scholarly journals Persistence and accumulation of environmental DNA from an endangered dragonfly

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kristie J. Schmidt ◽  
Daniel A. Soluk ◽  
Sarah E. Mays Maestas ◽  
Hugh B. Britten
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Environmental Dna ◽  
Target Species ◽  
Calcareous Fens ◽  
Training Time ◽  
Different Temperatures ◽  
Terrestrial Environments ◽  
Experimental Mesocosms ◽  
Polymerase Chain ◽  
Hine's Emerald Dragonfly

AbstractDetection of environmental DNA (eDNA) has become a commonly used surveillance method for threatened or invasive vertebrates in both aquatic and terrestrial environments. However, most studies in this field favor vertebrate target species. Environmental DNA protocols can be especially useful for endangered invertebrates such as the Hine’s emerald dragonfly (Somatochlora hineana) where conservation efforts have been greatly hindered by training, time, overall costs, and environmental impacts associated with conducting surveys in the calcareous fens occupied by this species. An essential step in developing such a protocol is to evaluate the dynamics of eDNA concentration under controlled conditions. We used the quantitative polymerase chain reaction (qPCR) to examine seasonal shifts in the persistence and net-accumulation of eDNA from captive S. hineana larvae in experimental mesocosms at temperatures corresponding with their overwintering (5.0 °C) and active (16.0 °C) seasons. Environmental DNA persisted longer at 5.0 °C but accumulated more readily at 16.0 °C. Differences in the accumulation and persistence of eDNA reflect differences in the longevity of eDNA at different temperatures and seasonal differences in larval S. hineana behavior. This study highlights the importance of considering how seasonal changes in temperature influence not only the speed of eDNA degradation but also the target species’ eDNA shedding rates.

scholarly journals The update and optimization of an eDNA assay to detect the invasive rusty crayfish (Faxonius rusticus)

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0259084
Author(s):  
Stephanie S. Coster ◽  
Megan N. Dillon ◽  
William Moore ◽  
George T. Merovich
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Environmental Dna ◽  
Extraction Methods ◽  
Noninvasive Detection ◽  
Rusty Crayfish ◽  
Central Pennsylvania ◽  
Presence Data ◽  
Invasion Fronts ◽  
Polymerase Chain ◽  
Dna Extraction Methods

Environmental DNA (eDNA) is nuclear or mitochondrial DNA shed into the environment, and amplifying this DNA can serve as a reliable, noninvasive way to monitor aquatic systems for the presence of an invasive species. Assays based on the collection of eDNA are becoming increasingly popular, and, when optimized, can aid in effectively and efficiently tracking invasion fronts. We set out to update an eDNA assay to detect the invasive rusty crayfish, Faxonius rusticus. We tested for species specificity compared to other stream crayfish and field tested the assay at sites with known presence (N = 3) and absence (N = 4) in the Juniata River watershed in central Pennsylvania, USA. To maximize sensitivity, we field tested different storage buffers (Longmire’s buffer and ethanol), DNA extraction methods (Qiagen’s DNEasy and PowerWater kits), and quantitative polymerase chain reaction (qPCR) chemistries (TaqMan and SYBR green). Our assay confirmed the presence data and performed optimally when filter samples were stored in Longmire’s buffer, DNA was extracted with DNeasy Blood and Tissue Kit, and TaqMan qPCR chemistry was utilized. With proper sample processing, our assay allows for accurate, noninvasive detection of F. rusticus in streams.

scholarly journals qPCR Quantification of Pathogenic Guignardia citricarpa and Nonpathogenic G. mangiferae in Citrus

Plant Disease ◽  
2014 ◽  
Vol 98 (1) ◽  
pp. 112-120 ◽  
Author(s):  
Jiahuai Hu ◽  
Evan G. Johnson ◽  
Nan-Yi Wang ◽  
Tiago Davoglio ◽  
Megan M. Dewdney
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Black Spot ◽  
Environmental Dna ◽  
Epidemiological Data ◽  
Fungal Endophyte ◽  
Guignardia Citricarpa ◽  
Polymerase Chain ◽  
Qpcr Quantification ◽  
Citrus Disease ◽  
Single Set

