Non-destructive DNA Extraction Method Research Articles

A morphological and high throughput sequencing workflow to identify Australian ants (Hymenoptera, Formicidae): a new tool for biosecurity and biodiversity

Ants are often the most ubiquitous and ecologically influential components of terrestrial systems, exhibiting an exceptionally high level of endemic diversity. Simultaneously, exotic ants can be amongst the most environmentally and economically devastating biological invaders. Distinguishing between exotic and related natives is essential for early detection and to reduce the chance of establishment, but at the species-level there remains a great deal of uncertainty among several of the most diverse, widespread and ecologically dominant genera. This taxonomic impediment negatively impacts the use of molecular techniques relying on reference databases, due to the poor state of ant DNA sequences in publicly available repositories, with many species not represented at all and others incorrectly identified. As a result, biosecurity screening for targeted exotic ants and assigning ant species-level identifications for biodiversity remain two separate areas of focus in Australia. Here we propose a workflow for the identification of ants that enables simultaneous processing of large numbers of “bulk” ant samples each containing many individuals, increasing resolution for taxonomic assignment, and generating a curated database linked to voucher specimens. We use a non-destructive DNA extraction method and compare two high throughput sequencing (HTS) metabarcoding platforms – MiSeq (Illumina) and MinION (Oxford Nanopore Technologies) – processing up to 180 bulk mixed ant samples per run. This approach allowed for the acquisition of curated DNA sequences from voucher specimens morphologically examined by expert taxonomists. This work highlights the advantages and current limitations of DNA-based identifications, the needs for standardisation, as well as the importance of a taxonomy-based curation of DNA database for both biodiversity and biosecurity.

Redescription of Taeniacanthus aulacocephali (Copepoda: Cyclopoida: Taeniacanthidae) parasitic on Uranoscopus japonicus Houttuyn (Uranoscopidae) from Pacific coast, Japan, with adaptation of the non-destructive DNA technique.

Taeniacanthus aulacocephali Izawa, 2021 (Copepoda: Cyclopoida: Taeniacanthidae) was redescribed from the branchial cavity and gill filaments of Uranoscopus japonicus Houttuyn (Perciformes: Uranoscopidae) collected from the Pacific coast of the Kochi and Wakayama prefectures, Japan. This is the second record of the copepod, and the finding from U. japonicus represents the new host record. The species is characterized by several distinguishing features: 1) a decrease in the width of the habitus between the second and fourth pedigerous segments; 2) the ratio of prosome/body length; 3) the presence of eight setae on the exopodal terminal segment of leg 2; 4) an un-bifurcated maxilliped claw surrounded by 14-28 transverse ridges; and 5) the presence of an inner coxal seta on legs 2 and 3. The newly collected specimens were subjected to a modified non-destructive DNA extraction method and morphological description based on the same copepod individual, while preserving a morphologically describable specimen. Sequences of 18S rDNA, 28S rDNA and the mitochondrial cytochrome c oxidase subunit 1 mitochondrial gene (cox1) were obtained.

Phylogeny and genetic diversity of Echinometra sea urchin in Taiwan

ABSTRACT Four closely related Echinometra species exist in the Indo-Pacific region, but little is known about their population genetics in Taiwan, an area rich in marine biodiversity. Echinometra dominates the Douziping coral reef off Liuqiu Island, where urchin barrens are also present. To understand the relationship between Echinometra species and urchin barrens, we investigated the genetic diversity of Echinometra species in Taiwan by using a nondestructive DNA extraction method on the spines of sea urchins. Mitochondrial DNA results showed that E. sp. C is the dominant species in Taiwan, with relatively high interspecific genetic distance. The E. sp. C is the only species in Douziping, and its population is expanding in accordance with the significantly low R2 value, the significantly negative Fu’s Fs values, and mismatch distribution analysis results. Gene flow estimates between the urchin populations in Taiwan and those in the neighbouring waters of Okinawa suggested that the Kuroshio Current could play an important role in Echinometra dispersal. This implies strongly connectivity between these populations. This study reports the first case of E. sp. C-associated urchin barrens in the tropics.

