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 consumer's 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.

Accounting for environmental and observer effects in estimating abundance of southern bluefin tuna from aerial survey data.

Southern bluefin tuna (SBT) is a valuable species that has been subject to high exploitation rates since the 1950s. In 2011, the spawning stock biomass was estimated to be at a historically low level, at only 5% of pre-fished biomass. A key component for managing and rebuilding the stock is having reliable, fishery-independent estimates of juvenile abundance. This paper describes how such estimates have been constructed from aerial surveys of juvenile (age 2-4) SBT conducted annually in the Great Australian Bight from 1993-2000 and 2005-2009. During these surveys, observers flew along pre-set transect lines searching for surface schools of SBT. Data were collected on the location and biomass of SBT sightings, and on the environmental conditions present during the survey. Sea surface temperature (SST) was found to correlate with the size (biomass) of schools, and several environmental variables, SST and wind speed in particular, were found to correlate with the number of sightings (presumably by affecting the ability of observers to see surface schools as well as whether fish were present at the surface). In addition, observers changed over time and differed in their aptitude for spotting tuna. Thus, generalized linear mixed models (GLMMs) were used to standardize the sightings and biomass data to a common set of observers and environmental conditions in order to produce an annual time series of relative abundance estimates. These estimates, which form one of two key inputs to the management procedure used by the international Commission for the Conservation of Southern Bluefin Tuna to set the global catch quota, suggest juvenile abundance was highest in the first years of the survey (1993-1996), after which it declined and fluctuated around a level about four times lower.

Migration dynamics of juvenile southern bluefin tuna.

Large scale migrations are a key component of the life history of many marine species. We quantified the annual migration cycle of juvenile southern bluefin tuna (Thunnus maccoyii; SBT) and spatiotemporal variability in this cycle, based on a multi-decadal electronic tagging dataset. Behaviour-switching models allowed for the identification of cohesive areas of residency and classified the temporal sequence of movements within a migration cycle from austral summer foraging grounds in the Great Australian Bight (GAB) to winter foraging grounds in the Indian Ocean and Tasman Sea and back to the GAB. Although specific regions within the Indian Ocean were frequented, individuals did not always return to the same area in consecutive years. Outward migrations from the GAB were typically longer than return migrations back to the GAB. The timing of individual arrivals to the GAB, which may be driven by seasonality in prey availability, was more cohesive than the timing of departures from the GAB, which may be subject to the physiological condition of SBT. A valuable fishery for SBT operates in the GAB, as do a number of scientific research programs designed to monitor SBT for management purposes; thus, understanding SBT migration to and from the area is of high importance to a number of stakeholders.

Decadal-Scale Forecasting of Climate Drivers for Marine Applications.

Climate influences marine ecosystems on a range of time scales, from weather-scale (days) through to climate-scale (hundreds of years). Understanding of interannual to decadal climate variability and impacts on marine industries has received less attention. Predictability up to 10 years ahead may come from large-scale climate modes in the ocean that can persist over these time scales. In Australia the key drivers of climate variability affecting the marine environment are the Southern Annular Mode, the Indian Ocean Dipole, the El Niño/Southern Oscillation, and the Interdecadal Pacific Oscillation, each has phases that are associated with different ocean circulation patterns and regional environmental variables. The roles of these drivers are illustrated with three case studies of extreme events-a marine heatwave in Western Australia, a coral bleaching of the Great Barrier Reef, and flooding in Queensland. Statistical and dynamical approaches are described to generate forecasts of climate drivers that can subsequently be translated to useful information for marine end users making decisions at these time scales. Considerable investment is still needed to support decadal forecasting including improvement of ocean-atmosphere models, enhancement of observing systems on all scales to support initiation of forecasting models, collection of important biological data, and integration of forecasts into decision support tools. Collaboration between forecast developers and marine resource sectors-fisheries, aquaculture, tourism, biodiversity management, infrastructure-is needed to support forecast-based tactical and strategic decisions that reduce environmental risk over annual to decadal time scales.

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.

Demographic structure, sex ratio and growth rates of southern bluefin tuna (Thunnus maccoyii) on the spawning ground.