Citrus black spot, a major citrus disease caused by Guignardia citricarpa, was recently introduced in Florida. The nonpathogenic fungal endophyte G. mangiferae is commonly found in the same citrus tissues as G. citricarpa. Quantitative polymerase chain reaction (qPCR) assays based on internal transcribed spacer (ITS)-1 genes were developed to detect, quantify, and distinguish between these morphologically similar organisms in environmental samples. The primer/probe sets GCITS and GMITS were more than 95% efficient in single-set reactions in complex environmental DNA samples. Detection of 10 fg of G. citricarpa and G. mangiferae DNA was possible. Pycnidiospore disruption resulted in detection of single pycnidiospores with 78 (59 to 102; 95% confidence interval [CI]) and 112 (92 to 136; 95% CI) ITS copies for G. citricarpa and G. mangiferae, respectively. Detection was from partially decomposed leaves where fruiting bodies cannot be morphologically distinguished. Temperature and wetting period have significant effects on Guignardia spp. pseudothecia production in leaf litter. Based on relative biomass or the proportion of nuclei detected, G. citricarpa and G. mangiferae respond more strongly to wetting period than temperature. This qPCR assay will provide additional epidemiological data on black spot in tissues where G. citricarpa and G. mangiferae are not easily distinguished.

scholarly journals Monitoring a loss: Detection of the semi‐aquatic crocodile lizard (Shinisaurus crocodilurus) in inaccessible habitats via environmental DNA

2021 ◽  
Vol 4 ◽  
Author(s):  
Patrick Fink ◽  
Timm Reinhardt ◽  
Mona van Schingen ◽  
Heidrun Windisch ◽  
Truong Nguyen ◽  
...  
Keyword(s):  
Census Data ◽  
Southern China ◽  
Quantitative Polymerase Chain Reaction ◽  
Conservation Status ◽  
Environmental Dna ◽  
Evergreen Forest ◽  
Running Water ◽  
Field Surveys ◽  
North East ◽  
Polymerase Chain

Assessing the conservation status of a species is strongly dependent upon data on species distribution and abundance. With the emergence of novel methods for species monitoring – such as the use of environmental DNA (eDNA) – monitoring success can be improved at reduced expenditure in the field, particularly in remote regions and terrains where access is difficult or dangerous. The highly endangered crocodile lizard (Shinisaurus crocodilurus Ahl, 1930) inhabits fragmented sites of the remaining evergreen forest with running water systems in a narrow distribution range in southern China and north‐east Vietnam. Crocodile lizards spend most of the day within or above water bodies, which are commonly remote and inaccessible. To monitor recent spatial occurrences, and to confirm the persistence or extinction of previously reported populations (especially in heavily altered habitats), the suitability of using eDNA and quantitative polymerase chain reaction (qPCR) was tested as an alternative method for monitoring this semiaquatic lizard. To assess the accuracy and limitations of this method, eDNA results from the field were compared with eDNA data from mesocosms and census data on the actual abundance of this species in the field. Environmental DNA of the crocodile lizard was detected in all of the positive controls, and in four of six natural sites; thus, all data collected using traditional field surveys were confirmed with eDNA results. eDNA monitoring was found to be a reliable method for assessing the viability of populations; we suggest that it should be developed as a tool for efficient wildlife management, particularly under difficult field and funding conditions.

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

Pim-1 is up-regulated by constitutively activated FLT3 and plays a role in FLT3-mediated cell survival

Blood ◽  
2005 ◽  
Vol 105 (4) ◽  
pp. 1759-1767 ◽  
Author(s):  
Kyu-Tae Kim ◽  
Kristin Baird ◽  
Joon-Young Ahn ◽  
Paul Meltzer ◽  
Michael Lilly ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Quantitative Polymerase Chain Reaction ◽  
Colony Growth ◽  
Dominant Negative ◽  
Internal Tandem Duplication ◽  
Downstream Target ◽  
Before And After ◽  
Flt3 Expression ◽  
Polymerase Chain ◽  
Expression Microarrays

AbstractConstitutively activating internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) play an important role in leukemogenesis, and their presence is associated with poor prognosis in acute myeloid leukemia (AML). To better understand FLT3 signaling in leukemogenesis, we have examined the changes in gene expression induced by FLT3/ITD or constitutively activated wild-type FLT3 expression. Microarrays were used with RNA harvested before and after inhibition of FLT3 signaling. Pim-1 was found to be one of the most significantly down-regulated genes upon FLT3 inhibition. Pim-1 is a proto-oncogene and is known to be up-regulated by signal transducer and activator of transcription 5 (STAT5), which itself is a downstream target of FLT3 signaling. Quantitative polymerase chain reaction (QPCR) confirmed the microarray results and demonstrated approximately 10-fold decreases in Pim-1 expression in response to FLT3 inhibition. Pim-1 protein also decreased rapidly in parallel with decreasing autophosphorylation activity of FLT3. Enforced expression of either the 44-kDa or 33-kDa Pim-1 isotypes resulted in increased resistance to FLT3 inhibition-mediated cytotoxicity and apoptosis. In contrast, expression of a dominant-negative Pim-1 construct accelerated cytotoxicity in response to FLT3 inhibition and inhibited colony growth of FLT3/ITD-transformed BaF3 cells. These findings demonstrate that constitutively activated FLT3 signaling up-regulates Pim-1 expression in leukemia cells. This up-regulation contributes to the proliferative and antiapoptotic pathways induced by FLT3 signaling. (Blood. 2005;105: 1759-1767)

scholarly journals Identification of Phytoplasmas Representing Multiple New Genetic Lineages from Phloem-Feeding Leafhoppers Highlights the Diversity of Phytoplasmas and Their Potential Vectors

