Austen C. Thomas

Quantifying sequence proportions in a DNA-based diet study using Ion Torrent amplicon sequencing: which counts count?

A goal of many environmental DNA barcoding studies is to infer quantitative information about relative abundances of different taxa based on sequence read proportions generated by high‐throughput sequencing. However, potential biases associated with this approach are only beginning to be examined. We sequenced DNA amplified from faeces (scats) of captive harbour seals (Phoca vitulina) to investigate whether sequence counts could be used to quantify the seals’ diet. Seals were fed fish in fixed proportions, a chordate‐specific mitochondrial 16S marker was amplified from scat DNA and amplicons sequenced using an Ion Torrent PGM™. For a given set of bioinformatic parameters, there was generally low variability between scat samples in proportions of prey species sequences recovered. However, proportions varied substantially depending on sequencing direction, level of quality filtering (due to differences in sequence quality between species) and minimum read length considered. Short primer tags used to identify individual samples also influenced species proportions. In addition, there were complex interactions between factors; for example, the effect of quality filtering was influenced by the primer tag and sequencing direction. Resequencing of a subset of samples revealed some, but not all, biases were consistent between runs. Less stringent data filtering (based on quality scores or read length) generally produced more consistent proportional data, but overall proportions of sequences were very different than dietary mass proportions, indicating additional technical or biological biases are present. Our findings highlight that quantitative interpretations of sequence proportions generated via high‐throughput sequencing will require careful experimental design and thoughtful data analysis.

Improving accuracy of DNA diet estimates using food tissue control materials and an evaluation of proxies for digestion bias.

Ecologists are increasingly interested in quantifying consumer diets based on food DNA in dietary samples and high‐throughput sequencing of marker genes. It is tempting to assume that food DNA sequence proportions recovered from diet samples are representative of consumers diet proportions, despite the fact that captive feeding studies do not support that assumption. Here, we examine the idea of sequencing control materials of known composition along with dietary samples in order to correct for technical biases introduced during amplicon sequencing and biological biases such as variable gene copy number. Using the Ion Torrent PGM©, we sequenced prey DNA amplified from scats of captive harbour seals (Phoca vitulina) fed a constant diet including three fish species in known proportions. Alongside, we sequenced a prey tissue mix matching the seals’ diet to generate tissue correction factors (TCFs). TCFs improved the diet estimates (based on sequence proportions) for all species and reduced the average estimate error from 28 ± 15% (uncorrected) to 14 ± 9% (TCF‐corrected). The experimental design also allowed us to infer the magnitude of prey‐specific digestion biases and calculate digestion correction factors (DCFs). The DCFs were compared with possible proxies for differential digestion (e.g. fish protein%, fish lipid%) revealing a strong relationship between the DCFs and percent lipid of the fish prey, suggesting prey‐specific corrections based on lipid content would produce accurate diet estimates in this study system. These findings demonstrate the value of parallel sequencing of food tissue mixtures in diet studies and offer new directions for future research in quantitative DNA diet analysis.

Quantitative DNA metabarcoding: improved estimates of species proportional biomass using correction factors derived from control material

DNA metabarcoding is a powerful new tool allowing characterization of species assemblages using high‐throughput amplicon sequencing. The utility of DNA metabarcoding for quantifying relative species abundances is currently limited by both biological and technical biases which influence sequence read counts. We tested the idea of sequencing 50/50 mixtures of target species and a control species in order to generate relative correction factors (RCFs) that account for multiple sources of bias and are applicable to field studies. RCFs will be most effective if they are not affected by input mass ratio or co‐occurring species. In a model experiment involving three target fish species and a fixed control, we found RCFs did vary with input ratio but in a consistent fashion, and that 50/50 RCFs applied to DNA sequence counts from various mixtures of the target species still greatly improved relative abundance estimates (e.g. average per species error of 19 ± 8% for uncorrected vs. 3 ± 1% for corrected estimates). To demonstrate the use of correction factors in a field setting, we calculated 50/50 RCFs for 18 harbour seal (Phoca vitulina) prey species (RCFs ranging from 0.68 to 3.68). Applying these corrections to field‐collected seal scats affected species percentages from individual samples (Δ 6.7 ± 6.6%) more than population‐level species estimates (Δ 1.7 ± 1.2%). Our results indicate that the 50/50 RCF approach is an effective tool for evaluating and correcting biases in DNA metabarcoding studies. The decision to apply correction factors will be influenced by the feasibility of creating tissue mixtures for the target species, and the level of accuracy needed to meet research objectives.

