scholarly journals Culicidae-centric metabarcoding through targeted use of D2 ribosomal DNA primers

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9057 ◽  
Author(s):  
Pedro M. Pedro ◽  
Jandui Amorim ◽  
Martha V.R. Rojas ◽  
Ivy Luizi Sá ◽  
Allan Kardec Ribeiro Galardo ◽  
...  
Keyword(s):  
Sampling Methods ◽  
Mosquito Species ◽  
Pcr Primers ◽  
Degraded Dna ◽  
Ribosomal Operon ◽  
Dna Pools ◽  
Current Sampling ◽  
High Concentrations ◽  
Dna Primers ◽  
Generation Sequencing

A practical limitation to many metabarcoding initiatives is that sampling methods tend to collect many non-target taxa, which become “amplicon noise” that can saturate Next Generation Sequencing results and lead to both financial and resource inefficiencies. An available molecular tool that can significantly decrease these non-target amplicons and decrease the need for pre-DNA-extraction sorting of bycatch is the design of PCR primers tailored to the taxa under investigation. We assessed whether the D2 extension segment of the 28S ribosomal operon can limit this shortcoming within the context of mosquito (Culicidae) monitoring. We designed PCR primers that are fully conserved across mosquitos and exclude from amplification most other taxa likely to be collected with current sampling apparatuses. We show that, given enough sequencing depth, D2 is an effective marker for the detection of mosquito sequences within mock genomic DNA pools. As few as 3,050 quality-filtered Illumina reads were able to recover all 17 species in a bulk pool containing as little as 0.2% of constituent DNA from single taxa. We also mixed these mosquito DNA pools with high concentrations of non-Culicidae bycatch DNA and show that the component mosquito species are generally still recoverable and faithful to their original relative frequencies. Finally, we show that there is little loss of fidelity in abundance parameters when pools from degraded DNA samples were sequenced using the D2 primers.

Transcriptional control of delta-crystallin gene expression in the chicken embryo lens: demonstration by a new method for measuring mRNA metabolism

1993 ◽  
Vol 13 (6) ◽  
pp. 3282-3290
Author(s):  
X Li ◽  
D C Beebe
Keyword(s):  
Chicken Embryo ◽  
Transcriptional Control ◽  
Cultured Cells ◽  
Pcr Primers ◽  
High Refractive Index ◽  
In Vitro Transcription ◽  
Equilibrium Density ◽  
Specific Pcr ◽  
High Concentrations

Crystallins are proteins that accumulate to very high concentrations in the fiber cells of the lens of the eye. Crystallins are responsible for the transparency and high refractive index that are essential for lens function. In the chicken embryo, delta-crystallin accounts for more than 70% of the newly synthesized lens proteins. We used density labeling and gene-specific polymerase chain reaction (PCR) to determine the mechanism regulating the expression of the two very similar delta-crystallin genes. Newly synthesized RNA was separated from preexisting RNA by incubating the lenses with 15N- and 13C-labeled ribonucleosides and then separating newly synthesized, density-labeled RNA from the bulk of light RNA by equilibrium density centrifugation in NaI-KI gradients. The relative abundances of the two crystallin mRNAs in the separated fractions were then determined by PCR. This method permitted the quantitation of newly synthesized processed and unprocessed delta-crystallin mRNAs. Additional studies used intron- and gene-specific PCR primers to determine the relative expression of the two delta-crystallin genes in processed RNA and unprocessed RNA extracted from different regions of the embryonic lens. Results of these tests indicated that the differential expression of the delta-crystallin genes was regulated primarily at the level of transcription. This outcome was not expected on the basis of the results of previous studies, which used in vitro transcription and transfection methods to evaluate the relative strengths of delta-crystallin promoter and enhancer sequences. Our data suggest that the cultured cells used in these earlier studies may not have provided an accurate view of delta-crystallin regulation in the intact lens.

scholarly journals Isolating Chromosomes of the Komodo Dragon: New Tools for Comparative Mapping and Sequence Assembly

