DIETARY ANALYSIS: ARTHROPODS

ARACHNIDS

Sint D., Thurner I., Kaufmann R., Traugott M. (2015): Sparing spiders: faeces as a non-invasive source of DNA. Frontiers in Zoology 12: 3.
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Spiders are important arthropod predators in many terrestrial ecosystems, and molecular tools have boosted our ability to investigate this taxon, which can be difficult to study with conventional methods. Nonetheless, it has typically been necessary to kill spiders to obtain their DNA for molecular applications, especially when studying their diet. We successfully tested the novel approach of employing spider faeces as a non-invasive source of DNA for species identification and diet analysis. Although the overall concentration of DNA in the samples was very low, consumer DNA, suitable for species identification, was amplified from 84% of the faecal pellets collected from lycosid spiders. Moreover, the most important prey types detected in the gut content of the lycosids were also amplified from the faecal samples. The ability to amplify DNA from spider faeces with specific and general primers suggests that this sample type can be used for diagnostic PCR and sequence-based species and prey identification such as DNA barcoding and next generation sequencing, respectively. These findings demonstrate that faeces provide a non-invasive alternative to full-body DNA extracts for molecular studies on spiders when killing or injuring the animal is not an option.

Xu C. C., Yen I. J., Bowman D., Turner C. R. (2015): Spider web DNA: a new spin on noninvasive genetics of predator and prey. Plos One 10: e0142503.
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Noninvasive genetic sampling enables biomonitoring without the need to directly observe or disturb target organisms. This paper describes a novel and promising source of noninvasive spider and insect DNA from spider webs. Using black widow spiders (Latrodectus spp.) fed with house crickets (Acheta domesticus), we successfully extracted, amplified, and sequenced mitochondrial DNA from spider web samples that identified both spider and prey to species. Detectability of spider DNA did not differ between assays with amplicon sizes from 135 to 497 base pairs. Spider and prey DNA remained detectable at least 88 days after living organisms were no longer present on the web. Spider web DNA as a proof-of-concept may open doors to other practical applications in conservation research, pest management, biogeography studies, and biodiversity assessments.

Powell E. C., Painting C. J., Hickey A. J., Machado G., Holwell G. I. (2021): Diet, predators, and defensive behaviors of New Zealand harvestmen (Opiliones: Neopilionidae). The Journal of Arachnology 49: 122-140.
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The Neopilionidae is a highly diversified harvestman family in New Zealand, comprising eight genera and 28 species. Although individuals of many species are abundant in the field, basic information on their natural history is absent. Here we describe the diet, predators, and defensive behaviors of 13 species across three genera, Forsteropsalis Taylor, 2013, Mangatangi Taylor, 2013, and Pantopsalis Simon, 1879. Using three years of field observations, we first identify food items for this family, finding that New Zealand neopilionids are opportunistic, generalist foragers with a diet composed of a wide variety of prey and scavenged soft-bodied invertebrates, including worms, amphipods, species from nine orders of insects, and two orders of arachnids (including conspecifics). We then describe the first known invertebrate predators of New Zealand harvestmen, including seven spider species, and conduct a review of the literature to collate a list of 32 species of native and non-native vertebrates (frogs, lizards, fish, birds, and mammals) that prey on harvestmen, including neopilionids. Finally, we describe the defensive behaviors of neopilionids, providing the first reports of autotomy and thanatosis in the family. In general, the diet of New Zealand neopilionids is similar to other harvestman species, and the list of predators includes mostly insectivorous taxa known to feed on harvestmen elsewhere. The defensive repertoire of neopilionids includes behaviors recorded for other species of Eupnoi, such as leg autotomy, but also unique behaviors that are only known for species of Dyspnoi and Laniatores, such as thanatosis.

