In conservation science, observation-based methods are generally applied to wildlife monitoring. While useful, such approaches are often restricted to well-characterized and conspicuous species. Environmental DNA (eDNA) can complement observation-dependent surveys, as sample collection is generally less labor-intensive. Furthermore, eDNA can be used to simultaneously detect multiple taxa in various habitats. Most eDNA applications rely on short PCR product-based meta-barcoding approaches. However, such approaches may be less robust when applied to genetically uncharacterized species. Hybridization capture techniques while less sensitive than metabarcoding, can identifying divergent sequences, especially those obtained from highly degraded DNA. To assess eDNA based methods for surveillance in a conservation context, we collected samples during the dry season from randomly selected waterholes from a protected area in eastern Cambodia. We applied both hybridization capture enrichment and metabarcoding targeting mammal mitogenomes to water (N = 46) and sediment (N = 10) samples. Seventeen species were detected, including 11 mammals, three amphibians, two reptiles, and one bird. Six species overlapped between the two applied methods. Seven species were hybridization capture-specific detections, and four were metabarcoding-specific. Metabarcoding was more sensitive to abundant or large body-size species while hybridization capture provided more mitogenomic information. While both methods have some advantages over observational approaches, combining them may improve the sensitivity, number of species detected and amount of genetic information obtained from eDNA. We demonstrate that eDNA from tropical forest waterholes can be used to determine the presence of wildlife that may be difficult to detect using other observational approaches.

AbstractSampling genetic material from rare and often secretive mammals can be difficult in challenging environments such as tropical rainforests. Large‐scale sampling is important however for resolving species' taxonomic uncertainties, as well as to help provide genetic material for reference databases that can be used with DNA forensics to combat the illegal wildlife trade. Environmental DNA (eDNA) and invertebrate‐derived DNA (iDNA) offer a promising way to overcome this sampling bottleneck, though to date e/iDNA studies have been primarily used to obtain information about species presence using barcoding or metabarcoding approaches. In this study, we collected 4,710 terrestrial leeches across six protected areas in the Annamite Mountains of Viet Nam and Laos and sequenced fragments (223–694 bp) of mt‐Cytb and/or mt‐Dcr of the mitogenomes from six species that are frequently found in the illegal wildlife trade. Although amplification success was generally low (<50%) because DNA was highly degraded, we showed that e/iDNA samples can be an important source of genetic material for difficult to sample species and show promise as a way to fill existing sampling gaps. Because e/iDNA sampling has the potential to generate much‐needed genetic data to improve taxonomic assessments, this approach can be used to support conservation management by contributing to genetic databases that are necessary to determine the origin of wildlife products or to inform the reintroduction of confiscated animals.

Convolutional neural networks (CNNs) and deep learning are powerful and robust tools for ecological applications. CNNs can perform very well in various tasks, especially for visual tasks and image data. Image segmentation (the classification of all pixels in images) is one such task and can for example be used to assess forest vertical and horizontal structure. While such methods have been suggested, widespread adoption in ecological research has been slow, likely due to technical difficulties in implementation of CNNs and lack of toolboxes for ecologists. Here, we present R package imageseg which implements a workflow for general-purpose image segmentation using CNNs and the U-Net architecture in R. The workflow covers data (pre)processing, model training, and predictions. We illustrate the utility of the package with two models for forest structural metrics: tree canopy density and understory vegetation density. We trained the models using large and diverse training data sets from a variety of forest types and biomes, consisting of 3288 canopy images (both canopy cover and hemispherical canopy closure photographs) and 1468 understory vegetation images. Overall classification accuracy of the models was high with a Dice score of 0.91 for the canopy model and 0.89 for the understory vegetation model (assessed with 821 and 367 images, respectively), indicating robustness to variation in input images and good generalization strength across forest types and biomes. The package and its workflow allow simple yet powerful assessments of forest structural metrics using pre-trained models. Furthermore, the package facilitates custom image segmentation with multiple classes and based on color or grayscale images, e.g. in cell biology or for medical images. Our package is free, open source, and available from CRAN. It will enable easier and faster implementation of deep learning-based image segmentation within R for ecological applications and beyond.

