provide more efficient estimates of the timing, magnitude, and spatial scale of reproductive events than conventional methods for a wide range of broadcast spawners.

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“…They will also prove useful in screening Lugol's preserved, bulk, field samples as described in Vandersea et al. (, ) and Darius et al. ().…”

Section: Average Toxicity Estimates For Gambierdiscus Excentricus Andmentioning

confidence: 99%

“…One isolate of G. silvae was obtained from Curacao (N 12°05.173, W 68°54.004; Curacao Gam11). Genomic DNA was extracted from each isolate and the ITS/5.8S and D1-D3 LSU rDNA regions were PCR amplified and sequenced using the methods described in Vandersea et al (2012Vandersea et al ( , 2017. The resulting ITS and D1-D3 LSU rDNA sequences were assembled using Vector NTI Advance 11 software and submitted to GenBank (MG981052-MG981061).…”

mentioning

confidence: 99%

“…These results indicate the PCR primer sets will be valuable tools for identifying cultured cell isolates and for potentially identifying single cells selected from field samples. They will also prove useful in screening Lugol's preserved, bulk, field samples as described in Vandersea et al (2012Vandersea et al ( , 2017 and Darius et al (2017). Broader screening of environmental samples will allow better characterization of the distribution and habitat preferences of G. excentricus and G. silvae.…”

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confidence: 99%

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Species‐specific PCR assays for Gambierdiscus excentricus and Gambierdiscus silvae (Gonyaulacales, Dinophyceae)

Litaker

Tester

Vandersea

2019

Journal of Phycology

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The two most toxic Gambierdiscus species identified from the Caribbean are G. excentricus and G. silvae. These species are the primary causes of ciguatera fish poisoning and likely contribute disproportionately to the toxicity of marine food webs. While Gambierdiscus species are difficult to distinguish using light or scanning electron microscopy, reliable species‐specific molecular identification methods have been developed and used successfully to identify a number of other Gambierdiscus species. Corresponding species‐specific assays are not yet available for G. excentricus and G. silvae, which imposes limitations on species identification and related ecological studies. The following note describes species‐specific polymerase chain reaction assays for G. excentricus and G. silvae that can be used for these purposes.

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qPCR assays for Alexandrium fundyense and A. ostenfeldii (Dinophyceae) identified from Alaskan waters and a review of species-specific Alexandrium molecular assays

Cited by 33 publications

References 90 publications

Tracking Triploid Mortalities of Eastern Oysters Crassostrea virginica in the Virginia Portion of the Chesapeake Bay

Tracking Triploid Mortalities of Eastern Oysters Crassostrea virginica in the Virginia Portion of the Chesapeake Bay

Developing an eDNA toolkit to quantify broadcast spawning events of the sea scallop Placopecten magellanicus: moving beyond fertilization assays

Species‐specific PCR assays for Gambierdiscus excentricus and Gambierdiscus silvae (Gonyaulacales, Dinophyceae)

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