CRUSTACEANS

Redd K. S., Ling S. D., Frusher S. D., Jarman S., Johnson C. R. (2014): Using molecular prey detection to quantify rock lobster predation on barrens‐forming sea urchins. Molecular Ecology 23: 3849-3869.
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We apply qPCR molecular techniques to detect in situ rates of consumption of sea urchins (Centrostephanus rodgersii and Heliocidaris erythrogramma) by rock lobsters (Jasus edwardsii). A non‐lethal method was used to source faecal samples from trap‐caught lobsters over 2 years within two no‐take research reserves. There was high variability in the proportion of lobsters with faeces positive for sea urchin DNA across years and seasons dependent on lobster size. Independent estimates of lobster predation rate on sea urchins (determined from observed declines in urchin abundances in the reserves relative to control sites) suggest that rates of molecular prey detection generally overestimated predation rates. Also, small lobsters known to be incapable of directly predating emergent sea urchins showed relatively high rates of positive tests. These results indicate that some lobsters ingest non‐predatory sources of sea urchin DNA, which may include (i) ingestion of C. rodgersii DNA from the benthos (urchin DNA is detectable in sediments and some lobsters yield urchin DNA in faeces when fed urchin faeces or sediment); (ii) scavenging; and/or predation by rock lobsters on small pre‐emergent urchins that live cryptically within the reef matrix (although this possibility could not be assessed). While the DNA‐based approach and direct monitoring of urchin populations both indicate high predation rates of large lobsters on emergent urchins, the study shows that in some cases absolute predation rates and inferences of predator–prey interactions cannot be reliably estimated from molecular signals obtained from the faeces of benthic predators. At a broad semi‐quantitative level, the approach is useful to identify relative magnitudes of predation and temporal and spatial variability in predation.

Nakamura Y., Tuji A., Makino W., Nagata N., Nakagawa M., Takamura N. (2020): Feeding ecology of a mysid species, Neomysis awatschensis in the Lake Kasumigaura: combining approach with microscopy, stable isotope analysis and DNA metabarcoding. Plankton and Benthos Research 15: 44-54.
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Neomysis awatschensis is an important prey item for various fishes in Lake Kasumigaura, Japan. There is, however, a contradiction concerning the major food sources of this mysid species: whether “bottom mud”, “particulate organic matter (POM) including phytoplankton”, or “mesozooplankton” is the main diet, and this uncertainty may be due to differences in the methodology used to determine the prey items in previous studies. This study examined the main food sources of N. awatschensis by combining three methods to eliminate methodological biases: DNA metabarcoding, microscopy and stable isotope analysis. Planktonic diatoms and green algae sequences were the main taxa detected by DNA metabarcoding and microscopy on the fecal pellets. The δ15N values of the mysids were similar to those of phytoplankton feeders rather than carnivorous planktonic crustaceans. These results suggest that diatoms and green algae were the major food sources for N. awatschensis in Lake Kasumigaura during the investigation period, and that its trophic level is as low as that of herbivores. However, the partial contribution of other “POM” (e.g., benthic diatoms) is also implied, considering the dispersion in δ13C values. Mesozooplankton prey, such as copepods, were detected both using DNA metabarcoding and microscopy, but they could not be a major food source due to the low nitrogen stable isotopic signature of the mysids. The opportunistic feeding habits of N. awatschensis possibly explain the consistent dominance of this species in the lake, where the ecosystem structure was substantially changed because of successive desalination.

