scholarly journals Combining metabarcoding and morphological approaches to identify phytoplankton taxa associated with harmful algal blooms

2019 ◽  
Author(s):  
Svetlana Esenkulova ◽  
Ben J.G. Sutherland ◽  
Amy Tabata ◽  
Nicola Haigh ◽  
Christopher M. Pearce ◽  
...  
Keyword(s):  
Harmful Algal Blooms ◽  
Algal Blooms ◽  
Quantitative Polymerase Chain Reaction ◽  
Large Subunit ◽  
Molecular Techniques ◽  
Alexandrium Tamarense ◽  
Morphological Identification ◽  
Multiple Markers ◽  
Molecular Approaches ◽  
Polymerase Chain

AbstractMolecular techniques are expected to be highly useful in detecting taxa causing harmful algal blooms (HABs). This is the first report in Canada evaluating HABs-related species identification using a combination of morphological and molecular approaches. Microscopy, quantitative polymerase chain reaction (qPCR), and metabarcoding with multiple markers (i.e. 16S, 18S-dinoflagellate and 18S-diatom, large subunit (28S) rDNA) were applied on samples (n=54) containing suspected harmful algae (e.g. Alexandrium spp., Chattonella sp., Chrysochromulina spp., Dictyocha spp., Heterosigma akashiwo, Protoceratium reticulatum, Pseudochattonella verruculosa, Pseudo-nitzschia spp., Pseudopedinella sp.). Due to methodology limitations, qPCR result interpretation was limited, although good detectability occurred using previously published assays for Alexandrium tamarense, H. akashiwo, and P. verruculosa. Overall, the multiple-marker metabarcoding results were superior to the morphology-based methods, with the exception of taxa from the silicoflagellate group. The combined results using both 18S markers and the 28S marker together closely corresponded with morphological identification of targeted species, providing the best overall taxonomic coverage and resolution. The most numerous unique taxa were identified using the 18S-dinoflagellate amplicon, and the best resolution to the species level occurred using the 28S amplicon. Molecular techniques are therefore highly useful for HABs taxa detection, but currently depend on deploying multiple markers for metabarcoding.

scholarly journals Comparing metabarcoding and morphological approaches to identify phytoplankton taxa associated with harmful algal blooms

FACETS ◽  
2020 ◽  
Vol 5 (1) ◽  
pp. 784-811
Author(s):  
Svetlana Esenkulova ◽  
Ben J.G. Sutherland ◽  
Amy Tabata ◽  
Nicola Haigh ◽  
Christopher M. Pearce ◽  
...  
Keyword(s):  
Harmful Algal Blooms ◽  
Algal Blooms ◽  
Quantitative Polymerase Chain Reaction ◽  
Large Subunit ◽  
Molecular Techniques ◽  
Alexandrium Tamarense ◽  
Morphological Identification ◽  
Multiple Markers ◽  
Molecular Approaches ◽  
Polymerase Chain

Molecular techniques are expected to be highly useful in detecting taxa causing harmful algal blooms (HABs). This is the first report in Canada evaluating HABs-related species identification using a combination of morphological and molecular approaches. Microscopy, quantitative polymerase chain reaction (qPCR), and metabarcoding with multiple markers (i.e., 16S, 18S-dinoflagellate and 18S-diatom, large subunit (28S) rDNA) were applied on samples ( n = 54) containing suspected harmful algae (e.g., Alexandrium spp., Chattonella sp., Chrysochromulina spp., Dictyocha spp., Heterosigma akashiwo, Protoceratium reticulatum, Pseudochattonella verruculosa, Pseudo-nitzschia spp., Pseudopedinella sp.). Owing to methodology limitations, qPCR result interpretation was limited, although good detectability occurred using previously published assays for Alexandrium tamarense, H. akashiwo, and P. verruculosa. Overall, the multiple-marker metabarcoding results were superior to the morphology-based methods, with the exception of taxa from the silicoflagellate group. The combined results using both 18S markers and the 28S marker together closely corresponded with morphological identification of targeted species, providing the best overall taxonomic coverage and resolution. The most numerous unique taxa were identified using the 18S-dinoflagellate amplicon, and the best resolution to the species level occurred using the 28S amplicon. Molecular techniques are therefore promising for HABs taxa detection but currently depend on deploying multiple markers for metabarcoding.

