A new cost‐effective and fast direct PCR protocol for insects based on PBS buffer

Insect DNA barcoding is a species identification technique used in biodiversity assessment and ecological studies. However, DNA extraction can result in the loss of up to 70% of DNA. Recent research has reported that direct PCR can overcome this issue. However, the success rates could still be improved, and tissues used for direct PCR could not be reused for further genetic studies. Here, we developed a direct PCR workflow that incorporates a 2‐min sample preparation in PBS‐buffer step for fast and effective universal insect species identification. The developed protocol achieved 100% success rates for amplification in six orders: Mantodea, Phasmatodea, Neuroptera, Odonata, Blattodea and Orthoptera. High and moderate success rates were obtained for five other species: Lepidoptera (97.3%), Coleoptera (93.8%), Diptera (90.5%), Hemiptera (81.8%) and Hymenoptera (75.0%). High‐quality sequencing data were also obtained from these amplifiable products, allowing confidence in species identification. The method was sensitive down to 1/4th of a 1‐mm fragment of leg or body and its success rates with oven‐dried, ethanol‐preserved, food, bat guano and museum specimens were 100%, 98.6%, 90.0%, 84.0% and 30.0%, respectively. In addition, the pre‐PCR solution (PBS with insect tissues) could be used for further DNA extraction if needed. The workflow will be beneficial in the fields of insect taxonomy and ecological studies due to its low cost, simplicity and applicability to highly degraded specimens.     

Development of an apparatus for rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in chondroitin sulfate-type proteoglycans

An apparatus, AutoGlycoCutter (AGC), was developed as a tool for rapid release of O-linked-type glycans under alkaline conditions. This system allowed rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in proteoglycans (PGs). After digestion of PGs with chondroitinase ABC, the oligosaccharides at the linkage region were successfully released from the protein core by AGC within 3 min. The reducing ends of the released oligosaccharides were labeled with 2-aminobenzoic acid and analyzed by a combination of capillary electrophoresis (CE) and matrix-assisted laser desorption time-of-flight mass spectrometry. In addition, the unsaturated disaccharides produced by chondroitinase ABC derived from the outer parts of the glycans were labeled with 2-aminoacridone and analyzed by CE to determine the disaccharide compositions. We evaluated AGC as a method for structural analysis of glycosaminoglycans in some chondroitin-sulfate-type PGs (urinary trypsin inhibitor, bovine nasal cartilage PG, bovine aggrecan, bovine decorin, and bovine biglycan). Recoveries of the released oligosaccharides were 57-73% for all PGs tested in the present study. In particular, we emphasize that the use of AGC achieved ca. 1000-fold rapid release of O-glycans compared with the conventional method.     

Biology and systematics of the New World Phyllocnistis Zeller leafminers of the avocado genus Persea (Lepidoptera, Gracillariidae)

Four New World species of Phyllocnistis Zeller are described from serpentine mines in Persea (Family Lauraceae). Phyllocnistis hyperpersea,new species, mines the upper leaf surfaces of avocado, Persea americana Mill., and red bay, Persea borbonia (L.) Spreng. and ranges over much of the southeastern United States into Central America. Phyllocnistis subpersea,new species, mines the underside and occasionally upper sides of new leaves of Persea borbonia in southeastern United States. Phyllocnistis longipalpa, new species, known only from southern Florida also mines the undersides of new leaves of Persea borbonia. Phyllocnistis perseafolia,new species, mines both leaf surfaces and possibly fruits of Persea americana in Colombia, South America. As in all known species of Phyllocnistis, the early instars are subepidermal sapfeeders in young (not fully hardened) foliage, and the final instar is an extremely specialized, nonfeeding larval form, whose primary function is to spin the silken cocoon, at the mine terminus, prior to pupation. Early stages are illustrated and described for three of the species. The unusual morphology of the pupae, particularly the frontal process of the head, is shown to be one of the most useful morphological sources of diagnostic characters for species identification of Phyllocnistis. COI barcode sequence distances are provided for the four proposed species and a fifth, undescribed species from Costa Rica.     

