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The rapidly growing field of molecular diet analysis is becoming increasingly popular among ecologists, especially when investigating methodologically challenging groups, such as invertebrate generalist predators. Prey DNA detection success is known to be affected by multiple factors; however, the type of dietary sample has rarely been considered. Here, we address this knowledge gap by comparing prey DNA detection success from three types of dietary samples. In a controlled feeding experiment, using the carabid beetle Pterostichus melanarius as a model predator, we collected regurgitates, faeces and whole consumers (including their gut contents) at different time points postfeeding. All dietary samples were analysed using multiplex PCR, targeting three different length DNA fragments (128, 332 and 612 bp). Our results show that both the type of dietary sample and the size of the DNA fragment contribute to a significant part of the variation found in the detectability of prey DNA. Specifically, we observed that in both regurgitates and whole consumers, prey DNA was detectable significantly longer for all fragment sizes than for faeces. Based on these observations, we conclude that prey DNA detected from regurgitates and whole consumers DNA extracts are comparable, whereas prey DNA detected from faeces, though still sufficiently reliable for ecological studies, will not be directly comparable to the former. Therefore, regurgitates and faeces constitute a useful, nonlethal source for dietary information that could be applied to field studies in situations when invertebrate predators should not be killed.

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Background Herbivores modify the structure and function of tundra ecosystems. Understanding their impacts is necessary to assess the responses of these ecosystems to ongoing environmental changes. However, the effects of herbivores on plants and ecosystem structure and function vary across the Arctic. Strong spatial variation in herbivore effects implies that the results of individual studies on herbivory depend on local conditions, i.e., their ecological context. An important first step in assessing whether generalizable conclusions can be produced is to identify the existing studies and assess how well they cover the underlying environmental conditions across the Arctic. This systematic map aims to identify the ecological contexts in which herbivore impacts on vegetation have been studied in the Arctic. Specifically, the primary question of the systematic map was: “What evidence exists on the effects of herbivores on Arctic vegetation?”. Methods We used a published systematic map protocol to identify studies addressing the effects of herbivores on Arctic vegetation. We conducted searches for relevant literature in online databases, search engines and specialist websites. Literature was screened to identify eligible studies, defined as reporting primary data on herbivore impacts on Arctic plants and plant communities. We extracted information on variables that describe the ecological context of the studies, from the studies themselves and from geospatial data. We synthesized the findings narratively and created a Shiny App where the coded data are searchable and variables can be visually explored. Review findings We identified 309 relevant articles with 662 studies (representing different ecological contexts or datasets within the same article). These studies addressed vertebrate herbivory seven times more often than invertebrate herbivory. Geographically, the largest cluster of studies was in Northern Fennoscandia. Warmer and wetter parts of the Arctic had the largest representation, as did coastal areas and areas where the increase in temperature has been moderate. In contrast, studies spanned the full range of ecological context variables describing Arctic vertebrate herbivore diversity and human population density and impact. Conclusions The current evidence base might not be sufficient to understand the effects of herbivores on Arctic vegetation throughout the region, as we identified clear biases in the distribution of herbivore studies in the Arctic and a limited evidence base on invertebrate herbivory. In particular, the overrepresentation of studies in areas with moderate increases in temperature prevents robust generalizations about the effects of herbivores under different climatic scenarios.

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24The rapidly growing field of molecular diet analysis is becoming increasingly popular 25 among ecologists, especially when investigating methodologically challenging groups 26 such as invertebrate generalist predators. Prey DNA detection success is known to be 27 affected by multiple factors, however the type of dietary sample has rarely been 28 considered. Here, we address this knowledge gap by comparing prey DNA detection 29 success from three types of dietary samples. In a controlled feeding experiment, using 30 the carabid beetle Pterostichus melanarius as a model predator, we collected 31 regurgitates, feces and whole consumers (including their gut contents) at different time 32 points post-feeding. All dietary samples were analyzed using multiplex PCR targeting 33 three different length DNA fragments (128 bp, 332 bp and 612 bp). Our results show 34 that both the type of dietary sample and the size of the DNA fragment contribute to a 35 significant part of the variation found in the detectability of prey DNA. Specifically, we 36 observed that in both regurgitates and whole consumers prey DNA was detectable 37 significantly longer for all fragment sizes than for feces. Based on these observations, 38we conclude that prey DNA detected from regurgitates and whole consumers DNA 39 extracts are comparable, whereas prey DNA detected from feces, though still 40 sufficiently reliable for ecological studies, will not be directly comparable to the former. 41

Annual monitoring of mortality agents in the course of a spruce budworm (Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae)) population cycle is essential to understanding the factors governing the rise and collapse of outbreaks. To date, assessments of causes of budworm mortality have relied on laboratory rearing of field-collected larvae, followed by visual identification of emerging parasitoids and/or microscopic analysis of pathogens in larval carcasses. Although this approach has provided vital information on the abundance and identity of mortality agents, the procedure is labor-intensive and has limits in terms of accuracy. To overcome these shortcomings, we developed a molecular identification tool that makes use of real-time quantitative PCR (qPCR) and TaqMan® technologies. The tool relies on taxon-specific molecular variants (single nucleotide polymorphism [SNP] markers) found in mitochondrial (COI) and nuclear (28S rDNA) genes, for parasitoids, and in the nuclear SSU rDNA gene for microsporidian pathogens; these are then used as molecular signatures targeted by qPCR primers and TaqMan probes. Thus, the design of several sets of primers and probes deployed in multiplex format enables the identification of natural enemies via a molecular sorting process, bypassing barcode sequencing. Crude budworm DNA extracts are processed through a first module that detects dipteran and hymenopteran parasitoids, and microsporidian infections. Positive samples are then processed for species determination using three additional modules, enabling the identification of 20 common natural enemies of the spruce budworm. The tool has been fully validated using DNA samples from all comprised taxa, and both its sensitivity and accuracy compared favorably with the rearing-based method in an analysis of field-collected budworms. Using this tool, sample processing can be completed within two days, does not require larval rearing, provides accurate species identification, and can be conducted by technical staff without extensive molecular biology or insect taxonomy training.