scholarly journals Mapping the Distribution of Cysts from the Toxic Dinoflagellate Cochlodinium polykrikoides in Bloom-Prone Estuaries by a Novel FluorescenceIn SituHybridization Assay

2015 ◽  
Vol 82 (4) ◽  
pp. 1114-1125 ◽  
Author(s):  
Theresa K. Hattenrath-Lehmann ◽  
Yu Zhen ◽  
Ryan B. Wallace ◽  
Ying-Zhong Tang ◽  
Christopher J. Gobler
Keyword(s):  
Harmful Algal Blooms ◽  
Algal Blooms ◽  
Large Subunit ◽  
Cross Reactivity ◽  
Epifluorescence Microscopy ◽  
Lsu Rdna ◽  
Cochlodinium Polykrikoides ◽  
Content Type ◽  
Ecosystem Studies ◽  
Resting Cysts

ABSTRACTCochlodinium polykrikoidesis a cosmopolitan dinoflagellate that is notorious for causing fish-killing harmful algal blooms (HABs) across North America and Asia. While recent laboratory and ecosystem studies have definitively demonstrated thatCochlodiniumforms resting cysts that may play a key role in the dynamics of its HABs, uncertainties regarding cyst morphology and detection have prohibited even a rudimentary understanding of the distribution ofC. polykrikoidescysts in coastal ecosystems. Here, we report on the development of a fluorescencein situhybridization (FISH) assay using oligonucleotide probes specific for the large subunit (LSU) ribosomal DNA (rDNA) ofC. polykrikoides. The LSU rDNA-targeted FISH assay was used with epifluorescence microscopy and was iteratively refined to maximize the fluorescent reaction withC. polykrikoidesand minimize cross-reactivity. The final LSU rDNA-targeted FISH assay was found to quantitatively recover cysts made by North American isolates ofC. polykrikoidesbut not cysts formed by other common cyst-forming dinoflagellates. The method was then applied to identify and mapC. polykrikoidescysts across bloom-prone estuaries. Annual cyst and vegetative cell surveys revealed that elevated densities ofC. polykrikoidescysts (>100 cm−3) during the spring of a given year were spatially consistent with regions of dense blooms the prior summer. The identity of cysts in sediments was confirmed via independent amplification ofC. polykrikoidesrDNA. This study mappedC. polykrikoidescysts in a natural marine setting and indicates that the excystment of cysts formed by this harmful alga may play a key role in the development of HABs of this species.

scholarly journals Study on the metabolites of DH-e, a Halomonas marine bacterium, against three toxic dinoflagellate species

2018 ◽  
Vol 78 (7) ◽  
pp. 1535-1544 ◽  
Author(s):  
Di Wang ◽  
Liling Xie ◽  
Xingbiao Zhu ◽  
Xiao Bi ◽  
Yuzhong Zheng ◽  
...  
Keyword(s):  
Harmful Algal Blooms ◽  
Algal Blooms ◽  
Morphological Characteristics ◽  
Alexandrium Tamarense ◽  
Algicidal Bacteria ◽  
Bacterial Strains ◽  
16S Rdna Sequence ◽  
Algicidal Bacterium ◽  
16S Rdna Sequence Analysis ◽  
Acetone Peroxide

