Wolbachia and DNA barcoding insects: patterns, potential and problems

Wolbachia is a genus of bacterial endosymbionts that impacts the breeding systems of their hosts. Wolbachia can confuse the patterns of mitochondrial variation, including DNA barcodes, because it influences the pathways through which mitochondria are inherited. We examined the extent to which these endosymbionts are detected in routine DNA barcoding, assessed their impact upon the insect sequence divergence and identification accuracy, and considered the variation present in Wolbachia COI. Using both standard PCR assays (Wolbachia surface coding protein – wsp), and bacterial COI fragments we found evidence of Wolbachia in insect total genomic extracts created for DNA barcoding library construction. When >2 million insect COI trace files were examined on the Barcode of Life Datasystem (BOLD) Wolbachia COI was present in 0.16% of the cases. It is possible to generate Wolbachia COI using standard insect primers; however, that amplicon was never confused with the COI of the host. Wolbachia alleles recovered were predominantly Supergroup A and were broadly distributed geographically and phylogenetically. We conclude that the presence of the Wolbachia DNA in total genomic extracts made from insects is unlikely to compromise the accuracy of the DNA barcode library; in fact, the ability to query this DNA library (the database and the extracts) for endosymbionts is one of the ancillary benefits of such a large scale endeavor – for which we provide several examples. It is our conclusion that regular assays for Wolbachia presence and type can, and should, be adopted by large scale insect barcoding initiatives. While COI is one of the five multi-locus sequence typing (MLST) genes used for categorizing Wolbachia, there is limited overlap with the eukaryotic DNA barcode region

Marine nematode taxonomy in the DNA age: the present and future of molecular tools to access their biodiversity

Molecular taxonomy is one of the most promising yet challenging fields of biology. Molecular markers such as nuclear and mitochondrial genes are being used in a variety of studies surveying marine nematode taxa. Sequences from more than 600 species have been deposited to date in online databases. These barcode sequences are assigned to 150 nominal species from 104 genera. There are 41 species assigned to Enoplea and 109 species to Chromadorea. Morphology-based surveys are greatly limited by processing speed, while barcoding approaches for nematodes are hampered by difficulties in matching sequence data with morphology-based taxonomy. DNA barcoding is a promising approach because some genes contain variable regions that are useful to discriminate species boundaries, discover cryptic species, quantify biodiversity and analyse phylogeny. We advocate a combination of several approaches in studies of molecular taxonomy, DNA barcoding and conventional taxonomy as a necessary step to enhance the knowledge of biodiversity of marine nematodes

A new molecular diagnostic tool for surveying and monitoring Triops cancriformis populations

© 2017 Sellers et al. The tadpole shrimp, Triops cancriformis, is a freshwater crustacean listed as endangered in the UK and Europe living in ephemeral pools. Populations are threatened by habitat destruction due to land development for agriculture and increased urbanisation. Despite this, there is a lack of efficient methods for discovering and monitoring populations. Established macroinvertebrate monitoring methods, such as net sampling, are unsuitable given the organism's life history, that include long lived diapausing eggs, benthic habits and ephemerally active populations. Conventional hatching methods, such as sediment incubation, are both time consuming and potentially confounded by bet-hedging hatching strategies of diapausing eggs. Here we develop a new molecular diagnostic method to detect viable egg banks of T. cancriformis, and compare its performance to two conventional monitoring methods involving diapausing egg hatching. We apply this method to a collection of pond sediments from the Wildfowl & Wetlands Trust Caerlaverock National Nature Reserve, which holds one of the two remaining British populations of T. cancriformis. DNA barcoding of isolated eggs, using newly designed species-specific primers for a large region of mtDNA, was used to estimate egg viability. These estimates were compared to those obtained by the conventional methods of sediment and isolation hatching. Our method outperformed the conventional methods, revealing six ponds holding viable T. cancriformis diapausing egg banks in Caerlaverock. Additionally, designed species-specific primers for a short region of mtDNA identified degraded, inviable eggs and were used to ascertain the levels of recent mortality within an egg bank. Together with efficient sugar flotation techniques to extract eggs from sediment samples, our molecular method proved to be a faster and more powerful alternative for assessing the viability and condition of T. cancriformis diapausing egg banks

124-Color Super-resolution Imaging by Engineering DNA-PAINT Blinking Kinetics

Optical super-resolution techniques reach unprecedented spatial resolution down to a few nanometers. However, efficient multiplexing strategies for the simultaneous detection of hundreds of molecular species are still elusive. Here, we introduce an entirely new approach to multiplexed super-resolution microscopy by designing the blinking behavior of targets with engineered binding frequency and duration in DNA-PAINT. We assay this kinetic barcoding approach in silico and in vitro using DNA origami structures, show the applicability for multiplexed RNA and protein detection in cells, and finally experimentally demonstrate 124-plex super-resolution imaging within minutes.We thank Martin Spitaler and the imaging facility of the MPI of Biochemistry for confocal imaging support

Evaluating detection limits of next-generation sequencing for the surveillance and monitoring of international marine pests

Most surveillance programmes for marine invasive species (MIS) require considerable taxonomic expertise, are laborious, and are unable to identify species at larval or juvenile stages. Therefore, marine pests may go undetected at the initial stages of incursions when population densities are low. In this study, we evaluated the ability of the benchtop GS Junior™ 454 pyrosequencing system to detect the presence of MIS in complex sample matrices. An initial in-silico evaluation of the mitochondrial cytochrome c oxidase subunit I (COI) and the nuclear small subunit ribosomal DNA (SSU) genes, found that multiple primer sets (targeting a ca. 400 base pair region) would be required to obtain species level identification within the COI gene. In contrast a single universal primer set was designed to target the V1–V3 region of SSU, allowing simultaneous PCR amplification of a wide taxonomic range of MIS. To evaluate the limits of detection of this method, artificial contrived communities (10 species from 5 taxonomic groups) were created using varying concentrations of known DNA samples and PCR products. Environmental samples (water and sediment) spiked with one or five 160 hr old Asterias amurensis larvae were also examined. Pyrosequencing was able to recover DNA/PCR products of individual species present at greater than 0.64% abundance from all tested contrived communities. Additionally, single A. amurensis larvae were detected from both water and sediment samples despite the co-occurrence of a large array of environmental eukaryotes, indicating an equivalent sensitivity to quantitative PCR. NGS technology has tremendous potential for the early detection of marine invasive species worldwide

Categorization of species as native or nonnative using DNA sequence signatures without a complete reference library.