HORSESHOE CRABS

Lee B. Y., Wakabayashi K., Sin S. Y. W., Ohtsuka S., Tsang L. M. (2021): DNA metabarcoding revealed interspecific dietary difference and prey selectivity in juvenile horseshoe crabs Carcinoscorpius rotundicauda and Tachypleus tridentatus from Hong Kong. Frontiers in Marine Science 8: 752806.
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Horseshoe crabs provide important ecological services including bioturbation and linking of food web in the shallow waters, but their populations are declining globally, leading to major concerns on conservation of these iconic animals. Baseline information of horseshoe crab ecology, such as their trophic role and food source, is pre-requisite for habitat protection plan and captive restocking program. Trophic ecology of Asian horseshoe crabs is relatively poorly understood and previous studies on their juveniles have suggested that they are selective feeders rather than opportunistic generalists. This study demonstrates a non-invasive approach, using DNA metabarcoding analyses of the nuclear 18S rRNA gene on fecal samples to assess the dietary compositions of Carcinoscorpius rotundicauda and Tachypleus tridentatus juveniles to (1) determine their dietary compositions and trophic roles in their ecosystem, (2) determine any prey selectivity, and (3) distinguish the interspecific dietary differences with potential implications on the habitat requirement and ecological partitioning between these two horseshoe crab species. The results based on relative read abundance (RRA) suggested that oligochaetes were the major prey items for both C. rotundicauda (41.6%) and T. tridentatus (32.4%). Bivalves and crustaceans were second major prey groups for C. rotundicauda (8.6 and 8.4%, respectively). Surprisingly, anthozoans contributed a considerable portion of T. tridentatus’s diet (22.8%), which has never been reported. Furthermore, the major prey groups identified in the fecal samples were not the dominant benthic organisms in the studied area as revealed by environmental DNA (eDNA) analyses on the sediment samples, implying that both species are selective feeders rather than dietary generalists. Significant differences observed in the dietary compositions of the two species might be partially due to the difference in habitat preference between the two species. This study provides new insights into the trophic ecology of the two Asian horseshoe crab species in the estuarine habitat and establishes a new framework for future detailed molecular dietary analyses on all developmental stages of horseshoe crabs around the world, which will allow us to evaluate the food sources needed for the survival of horseshoe crabs and facilitate future conservation strategies without killing the animals.

Waldner T., Traugott M. (2012): DNA‐based analysis of regurgitates: a noninvasive approach to examine the diet of invertebrate consumers. Molecular Ecology Resources 12: 669-675.
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DNA‐based gut content analysis has become an important tool for unravelling feeding interactions in invertebrate communities under natural conditions. It usually implies killing of the consumer and extracting the DNA from its food, using either the whole animal or its dissected gut. This post‐mortem approach, however, is not suitable for investigating the diet of rare or protected species and also prohibits tracking individual dietary preferences as each consumer can provide trophic information only once. Moreover, removing large numbers of consumers from a habitat for analysis might critically change population densities and affect species interactions. Here, we present DNA‐based analysis of invertebrate regurgitates, a novel approach to overcome these limitations. Conducting feeding experiments where adult Poecilus cupreus (Coleoptera: Carabidae) were fed with larvae of Amphimallon solstitiale (Coleoptera: Scarabaeidae), we show that detection success in regurgitates compared to samples prepared from whole beetles was similar or significantly enhanced for small/medium and large prey DNA fragments, respectively. Prey DNA detection success remained high in regurgitates stored in ethanol for 21 months at room temperature prior to DNA extraction. We conclude that in those invertebrates where regurgitates can be obtained, examination of food DNA in regurgitates offers many advantages over conventional post‐mortem gut content analysis.

Wallinger C., Sint D., Baier F., Schmid C., Mayer R., Traugott M. (2015): Detection of seed DNA in regurgitates of granivorous carabid beetles. Bulletin of Entomological Research 105: 728.
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Granivory can play a pivotal role in influencing regeneration, colonization as well as abundance and distribution of plants. Due to their high abundance, nutrient content and longevity, seeds are an important food source for many animals. Among insects, carabid beetles consume substantial numbers of seeds and are thought to be responsible for a significant amount of seed loss. However, the processes that govern which seeds are eaten and are therefore prevented from entering the seedbank are poorly understood. Here, we assess if DNA-based diet analysis allows tracking the consumption of seeds by carabids. Adult individuals of Harpalus rufipes were fed with seeds of Taraxacum officinale and Lolium perenne allowing them to digest for up to 3 days. Regurgitates were tested for the DNA of ingested seeds at eight different time points post-feeding using general and species-specific plant primers. The detection of seed DNA decreased with digestion time for both seed species, albeit in a species-specific manner. Significant differences in overall DNA detection rates were found with the general plant primers but not with the species-specific primers. This can have implications for the interpretation of trophic data derived from next-generation sequencing, which is based on the application of general primers. Our findings demonstrate that seed predation by carabids can be tracked, molecularly, on a species-specific level, providing a new way to unravel the mechanisms underlying in-field diet choice in granivores.