2019 ◽  
Vol 157 (1-2) ◽  
pp. 123-131 ◽  
Author(s):  
Alessio Iannucci ◽  
Marie Altmanová ◽  
Claudio Ciofi ◽  
Malcolm Ferguson-Smith ◽  
Jorge C. Pereira ◽  
...  
Keyword(s):  
Karyotype Evolution ◽  
Sequencing Technology ◽  
Next Generation Sequencing Technology ◽  
Flow Karyotype ◽  
Flow Sorting ◽  
Dna Pools ◽  
Phylogenomic Analyses ◽  
Cross Species Chromosome Painting ◽  
Chromosomal Map ◽  
Generation Sequencing

We developed new tools to build a high-quality chromosomal map of the Komodo dragon (Varanus komodoensis) available for cross-species phylogenomic analyses. First, we isolated chromosomes by flow sorting and determined the chromosome content of each flow karyotype peak by FISH. We then isolated additional Komodo dragon chromosomes by microdissection and amplified chromosome-specific DNA pools. The chromosome-specific DNA pools can be sequenced, assembled, and mapped by next-generation sequencing technology. The chromosome-specific paint probes can be used to investigate karyotype evolution through cross-species chromosome painting. Overall, the set of chromosome-specific DNA pools of V. komodoensis provides new tools for detailed phylogenomic analyses of Varanidae and squamates in general.

scholarly journals Analysis of the Type IV Fimbrial-Subunit GenefimA of Xanthomonas hyacinthi: Application in PCR-Mediated Detection of Yellow Disease in Hyacinths

2001 ◽  
Vol 67 (2) ◽  
pp. 598-607 ◽  
Author(s):  
J. van Doorn ◽  
T. C. Hollinger ◽  
B. Oudega
Keyword(s):  
Xanthomonas Campestris ◽  
Pcr Primers ◽  
Structural Type ◽  
Nucleotide Sequences ◽  
Amino Acid Sequences ◽  
Fimbrial Subunit ◽  
Type Iv ◽  
Moraxella Bovis ◽  
Xanthomonas Translucens ◽  
Dna Primers

ABSTRACT A sensitive and specific detection method was developed forXanthomonas hyacinthi; this method was based on amplification of a subsequence of the type IV fimbrial-subunit genefimA from strain S148. The fimA gene was amplified by PCR with degenerate DNA primers designed by using the N-terminal and C-terminal amino acid sequences of trypsin fragments of FimA. The nucleotide sequence of fimA was determined and compared with the nucleotide sequences coding for the fimbrial subunits in other type IV fimbria-producing bacteria, such as Xanthomonas campestris pv. vesicatoria, Neisseria gonorrhoeae, and Moraxella bovis. In a PCR internal primers JAAN and JARA, designed by using the nucleotide sequences of the variable central and C-terminal region of fimA, amplified a 226-bp DNA fragment in all X. hyacinthi isolates. This PCR was shown to be pathovar specific, as assessed by testing 71Xanthomonas pathovars and bacterial isolates belonging to other genera, such as Erwinia and Pseudomonas. Southern hybridization experiments performed with the labelled 226-bp DNA amplicon as a probe suggested that there is only one structural type IV fimbrial-gene cluster in X. hyacinthi. Only twoXanthomonas translucens pathovars cross-reacted weakly in PCR. Primers amplifying a subsequence of the fimA gene ofX. campestris pv. vesicatoria (T. Ojanen-Reuhs, N. Kalkkinen, B. Westerlund-Wikström, J. van Doorn, K. Haahtela, E.-L. Nurmiaho-Lassila, K. Wengelink, U. Bonas, and T. K. Korhonen, J. Bacteriol. 179: 1280–1290, 1997) were shown to be pathovar specific, indicating that the fimbrial-subunit sequences are more generally applicable in xanthomonads for detection purposes. Under laboratory conditions, approximately 1,000 CFU of X. hyacinthi per ml could be detected. In inoculated leaves of hyacinths the threshold was 5,000 CFU/ml. The results indicated that infected hyacinths with early symptoms could be successfully screened for X. hyacinthi with PCR.