Abstract Algicidal bacteria play an important role in mitigating harmful algal blooms (HABs). In the study, five bacterial strains were isolated from the East China Sea. One strain of algicidal bacterium, named DH-e, was found to selectively inhibit the motor ability of Prorocentrum donghaiense, Alexandrium tamarense (ATDH-47) and Karenia mikimotoi Hansen. Both 16S rDNA sequence analysis and morphological characteristics revealed that the algicidal DH-e bacterium belonged to Halomonas. Furthermore, results showed that the metabolites in the DH-e cell-free filtrate could kill algae directly, and the minimum inhibitory concentrations (MICs) of the bacterial metabolites on the cells of the three dinoflagellate species ranged from 35.0–70.0 μg/mL. Following short-term inhibitory tests, the dinoflagellates in mixed crude extract solution (0.7 mg/mL) ceased movement after 5 min. The algicidal mechanism of the metabolites was investigated through enzyme activities, including that of catalase (CAT), alkaline phosphatase (AKP), acetone peroxide (T-ATP) synthetase and nitrite reductase (NR). Results indicated that metabolites did not disrupt the energy or nutrient routes of the algae (P > 0.05), but did initiate an increase in free radicals in the algal cells, which might explain the subsequent death of sensitive algae. Thus, the metabolites of the DH-e bacterium showed promising potential for controlling HABs.

Molecular diagnosis of fungal disease

2017 ◽  
Author(s):  
P. Lewis White ◽  
Rosemary A. Barnes
Keyword(s):  
Invasive Fungal Disease ◽  
Fungal Disease ◽  
Molecular Techniques ◽  
Disease Manifestation ◽  
Molecular Tests ◽  
Molecular Approaches ◽  
Widespread Acceptance ◽  
Recent Developments ◽  
Emerging Issues ◽  
Polymerase Chain

Molecular techniques to aid in the diagnosis of fungal disease have been in use for over two decades. However, for polymerase chain reaction (PCR) to gain widespread acceptance outside of specialist centres, methodology must be standardized and in line with general microbiological molecular diagnostics assays, yet for infections other than fungal disease. Apart from Aspergillus PCR, standardized methodology is lacking. It is also essential to identify the optimal role for an assay. Whether this is to confirm a specific disease in symptomatic patients or to exclude disease and prevent the unnecessary use of antifungals will, in part, be determined by prevalence, but will also, along with the disease manifestation, dictate specimen choice and subsequent methodological procedure. This chapter will focus on disease processes determining optimal sample types, before concentrating on the clinical validation of molecular tests for the diagnosis of the main causes of invasive fungal disease, concluding with recent developments. The clinical utility of molecular approaches and potential future benefits that can address emerging issues, such as azole resistance, will also be discussed.

scholarly journals A Brief History of Endophyte Detection Techniques in Grasses

2019 ◽  
Vol 8 (3) ◽  
pp. 66
Author(s):  
Rana Zaheer Ahmad ◽  
Fuad Ameen ◽  
Rida Khalid ◽  
Mousa A. Alghuthaymi ◽  
Reem Alsalmi ◽  
...  
Keyword(s):  
Molecular Phylogenetics ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Economic Value ◽  
Molecular Techniques ◽  
Morphological Identification ◽  
Detection Techniques ◽  
Molecular Approaches ◽  
Gradient Gel Electrophoresis ◽  
History Of ◽  
Traditional Approaches

Endophytes are the plant mutualists that live asymptomatically inside plant tissue and are found in nearly whole plant kingdom. Endophytic fungi receive shelter and nutrition from host plants and in return provide great advantages to the host. Grasses are a useful forage species and are of great agricultural and socio-economic value. The presence of endophytes in these grasses provide protection, persistence and improved yield against herbivores, insects, pathogens, drought and several other biotic and abiotic stresses. This review summarizes traditional and modern molecular techniques to identify endophytes from turf and forage grasses. Traditional approaches include direct observation, staining, laser micro dissection and pressure catapulting and cultivation-dependent methods that provide a morphological identification of endophytic mycobiota in grass tissues. Earlier studies on endophytes using these methods resulted in several technical implications which molecular approaches are able to solve now-a-days. Molecular approaches include DNA extraction, PCR based DNA Fingerprinting techniques, Denaturing Gradient Gel Electrophoresis, Sanger sequencing, Pyrosequencing, Immunoblot assay, Biosensors, DNA Barcoding and Molecular Phylogenetics etc. A comparison of these detection techniques will facilitate other researchers as well to develop new ways for the detection of endophytes that will contribute to the improvement of grassland in future.