Abstract Environmental DNA (eDNA) and invertebrate‐derived DNA (iDNA) are used to survey biodiversity non‐invasively to mitigate difficulties in obtaining wildlife samples, particularly in remote areas or for rare species. Recently, eDNA/iDNA were used to monitor known wildlife pathogens; however, most wildlife pathogens are unknown and often evolutionarily divergent. To detect and identify known and novel mammalian viruses from eDNA/iDNA, we used a curated set of RNA oligonucleotides as viral baits in a hybridization capture system coupled with high‐throughput sequencing. We detected multiple known and novel mammalian RNA and DNA viruses from multiple viral families from both waterhole eDNA and leech‐derived iDNA. Congruence was found between detected hosts and viruses identified in leeches and waterholes. Our results demonstrate that eDNA/iDNA samples represent an effective non‐invasive resource for studying wildlife viral diversity and for detecting novel potentially zoonotic viruses prior to their emergence.

AbstractInformation on geographic patterns of species genetic diversity is often needed to inform conservation strategies. Obtaining data over large geographic areas can be difficult, especially for rare or elusive species. Here, we explore the use of invertebrate‐derived DNA (iDNA) obtained from terrestrial leeches to assess genetic diversity of the Annamite striped rabbit Nesolagus timminsi, a little‐known and highly threatened lagomorph endemic to the Annamites ecoregion of Vietnam and Laos. We collected 4,877 leeches pooled into 651 leech bulk samples from eight study areas across the northern and central Annamites. Using metabarcoding, we detected Annamite striped rabbit DNA in 11 leech bulk samples from five of the study areas. We amplified up to 3,000 bp of six mitochondrial DNA loci of Annamite striped rabbit from nine of the 11 bulk samples. Haplotype network analyses showed five shared haplotypes among these samples. Phylogenetic trees constructed using maximum likelihood and Bayesian inference showed little geographic structuring among the Annamite striped rabbit populations across the five study areas. Our results provide information that can be directly used to inform planned ex situ conservation measures for the Annamite striped rabbit. We discuss our results within the context of using iDNA to noninvasively obtain data to assess species genetic diversity, and the potential of this approach to be applied to other species and systems.

Aim Unsustainable hunting is leading to widespread defaunation across the tropics. To mitigate against this threat with limited conservation resources, stakeholders must make decisions on where to focus anti-poaching activities. Identifying priority areas in a robust way allows decision-makers to target areas of conservation importance, therefore maximizing the impact of conservation interventions. Location Annamite mountains, Vietnam and Laos. Methods We conducted systematic landscape-scale surveys across five study sites (four protected areas, one unprotected area) using camera-trapping and leech-derived environmental DNA. We analyzed detections within a Bayesian multi-species occupancy framework to evaluate species responses to environmental and anthropogenic influences. Species responses were then used to predict occurrence to unsampled regions. We used predicted species richness maps and occurrence of endemic species to identify areas of conservation importance for targeted conservation interventions. Results Analyses showed that habitat-based covariates were uninformative. Our final model therefore incorporated three anthropogenic covariates as well as elevation, which reflects both ecological and anthropogenic factors. Conservation-priority species tended to found in areas that are more remote now or have been less accessible in the past, and at higher elevations. Predicted species richness was low and broadly similar across the sites, but slightly higher in the more remote site. Occupancy of the three endemic species showed a similar trend. Main conclusion Identifying spatial patterns of biodiversity in heavily-defaunated landscapes may require novel methodological and analytical approaches. Our results indicate to build robust prediction maps it is beneficial to sample over large spatial scales, use multiple detection methods to increase detections for rare species, include anthropogenic covariates that capture different aspects of hunting pressure, and analyze data within a Bayesian multi-species framework. Our models further suggest that more remote areas should be prioritized for anti-poaching efforts to prevent the loss of rare and endemic species.

Invertebrate-derived DNA (iDNA), in combination with high throughput sequencing, has been proposed as a cost-efficient and powerful tool to survey vertebrate species. Previous studies, however, have only provided evidence that vertebrates can be detected using iDNA, but have not taken the next step of placing these detection events within a statistical framework that allows for robust biodiversity assessments. Here, we compare concurrent iDNA and camera-trap surveys. Leeches were repeatedly collected in close vicinity to 64 camera-trap stations in Sabah, Malaysian Borneo. We analyse iDNA-derived mammalian detection events in a modern occupancy model that accounts for imperfect detection and compare the results with those from occupancy models parameterised with camera-trap-derived detection events. We also combine leech-iDNA and camera-trap data in a single occupancy model. We found consistent estimates of occupancy probabilities produced by our camera-trap and leech datasets. This indicates that the metabarcoding of leech-iDNA method provides reasonable estimates of occupancy and may be a suitable method for studying and monitoring mammal species in tropical rainforests. However, we also show that a more extensive collection of leeches would be needed to assess mammal biodiversity with a robustness similar to that of camera traps. As certain taxa were only detected in leeches, we see great potential in complementing camera-trap studies with the iDNA approach, as long as the collection of leeches follows a robust and standardised sampling scheme. Synthesis and applications. Here, we describe an approach to analyse detection records of mammals derived from leech samples using an occupancy framework that accounts for leech-specific factors influencing the detection probability. We further combined camera trap and leech data, which lead to increased confidence in occupancy estimates. Our approach is not restricted to the processing of leech samples, but can be used for the analysis of other invertebrate DNA and environmental DNA data. Our study is the first step to shift the application of invertebrate DNA studies from opportunistic ad-hoc collections to the systematic surveys required for long-term management of wildlife populations.