A Droplet Digital PCR (ddPCR) Assay to Detect Phthorimaea absoluta (Lepidoptera: Gelechiidae) in Bulk Trap Samples.

The moth species Phthorimaea absoluta (Meyrick) (formerly Tuta absoluta) is serious threat to tomato and other Solanaceous crops worldwide and is invasive throughout Europe, Asia, and Africa. While P. absoluta has not yet been found in the U.S. recent detections in the Caribbean have raised concerns that the species could be introduced to mainland North America. To improve detection capacity, a droplet digital PCR (ddPCR) assay was developed that employs a nondestructive bulk DNA extraction method able to detect one P. absoluta sample among 200 nontargets. Such high-throughput and sensitive molecular assays are essential to preventing introductions through early detection and response. This assay can also be used in areas where P. absoluta is established to monitor outbreaks and track migratory patterns.

A LAMP assay for the detection of Bactrocera tryoni Queensland fruit fly (Diptera: Tephritidae)

LAMP assays are targeted molecular tests for the rapid detection of species in the laboratory and field. We developed a LAMP assay for an economically important fruit fly species, Queensland fruit fly, Bactrocera tryoni. This assay was assessed against a broad panel of target and non-target species and found to be specific, only amplifying the target species and closest relatives, in a portable real-time fluorometer (Genie III) in under 15 minutes with an anneal derivative temperature of 82.5 oC. The assay is sensitive to low levels of target DNA (>0.016 ng/µl), performing equally to the existing qPCR test. To enable retention of a physical voucher specimen, for potential morphological confirmation of LAMP results, a novel whole-specimen non-destructive DNA extraction method was developed, suitable for LAMP in the field. The stability of DNA extraction and LAMP reagents was tested under simulated and actual field conditions and shown to be robust. Our new assay now provides a portable molecular tool for the detection of this significant tephritid fruit fly pest species of biosecurity/quarantine concern. This has already proven invaluable for in-field diagnostics, providing real-time support influencing immediate actions, with negative results allowing the release of fruit produce, and positive results initiating fruit fly control measures.

Non-destructive DNA extraction method for identification of Bradysia odoriphaga (Diptera: Sciaridae), a pest of Welsh onion, carrot, and Chinese chive in Japan

We examined the utility of a non-destructive DNA extraction method for identification of Bradysia odoriphaga Yang and Zhang, 1985 (Diptera: Sciaridae) based on PCR using species-specific primers and DNA barcoding targeting the mitochondrial cytochrome c oxidase subunit I gene. Using species-specific primers, PCR success rates for adults and pupae were more than 90%, while those for larvae and eggs were 51% and 13%, respectively. The success rates of PCR for DNA barcode region were slightly higher than those of PCR using species-specific primers. In all sequenced samples, the 658 bp of sequences in the DNA barcode region were identical to the sequences from B. odoriphaga that we previously obtained using a destructive DNA extraction method. The adults were successfully identified based on morphological characters after non-destructive DNA extraction. The non-destructive DNA extraction method examined in this study is useful for the identification of adults and pupae of B. odoriphaga collected in the fields.

Species identification and phylogenetic relationships among five species of psocids from Chinese herbal medicines

The main objectives of the study were to establish an accurate multiplex PCR identification technique for five species of psocids from Chinese herbal medicines and to discuss the phylogenetic relationships among those species. Five species of booklice (Liposcelis bostrychophila, L. entomophila, L. tricolor, L. pearmani and L. rufa) were collected from Chinese herbal medicines. Genomic DNA of individual booklice was obtained via a nondestructive DNA extraction method. The internal transcribed spacer (ITS)1-5.8S-ITS2 gene sequences were obtained by PCR amplifying and sequencing. The primers were designed, and the phylogenetic trees were constructed using the NCBI website and MEGA software. In this research, a Multiplex PCR identification technique was established based on the ITS2 gene for the five species of booklice. The phylogenetic relationships among the five booklice species were analyzed and discussed based on the ITS gene sequences.