Molecular identification of zoonotic hookworm species in dog faeces from Tanzania

2018 ◽  
Vol 93 (3) ◽  
pp. 313-318 ◽  
Author(s):  
A. Merino-Tejedor ◽  
P. Nejsum ◽  
E.M. Mkupasi ◽  
M.V. Johansen ◽  
Annette Olsen
Keyword(s):  
Sanger Sequencing ◽  
Molecular Techniques ◽  
Health Concern ◽  
Specific Pcr ◽  
Molecular Approaches ◽  
Faecal Samples ◽  
Pcr Rflp ◽  
Veterinary Importance ◽  
Polymerase Chain ◽  
Species Specific

AbstractThe presence and distribution of various species of canine hookworms in Africa are poorly known. The main objective of this study, therefore, was to identify the hookworm species present in canine faecal samples from Morogoro, Tanzania, using molecular techniques. Faecal samples from 160 local dogs were collected and hookworm positive samples processed to recover larvae for further molecular characterization. DNA was extracted from pools of larvae from individual samples (n = 66), which were analysed subsequently using two different molecular approaches, polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) and species-specific PCR coupled with Sanger sequencing. The PCR-RFLP technique detected only the presence of the ubiquitousAncylostoma caninumin the 66 samples. However, by species-specific PCR coupled with Sanger sequencing we identified ten samples withA. braziliense, two withUncinaria stenocephalaand five withA. ceylanicum. Thus, all four known species of canine hookworms were identified in Morogoro, Tanzania. To our knowledge this is the first report of the detection of the presence ofU. stenocephalaandA. ceylanicumin Africa using molecular techniques. In addition to their veterinary importance, canine hookworms have zoonotic potential and are of public health concern.

scholarly journals Intraspecific facilitation by allelochemical mediated grazing protection within a toxigenic dinoflagellate population

2015 ◽  
Vol 282 (1798) ◽  
pp. 20141268 ◽  
Author(s):  
Uwe John ◽  
Urban Tillmann ◽  
Jennifer Hülskötter ◽  
Tilman J. Alpermann ◽  
Sylke Wohlrab ◽  
...  
Keyword(s):  
Harmful Algal Blooms ◽  
Algal Blooms ◽  
Phenotypic Diversity ◽  
Marine Ecosystem ◽  
Paralytic Shellfish Poisoning ◽  
Alexandrium Tamarense ◽  
Shellfish Poisoning ◽  
Coastal Marine ◽  
The Impact ◽  
Intraspecific Facilitation

Dinoflagellates are a major cause of harmful algal blooms (HABs), with consequences for coastal marine ecosystem functioning and services. Alexandrium fundyense (previously Alexandrium tamarense ) is one of the most abundant and widespread toxigenic species in the temperate Northern and Southern Hemisphere and produces paralytic shellfish poisoning toxins as well as lytic allelochemical substances. These bioactive compounds may support the success of A. fundyense and its ability to form blooms. Here we investigate the impact of grazing on monoclonal and mixed set-ups of highly (Alex2) and moderately (Alex4) allelochemically active A. fundyense strains and a non-allelochemically active conspecific (Alex5) by the heterotrophic dinoflagellate Polykrikos kofoidii . While Alex4 and particularly Alex5 were strongly grazed by P. kofoidii when offered alone, both strains grew well in the mixed assemblages (Alex4 + Alex5 and Alex2 + Alex5). Hence, the allelochemical active strains facilitated growth of the non-active strain by protecting the population as a whole against grazing. Based on our results, we argue that facilitation among clonal lineages within a species may partly explain the high genotypic and phenotypic diversity of Alexandrium populations. Populations of Alexandrium may comprise multiple cooperative traits that act in concert with intraspecific facilitation, and hence promote the success of this notorious HAB species.

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