Cheng Y. C., Lin C. P. (2016): Dietary niche partitioning of Euphaea formosa and Matrona cyanoptera (Odonata: Zygoptera) on the basis of DNA barcoding of larval feces. Journal of Insect Science 16: 73.
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Odonate larvae are commonly considered opportunistic general predators in freshwater ecosystems. However, the dietary breadth of most odonate larvae in forest streams is still poorly documented. We characterized the prey species and estimated the level of dietary niche overlap of two damselflies, Euphaea formosa Hagen 1869 and Matrona cyanoptera Hämäläinen and Yeh, 2000 in a forest stream of central Taiwan on the basis of DNA barcoding of larval feces. A collection of 23 successfully identified cytochrome c oxidase 1 (CO1) barcoding sequences suggested that the mayflies (Ephemeroptera), caddisflies (Trichoptera), and midges (Diptera) comprise the majority (43%, 6/14) of prey species consumed by E. formosa larvae, whereas the identified prey for M. cyanoptera were mainly zooplankton (56%, 5/9). Statistical analysis of dietary overlap indicated that these two species occupy different dietary niches (Pianka’s index = 0.219). DNA barcoding analysis of damselfly larval feces was effective in detecting less sclerotized prey such as vertebrates (fish and frog) and small zooplankton. However, a moderately successful rate (<70%) of PCR amplification by universal CO1 primers and a low percentage (<60%) of identifiable sequences in public databases indicate the limitations of naive DNA barcoding in fecal analysis.

Kaunisto K. M., Roslin T., Sääksjärvi I. E., Vesterinen E. J. (2017): Pellets of proof: first glimpse of the dietary composition of adult odonates as revealed by metabarcoding of feces. Ecology and Evolution 7: 8588-8598.
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Recent advances in molecular techniques allow us to resolve the diet of unstudied taxa. Odonates are potentially important top‐down regulators of many insects. Yet, to date, our knowledge of odonate prey use is based mainly on limited observations of odonates catching or eating their prey. In this study, we examine the potential use of metabarcoding in establishing the diet of three adult odonate species (Lestes sponsa, Enallagma cyathigerum, and Sympetrum danae) at a site in southwestern Finland. To this purpose, we compared three different methods for extracting DNA from fecal samples: the Macherey‐Nagel Nucleospin XS kit, a traditional salt extraction, and the Zymo Research Fecal Microprep kit. From these extracts, we amplified group‐specific mitochondrial markers (COI and 16S rRNA) from altogether 72 odonate individuals, and compared them to comprehensive reference libraries. The three odonate species show major overlap in diet, with no significant differences between individuals of different size and/or gender, reflecting opportunistic foraging of adult odonates. Of a total of 41 different prey species detected, the most frequently consumed ones were Diptera, with additional records of six other orders. Based on our data, the best DNA extraction method is the traditional salt extraction, as it provides the most information on prey content while also being the most economical. To our knowledge, this is the first study to resolve the species‐level diet of adult odonates. Armed with the appropriate methodological caveats, we are ready to examine the ecological role of odonates in both terrestrial and aquatic food webs, and in transferring subsidies between these two realms.

Kamenova S., Mayer R., Rubbmark O. R., Coissac E., Plantegenest M., Traugott M. (2018): Comparing three types of dietary samples for prey DNA decay in an insect generalist predator. Molecular Ecology Resources 18: 966-973.
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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.

Kerley G. I., Landman M., Ficetola G. F., Boyer F., Bonin A., Rioux D., Taberlet P., Coissac E. (2018): Diet shifts by adult flightless dung beetles Circellium bacchus, revealed using DNA metabarcoding, reflect complex life histories. Oecologia 188: 107-115.
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Life history changes may change resource use. Such shifts are not well understood in the dung beetles, despite recognised differences in larval and adult feeding ability. We use the flightless dung beetle Circellium bacchus to explore such shifts, identifying dung sources of adults using DNA metabarcoding, and comparing these with published accounts of larval dung sources. C. bacchus is traditionally considered to specialise on the dung of large herbivores for both larval and adult feeding. We successfully extracted mammal DNA from 151 adult C. bacchus fecal samples, representing 16 mammal species (ranging from elephants to small rodents), many of which are hitherto undescribed in the diet. Adult C. bacchus showed clear dung source preferences, especially for large herbivores inhabiting dense-cover vegetation. Our approach also confirmed the presence of cryptic taxa in the study area, and we propose that this may be used for biodiversity survey and monitoring purposes. Murid rodent feces were the most commonly fed-upon dung source (77.5%) for adult C. bacchus, differing markedly from the large and megaherbivore dung sources used for larval rearing. These findings support the hypothesis of life history-specific shifts in resource use in dung beetles, and reveal a hitherto unsuspected, but ecologically important, role of these dung beetles in consuming rodent feces. The differences in feeding abilities of the larval and adult life history stages have profound consequences for their resource use and foraging strategies, and hence the ecological role of dung beetles. This principle and its ecological consequences should be explored in other scarabaeids.