Charge-Dependent Regulation in DNA Adsorption on 2D Clay Minerals

DNA purification is essential for the detection of human clinical specimens. A non-destructive, controllable, and low reagent consuming DNA extraction method is described. Negatively charged DNA is absorbed onto a negatively charged montmorillonite to achieve non-destructive DNA extraction based on cation bridge construction and electric double layer formation. Different valence cation modified montmorillonite forms were used to validate the charge-dependent nature of DNA adsorption on montmorillonite. Electric double layer thickness thinning/thickening with the high/lower valence cations exists, and the minerals tended to be sedimentation-stable due to the Van der Waals attraction/electrostatic repulsion. Li-modified montmorillonite with the lowest charge states showed the best DNA adsorption efficiency of 8–10 ng/μg. Charge-dependent regulating research provides a new perspective for controllable DNA extraction and a deep analysis of interface engineering mechanisms.

Non-destructive DNA extraction from aphids: the application in virus - vector studies of Banana bunchy top virus (BBTV)

Banana bunchy top virus (BBTV), one of the most devastating viruses of banana, is transmitted by the banana aphid, Pentalonia nigronervosa. The high degree of morphological similarity between P. nigronervosa and the related experimental vector P. caladii makes it difficult to distinguish the two species. DNA barcoding can be used as an alternative for the rapid identification of these aphid species. However, standard molecular identification techniques for small insects usually require maceration of the sample to obtain sufficient amounts of DNA, resulting in the loss of voucher specimens. In this study, a non-destructive DNA extraction method for aphids was optimised, allowing the specimens to remain intact for use as vouchers for further morphological studies. Sufficient DNA concentrations were obtained from the aphid extractions and were used for species identification through direct sequencing of PCR products. Extracted DNA was further used to detect BBTV in the insect vector, P. nigronervosa. Approximately 78% of the aphids collected from symptomatic plants in infected plantations in KwaZulu-Natal tested positive for BBTV, as well as some aphids collected in other African countries. The BBTV viral strain identified from South Africa grouped with the “South Pacific” clade. This non-destructive DNA extraction method can be used in an early detection management strategy, especially in epidemiology and virus-vector studies of Banana bunchy top disease (BBTD).

Unraveling the intricate biodiversity of the benthic harpacticoid genus Nannopus (Copepoda, Harpacticoida, Nannopodidae) in Korean waters

Nannopus (Harpacticoida, Nannopodidae) species are abundant and widely distributed throughout the world across a variety of habitats. Nannopus is well known for high frequencies of misidentifications and thus may comprise several cryptic complexes and morphologically distinct species. Cryptic taxa are common in meiofauna communities. In this study, we aimed to identify Nannopus species using an integrative approach including molecular taxonomy. We adopted a non-destructive DNA extraction method so that morphological and molecular data could be obtained from the same specimen. We analyzed the molecular diversity and distributions of Nannopus using a total of 190 individuals. We sequenced the 190 mtCOI, 53 mtCYTB, 25 18SrDNA, and 43 28SrDNA genes from 190 individuals. Several species delimitation approaches were applied, including uncorrected p-distances for mtCOI, mtCYTB, 18SrDNA, and 28SrDNA, and Automatic Barcode Gap Discovery and Bayesian implemented Poisson tree processes for mtCOI and mtCYTB data. The maximum likelihood and Bayesian approaches were used to examine the phylogenetic relationships among individuals using the combined set of all four genes. Our species delimitation and phylogenetic analyses indicated the presence of three cryptic and six morphologically distinct species. All species are sympatric and widely distributed across mudflats ranging from the Yellow Sea to the South Sea in Korea. The divergence patterns of the four genes were not congruent. A phylogenetic tree based on the concatenated dataset was the most robust, was congruent with morphology, and suggested two major clades. We considered the validity of reinstating the genus Ilyophilus (Lilljeborg, 1902) and ultimately concluded that including all congeners in Nannopus until the type species (N. palustris Brady, 1880) is re-described was the most prudent approach.