The demographics of the southern bluefin tuna (SBT) Thunnus maccoyii spawning stock were examined through a large-scale monitoring program of the Indonesian longline catch on the spawning ground between 1995 and 2012. The size and age structure of the spawning population has undergone significant changes since monitoring began. There has been a reduction in the relative abundance of larger/older SBT in the catch since the early 2000s, and a corresponding decrease in mean length and age, but there was no evidence of a significant truncation of the age distribution. Pulses of young SBT appear in the catches in the early- and mid-2000s and may be the first evidence of increased recruitment into the spawning stock since 1995. Fish in these two recruitment pulses were spawned around 1991 and 1997. Size-related variations in sex ratio were also observed with female bias for fish less than 170 cm FL and male bias for fish greater than 170 cm FL. This trend of increasing proportion of males with size above 170 cm FL is likely to be related to sexual dimorphism in growth rates as male length-at-age is greater than that for females after age 10 years. Mean length-at-age of fish aged 8-10 years was greater for both males and females on the spawning ground than off the spawning ground, suggesting that size may be the dominant factor determining timing of maturation in SBT. In addition to these direct results, the data and samples from this program have been central to the assessment and management of this internationally harvested stock.

Maturity ogives for South Pacific albacore tuna (Thunnus alalunga) that account for spatial and seasonal variation in the distributions of mature and immature fish.

Length and age at maturity are important life history parameters for estimating spawning stock biomass and reproductive potential of fish stocks. Bias in estimates of size and age at maturity can arise when disparate distributions of mature and immature fish within a population are not accounted for in the analysis. Here we investigate the spatial and temporal variability in observed size and age at maturity of female albacore tuna, Thunnus alalunga, using samples collected across the South Pacific. Maturity status was identified using consistent histological criteria that were precise enough to allow for mature but regenerating females to be distinguished from immature females during the non-spawning season, permitting year-round sampling for maturity estimation in albacore. Using generalised linear mixed models, we found that the proportion of mature females at length varied significantly with latitude and time of year. Specifically, females at northern latitudes (∼10-20°S, where spawning occurs) were mature at significantly smaller lengths and ages than females at southern latitudes (∼20-40°S), particularly during the spawning season (October-March). This variation was due to different geographic distributions of mature and immature fish during the year. We present a method for estimating an unbiased maturity ogive that takes into account the latitudinal variation in proportion mature at length during a given season (spawning or non-spawning). Applying this method to albacore samples from the western region of the South Pacific gave a predicted length at 50% mature of ∼87 cm fork length (4.5 years).

Co-overexpression of Bag-1 and heat shock protein 70 in human epidermal squamous cell carcinoma: Bag-1-mediated resistance to 5-fluorouracil-induced apoptosis.

BACKGROUND: The aim was to determine whether Bcl-2-associated athanogene-1 (Bag-1) and/or its binding protein heat shock protein-70 (Hsp70) exhibit deregulated expression in epidermal squamous cell carcinoma (SCC) and whether Bag-1 confers apoptosis resistance. METHOD: Immunohistochemistry for Bag-1 and Hsp70 was performed on 60 epidermal SCC and 10 normal skin samples. The epidermal SCC cell line SCC-13 was treated with 5-fluorouracil (5-FU) after Bag-1 knockdown to determine whether high Bag-1 levels contribute to growth and/or apoptosis resistance. RESULTS: Normal epithelium expressed primarily nuclear Bag-1. Most tumours showed reduced nuclear Bag-1 staining, but a subset exhibited strong Bag-1 staining, with cytoplasmic Bag-1 staining intensity correlating with cytoplasmic Hsp70 staining intensity (r(s)=0.462; P<0.001) and less differentiation (P<0.001). Bag-1 knockdown resulted in markedly reduced SCC-13 cell yield, increased spontaneous apoptosis and enhanced sensitivity to 5-FU-induced apoptosis. Apoptosis induced by 5-FU in the Bag-1-knockdown cells was significantly greater than the additive apoptotic effect of 5-FU or Bag-1 knockdown alone. CONCLUSIONS: Overexpression of Bag-1 and Hsp70 in poorly differentiated SCC may confer both enhanced tumour cell growth and apoptosis resistance. Bag-1 may contribute to the resistance of more advanced epidermal SCC to chemotherapy.

Quantification of damage in DNA recovered from highly degraded samples--a case study on DNA in faeces.