BASELINE HEALTH PARAMETERS AND SPECIES COMPARISONS AMONG FREE-RANGING ATLANTIC SHARPNOSE (RHIZOPRIONODON TERRAENOVAE), BONNETHEAD (SPHYRNA TIBURO), AND SPINY DOGFISH (SQUALUS ACANTHIAS) SHARKS IN GEORGIA, FLORIDA, AND WASHINGTON, USA

Sharks are of commercial, research, conservation, and exhibition importance but we know little regarding health parameters and population status for many species. Here we present health indicators and species comparisons for adults of three common wild-caught species: 30 Atlantic sharpnose sharks (Rhizoprionodon terraenovae) and 31 bonnethead sharks (Sphyrna tiburo) from the western Atlantic, and 30 spiny dogfish sharks (Squalus acanthias) from the eastern Pacific. All animals were captured during June–July 2009 and 2010. Median values and preliminary reference intervals were calculated for hematology, plasma biochemistry, trace nutrients, and vitamin A, E, and D concentrations. Significant differences, attributable to physiologic differences among the species, were found in the basic hematologic and plasma biochemistry variables. Significant species differences in arsenic and selenium plasma concentrations were found and appear to coincide with diet and habitat variability among these three species. Vitamin E was significantly higher in the bonnethead shark, again related to the foraging ecology and ingestion of plant material by this species. The Atlantic sharpnose had significantly higher vitamin A concentrations, supported by the higher proportion of teleosts in the diet. Vitamin D was below the limit of quantification in all three species. These preliminary reference intervals for health variables can be used to assess and monitor the population health and serve as indicators of nutritional status in these populations of wild elasmobranchs.

GREAT SHEARWATER (PUFFINUS GRAVIS) MORTALITY EVENTS ALONG THE EASTERN COAST OF THE UNITED STATES

The Great Shearwater (Puffinus gravis) is an abundant pelagic seabird that undertakes transequatorial migrations between the North and South Atlantic Ocean. This species is a useful indicator of large-scale alterations in marine dynamics due to its wide geographic range, long-distance migrations, and relative abundance. From 1993 to 2011, 12 separate mortality events, with 4,961 Great Shearwaters recovered, were documented along the eastern coast of the United States. Of these, seven events (n=4,885) occurred in the Southeast (SE) and five (n=76) in the Northeast (NE) United States. The cause of death was determined either by necropsy (n=60) or external examination (n=4,901). All Great Shearwaters stranded along the SE United States were emaciated while 58% were emaciated in the NE United States. No plastic was observed in Great Shearwaters in the SE US (n=27), but the gastrointestinal tract of 82% (n=27) of all stranded birds along the NE United States had at least one plastic bead. There was no evidence of infectious disease or heavy metals in stranded Great Shearwaters examined (n=14, from the 2005 SE event). Stable isotope analysis of feathers (n=9, from a 2007 SE event) suggests dietary differences between emaciated stranded birds and live-caught healthy birds. The temporal distribution of stranding detections suggests a general increase in the number of observed Great Shearwater strandings over the past two decades. From 1993 to 2000 there were a total of three mortality events with 296 individual Great Shearwaters. However, there was a threefold increase in the number of mortality events from 2001 to 2011 (nine events involving 4,665 individuals). The causes of this apparent increase in strandings are unknown but may be due to an increase in reporting effort over the past two decades combined with changing oceanographic conditions in the South Atlantic Ocean, leading to large-scale mortality of emaciated Great Shearwaters along the east coast of the United States.

Competing tradeoffs between increasing marine mammal predation and fisheries harvest of Chinook salmon

Many marine mammal predators, particularly pinnipeds, have increased in abundance in recent decades, generating new challenges for balancing human uses with recovery goals via ecosystem-based management. We used a spatio-temporal bioenergetics model of the Northeast Pacific Ocean to quantify how predation by three species of pinnipeds and killer whales (Orcinus orca) on Chinook salmon (Oncorhynchus tshawytscha) has changed since the 1970s along the west coast of North America, and compare these estimates to salmon fisheries. We find that from 1975 to 2015, biomass of Chinook salmon consumed by pinnipeds and killer whales increased from 6,100 to 15,200 metric tons (from 5 to 31.5 million individual salmon). Though there is variation across the regions in our model, overall, killer whales consume the largest biomass of Chinook salmon, but harbor seals (Phoca vitulina) consume the largest number of individuals. The decrease in adult Chinook salmon harvest from 1975–2015 was 16,400 to 9,600 metric tons. Thus, Chinook salmon removals (harvest + consumption) increased in the past 40 years despite catch reductions by fisheries, due to consumption by recovering pinnipeds and endangered killer whales. Long-term management strategies for Chinook salmon will need to consider potential conflicts between rebounding predators or endangered predators and prey.

Counting with DNA in metabarcoding studies: How should we convert sequence reads to dietary data?

Advances in DNA sequencing technology have revolutionized the field of molecular analysis of trophic interactions, and it is now possible to recover counts of food DNA sequences from a wide range of dietary samples. But what do these counts mean? To obtain an accurate estimate of a consumers diet should we work strictly with data sets summarizing frequency of occurrence of different food taxa, or is it possible to use relative number of sequences? Both approaches are applied to obtain semi‐quantitative diet summaries, but occurrence data are often promoted as a more conservative and reliable option due to taxa‐specific biases in recovery of sequences. We explore representative dietary metabarcoding data sets and point out that diet summaries based on occurrence data often overestimate the importance of food consumed in small quantities (potentially including low‐level contaminants) and are sensitive to the count threshold used to define an occurrence. Our simulations indicate that using relative read abundance (RRA) information often provides a more accurate view of population‐level diet even with moderate recovery biases incorporated; however, RRA summaries are sensitive to recovery biases impacting common diet taxa. Both approaches are more accurate when the mean number of food taxa in samples is small. The ideas presented here highlight the need to consider all sources of bias and to justify the methods used to interpret count data in dietary metabarcoding studies. We encourage researchers to continue addressing methodological challenges and acknowledge unanswered questions to help spur future investigations in this rapidly developing area of research.

