Now back to Monday reads on a Monday. Some new studies for you to catch up.
An rbcL Reference Library to Aid in the Identification of Plant Species Mixtures by DNA Metabarcoding
Premise of the study: DNA metabarcoding has broad-ranging applications in ecology, aerobiology, biosecurity, and forensics. A bioinformatics pipeline has recently been published for identification using a comprehensive database of ITS2, one of the common plant DNA barcoding markers. There is, however, no corresponding database for rbcL, the other primary marker used in plants.
Methods: Using publicly available data, we compiled a reference library of rbcL sequences and trained databases for use with UTAX and RDP classifier algorithms. We used this reference library, along with the existing bioinformatics pipeline and ITS2 reference library, to identify species in an artificial mixture of nine species of pollen. We have made this database publicly available in multiple formats, to allow use with multiple bioinformatics pipelines, now and in the future.
Results: Using the rbcL database, in addition to the ITS2 database, we succeeded in making species-level identifications for eight species and a family-level identification of the ninth species. This is an improvement on ITS2 sequence alone.
Discussion: The reference library described here will assist with identification of plant species using rbcL. By making another gene region available for standard barcoding, this will increase the resolution and accuracy of identifications.
Feeding by Leucopis argenticollis and Leucopis piniperda (Diptera: Chamaemyiidae) from the western USA on Adelges tsugae (Hemiptera: Adelgidae) in the eastern USA
Leucopis argenticollis (Zetterstedt) and Leucopis piniperda (Malloch) are known to feed on the lineage of Adelges tsugae Annand that is native to western North America, but it is not known if they will survive on the lineage that was introduced from Japan to the eastern USA. In 2014, western Leucopis spp. larvae were brought to the laboratory and placed on A. tsugae collected in either Washington (North American A. tsugae lineage) or Connecticut (Japanese lineage). There were no significant differences in survival or developmental times between flies reared on the two different adelgid lineages. In 2015 and 2016, western Leucopis spp. adults were released at two different densities onto enclosed branches of A. tsugae infested eastern hemlock (Tsuga canadensis (L.) Carr.) in Tennessee and New York. Cages were recovered and their contents examined 4 weeks after release at each location. Leucopis spp. larvae and puparia of the F1 generation were recovered at both release locations and adults of the F1 generation were collected at the Tennessee location. The number of Leucopis spp. offspring collected increased with increasing adelgid density, but did not differ by the number of adult flies released. Flies recovered from cages and flies collected from the source colony were identified as L.argenticollis and L. piniperda using DNA barcoding. These results demonstrate that Leucopis spp. from the Pacific Northwest are capable of feeding and developing to the adult stage on A. tsugae in the eastern USA and they are able to tolerate environmental conditions during late spring and early summer at the southern and northern extent of the area invaded by A. tsugae in the eastern USA.
Despite the high value of decapod crustaceans, relatively little research has focused on assessing the transparency in the marketing of these species. This study represents the first comprehensive evaluation of the quality of labelling, and the extent of mislabelling, of decapod crustacean products on the South African market. Data collected through surveys of supermarkets and seafood shops in three provinces (KwaZulu-Natal [KZN], Western Cape [WC] and Gauteng [GP]), indicated that the large majority of domestically available crustacean products were imported, but that 18% of these failed to comply with locally applicable country of origin labelling regulations. Voluntary information relating to the scientific name, production method (wild caught or farmed), and capture method of the species was supplied more frequently in supermarkets than in seafood shops, more frequently in the WC and GP than in KZN, and more frequently on shrimp products than on crab and lobster products. DNA sequencing of 77 products collected from the surveyed outlets revealed that 24 (31%) were misrepresented in some way. Species misrepresentations were most pronounced for shrimps, with Litopenaeus vannamei and Pleoticus muelleri being confirmed as the most common substitute species. One shrimp product was found to contain at least three different species, none of which matched the declared species, whereas a product labelled as crab turned out to be a member of the phylum Mollusca rather than the subphylum Crustacea. Overall, these findings demonstrate that the misrepresentation of crustaceans is commonplace on the South African market, signalling the need for a revision of the current seafood labelling and traceability legislation, as well as monitoring and enforcement efforts.