Can non-destructive DNA extraction of bulk invertebrate samples be used for metabarcoding?

BackgroundHigh throughput DNA sequencing of bulk invertebrate samples or metabarcoding is becoming increasingly used to provide profiles of biological communities for environmental monitoring. As metabarcoding becomes more widely applied, new reference DNA barcodes linked to individual specimens identified by taxonomists are needed. This can be achieved through using DNA extraction methods that are not only suitable for metabarcoding but also for building reference DNA barcode libraries.MethodsIn this study, we test the suitability of a rapid non-destructive DNA extraction method for metabarcoding of freshwater invertebrate samples.ResultsThis method resulted in detection of taxa from many taxonomic groups, comparable to results obtained with two other tissue-based extraction methods. Most taxa could also be successfully used for subsequent individual-based DNA barcoding and taxonomic identification. The method was successfully applied to field-collected invertebrate samples stored for taxonomic studies in 70% ethanol at room temperature, a commonly used storage method for freshwater samples.DiscussionWith further refinement and testing, non-destructive extraction has the potential to rapidly characterise species biodiversity in invertebrate samples, while preserving specimens for taxonomic investigation.

Molecular detection and identification of Rickettsia in psocids collected from herbs

The aims of this study were to assess the psocids that potentially carry Rickettsia and to characterize Rickettsia species. We collected booklice from dried fragments of Chinese medicinal herbs for retail. Species identification and Rickettsia detection were performed by using nondestructive DNA extraction method and PCR. Of the 224 booklice samples collected, the following species were identified: Liposcelis pearmani (50.89%), L. bostrychophila (25.89%) and L. entomophila (23.21%). Twenty-four samples were found to be positive for Rickettsia (10.71%, 24/224), and the number of Rickettsia-positive samples per species was as follows: L. pearmani (n = 10), L. bostrychophila (n = 8) and L. entomophila (n = 6). The phylogenetic tree revealed that the rickettsial agents found in booklice cluster along with Rickettsia felis belong to the spotted fever group (SFG). Mixed booklice contamination occurs in herbal fragments. The presence of Rickettsia-infected booklice suggests that there is a risk of louse-borne rickettsioses to humans in contact with herbs. These findings can contribute to the development of a disease control program that assists relevant practitioners and promotes public health.

An efficient and non-destructive DNA extraction method for oribatid mites

Molecular methods play an important role in systematic acarology and DNA extraction is the first step in this ground. Nowadays, the varieties of methods are being used for extraction of DNA, but most of them destroy the important external characters that are essential for morphological identification. In order to overcome the problem of associating molecular and morphological information, we have developed a simple, efficient and non-destructive DNA extraction method for oribatid mites. The non-destructive method was tested on three different species [Oppia nitens C.L. Koch, 1836 (Oppiidae), Acrotritia ardua C.L. Koch, 1841 (Euphthiracaridae), and Amerus polonicus Kulczynski, 1902 (Ameridae)] and exoskeleton of specimens were preserved in Hoyer’s medium on glass microscopic slides for further identification via morphological studies. This method is more time consuming than commercial kits, but is easier and cheaper, meanwhile, allows systematic analysis with linking the morphological and genetic traits from a single mite. The most important advantage of TNES method is that after using this non-destructive method, specimens preserve all of their morphological features.

Where Taxonomy Based on Subtle Morphological Differences Is Perfectly Mirrored by Huge Genetic Distances: DNA Barcoding in Protura (Hexapoda)