New genetic diagnostic approaches have greatly aided efforts to document global biodiversity and improve biosecurity. This is especially true for organismal groups in which species diversity has been underestimated historically due to difficulties associated with sampling, the lack of clear morphological characteristics, and/or limited availability of taxonomic expertise. Among these methods, DNA sequence barcoding (also known as "DNA barcoding") and by extension, meta-barcoding for biological communities, has emerged as one of the most frequently utilized methods for DNA-based species identifications. Unfortunately, the use of DNA barcoding is limited by the availability of complete reference libraries (i.e., a collection of DNA sequences from morphologically identified species), and by the fact that the vast majority of species do not have sequences present in reference databases. Such conditions are critical especially in tropical locations that are simultaneously biodiversity rich and suffer from a lack of exploration and DNA characterization by trained taxonomic specialists. To facilitate efforts to document biodiversity in regions lacking complete reference libraries, we developed a novel statistical approach that categorizes unidentified species as being either likely native or likely nonnative based solely on measures of nucleotide diversity. We demonstrate the utility of this approach by categorizing a large sample of specimens of terrestrial insects and spiders (collected as part of the Moorea BioCode project) using a generalized linear mixed model (GLMM). Using a training data set of known endemic (n = 45) and known introduced species (n = 102), we then estimated the likely native/nonnative status for 4,663 specimens representing an estimated 1,288 species (412 identified species), including both those specimens that were either unidentified or whose endemic/introduced status was uncertain. Using this approach, we were able to increase the number of categorized specimens by a factor of 4.4 (from 794 to 3,497), and the number of categorized species by a factor of 4.8 from (147 to 707) at a rate much greater than chance (77.6% accuracy). The study identifies phylogenetic signatures of both native and nonnative species and suggests several practical applications for this approach including monitoring biodiversity and facilitating biosecurity

DNA barcoding Indonesian Acanthopleurinae (Polyplacophora)

The approximately 14 recognized species of Acanthopleurinae worldwide include conspicuous and large tropical shore chitons of the genera Acanthopleura, Liolphura, and Squamopleura. They are well studied for their impressive homing behavior, shell eyes (ocelli), radular biomineralization, and bioerosion activities, but have a relatively shallow fossil record, not recorded from before the Miocene. The accessibility of these chitons on the shores of Indonesia made them an excellent test case for new efforts to DNA barcode biodiversity, training and employing Indonesians with the objective of initiating a more complete assessment of biodiversity throughout the Coral Triangle. The latest monographic treatment including Acanthopleurinae was published in 2006 and reports only three members of this taxon in the vicinity of Indonesia: Acanthopleura spinosa (Bruguière 1792), A. gemmata (De Blainville 1825), and Squamopleura miles (Carpenter in Plsbry 1893). Here we apply current and accepted DNA barcoding methods to assess biodiversity in Acanthopleurinae throughout Indonesia, also employing available published or unpublished relevant sequences. Because Indonesian marine research has been historically underrepresented in the international scientific community until recent years, we hypothesized that we would discover new operational taxonomic units (OTUs), which could represent previously-undescribed species. If the Coral Triangle acts as a center of origin of chiton biodiversity, we hypothesized that the phylogenetic positions of the Indonesian chitons will be more derived than described species. Our combined analysis of mitochondrial COI and 16S rDNA gene portions for over 200 Acanthopleurinae from Indonesia has confirmed this expectation. Our preliminary analyses have identified as many as 11 OTUs, indicating that either cryptic species or strong phylogeographic structure are to be expected in this region of known high endemism. We conclude by making recommendations for future intertidal research in Indonesia and the Coral Triangle

Testing use of mitochondrial COI sequences for the identification and phylogenetic analysis of New Zealand caddisflies (Trichoptera)

We tested the hypothesis that cytochrome c oxidase subunit 1 (COI) sequences would successfully discriminate recognised species of New Zealand caddisflies. We further examined whether phylogenetic analyses, based on the COI locus, could recover currently recognised superfamilies and suborders. COI sequences were obtained from 105 individuals representing 61 species and all 16 families of Trichoptera known from New Zealand. No sequence sharing was observed between members of different species, and congeneric species showed from 2.3 to 19.5% divergence. Sequence divergence among members of a species was typically low (mean = 0.7%; range 0.0–8.5%), but two species showed intraspecific divergences in excess of 2%. Phylogenetic reconstructions based on COI were largely congruent with previous conclusions based on morphology, although the sequence data did not support placement of the purse-cased caddisflies (Hydroptilidae) within the uncased caddisflies, and, in particular, the Rhyacophiloidea. We conclude that sequence variation in the COI gene locus is an effective tool for the identification of New Zealand caddisfly species, and can provide preliminary phylogenetic inferences. Further research is needed to ascertain the significance of the few instances of high intra-specific divergence and to determine if any instances of sequence sharing will be detected with larger sample sizes