Background: The use of environmental DNA for species detection via metabarcoding is growing rapidly. We present a co-designed lab workflow and bioinformatic pipeline to mitigate the 2 most important risks of environmental DNA use: sample contamination and taxonomic misassignment. These risks arise from the need for polymerase chain reaction (PCR) amplification to detect the trace amounts of DNA combined with the necessity of using short target regions due to DNA degradation. Findings: Our high-throughput workflow minimizes these risks via a 4-step strategy: (i) technical replication with 2 PCR replicates and 2 extraction replicates; (ii) using multi-markers (12S, 16S, CytB); (iii) a "twin-tagging," 2-step PCR protocol; and (iv) use of the probabilistic taxonomic assignment method PROTAX, which can account for incomplete reference databases. Because annotation errors in the reference sequences can result in taxonomic misassignment, we supply a protocol for curating sequence datasets. For some taxonomic groups and some markers, curation resulted in >50% of sequences being deleted from public reference databases, owing to (i) limited overlap between our target amplicon and reference sequences, (ii) mislabelling of reference sequences, and (iii) redundancy. Finally, we provide a bioinformatic pipeline to process amplicons and conduct PROTAX assignment and tested it on an invertebrate-derived DNA dataset from 1,532 leeches from Sabah, Malaysia. Twin-tagging allowed us to detect and exclude sequences with non-matching tags. The smallest DNA fragment (16S) amplified most frequently for all samples but was less powerful for discriminating at species rank. Using a stringent and lax acceptance criterion we found 162 (stringent) and 190 (lax) vertebrate detections of 95 (stringent) and 109 (lax) leech samples. Conclusions: Our metabarcoding workflow should help research groups increase the robustness of their results and therefore facilitate wider use of environmental and invertebrate-derived DNA, which is turning into a valuable source of ecological and conservation information on tetrapods.

Surveillance of wild vertebrates can be challenging in remote and inaccessible areas such as tropical rainforests. Blood-feeding parasites, such as leeches, can facilitate wild vertebrate monitoring by targeting residual DNA from the animals the leeches feed on. Successes in detecting host DNA from leeches suggest that host viruses may also be detectable. To systematically test this hypothesis, we performed a proof of concept study using quantitative PCR (qPCR) to detect DNA viruses (bovine herpesvirus [BHV], human adenovirus [HAdV]) and RNA viruses (influenza A [InfA] and measles morbillivirus [MeV]) from nucleic acids extracted from medicinal leeches fed with blood spiked with each virus. All viruses except BHV showed a gradual decline in concentration from day 1 to 50, and all except BHV were detectable in at least half of the samples even after 50 days. BHV exhibited a rapid decline at day 27 and was undetectable at day 50. Our findings in medicinal leeches indicate that leeches collected in the wild might be an untapped resource for detecting vertebrate viruses and could provide new opportunities to study wildlife viral diseases of rare species in challenging environments, where capturing and handling of animals is difficult.

Combination strategies involving chemotherapy and monoclonal antibodies (mAb) are commonly used in attempts to produce better clinical outcomes. This practice has led to new and ongoing toxicities that may lead to reductions in dose or noncompliance, limiting the effectiveness of treatment. Viscum album L (VA) preparations are widely used in Europe as additive therapy and have been associated with reduced chemotherapy-related adverse reactions and increased health-related quality of life. Concomitant VA therapy might also reduce toxicity related to mAb. This retrospective study investigated the safety of combined treatment with VA and mAb in cancer patients. A total of 43 patients had combined therapy (474 exposures); 12 had VA without mAb (129 exposures), and 8 had mAb without VA (68 exposures). Most patients (89.3%) received concomitant chemotherapy or supportive therapies. A total of 34 patients (60.7%) experienced 142 adverse events (AEs). Leucopenia (14.1% of all events), acneiform rash (8.5%), and stomatitis (6.3%) occurred most frequently. Longitudinal logistic regression analysis suggested a nearly 5 times higher odds of experiencing an AE following treatment with mAb compared with mAb plus VA (95% CI = 1.53-16.14). Our results, together with theoretical consideration of potential botanical-drug interactions, suggest that combined treatment with VA and mAb is safe.