Yamamoto S., Uchida K. (2018): A generalist herbivore requires a wide array of plant species to maintain its populations. Biological Conservation 228: 167-174.
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Generalist herbivores are less susceptible to changes in the plant composition of their habitats than specialists are because generalists can consume a diverse array of plants. However, even generalists exhibit dietary choices, for example because they need to balance their nutritional intake for both growth and reproduction. In this study, we showed that an endangered generalist herbivorous grasshopper (Celes akitanus; Orthoptera: Acrididae) actively chooses which plants to include in its diet. Moreover, we found that grasshopper abundance is correlated with host plant abundance and richness. This grasshopper has been reported to occur mainly in traditionally managed grasslands that harbour more diverse plant species than other, nearby grasslands. To elucidate the links between this grasshopper and plant richness, we surveyed grasshopper abundance in grasslands under traditional and other management practices. Plant DNA barcoding of faecal samples demonstrated that this grasshopper is a generalist herbivore while also showing that it makes active dietary choices. Furthermore, although the grasshopper’s host plants occurred in all grasslands, the grasshopper itself was found only in species-rich grasslands. In addition, grasshopper abundance was positively related to the abundance and richness of host plants. Our findings suggest that this endangered herbivore requires a wide array of host plants to maintain its populations.

Nboyine J. A., Boyer S., Saville D. J., Wratten S. D. (2019): Identifying plant DNA in the faeces of a generalist insect pest to inform trap cropping strategy. Agronomy for Sustainable Development 39: 57.
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Monocropping elevates many insects to the status of economic pests. In these agroecosystems, non-crop habitats are sometimes deployed as trap crops to reduce pest damage. This environmentally friendly alternative to pesticides can be particularly fitting when dealing with native invaders that may be afforded legal protection or enjoy public sympathy as is the case for the ground wētā Hemiandrus sp. ‘promontorius’ (Orthoptera) in New Zealand. However, this approach requires knowledge of the insects’ diet to select the most appropriate plant species for trap cropping. Here, ingested plant DNA in the faeces of wētā was analysed to help develop strategies for mitigating its damage in New Zealand vineyards. DNA was extracted from faeces of wētā collected from six different vineyards over four seasons. Using a DNA metabarcoding approach, we amplified the rbcL gene region and sequenced the amplicons on an Illumina MiSeq platform. The identity of plants in the diet of this insect was determined by comparing the sequences generated with those available in the GenBank database and cross-checking the results with a database of plants known to be present in New Zealand. A total of 47 plant families and 79 genera were detected. Of the genera identified, Vitis, Poa, Festuca, Anthoxanthum, Anagallis, Camelina, Epilobium, Menyanthes, Pedicularis, Urtica, Garrya, Pinus and Tilia were the major ones (i.e. they were present in more than 50% of the faecal samples). The composition of the above plant taxa in faecal materials was significantly different between collection sites or dates, except for Menyanthes. The occurrence of the latter was significantly different between collection sites. These results indicate that effectively mitigating wētā damage to vines requires the use of a diverse mix of plant species for trap cropping as wētā seem to be highly generalist in their feeding behaviour even when plant diversity is relatively low.

