Using low-coverage whole genome sequencing (genome skimming) to delineate three introgressed species of buffalofish (Ictiobus).

Consumption of buffalofish has been sporadically associated with Haff disease-like illnesses involving sudden onset muscle pain and weakness due to skeletal muscle rhabdomyolysis, but determination of precisely which species are associated with these illnesses has been impeded by a lack of species-specific DNA-based markers. Here, three closely related species of buffalofish native to the Mississippi River Basin (Ictiobus bubalus, Ictiobus cyprinellus and Ictiobus niger) that have previously proven genetically indistinguishable using both mitochondrial and nuclear single-locus sequencing were reliably discriminated using low-coverage whole genome sequencing ('genome skimming'). Using 44 specimens representing the three species collected from the mid/upper (Missouri) and lower (Louisiana) regions of the species' native ranges, the SISRS (Site Identification from Short Read Sequences) bioinformatics pipeline was adapted to (1) identify over 620Mbp of putatively homologous nuclear sequence data and (2) isolate over 140,000 single-nucleotide polymorphisms (SNPs) that supported accurate species delimitation, all without the use of a reference genome or annotation data. These sites were used to classify Ictiobus spp. samples with genome-skim data, along with a larger set (n = 67) where ultraconserved elements (UCEs) were sequenced. Analyses of whole mitochondrial data revealed more limited signal. Nearly all samples matched their purported species based on morphologic identification, but two Missouri samples morphologically identified as I. niger grouped with samples of I. bubalus, albeit with significant enrichment of I. niger SNPs. To our knowledge this is the first report of a DNA-based tool to reliably discriminate these three morphologically distinct species.

Codependence of individuals in the Nephromyces species swarm requires heterospecific bacterial endosymbionts.

Symbiosis is one of the most important evolutionary processes shaping the biodiversity on Earth. Symbiotic associations often bring together organisms from different domains of life, which can provide an unparalleled route to evolutionary innovation.1-4 The phylum Apicomplexa encompasses 6,000 ubiquitous animal parasites; however, species in the recently described apicomplexan family, Nephromycidae, are reportedly non-virulent.5,6 The members of the genus Nephromyces live within a specialized organ of tunicates, called the renal sac, in which they use concentrated uric acid as a primary nitrogen source.7,8 Here, we report genomic and transcriptomic data from the diverse genus Nephromyces, as well as the three bacterial symbionts that live within this species complex. We show that the diversity of Nephromyces is unexpectedly high within each renal sac, with as many as 20 different species inhabiting the renal sacs in wild populations. The many species of Nephromyces can host three different types of bacterial endosymbionts; however, FISH microscopy allowed us to demonstrate that each individual Nephromyces cell hosts only a single bacterial type. Through the reconstruction and analyses of the endosymbiont bacterial genomes, we infer that each bacterial type supplies its host with different metabolites. No individual species of Nephromyces, in combination with its endosymbiont, can produce a complete set of essential amino acids, and culture experiments demonstrate that individual Nephromyces species cannot form a viable infection. Therefore, we hypothesize that Nephromyces spp. depend on co-infection with congeners containing different bacterial symbionts in order to exchange metabolites to meet their needs.

Screening the PRISM Library against Staphylococcus aureus Reveals a Sesquiterpene Lactone from Liriodendron tulipifera with Inhibitory Activity.

Infections caused by the bacterium Staphylococcus aureus continue to pose threats to human health and put a financial burden on the healthcare system. The overuse of antibiotics has contributed to mutations leading to the emergence of methicillin-resistant S. aureus, and there is a critical need for the discovery and development of new antibiotics to evade drug-resistant bacteria. Medicinal plants have shown promise as sources of new small-molecule therapeutics with potential uses against pathogenic infections. The principal Rhode Island secondary metabolite (PRISM) library is a botanical extract library generated from specimens in the URI Youngken Medicinal Garden by upper-division undergraduate students. PRISM extracts were screened for activity against strains of methicillin-susceptible S. aureus (MSSA). An extract generated from the tulip tree (Liriodendron tulipifera) demonstrated growth inhibition against MSSA, and a bioassay-guided approach identified a sesquiterpene lactone, laurenobiolide, as the active constituent. Intriguingly, its isomers, tulipinolide and epi-tulipinolide, lacked potent activity against MSSA. Laurenobiolide also proved to be more potent against MSSA than the structurally similar sesquiterpene lactones, costunolide and dehydrocostus lactone. Laurenobiolide was the most abundant in the twig bark of the tulip tree, supporting the twig bark's historical and cultural usage in poultices and teas.