BACKGROUND: Poorly preserved biological tissues have become an important source of DNA for a wide range of zoological studies. Measuring the quality of DNA obtained from these samples is often desired; however, there are no widely used techniques available for quantifying damage in highly degraded DNA samples. We present a general method that can be used to determine the frequency of polymerase blocking DNA damage in specific gene-regions in such samples. The approach uses quantitative PCR to measure the amount of DNA present at several fragment sizes within a sample. According to a model of random degradation the amount of available template will decline exponentially with increasing fragment size in damaged samples, and the frequency of DNA damage (lambda) can be estimated by determining the rate of decline. RESULTS: The method is illustrated through the analysis of DNA extracted from sea lion faecal samples. Faeces contain a complex mixture of DNA from several sources and different components are expected to be differentially degraded. We estimated the frequency of DNA damage in both predator and prey DNA within individual faecal samples. The distribution of fragment lengths for each target fit well with the assumption of a random degradation process and, in keeping with our expectations, the estimated frequency of damage was always less in predator DNA than in prey DNA within the same sample (mean lambda(predator) = 0.0106 per nucleotide; mean lambda(prey) = 0.0176 per nucleotide). This study is the first to explicitly define the amount of template damage in any DNA extracted from faeces and the first to quantify the amount of predator and prey DNA present within individual faecal samples. CONCLUSION: We present an approach for characterizing mixed, highly degraded PCR templates such as those often encountered in ecological studies using non-invasive samples as a source of DNA, wildlife forensics investigations and ancient DNA research. This method will allow researchers to measure template quality in order to evaluate alternate sources of DNA, different methods of sample preservation and different DNA extraction protocols. The technique could also be applied to study the process of DNA decay.

Sialosis and necrotising sialometaplasia in bulimia; a case report.

Salivary gland involvement, particularly salivary gland enlargement (sialosis), is a recognised complication in bulimia. We report the rare association of sialosis and necrotising sialometaplasia with bulimia in the same patient. The association of sialosis and necrotising sialometaplasia in the same patient with bulimia has been reported previously in two patients and may be coincidental, but the appearance in this additional patient suggests it may be prudent to explore this further.

Metastatic dissemination of human malignant oral keratinocyte cell lines following orthotopic transplantation reflects response to TGF-beta 1.

This study examined the behaviour of nine human malignant oral keratinocyte cell lines following orthotopic transplantation to the floor of the mouth of athymic mice. Tumourigenesis, local spread, and metastatic dissemination were correlated with known cellular responses to transforming growth factor-beta 1 (TGF-beta 1). Six of nine cell lines were tumourigenic; four of these cell lines showed local spread which was characterized by vascular and bone invasion. Metastatic spread was uncommon, with only 9% of animals with primary tumours developing metastases and these were almost exclusively found in the regional lymph nodes; there was one pulmonary metastasis and no liver deposits. Tumour cell behaviour did not reflect the clinical stage of the original tumours. Cell lines that were resistant to TGF-beta 1-induced growth inhibition were more likely to form primary tumours, exhibit local spread, and metastasize than cells that were growth-inhibited by the ligand. The data demonstrate that tumourigenicity and tumour behaviour in this orthotopic mouse model varied between cell lines and that the pattern of local invasion and metastasis was similar to that seen in human oral cancer. Furthermore, cell lines that were refractory to the growth inhibitory effects of TGF-beta 1 behaved more aggressively than cells that underwent ligand-induced cell-cycle arrest.

Salivary gland basal cell adenocarcinoma: a report of cases in a cat and two dogs.

Basal cell adenocarcinoma of the salivary gland is described in a cat and two dogs; tumour tissue was characterized by cords and islands of epithelial cells with a distinct basal layer. The tumours were stained by various immunohistochemical methods. In addition to positive staining with cytokeratin 14 and pancytokeratin (CKs 5, 6, 8, 17 and 19), there was also staining with Jack bean agglutinin A (ConA) and soya bean agglutinin (SBA); this occurs in many other types of salivary gland tumours and is a feature of normal salivary gland acinar cells. In one dog there was also staining with SBA. This is the first report of this tumour in domestic animals; the immunohistochemical characteristics did not distinguish it from other salivary gland tumours.

Intramuscular capillary hamartoma of the tongue.

Intramuscular vascular lesions in the tongue are rare and are usually the capillary form of haemangioma. A case is described of a lingual mass that resembled intramuscular haemangioma but had unique histological features that do not appear to have been described previously. The term intramuscular capillary hamartoma is suggested to describe it.

Oral epithelial dysplasia: clinical characteristics of western European residents.