Kaunisto K. M., Roslin T., Forbes M. R., Morrill A., Sääksjärvi I. E., Puisto A. I., Vesterinen E. J. (2020): Threats from the air: Damselfly predation on diverse prey taxa. Journal of Animal Ecology 89: 1365-1374.
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To understand the diversity and strength of predation in natural communities, researchers must quantify the total amount of prey species in the diet of predators. Metabarcoding approaches have allowed widespread characterization of predator diets with high taxonomic resolution. To determine the wider impacts of predators, researchers should combine DNA techniques with estimates of population size of predators using mark–release–recapture (MRR) methods, and with accurate metrics of food consumption by individuals. Herein, we estimate the scale of predation exerted by four damselfly species on diverse prey taxa within a well-defined 12-ha study area, resolving the prey species of individual damselflies, to what extent the diets of predatory species overlap, and which fraction of the main prey populations are consumed. We identify the taxonomic composition of diets using DNA metabarcoding and quantify damselfly population sizes by MRR. We also use predator-specific estimates of consumption rates, and independent data on prey emergence rates to estimate the collective predation pressure summed over all prey taxa and specific to their main prey (non-biting midges or chironomids) of the four damselfly species. The four damselfly species collectively consumed a prey mass equivalent to roughly 870 (95% CL 410–1,800) g, over 2 months. Each individual consumed 29%–66% (95% CL 9.4–123) of its body weight during its relatively short life span (2.1–4.7 days; 95% CL 0.74–7.9) in the focal population. This predation pressure was widely distributed across the local invertebrate prey community, including 4 classes, 19 orders and c. 140 genera. Different predator species showed extensive overlap in diets, with an average of 30% of prey shared by at least two predator species. Of the available prey individuals in the widely consumed family Chironomidae, only a relatively small proportion (0.76%; 95% CL 0.35%–1.61%) were consumed. Our synthesis of population sizes, per-capita consumption rates and taxonomic distribution of diets identifies damselflies as a comparatively minor predator group of aerial insects. As the next step, we should add estimates of predation by larger odonate species, and experimental removal of odonates, thereby establishing the full impact of odonate predation on prey communities.

Lefort M. C., Beggs J. R., Glare T. R., Saunders T. E., Doyle E. J., Stephane B. (2020): A molecular approach to study Hymenoptera diets using polistine wasps. NeoBiota 63: 57-79.
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The study of animal diets has benefited from the rise of high-throughput DNA sequencing applied to stomach content or faecal samples. The latter can be fresh samples used to describe recent meals, or older samples, which can inform about past feeding activities. For most invertebrates, however, it is difficult to access ‘historical’ samples, due to the small size of the animals and the absence of permanent defecation sites. Therefore, sampling must be repeated to account for seasonal variation and to capture the overall diet of a species. This study develops a method to describe the overall diet of nest-building Hymenoptera based on a single sampling event, by analysing prey DNA from faeces accumulated in brood cells. We collected 48 nests from two species of introduced paper wasps (Polistes chinensis, and P. humilis) in the urban and peri-urban areas of Auckland, New Zealand, and selected two samples per nest. One from brood cells in the outer layer of the nest to represent the most recent diet, and one from brood cells in an inner layer to represent older diet. Diet differed between species, although both fed mainly on Thysanoptera, Lepidoptera and Acariformes. Prey taxa identified to species level included both agricultural pests and native species. Prey communities consumed were significantly different between inner and outer nest samples suggesting seasonal variation in prey availability and/or a diversification of the wasps’ diet as the colony grows. We also show for the first time potential predation of marine organisms by Polistes wasps. Our study provides field evidence that polistine wasps feed on agricultural pests, supporting the hypothesis that some social wasp species could have a suppressing effect on agricultural pests. The proposed methodology is readily applicable to other nest-building Hymenoptera and has the potential to provide comprehensive knowledge about their diet with minimum sampling effort. Such knowledge is Essential to measure the ecological impact of invasive Vespidae and support the conservation of native invertebrate biodiversity.