Application of real-time quantitative PCR assays for detecting marine Brucella spp. in fish:

Brucella ceti and Brucella pinnipedialis have been documented as occurring in marine mammals, and B. ceti has been identified in 3 naturally acquired human cases. Seroconversion and infection patterns in Pacific Northwest harbor seals (Phoca vitulina richardii) and North Atlantic hooded seals (Cystophora cristata) indicate post-weaning exposure through prey consumption or lungworm infection, suggesting fish and possibly invertebrates play an epizootiologic role in marine Brucella transmission and possible foodborne risk to humans. We determined if real-time quantitative PCR (qPCR) assays can detect marine Brucella DNA in fish DNA. Insertion sequence (IS)711 gene and sequence type (ST)27 primer–probe sets were used to detect Brucella associated with marine mammals and human zoonotic infections, respectively. First, DNA extracts from paired-species fish (containing 2 species) samples were tested and determined to be Brucella DNA negative using both IS711 and ST27 primer–probe sets. A representative paired-species fish DNA sample was spiked with decreasing concentrations of B. pinnipedialis DNA to verify Brucella detection by the IS711 primer–probe within fish DNA. A standard curve, developed using isolated DNA from B. pinnipedialis, determined the limit of detection. Finally, the IS711 primer–probe was used to test Atlantic cod (Gadus morhua) DNA extracts experimentally infected with the B. pinnipedialis hooded seal strain. In culture-positive cod tissue, the IS711 limit of detection was ~1 genome copy of Brucella. Agreement between culture and PCR results for the 9 positive and 9 negative cod tissues was 100%. Although a larger sample set is required for validation, our study shows that qPCR can detect marine Brucella in fish.

Large-scale molecular diet analysis in a generalist marine mammal reveals male preference for prey of conservation concern

Abstract Sex‐specific diet information is important in the determination of predator impacts on prey populations. Unfortunately, the diet of males and females can be difficult to describe, particularly when they are marine predators. We combined two molecular techniques to describe haul‐out use and prey preferences of male and female harbor seals (Phoca vitulina) from Comox and Cowichan Bay (Canada) during 2012–2013. DNA metabarcoding quantified the diet proportions comprised of prey species in harbor seal scat, and qPCR determined the sex of the individual that deposited each scat. Using 287 female and 260 male samples, we compared the monthly sex ratio with GLMs and analyzed prey consumption relative to sex, season, site, and year with PERMANOVA. The sex ratio between monthly samples differed widely in both years (range = 12%–79% males) and showed different patterns at each haul‐out site. Male and female diet differed across both years and sites: Females consumed a high proportion of demersal fish species while males consumed more salmonid species. Diet composition was related to both sex and season (PERMANOVA: R 2 = 27%, p < 0.001; R 2 = 24%, p < 0.001, respectively) and their interaction (PERMANOVA: R 2 = 11%, p < 0.001). Diet differences between males and females were consistent across site and year, suggesting fundamental foraging differences, including that males may have a larger impact on salmonids than females. Our novel combination of techniques allowed for both prey taxonomic and spatiotemporal resolution unprecedented in marine predators.

Can sex-specific consumption of prey be determined from DNA in predator scat?

Sex-biased predation, a predator’s bias for one prey sex over the other, can have important demographic impacts on prey species of conservation concern. Yet, it is difficult to measure in the wild. Molecular scatology has been used to indirectly determine the proportion of prey items consumed in the diet, and it may be possible to apply similar approaches to determine the sex-biased consumption of prey items. We developed a molecular method to indirectly examine sex-specific predation employing scat, focusing on predator–prey interactions between Chinook salmon (Oncorhynchus tshawytscha) and harbor seals (Phoca vitulina richardii). We established that the proportions of male and female Chinook DNA can be determined in a controlled sample by measuring a Y-linked marker, growth hormone pseudogene, using qPCR. We then applied the assay to harbor seal scat samples. Although the assay amplified in 83% of scat samples, 27% of samples quantified had an estimated male proportion > 1, which may have been due to a lack of robustness of the PCR assay in samples. Lastly, we constructed a biomass calibration curve to determine whether DNA measurements could estimate the proportions of male and female biomass consumed. The calibration curve was skewed by high male DNA density precluding our ability to quantify the relative amounts. We demonstrated that nuclear prey markers can be amplified in predator scat, however, contamination and extreme DNA density differences between the prey sexes may pose practical difficulties to estimate the relative amounts of male to female biomass consumed.