COI barcoding of true bugs (Insecta, Heteroptera)

Several recent studies have proposed that partial DNA sequences of the cytochrome c oxidase I (COI) mitochondrial gene might serve as DNA barcodes for identifying and differentiating between animal species, such as birds, fish and insects. In this study, we tested the effectiveness of a COI barcode to identify true bugs from 139 species collected from Korea and adjacent regions (Japan, Northeastern China and Fareast Russia). All the species had a unique COI barcode sequence except for the genus Apolygus (Miridae), and the average interspecific genetic distance between closely related species was about 16 times higher than the average intraspecific genetic distance. DNA barcoding identified one probable new species of true bug and revealed identical or very recently divergent species that were clearly distinguished by morphological characteristics. Therefore, our results suggest that COI barcodes can reveal new cryptic true bug species and are able to contribute for the exact identification of the true bugs.     

Redescription of arenicolous dipluran Parajapyx pauliani (Diplura,Parajapygidae) and DNA barcoding analyses of Parajapyx from China

Littoral dipluran Parajapyx pauliani Pagés, 1959 was redescribed based on the specimens collected in Hainan Island, South China. The littoral habitat was confirmed for the species, as the first report of arenicolous dipluran in China. DNA barcoding fragment was sequenced for five Parajapyx species (18 individuals) from China, and this is the first report on DNA barcodes used for dipluran identification. The mean intra- and interspecific divergencesare 1.9% and 19.1% respectively. Synonymy of Parajapyx paucidentis and Parajapyx isabellae was confirmed.     

High-throughput molecular identification of fish eggs using multiplex suspension bead arrays

The location and abundance of fish eggs provide information concerning the timing and location of spawning activities and can provide fishery-independent estimates of spawning biomass. However, the full value of egg and larval surveys is severely restricted because many species' eggs and larvae are morphologically similar, making species-level identification difficult. Recent efforts have shown that nearly all species of fish may be identified by mitochondrial DNA (mtDNA) sequences (e.g. via 'DNA barcoding'). By taking advantage of a DNA barcode database, we have developed oligonucleotide probes for 23 marine fish species that produce pelagic eggs commonly found in California waters. Probes were coupled to fluorescent microspheres to create a suspension bead array. Biotin-labelled primers were used to amplify the mitochondrial cytochrome oxidase subunit I (COI) and 16S ribosomal rRNA genes from individual fish eggs. The amplicons were then hybridized to the bead array, and after the addition of a reporter fluorophore, samples were analysed by flow cytometry with Luminex 100 instrumentation. Probes specifically targeted eggs that are abundant and/or from morphologically indistinguishable species pairs. Results showed that the 33 different probes designed for this study accurately identified all samples when PCR was successful. Suspension bead arrays have a number of benefits over other methods of molecular identification; these arrays permit high multiplexing, simple addition of new probes, high throughput and lower cost than DNA sequencing. The increasing availability of DNA barcode data for numerous fish faunas worldwide suggests that bead arrays could be developed and widely used for fish egg, larval and tissue identifications.     

Unraveling the plant diversity of the Amazonian canga through DNA barcoding

The canga of the Serra dos Carajás, in Eastern Amazon, is home to a unique open plant community, harboring several endemic and rare species. Although a complete flora survey has been recently published, scarce to no genetic information is available for most plant species of the ironstone outcrops of the Serra dos Carajás. In this scenario, DNA barcoding appears as a fast and effective approach to assess the genetic diversity of the Serra dos Carajás flora, considering the growing need for robust biodiversity conservation planning in such an area with industrial mining activities. Thus, after testing eight different DNA barcode markers (matK, rbcL, rpoB, rpoC1, atpF‐atpH, psbK‐psbI, trnH‐psbA, and ITS2), we chose rbcL and ITS2 as the most suitable markers for a broad application in the regional flora. Here we describe DNA barcodes for 1,130 specimens of 538 species, 323 genera, and 115 families of vascular plants from a highly diverse flora in the Amazon basin, with a total of 344 species being barcoded for the first time. In addition, we assessed the potential of using DNA metabarcoding of bulk samples for surveying plant diversity in the canga. Upon achieving the first comprehensive DNA barcoding effort directed to a complete flora in the Brazilian Amazon, we discuss the relevance of our results to guide future conservation measures in the Serra dos Carajás.     

ER stress transforms random olfactory receptor choice into axon targeting precision

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