Casanelles‐Abella J., Müller S., Keller A., Aleixo C., Alós Orti M., Chiron, F., Deguines N., Hallikma T., Laanisto L., Pinho P., Samson R., Tryjanowski P., Van Mensel A., Pellissier L., Moretti M. (2021): How wild bees find a way in European cities: Pollen metabarcoding unravels multiple feeding strategies and their effects on distribution patterns in four wild bee species. Journal of Applied Ecology 59: 457-470.
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Urban ecosystems can sustain populations of wild bees, partly because of their rich native and exotic floral resources. A better understanding of the urban bee diet, particularly at the larval stage, is necessary to understand biotic interactions and feeding behaviour in urban ecosystems, and to promote bees by improving the management of urban floral resources. We investigated the larval diet and distribution patterns of four solitary wild bee species with different diet specialization (i.e. Chelostoma florisomne, Osmia bicornis, Osmia cornuta and Hylaeus communis) along urban intensity gradients in five European cities (Antwerp, Paris, Poznan, Tartu and Zurich) using two complementary analyses. Specifically, using trap-nests and pollen metabarcoding techniques, we characterized the species’ larval diet, assessed diet consistency across cities and modelled the distribution of wild bees using species distribution models (SDMs). Our results demonstrate that urban wild bees display different successful strategies to exploit existing urban floral resources: not only broad generalism (i.e. H. communis) but also intermediate generalism, with some degree of diet conservatism at the plant family or genus level (i.e. O. cornuta and O. bicornis), or even strict specialization on widely available urban pollen hosts (i.e. C. florisomne). Furthermore, we detected important diet variation in H. communis, with a switch from an herbaceous pollen diet to a tree pollen diet with increasing urban intensity. Species distribution modelling indicated that wild bee distribution ranges inside urban ecosystems ultimately depend on their degree of specialization, and that broader diets result in less sensitivity to urban intensity. Satisfying larval dietary requirements is critical to preserving and enhancing wild bee distributions within urban gradients. For high to intermediate levels of feeding specialization, we found considerable consistency in the preferred plant families or genera across the studied cities, which could be generalized to other cities where these bees occur. Identifying larval floral preferences (e.g. using pollen metabarcoding) could be helpful for identifying key plant taxa and traits for bee survival and for improving strategies to develop bee-friendly cities.

Morrill A., Kaunisto K. M., Mlynarek J. J., Sippola E., Vesterinen E. J., Forbes M. R. (2021): Metabarcoding prey DNA from fecal samples of adult dragonflies shows no predicted sex differences, and substantial inter-individual variation, in diets. PeerJ 9: e12634.
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Sexes often differ in foraging and diet, which is associated with sex differences in size, trophic morphology, use of habitats, and/or life history tactics. Herein, strikingly similar diets were found for adult sexes of a dragonfly (Leucorrhinia intacta), based on comparing 141 dietary taxa identified from the metabarcoding of mitochondrial DNA archived in feces. Arthropods in > 5% of samples included five species of dipterans, two hemipterans, two spider species and one parasitic mite. The mite was not traditional prey as its presence was likely due to DNA contamination of samples arising through parasitism or possibly via accidental consumption during grooming, and therefore the mite was excluded from diet characterizations. Common prey species were found with statistically indistinguishable frequencies in male and female diets, with one exception of an aphid more often found in male diets, although this pattern was not robust to corrections for multiple statistical tests. While rare prey species were often found in diets of only one sex, instances of this were more frequent in the more oft-sampled females, suggesting sampling artefact. Sexes did not differ in the mean prey species richness in their diets. Overall, sexes showed statistically indistinguishable diets both on a prey species-by-species basis and in terms of multivariate characterizations of diet composition, derived from presence-absence data of prey species analyzed via PERMANOVA and accumulation curves. Males and females may have similar diets by being both opportunistic and generalist predators of arthropods, using the same foraging habitats and having similar sizes and flight agilities. Notably, similarities in diet between sexes occur alongside large interindividual differences in diet, within sexes. Researchers intending on explaining adaptive sex differences in diet should consider characteristics of species whose sexes show similar diets.