Background: Pancreatic cancer has a dire prognosis and is associated with a high mortality. Palliative patients have special needs and often seek help in integrative oncological concepts (IO) that combine conventional and complementary therapies. Nevertheless there are few recommendations regarding IO in current cancer guidelines. The aims of this study were to report on implementation of IO in everyday palliative care and to analyze patient survival in advanced pancreatic cancer.Methods: This multicenter observational study investigates the implementation of IO and length of survival of patients suffering from advanced pancreatic cancer (stage IV). We analyzed patient's survival by employing multivariable proportional hazard models using different parametric distribution functions and compared patients receiving chemotherapy only, a combination of chemotherapy and Viscum album (VA) treatment, and VA treatment only.Results: Records of 240 patients were analyzed. Complementary therapy showed high acceptance (93 %). Most frequent therapy was VA treatment (74 %) that was often administered concomitantly to chemotherapy (64 %). Both therapies had positive effects on patient survival as they had significant negative effects on the hazard in our log-normal model. A second analysis showed that patients with combined chemotherapy and VA therapy performed significantly better than patients receiving only chemotherapy (12.1 to 7.3 month). Patients receiving only VA therapy showed longer survival than those receiving neither chemotherapy nor VA therapy (5.4 to 2.5 months). Our data demonstrates that IO can be implemented in the everyday care of patients without disregarding conventional treatment. Patients combining VA with chemotherapy showed longest survival.Conclusions: Our data demonstrate the importance and potential of health services research showing that IO treatment can be successfully implemented in the every-day care of patients suffering from advanced pancreatic cancer. Patients combining VA with chemotherapy showed longest survival. To address patients' needs adequately, future cancer guidelines might increasingly include comments on complementary treatment options in addition to conventional therapies. Further studies should investigate the effect of complementary treatments on survival and quality of life in more detail.

Background. Intratumoral (IT) injection of European mistletoe (Viscum album L) preparations might induce local tumor response through combined cytotoxic and immunomodulatory actions of the preparations. Although promising in vitro and in vivo data, along with clinical case studies suggest the need for validation of this hypothesis in prospective trials, the safety of IT mistletoe injections has yet to be thoroughly assessed. Methods. The present study summarizes the practice and safety of off-label IT mistletoe therapy within the Network Oncology, a conjoint clinical registry of German hospitals and outpatients specialized in anthroposophic and integrative medicine. Demographic, diagnosis and treatment data of cancer patients who received IT mistletoe applications between 2007 and 2013 were assessed. Suspected adverse drug reactions (ADRs) were analyzed in terms of type, frequency, severity, seriousness and potential risk factors. Results. A total of 123 cancer patients received 862 IT mistletoe injections (preparations from Abnoba, Helixor and Iscucin). The most commonly applied preparations were Abnoba viscum Fraxini (71 patients) and Helixor Mali (54 patients). Of the total patients, 26 patients (21.1%) experienced 74 ADRs. All ADRs were in response to either Abnoba viscum Fraxini (25.4% of exposed patients) or Helixor Mali (18.5% of exposed patients). ADRs were mostly body temperature or immune related and of mild (83.8%) or moderate (14.9%) intensity. Only one possible ADR was described as severe (hypertension) and no serious ADRs occurred. The frequency of ADRs to IT mistletoe injections was 3 times and 5 times higher than has previously been found for subcutaneous and intravenous applications of mistletoe, respectively. Conclusion. IT injection of mistletoe preparations resulted in a relatively high frequency of ADRs. Nearly all ADRs were mild to moderate however, and no serious ADRs occurred. Furthermore, it is possible that immune-related ADRs such as pyrexia and local inflammatory reactions might be critical for tumor response. In light of these results, IT mistletoe therapy seems to be safe and prospective trials are recommended.