Variant identification in BARD1, PRDM9, RCC1, and RECQL in patients with ovarian cancer by targeted next-generation sequencing of DNA pools

2021 ◽  
pp. canprevres.0295.2021
Author(s):  
Malwina Suszynska ◽  
Magdalena Ratajska ◽  
Paulina Galka-Marciniak ◽  
Aleksandra Ryszkowska ◽  
Dariusz Wydra ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Next Generation Sequencing ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Dna Pools ◽  
Generation Sequencing ◽  
Variant Identification

Insights into Large Audit Firm Sampling Policies

2015 ◽  
Vol 9 (2) ◽  
pp. P7-P18 ◽  
Author(s):  
Brant E. Christensen ◽  
Randal J. Elder ◽  
Steven M. Glover
Keyword(s):  
Financial Reporting ◽  
Internal Control ◽  
Sampling Methods ◽  
Accounting Firms ◽  
Audit Firm ◽  
Sarbanes Oxley ◽  
Big 4 ◽  
Audit Firms ◽  
Current Sampling ◽  
Audit Sampling

SUMMARY Changes in the audit profession after Sarbanes-Oxley, including mandatory audits of internal control over financial reporting and PCAOB oversight and inspection of audit work, have potentially changed the nature and extent of audit sampling in the largest accounting firms. In our study, “Behind the Numbers: Insights into Large Audit Firm Sampling Policies” (Christensen, Elder, and Glover 2015), we administered an extensive, open-ended survey to the national offices of the Big 4 and two other international accounting firms regarding their firm's audit sampling policies. We find variation among the largest firms' policies in their use of different sampling methods and in inputs used in the sampling applications that could result in different sample sizes. We also provide evidence of some of the sampling topics firms find most problematic, as well as changes to firms' policies regarding revenue testing due to PCAOB inspections. Our evidence provides important insights into current sampling policies, which may be helpful to audit firms in evaluating their sampling inputs and overall sampling approaches.

Performance of a next generation sequencing SNP assay on degraded DNA

2015 ◽  
Vol 19 ◽  
pp. 1-9 ◽  
Author(s):  
Katherine Butler Gettings ◽  
Kevin M. Kiesler ◽  
Peter M. Vallone
Keyword(s):  
Next Generation Sequencing ◽  
Degraded Dna ◽  
Next Generation ◽  
Generation Sequencing

Lactate dehydrogenase and its isoenzymes in vaginal fluid in vaginitis/vaginosis cases and in healthy controls

2003 ◽  
Vol 14 (4) ◽  
pp. 270-273 ◽  
Author(s):  
O Niklasson ◽  
G Skude ◽  
P-A Mårdh
Keyword(s):  
Lactate Dehydrogenase ◽  
Sampling Methods ◽  
Lavage Fluid ◽  
Vaginal Fluid ◽  
Healthy Controls ◽  
Vaginal Lavage ◽  
Vaginal Ph ◽  
High Concentrations ◽  
Post Menopausal ◽  
Isoenzyme Patterns

The objective of the study was to measure the lactate dehydrogenase (LD) activity in vaginal lavage fluid of women with vaginitis/vaginosis and in healthy pre- and post-menopausal controls. Also to analyse the LD isoenzyme patterns in such samples and compare the influence on the LD activity by different storage and sampling methods. Twenty of the women studied, who had no signs of inflammation as evidenced from vaginal wet smears, were pre-menopausal and 8 post-menopausal. Fifty-eight non-pregnant patients with vaginitis/vaginosis or non-inflammatory gynaecological conditions were analysed for LD isoenzyme patterns. The LD activity was correlated to vaginal pH. Furthermore, the LD activity was determined in another 100 women screened for Chlamydia trachomatis. Finally, the influence on the LD activity when sampling was made by a cytological brush vs vaginal lavage and analysed after different storage periods, as studied. The LD activity was elevated, i.e. >2 µkat/L, in all but two of the women with leucorrhoea. Only women with bacterial vaginosis (BV) without leucorrhoea, had an increased LD activity. An increased vaginal pH correlated to the LD concentration. The LD activity was elevated in cases with vulvovaginal candidiasis, trichomoniasis, chlamydial cervicitis and senile colpitis. Storage of samples for up to six hours had no influence on the test outcome. Brush and lavage fluid samples did not differ with regard to the rate of positive LD tests. In healthy women, the LD activity is low and predominated by slow-migrating isoenzymes, i.e. LD 4 and 5. The LD activity is generally increased in cases of vaginitis and in women with BV and chlamydial cervicitis. In trichomoniasis, particularly high concentrations of LD 5, are found.