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 352
Author(s):  
Wei Wei ◽  
Valeria Trivellone ◽  
Christopher H. Dietrich ◽  
Yan Zhao ◽  
Kristi D. Bottner-Parker ◽  
...  
Keyword(s):  
Host Range ◽  
Quantitative Polymerase Chain Reaction ◽  
Rflp Analysis ◽  
Natural Habitats ◽  
Genetic Lineages ◽  
Genetic Subgroup ◽  
Polymerase Chain ◽  
Phloem Feeding ◽  
Fresh Insight

Phytoplasmas are obligate transkingdom bacterial parasites that infect a variety of plant species and replicate in phloem-feeding insects in the order Hemiptera, mainly leafhoppers (Cicadellidae). The insect capacity in acquisition, transmission, survival, and host range directly determines the epidemiology of phytoplasmas. However, due to the difficulty of insect sampling and the lack of follow-up transmission trials, the confirmed phytoplasma insect hosts are still limited compared with the identified plant hosts. Recently, quantitative polymerase chain reaction (qPCR)-based quick screening of 227 leafhoppers collected in natural habitats unveiled the presence of previously unknown phytoplasmas in six samples. In the present study, 76 leafhoppers, including the six prescreened positive samples, were further examined to identify and characterize the phytoplasma strains by semi-nested PCR. A total of ten phytoplasma strains were identified in leafhoppers from four countries including South Africa, Kyrgyzstan, Australia, and China. Based on virtual restriction fragment length polymorphism (RFLP) analysis, these ten phytoplasma strains were classified into four distinct ribosomal (16Sr) groups (16SrI, 16SrIII, 16SrXIV, and 16SrXV), representing five new subgroups (16SrI-AO, 16SrXIV-D, 16SrXIV-E, 16SrXIV-F, and 16SrXV-C). The results strongly suggest that the newly identified phytoplasma strains not only represent new genetic subgroup lineages, but also extend previously undiscovered geographical distributions. In addition, ten phytoplasma-harboring leafhoppers belonged to seven known leafhopper species, none of which were previously reported insect vectors of phytoplasmas. The findings from this study provide fresh insight into genetic diversity, geographical distribution, and insect host range of phytoplasmas. Further transmission trials and screening of new potential host plants and weed reservoirs in areas adjacent to collection sites of phytoplasma harboring leafhoppers will contribute to a better understanding of phytoplasma transmission and epidemiology.

scholarly journals Cyclo-oxygenase 2, a putative mediator of vessel remodeling, is expressed in the brain AVM vessels and associates with inflammation

2021 ◽  
Author(s):  
Sara Keränen ◽  
Santeri Suutarinen ◽  
Rahul Mallick ◽  
Johanna P. Laakkonen ◽  
Diana Guo ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Rank Correlation ◽  
Inflammatory Cells ◽  
Vessel Remodeling ◽  
Brain Avm ◽  
Inflammatory Drugs ◽  
Polymerase Chain ◽  
The Brain ◽  
Cox2 Expression

Abstract Background Brain arteriovenous malformations (bAVM) may rupture causing disability or death. BAVM vessels are characterized by abnormally high flow that in general triggers expansive vessel remodeling mediated by cyclo-oxygenase-2 (COX2), the target of non-steroidal anti-inflammatory drugs. We investigated whether COX2 is expressed in bAVMs and whether it associates with inflammation and haemorrhage in these lesions. Methods Tissue was obtained from surgery of 139 bAVMs and 21 normal Circle of Willis samples. The samples were studied with immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR). Clinical data was collected from patient records. Results COX2 expression was found in 78% (109/139) of the bAVMs and localized to the vessels’ lumen or medial layer in 70% (95/135) of the bAVMs. Receptors for prostaglandin E2, a COX2-derived mediator of vascular remodeling, were found in the endothelial and smooth muscle cells and perivascular inflammatory cells of bAVMs. COX2 was expressed by infiltrating inflammatory cells and correlated with the extent of inflammation (r = .231, p = .007, Spearman rank correlation). COX2 expression did not associate with haemorrhage. Conclusion COX2 is induced in bAVMs, and possibly participates in the regulation of vessel wall remodelling and ongoing inflammation. Role of COX2 signalling in the pathobiology and clinical course of bAVMs merits further studies.

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