BackgroundProtura is a group of tiny, primarily wingless hexapods living in soil habitats. Presently about 800 valid species are known. Diagnostic characters are very inconspicuous and difficult to recognize. Therefore taxonomic work constitutes an extraordinary challenge which requires special skills and experience. Aim of the present pilot project was to examine if DNA barcoding can be a useful additional approach for delimiting and determining proturan species.Methodology and Principal FindingsThe study was performed on 103 proturan specimens, collected primarily in Austria, with additional samples from China and Japan. The animals were examined with two markers, the DNA barcoding region of the mitochondrial COI gene and a fragment of the nuclear 28S rDNA (Divergent Domain 2 and 3). Due to the minuteness of Protura a modified non-destructive DNA-extraction method was used which enables subsequent species determination. Both markers separated the examined proturans into highly congruent well supported clusters. Species determination was performed without knowledge of the results of the molecular analyses. The investigated specimens comprise a total of 16 species belonging to 8 genera. Remarkably, morphological determination in all species exactly mirrors molecular clusters. The investigation revealed unusually huge genetic COI distances among the investigated proturans, both maximal intraspecific distances (0–21.3%), as well as maximal congeneric interspecifical distances (up to 44.7%).ConclusionsThe study clearly demonstrates that the tricky morphological taxonomy in Protura has a solid biological background and that accurate species delimitation is possible using both markers, COI and 28S rDNA. The fact that both molecular and morphological analyses can be performed on the same individual will be of great importance for the description of new species and offers a valuable new tool for biological and ecological studies, in which proturans have generally remained undetermined at species level.

TYLCV検定のための黄色粘着板に捕捉されたタバココナジラミ成虫からの簡易凍結DNA抽出

TYLCV検定のための黄色粘着板に捕捉されたタバココナジラミ成虫からの簡易凍結DNA抽出

A rapid non-destructive DNA extraction method for insects and other arthropods

Preparation of arthropods for morphological identification often damages or destroys DNA within the specimen. Conversely, DNA extraction methods often destroy the external physical characteristics essential for morphological identification. We have developed a rapid, simple and non-destructive DNA extraction technique for arthropod specimens. This technique was tested on four arthropod orders, using specimens that were fresh, preserved by air drying, stored in ethanol, or collected with sticky or propylene glycol traps. The technique could be completed in 20 min for Coleoptera, Diptera and Hemiptera, and 2 min for the subclass Acarina, without significant distortion, discolouration, or other damage to the specimens.

Non-Destructive Sampling of Ancient Insect DNA

BackgroundA major challenge for ancient DNA (aDNA) studies on insect remains is that sampling procedures involve at least partial destruction of the specimens. A recent extraction protocol reveals the possibility of obtaining DNA from past insect remains without causing visual morphological damage. We test the applicability of this protocol on historic museum beetle specimens dating back to AD 1820 and on ancient beetle chitin remains from permafrost (permanently frozen soil) dating back more than 47,000 years. Finally, we test the possibility of obtaining ancient insect DNA directly from non-frozen sediments deposited 3280-1800 years ago - an alternative approach that also does not involve destruction of valuable material.Methodology/Principal FindingsThe success of the methodological approaches are tested by PCR and sequencing of COI and 16S mitochondrial DNA (mtDNA) fragments of 77–204 base pairs (-bp) in size using species-specific and general insect primers.Conclusion/SignificanceThe applied non-destructive DNA extraction method shows promising potential on insect museum specimens of historical age as far back as AD 1820, but less so on the ancient permafrost-preserved insect fossil remains tested, where DNA was obtained from samples up to ca. 26,000 years old. The non-frozen sediment DNA approach appears to have great potential for recording the former presence of insect taxa not normally preserved as macrofossils and opens new frontiers in research on ancient biodiversity.

A nondestructive, rapid, reliable and inexpensive method to sample, store and extract high‐quality DNA from fish body mucus and buccal cells

AbstractA rapid, nondestructive, reproducible and cheap DNA extraction method from body mucus and buccal cells of northern pike and brown trout is described. Buccal cells and body mucus were sampled on FTA Cards; the captured DNA was used directly for microsatellite and polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analyses. A complete concordance with control DNA was found. The genotyping error rate for microsatellite ranged from 1.9% to 3.3% for the northern pike and brown trout, respectively. This methodology, using for the first time these materials as a fish DNA source, combines speed of sampling and processing, with a twofold to a threefold time and costs saving.