scholarly journals Mosquito Guttersnipe: A New Sampling Tool for Roof Gutters, Tree Holes, and Other Elevated Mosquito Habitats

2021 ◽  
Vol 37 (2) ◽  
pp. 109-112
Author(s):  
Mark P. Leonard ◽  
Jonathan D. Oliver
Keyword(s):  
Health Risks ◽  
Sampling Methods ◽  
Mosquito Control ◽  
Current Model ◽  
Cost Effective ◽  
Human Populations ◽  
Risk Of Infection ◽  
Tree Holes ◽  
Current Sampling

ABSTRACT Mosquitoes pose health risks to human populations by serving as vectors of diseases. Mosquito control organizations are responsible for inspecting and controlling vector populations to reduce the risk of infection of these diseases. Current sampling methods are effective for numerous types of mosquito habitat, but not conducive for sampling small overhead habitat such as roof gutters or tree holes. We have developed and tested a tool called the Mosquito GutterSnipe to sample these overhead habitats. Volumetric and larval capacity testing of the tool prototype demonstrated comparable sampling integrity to standard mosquito dipping methods. The GutterSnipe can be employed as a reliable way to sample previously overlooked mosquito habitat. Its current model is cost effective and easy to produce for mosquito control organizations and easy to use for inspectors.

Cananga odorata (Magnoliales: Annonaceae) Essential Oil Produces Significant Avoidance Behavior in Mosquitoes

2021 ◽  
Author(s):  
Chutipong Sukkanon ◽  
Jirod Nararak ◽  
Michael J Bangs ◽  
Theeraphap Chareonviriyaphap
Keyword(s):  
Essential Oil ◽  
Avoidance Behavior ◽  
Mosquito Species ◽  
Test Chamber ◽  
Physical Contact ◽  
Anopheles Dirus ◽  
Anopheles Mosquitoes ◽  
Primary Compounds ◽  
High Concentrations ◽  
Cananga Odorata

Abstract Essential oil of Cananga odorata Hook. F. & Tomson is a source of insect repellent, but contact irritancy and noncontact repellency actions that stimulate insect’s avoidance behavior (escape away from chemical source after direct physical contact or without making physical contact, respectively) have not been investigated. Therefore, an excito-repellency test chamber was used for measuring avoidance behavior of four insectary-reared mosquito species (Diptera: Culicidae) that escape from esposure to four concentrations (0.5, 1.0, 2.5, and 5.0% v/v) of C. odorata oil. The oil strongly repelled both Culex quinquefasciatus Say (85–97% escape) and Anopheles minimus Theobald (97–99%) at high concentrations (2.5–5.0%). For Anopheles dirus Peyton & Harrison and Aedes aegypti (L.), highest repellency (64 and 39% escape, respectively) was demonstrated at 2.5% concentration. For contact irritancy, the oil produced relatively high percent escape found in Cx. quinquefasciatus (90–100% escape) and An. minimus (83–100%). Whereas moderate contact irritancy was observed against An. dirus (40–50% escape) and Ae. aegypti (51–59%). The percent escape was then adjusted with repellency to estimate the effect of contact irritancy alone. We found that highest contact irritancy was presented at 0.5% concentration against An. minimus (67% escape). Knockdown and toxic actions were only found in Anopheles mosquitoes at 5.0% concentration. The results revealed that An. minimus and Cx. quinquefasciatus were more prone to be repelled by C. odorata oil. Detailed analysis of oil identified primary compounds as methyl benzoate (14.6%), α-gurjunene (12.8%), p-methyl-anisole (11.3%), and benzyl acetate (9.9%). Further investigations are needed to assess excito-repellency actions of these compounds alone or in combination.