To detail the clinical presentation of oral epithelial dysplasia in a large cohort of residents in western Europe. Descriptive statistical analysis of the data were calculated using chi-square and Fisher's exact tests. Oral epithelial dysplasia manifested typically as a white or mixed red and white lesion on the tongue, buccal mucosa or floor of mouth. The peak age of presentation of oral epithelial dysplasia was the 6th decade. Most clinically detected lesions had only mild oral epithelial dysplasia. Although uncommon, lesions with severe dysplasia were most likely to arise on the floor of mouth or lateral border of tongue. Oral epithelial dysplasia is likely to manifest as a solitary white patch, but it is not possible to accurately predict the likely degree of dysplasia from the clinical features of such lesions.

A study of the clinical characteristics of benign trigeminal sensory neuropathy.

PURPOSE: The purpose of this study was to describe the clinical characteristics of a series of patients presenting with benign trigeminal sensory neuropathy. PATIENTS AND METHODS: We conducted a retrospective analysis of the clinical and pathologic characteristics of 23 patients presenting with facial numbness of unknown etiology. RESULTS: Patients presented with diverse medical histories but could be grouped into those with a connective tissue disorder, neurologic disease, psychologic problems, or a medical history of unknown significance. The age of the patient, the severity and distribution of the trigeminal neuropathy, and symptoms other than neuropathy closely reflected the patient's medical history. The majority of patients underwent magnetic resonance imaging, but the results did not facilitate the diagnosis of the condition or reflect the extent and severity of the symptoms. In 60% of patients, the symptoms remained unchanged during the course of the study and outcome was not influenced by medical treatment. CONCLUSIONS: The diagnosis and management of benign trigeminal sensory neuropathy remain a significant clinical challenge.

Focal acantholytic dyskeratosis arising in an intraoral skin flap.

A case is described of a patient with a myocutaneous pectoralis major flap who presented with an oral lesion of focal acantholytic dyskeratosis that was clinically suspicious of recurrent tumour.

Sebaceous carcinoma of the salivary gland in a cat.

Sebaceous carcinoma of the submandibular salivary gland is described in a cat, tumour cells were characterized histologically by moderate amounts of pale eosinophilic or vacuolated cytoplasm. Tumour cells were stained with antibody to cytokeratins (CKs 5. 6, 8, 17 and 19) and with lectins Con A and wheat germ agglutinin (WGA); this occurs in many other types of salivary gland tumour and is a feature of normal salivary gland acinar cells.

Parotid Warthin's tumour Bristol Royal Infirmary (1985-1995): a study of histopathology in 33 cases.

This is a study of 33 (32 patients) confirmed Warthin's tumours (adenolymphomas) treated surgically at Bristol Royal Infirmary (1985--1995) focussing specifically upon 17 salient histopathological features together with capsular measurement by micrometry. Twenty-four out of 33 tumours had 'thin' capsules (< or = 200 microm), whereas 5/9 'thick' capsules were associated with gross tumour degeneration. The degenerative features including cystic change, squamous metaplasia, corpora amylacea like bodies, etc. were quantified. Tumours were classified into subtypes (typical, stroma poor, etc). These variations were not associated with age or sex. In 20/33 tumours a marginal sinus suggested a lymph node origin. Fine needle aspiration cytology (FNA) smears showing degeneration features are a diagnostic problem, but histopathological features are reflected in such smears and their recognition enhances diagnostic precision, enabling controlled surgical enucleation, the ideal operation, to be performed in most cases. In this study only 12 tumours were so treated; 11 others received parotidectomy. The theories of histogenesis are reviewed and discussed.

Pleomorphic adenomas of the major salivary glands: a study of the capsular form in relation to surgical management.

This was a retrospective study of 126 primary pleomorphic adenomas to correlate capsular characteristics with tumour histopathology in relation to current surgical debate (parotidectomy versus local excision). Capsular thickness was measured by micrometry and tumours classified into subtypes (1-4). Evidence of fine needle aspiration damage (needle tracks, infarction) was sought. Minimal changes were seen in eight tumours. Tumour growth features (bosselations, enveloping) were present in 57% and 33%, respectively, also microinvasion (42%) and tumour 'buds' (12%). Parotid lesions possessed thicker capsules than submandibular tumours. There was little correlation between capsular thickness and cellular structure. The significant exception was large (> 25 mm) hypocellular parotid tumours which had thinner capsules and could be vulnerable to operative rupture. In 110 standard operations (parotidectomy, submandibular gland excision), capsular exposure was evident in 81%. Field irrigation is recommended to lessen the risk of tumour seeding. This study reaffirms many elements of capsular weakness and suggests that parotidectomy is the operation of choice.