Background. In Europe, mistletoe extracts are widely used as a complementary cancer therapy. We assessed the safety of subcutaneous mistletoe as a conjunctive therapy in cancer patients within an anthroposophic medicine setting in Germany. Methods. A multicentre, observational study was performed within the Network Oncology. Suspected mistletoe adverse drug reactions (ADRs) were described by frequency, causality, severity, and seriousness. Potential risk factors, dose relationships and drug-drug interactions were investigated. Results. Of 1923 cancer patients treated with subcutaneous mistletoe extracts, 283 patients (14.7%) reported 427 expected effects (local reactions <5 cm and increased body temperature <38°C). ADRs were documented in 162 (8.4%) patients who reported a total of 264 events. ADRs were mild (50.8%), moderate (45.1%), or severe (4.2%). All were nonserious. Logistic regression analysis revealed that expected effects were more common in females, while immunoreactivity decreased with increasing age and tumour stage. No risk factors were identified for ADRs. ADR frequency increased as mistletoe dose increased, while fewer ADRs occurred during mistletoe therapy received concurrent with conventional therapies. Conclusion. The results of this study indicate that mistletoe therapy is safe. ADRs were mostly mild to moderate in intensity and appear to be dose-related and explained by the immune-stimulating, pharmacological activity of mistletoe.

Fragestellung: Neben den drei onkologischen Therapieverfahren chirurgische Resektion, Radiotherapie und pharmakologische Systemtherapie wird der Stellenwert von nicht-medikamentösen-system Interventionen (NPI) in der Therapie des Lungenkarzinom als eine vierte Modalität in der Behandlung diskutiert. NPIs wurden in der Studie von J. Temel (NEJM, 2010) in der palliativen Therapie des NSCLC ähnlich wirksam beschrieben wie eine palliative CTX. Um den Stellenwert der NPIs in der Therapie vom Lungenkarzinom zu evaluieren werden diese im Rahmen des Anthroposophisch-Integrativen onkologischen Konzeptes systematisch in einer klinischen Datenbank "Netzwerk Onkologie" (NO) erfasst. Methode: vom 30.06.2011 – 1.7.2013 wurden 384 Patienten mit SCLC und NSCLC in die Datenbank aufgenommen. Die NPI wurden in verbale (v-NPI) und non-verbale (nv-NPI) unterschieden. V-NPI umfassen Dimension der verbalen Krankheitsbewältigung (z.B. Arztgespräch, sozialdienstliche oder psycho-onkologische Betreuung). Nv-NPI umfassen Maßnahmen der körpernahen, oder kreativ-therapeutischen, bewegungstherapeutischen Krankheitsbewältigung. Ergebnisse: 199 Männer und 185 Frauen mit NSCLC oder SCLC wurden in den 24 Monaten aufgenommen. Mittleres Alter war 67 Jahre. 81 Patienten erhielten eine operative Resektion, 60 Patienten erhielten eine Strahlentherapie, 155 Patienten erhielten eine adjuvante oder eine palliative first-line CTX. 241 (63%) Patienten erhielten NPIs. Davon hatten 50% mehr als 3 NPI. Maximale Anzahl waren 13 NPI. 44% erhielten eine v-NPI und 59% mindestens eine nv-NPI. Schlussfolgerungen: Versorgungsdaten des NO kann eine Analyse der NPI im Sinne der Versorgungsforschung leisten. In der Anthroposophisch-Integrativen Therapie werden NPI häufig angewandt und es haben v-NPI und nv-NPI eine vergleichbar hohe Anwendungszahl. Eine Nachverfolgung der Patienten ermöglicht genaueren Aufschluss und Planung für prospektive RCTs über die bislang nicht ausreichend evaluierte vierte Therapiemodalität.

Background. Traditional mistletoe therapy in cancer patients involves subcutaneous applications of Viscum album L. preparations, with doses slowly increasing based on patient responses. Intravenous infusion of high doses may improve therapeutic outcomes and is becoming more common. Little is known about the safety of this “off-label” application of mistletoe. Methods. An observational study was performed within the Network Oncology. Treatment with intravenous mistletoe applications is described. The frequency of adverse drug reactions (ADRs) to intravenous mistletoe applications was calculated and compared to ADR data from a study on subcutaneous applications. Results. Of 475 cancer patients who received intravenous infusions of Helixor, Abnoba viscum, or Iscador mistletoe preparations, 22 patients (4.6%) reported 32 ADRs of mild (59.4%) or moderate severity (40.6%). No serious ADRs occurred. ADRs were more frequently reported to i.v. mistletoe administered alone (4.3%), versus prior to chemotherapy (1.6%). ADR frequency differed with respect to preparation type, with Iscador preparations showing a higher relative frequency, compared to Abnoba viscum and Helixor. Overall, patients were almost two times less likely to experience an ADR to intravenous compared to subcutaneous application of mistletoe. Conclusion. Intravenous mistletoe therapy was found to be safe and prospective studies for efficacy are recommended.