DNA metabarcoding for diet analysis and biodiversity: A case study using the endangered Australian sea lion (Neophoca cinerea)

The analysis of apex predator diet has the ability to deliver valuable insights into ecosystem health, and the potential impacts a predator might have on commercially relevant species. The Australian sea lion (Neophoca cinerea) is an endemic apex predator and one of the world’s most endangered pinnipeds. Given that prey availability is vital to the survival of top predators, this study set out to understand what dietary information DNA metabarcoding could yield from 36 sea lion scats collected across 1,500 km of its distribution in southwest Western Australia. A combination of PCR assays were designed to target a variety of potential sea lion prey, including mammals, fish, crustaceans, cephalopods, and birds. Over 1.2 million metabarcodes identified six classes from three phyla, together representing over 80 taxa. The results confirm that the Australian sea lion is a wide- ranging opportunistic predator that consumes an array of mainly demersal fauna. Further, the important commercial species Sepioteuthis australis (southern calamari squid) and Panulirus cygnus (western rock lobster) were detected, but were present in <25% of samples. Some of the taxa identified, such as fish, sharks and rays, clarify previous knowledge of sea lion prey, and some, such as eel taxa and two gastropod species, represent new dietary insights. Even with modest sample sizes, a spatial analysis of taxa and operational taxonomic units found within the scat shows significant differences in diet between many of the sample locations and identifies the primary taxa that are driving this variance. This study provides new insights into the diet of this endangered predator and confirms the efficacy of DNA metabarcoding of scat as a noninvasive tool to more broadly define regional biodiversity

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Experiment2

Fasta files of amplicon reads from Experiment 2 for both the MiSeq and Ion Torrent. All fasta files are non-dereplicated and have been separated into sample batches based on MID (index) tags and all primers have been removed. No further quality or abundance filtering has been performed on the fasta file

Experiment1

Fasta files of amplicon reads from Experiment 1 for both the GS Junior and Ion Torrent. All fasta files are non-dereplicated and have been separated into sample batches based on MID (index) tags and all primers have been removed. No further quality or abundance filtering has been performed on the fasta file

Experiment5

Fasta file of amplicon reads from Experiment 5 for the Ion Torrent. The file non-dereplicated and has only been separated based on MID (index) tag. Primers have not been removed and no quality or abundance filtering has been performed on the fasta file

Impact of analysis parameters on the numbers of taxonomic units obtained for a bulk-bone sample.

<p>A number of analysis parameters were used to analyse a complex mixture containing numerous taxa. Different quality and abundance filtering methods were used in addition to two taxonomy-independent measures of analysis, full definitions and explanations of which are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124671#pone.0124671.g001" target="_blank">Fig 1</a>. The spread in the numbers of taxonomic units obtained across the combinations of parameters chosen is seen. The radius of each semicircle represents the number of taxonomic units obtained given a set combination of the parameters used. The number of taxonomic units is also indicated above each semicircle. Each semicircle is proportional to all others. AFM—abundance filtering method; QFM—quality filtering method; TIM—taxonomy-independent method.</p

Average sequencing error rates across a single amplicon region.

<p>Average sequencing error rates are shown for multiple bird species across the whole of a short 12S rRNA gene region <b>(A)</b>. Additionally, the error profile across the gene region is shown for <i>Calyptorhynchus lathami</i> for both the Ion Torrent PGM <b>(B)</b> and MiSeq <b>(C)</b> with key. The error patterns observed were similar across all species sequenced. Error rates are shown across 5 bp segments and where error rates were above 1% for a single base this is indicated through the red circles.</p

Application of STR markers in wildlife forensic casework involving Australian black-cockatoos (Calyptorhynchus spp.)

Parrots and cockatoos are highly prized aviary birds and the demands for such species has fuelled their illegal trade and harvest from the wild. Here we report on three forensic case studies involving black-cockatoos (Calyptorhynchus spp.) endemic to Australia. These cases involve suspected poaching and illegal killing of endangered red- and white-tailed black-cockatoos. Through the prior development of 20 polymorphic microsatellite loci and population databases for white- and red-tailed black-cockatoos, the tools are available to conduct high-resolution paternity and individual identity testing. In one case, we matched a red-tailed black-cockatoo nestling to a tree hollow from which it was poached through the use of DNA from eggshell recovered from the nest. For the second case, we utilized our provenance population database (nest sites), and identified the kinship and geographic origin of a white-tailed black-cockatoo, which was illegally harvested from the wild. The third case determined the number individual white-tailed black-cockatoos allegedly shot at a fruit grower's orchard from body part remains. These genetic investigations highlight the significance and statistical confidence of DNA profiling and associated databases for endangered taxa, such as exotic birds. Our cockatoo population databases are the first of their kind in Australia, and demonstrate the efficacy of such approaches to identify such illegal activity. With a robust set of genetic markers and methodologies in place, we aim to broaden our population databases to include other cockatoo species of conservation concern

TCM_006

Animal and Plant Sequences from Traditional Chinese Medicine Sample No.

DNA metabarcoding for diet analysis and biodiversity A case study using the endangered Australian sea lion

Results from a dietary study. The file contains mostly unique filtered fasta files of DNA sequences extracted and amplified from the scat of the Australian Sea Lion (Neophoca cinerea). The assays used focused mainly on fish, crustaceans, and cephalopods