The aim of this study was to compare the performance of a DNA-barcode assay with fatty acid profile analysis to authenticate the botanical origin of olive oil. To achieve this aim, we performed a PCR-capillary electrophoresis (PCR-CE) approach on olive oil: seed oil blends using the plastid trnL (UAA) intron barcode. In parallel to genomic analysis, we subjected the samples to gas chromatography analysis of fatty acid composition. While the PCR-CE assay proved equally efficient as gas chromatography analysis in detecting adulteration with soybean, palm, rapeseed, sunflower, sesame, cottonseed and peanut oils, it was superior to the widely utilized analytical chemistry approach in revealing the adulterant species and detecting small quantities of corn and safflower oils in olive oil. Moreover, the DNA-based test correctly identified all tested olive oil: hazelnut oil blends whereas it was not feasible to detect hazelnut oil adulteration through fatty acid profile analysis. Thus, the present research has shown the feasibility of a PCR-CE barcode assay to detect adulteration in olive oil.
Molecular scatology and high-throughput sequencing reveal predominately herbivorous insects in the diets of adult and nestling Western Bluebirds (Sialia mexicana) in California vineyards
Determining the ecosystem function of high-order predators is critical for evaluation of food web interactions. Insectivorous birds are abundant predators in many ecosystems yet because they forage upon small taxa, it remains largely unknown whether birds are providing ecosystem services in the form of pest control or disservices by preying upon predaceous arthropod species. We extracted DNA from noninvasive fecal samples of adult and nestling Western Bluebirds (Sialia mexicana) in California vineyards. Using universal arthropod-specific primers, we sequenced prey items via massively parallel sequencing on the Illumina MiSeq platform. Bluebirds consumed a broad diet comprising 66 unique arthropod species from 6 orders and 28 families. Aedes sp. (mosquitoes: Culicidae), a previously unknown prey, was the most common item recovered, occurring in 49.5% of the fecal samples. Ectoparasitic bird blowfly (Protocalliphora) DNA was found in 7% of adult and 11% of nestling samples, presenting clear evidence of active feeding by the avian hosts on adult or larval ectoparasites. Herbivorous insects, primarily from the orders Hemiptera and Lepidoptera, represented over half (56%) of the prey items in bluebird diets. Intraguild predation (consumption of predator or parasitoid arthropods) represented only 3% of adult and nestling dietary items. Diets of adults were significantly different from nestlings as were diets from birds sampled in different vineyard blocks. Sex, date, number of young, and individual bird (based on resampled individuals) were all insignificant factors that did not explain diet variability. Nestling age was a significant factor in explaining a small amount of the variability in dietary components. In addition, our analysis of subsampling larger fecal samples and processing them independently revealed highly dissimilar results in all 10 trials and we recommend avoiding this common methodology. Molecular scatology offers powerfully informative techniques that can reveal the ecosystem function and services provided by abundant yet cryptic avian foragers.
A wide variety of DNA based methods have been developed to identify fish species, including those that employ a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. One such method developed by Dooley, Sage, Clarke, Brown, and Garrett (2005) used amplification of a portion of the mitochondrial cytochrome B (cytB) gene, with a three enzyme digestion, visualized and identified using the Agilent 2100 Bioanalyzer System. More recently this method was modified by Agilent Technologies to target a section of the cytochrome c oxidase 1 (CO1) gene, within the 655 base pair (bp) "barcoding" fragment, using a two enzyme digestion to increase sample throughput and to exploit publically available CO1 data generated through the Barcode of Life initiative (Mueller et al. 2015). Here we evaluate this method on fifteen different commercial fish species with five replicate specimens of each. DNA barcoding of the CO1 gene was used as an orthogonal confirmatory method and also to further understand the results found using the modified Agilent PCR-RFLP method.