Utilizing Big Data to Identify Tiny Toxic Components: Digitalis.

The botanical genus Digitalis is equal parts colorful, toxic, and medicinal, and its bioactive compounds have a long history of therapeutic use. However, with an extremely narrow therapeutic range, even trace amounts of Digitalis can cause adverse effects. Using chemical methods, the United States Food and Drug Administration traced a 1997 case of Digitalis toxicity to a shipment of Plantago (a common ingredient in dietary supplements marketed to improve digestion) contaminated with Digitalis lanata. With increased accessibility to next generation sequencing technology, here we ask whether this case could have been cracked rapidly using shallow genome sequencing strategies (e.g., genome skims). Using a modified implementation of the Site Identification from Short Read Sequences (SISRS) bioinformatics pipeline with whole-genome sequence data, we generated over 2 M genus-level single nucleotide polymorphisms in addition to species-informative single nucleotide polymorphisms. We simulated dietary supplement contamination by spiking low quantities (0-10%) of Digitalis whole-genome sequence data into a background of commonly used ingredients in products marketed for "digestive cleansing" and reliably detected Digitalis at the genus level while also discriminating between Digitalis species. This work serves as a roadmap for the development of novel DNA-based assays to quickly and reliably detect the presence of toxic species such as Digitalis in food products or dietary supplements using genomic methods and highlights the power of harnessing the entire genome to identify botanical species.

Metabolic Contributions of an Alphaproteobacterial Endosymbiont in the Apicomplexan Cardiosporidium cionae.

Apicomplexa is a diverse protistan phylum composed almost exclusively of metazoan-infecting parasites, including the causative agents of malaria, cryptosporidiosis, and toxoplasmosis. A single apicomplexan genus, Nephromyces, was described in 2010 as a mutualist partner to its tunicate host. Here we present genomic and transcriptomic data from the parasitic sister species to this mutualist, Cardiosporidium cionae, and its associated bacterial endosymbiont. Cardiosporidium cionae and Nephromyces both infect tunicate hosts, localize to similar organs within these hosts, and maintain bacterial endosymbionts. Though many other protists are known to harbor bacterial endosymbionts, these associations are completely unknown in Apicomplexa outside of the Nephromycidae clade. Our data indicate that a vertically transmitted α-proteobacteria has been retained in each lineage since Nephromyces and Cardiosporidium diverged. This α-proteobacterial endosymbiont has highly reduced metabolic capabilities, but contributes the essential amino acid lysine, and essential cofactor lipoic acid to C. cionae. This partnership likely reduces resource competition with the tunicate host. However, our data indicate that the contribution of the single α-proteobacterial endosymbiont in C. cionae is minimal compared to the three taxa of endosymbionts present in the Nephromyces system, and is a potential explanation for the virulence disparity between these lineages.

Suitability of DNA Sequencing Tools for Identifying Edible Seaweeds Sold in the United States.

Seaweeds have been consumed by billions of people around the world and are increasingly popular in United States (US) diets. Some seaweed species have been associated with adverse health effects-such as heavy metal toxicity-and higher priced seaweeds may be more prone to adulteration. Knowing which species of seaweeds are being marketed in the US is important for protecting human health and preventing economic adulteration. Therefore, the United States Food and Drug Administration is developing new DNA-based species identification tools to complement established chemical methods for verifying the accurate labeling of products. Here, seaweed products available in the United States were surveyed using a tiered approach to evaluate a variety of DNA extraction techniques followed by traditional DNA barcoding via Sanger sequencing; if needed, genome skimming of total extracted nuclear DNA via next-generation sequencing was performed. This two-tiered approach of DNA barcoding and genome skimming could identify most seaweed samples (41/46), even those in blends (2/2, 1 out of 3 labeled species in each). Only two commercial samples appeared to be mislabeled or to contain unintended algal species. Five samples, labeled as "hijiki" or "arame", could not be confirmed by these DNA-based identification methods.