Insect Repellents Mediate Species-Specific Olfactory Behaviours in Mosquitoes

2020 ◽  
Author(s):  
Ali Afify ◽  
Christopher John Potter
Keyword(s):  
Mosquito Species ◽  
Anopheles Coluzzii ◽  
Olfactory Neurons ◽  
Olfactory Responses ◽  
Close Proximity ◽  
Lemongrass Oil ◽  
Mosquito Repellents ◽  
Receptor Neurons ◽  
High Concentrations ◽  
Species Specific

Abstract Background The species-specific mode of action for DEET and many other mosquito repellents is often unclear. Confusion may arise for many reasons. First, the response of a single mosquito species is often used to represent all mosquito species. Second, behavioural studies usually test the effect of repellents on mosquito attraction towards human odorants, rather than their direct repulsive effect on mosquitoes. Third, the mosquito sensory neuron responses towards repellents are often not directly examined. Methods A close proximity response assay was used to test the direct repulsive effect of six mosquito repellents on Anopheles coluzzii, Aedes aegypti and Culex quinquefasciatus mosquitoes. Additionally, behavioural assay and calcium imaging recordings of antennae were used to test the response of An. coluzzii mosquitoes towards two human odorants (1-octen-3-ol and benzaldehyde) at different concentrations, and mixtures of the repellents lemongrass oil and p-menthane-3,8-diol (PMD) with DEET. Results An. coluzzii mosquitoes were repelled by lemongrass oil and PMD, while Ae. aegypti and Cx. quinquefasciatus mosquitoes were repelled by lemongrass oil, PMD, eugenol, and DEET. In addition, high concentrations of 1-octen-3-ol and benzaldehyde were repellent, and activated more olfactory receptor neurons on the An. coluzzii antennae than lower concentrations. Finally, changes in olfactory responses to repellent mixtures reflected changes in repulsive behaviours. Conclusions The findings described here suggest that different species of mosquitoes have different behavioural responses to repellents. The data further suggest that high-odour concentrations may recruit repellent-sensing neurons, or generally excite many olfactory neurons, yielding repellent behavioural responses. Finally, DEET can decrease the neuronal and behavioural response of An. coluzzii mosquitoes towards PMD but not towards lemongrass oil. Overall, these studies can help inform mosquito repellent choice by species, guide decisions on effective repellent blends, and could ultimately identify the olfactory neurons and receptors in mosquitoes that mediate repellency. Methods: Here, we used a close proximity response assay to test the direct repulsive effect of six mosquito repellents on Anopheles coluzzii , Aedes aegypti , and Culex quinquefasciatus mosquitoes. Additionally, we used this behavioral assay and calcium imaging recordings of antennae to test the response of An. coluzzii mosquitoes towards two human odorants (1-octen-3-ol and benzaldehyde) at different concentrations, and mixtures of the repellents lemongrass oil and p-menthane-3,8-diol (PMD) with DEET. Results: We found that An. coluzzii mosquitoes were repelled by lemongrass oil and PMD, while Ae. aegypti and Cx. Quinquefasciatus mosquitoes were repelled by lemongrass oil, PMD, eugenol, and DEET. In addition, we found that high concentrations of 1-octen-3-ol and benzaldehyde were repellent, and activated more olfactory receptor neurons on the An. coluzzii antenna than lower concentrations. Finally, we found that changes in olfactory responses to repellent mixtures reflected changes in repulsive behaviors. Conclusions: Our findings suggest that different species of mosquitoes have different behavioral responses to repellents. We further suggest that high-odor concentrations may recruit repellent-sensing neurons, or generally excite many olfactory neurons, yielding repellent behavioral responses. Finally, we show that DEET can decrease the neuronal and behavioral response of An. coluzzii mosquitoes towards PMD but not towards lemongrass oil. Overall, these studies can help inform mosquito repellent choice by species, guide decisions on effective repellent blends, and could ultimately identify the olfactory neurons and receptors in mosquitoes that mediate repellency.

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