Verdasca M. J., Godinho R., Rocha R. G., Portocarrero M., Carvalheiro L. G., Rebelo R., Rebelo H. (2022): A metabarcoding tool to detect predation of the honeybee Apis mellifera and other wild insects by the invasive Vespa velutina. Journal of Pest Science 95: 997-1007. 
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The invasive Vespa velutina has been widely referred as an effective predator of honeybees. Despite the potential risk to pollination services provision and honey production, there is no accurate quantification and assessment of its real consequences for honeybees. To date, the identification of the honeybee and other insects in the diet of V. velutina has been investigated by direct observation of adult foraging or examination of food pellets. To overcome these limitations, in this study we used a DNA metabarcoding approach to evaluate the usefulness of different types of sample (jaws and stomachs collected from workers and larval faecal pellets taken from the hornet comb) to investigate the predation of V. velutina upon honeybees, and potentially on other insects. Honeybee DNA was identified in all types of samples, but larval faecal pellets retrieved the higher number of reads of honeybee DNA and the largest diversity at all taxonomic levels. Over all samples we could identify 4 orders, 9 families, 6 genera and 1 species of prey. We estimate that collecting 6 workers is sufficient to identify honeybee predation by a colony using worker’s jaws. Stomachs were the least useful sample type to detect honeybee DNA. The presence of honeybee DNA in all analysed colonies irrespective of collection site, and the variety of insect orders detected in the diet support current concerns over the acknowledged negative impact of V. velutina on managed honeybees and its potential threat to pollination services provision.

Pitteloud C., Defossez E., Albouy C., Descombes P., Rasmann S., Pellissier L. (2022): DNA‐based networks reveal the ecological determinants of plant–herbivore interactions along environmental gradients. Molecular Ecology 32: 6436-6448. 
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Understanding the ecological rules structuring the organization of species interactions is a prerequisite to predicting how ecosystems respond to environmental changes. While the ecological determinants of single networks have been documented, it remains unclear whether network ecological rules are conserved along spatial and environmental gradients. To address this gap, we reconstructed 48 plant–herbivore interaction networks along six elevation gradients in the Central European Alps in Switzerland, using DNA metabarcoding on orthoptera feces. We developed hypotheses on the ecological mechanisms expected to structure interaction networks, based on plant phylogeny, plant abundance, leaf toughness, leaf nitrogen content and plant metabolomics. We show that plant phylogenetic relationships and species abundance have the greatest explanatory power regarding the structure of the ecological networks. Moreover, we found that leaf nitrogen content is a key determinant of interactions in warmer environments, while phenolic compounds and terpenoids are more important in colder environments, suggesting that determinants of species interactions can shift along environmental gradients. With this work, we propose an approach to study the mechanisms that structure the way species interact with each other between bioregions and ecosystems.

Allen W. J., Waller L. P., Barratt B. I., Dickie I. A. (2023): Puke or poop? Comparison of regurgitate and faecal samples to infer alpine grasshopper (Paprides nitidus Hutton) diet in experimental plant communities. Ecology and Evolution 13: e10444.
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Abstract
Characterising plant-herbivore interactions is important to understanding the processes that influence community structure and ecosystem functioning. Traditional methods used to identify plant-herbivore interactions are being superseded by non-destructive molecular approaches that can infer interactions with greater resolution and accuracy from environmental DNA (e.g. faeces and regurgitate). However, few studies have compared the success of using different sample types and whether they provide similar or contrasting information about species’ diet. Here we compared the success of DNA amplification and host plant species identification using restriction fragment length polymorphism (RFLP) applied to faecal and regurgitate samples collected from alpine grasshoppers Paprides nitidus Hutton during a grassland community mesocosm experiment. We found that DNA amplification success was 23% and 86% higher for faecal than regurgitate samples from female and male grasshoppers, respectively. In contrast, successful host plant identification using RFLP was 9% higher for regurgitate than faecal samples. The mean number of host plant species identified per sample (1.40) did not differ between sample types or grasshopper sexes. Of the 136 paired faecal-regurgitate samples, just 41% and 74% produced exactly or partially matching host plant identifications, respectively, indicating that different sample types provided complementary information about herbivore diet. Some plant species were more likely to be identified from faecal samples than expected by chance, and we found that this identification bias skewed towards plant species with higher investment in leaf tissue. We conclude that multiple sample types may be required to fully characterise an invertebrate herbivore species’ diet.