Molecular Gut Content Analysis Research Articles

What's on the menu? A novel molecular gut content analysis to investigate the feeding behavior of phytophagous insects.

The relationship between phytophagous insects and plants is a central aspect of food webs and ecosystem functioning. The introduction of new species into an environment can have significant impacts on the food web of a native ecosystem. In many cases, there is a lack of knowledge on the biology and feeding behavior of invasive species prior their introduction and in the invaded regions. Gut content analyses of insects have provided valuable information on the host spectrum of insects. However, current approaches are time-consuming and costly. Here, we describe a new molecular gut content analysis (GCA) approach using the Oxford Nanopore (ONT) Flongle sequencing platform to characterize the plant DNA present in the gut of the highly polyphagous insect species Halyomorpha halys. We demonstrate that this technique efficiently amplifies and correctly identifies plant DNA in a mock community. We performed a feeding experiment to determine the sensitivity of this approach and to assess how long the plant DNA can be detected. All plants used in the feeding experiment were correctly identified and detected after 56 days. Surprisingly, we also detected various plant genera that were not included in the feeding experiment and thus were likely ingested months before the experiment. Our study suggests that the GCA using the ONT Flongle sequencing platform represents a rapid and cost-efficient diagnosis of the dietary preferences, host range, and the diversity of consumed plant species of pest insects with high precision.

Unveiling the diet of two generalist stink bugs, Halyomorpha halys and Pentatoma rufipes (Hemiptera: Pentatomidae), through metabarcoding of the ITS2 region from gut content.

The use of DNA metabarcoding has become an increasingly popular technique to infer feeding relationships in polyphagous herbivores and predators. Understanding host plant preference of native and invasive herbivore insects can be helpful in establishing effective integrated pest management (IPM) strategies. The invasive Halyomorpha halys and native Pentatoma rufipes are piercing-sucking stink bug pests that are known to cause economic damage in commercial fruit orchards. In this study, we performed molecular gut content analysis (MGCA) on field-collected specimens of these two herbivorous pentatomids using next-generation amplicon sequencing (NGAS) of the internal transcribed spacer 2 (ITS2) barcode region. Additionally, a laboratory experiment was set up where H. halys was switched from a mixed diet to a monotypic diet, allowing us to determine the detectability of the initial diet in a time series of ≤3 days after the diet switch. We detected 68 unique plant species from 54 genera in the diet of two stink bug species, with fewer genera found per sample and a smaller diet breadth for P. rufipes than for H. halys. Both stink bug species generally prefer deciduous trees over gymnosperms and herbaceous plants. Landscape type significantly impacted the observed genera in the diet of both stink bug species, whereas season only had a significant effect on the diet of H. halys. This study provides further insights into the dietary composition of two polyphagous pentatomid pests and illustrates that metabarcoding can deliver a relevant species-level resolution of host plant preference. © 2024 Society of Chemical Industry.

Resource overlap and infrequent predation on key pests show vulnerability in cotton biological control services

Promoting arthropod biodiversity to increase ecosystem services through ecological intensification is a challenge for agriculture. And recent evidence suggests that standard pesticide applications not only harm natural enemies but may also fail to deliver long-term pest control solutions. To fuel ecological intensification and build predictive frameworks for pest management incorporating estimates of pest abundance, natural enemy abundance and their associated interactions is essential. Within this framework, there is a need to shift the focus from a single pest and predator to consider the community of predatory arthropods that interact with communities of prey. We took a network-based approach to investigate community interactions of predatory arthropods that feed on key pests and alternative prey in cotton over a three-year period. We merged prey activity, generalist predator communities collected from cotton canopies, and reconstructed trophic interactions with DNA detection frequencies estimated from molecular gut content analysis. Overall, many predator diets overlap, resulting in similar foraging patterns on groups of cotton pests. Moreover, predation on key cotton pests, such as stink bugs and white flies, was low. Therefore, ecological intensification that increases specialized arthropod predators within the community should improve biological control service delivery.

Diversified cropping strengthens herbivore regulation by providing seasonal resource continuity to predators

Abstract Agricultural practices shape arthropod communities in arable fields, consequently influencing their interactions and the resulting ecosystem services, in particular pest regulation. Predatory arthropods play a pivotal role by preying on herbivores, soil fauna, and on other predators. However, the intricate mechanisms through which agricultural practices shape the dietary preferences of predators, and regulate herbivore populations remain complex and inadequately understood. We assessed how fertilisation with organic fertiliser and extending crop rotations with perennial ley affected predation pressure across prey taxa. We mapped predator and prey trophic linkages with molecular analysis of carabid predator gut contents, and measured densities and taxonomic richness of predators, herbivores, and soil fauna in 19 cereal fields during three samplings across the growing season. We derived two food web structure metrics: prey vulnerability that is the average number of predators feeding on a selected prey, and predator trophic redundancy, that is dietary overlap. Prey vulnerability was compared among soil fauna, herbivores, and other predator species (that is interspecific intraguild predation) over the growing season, and across treatments. The mechanistic underpinnings of observed shifts in vulnerability of herbivorous prey at different crop stages were identified using information criteria to select among candidate variables related to the richness, density and interaction structure of the different guilds during both the current, and the previous crop stages. Agricultural diversification via organic fertilisation combined with perennial ley in the crop rotation decreased the vulnerability of both intraguild prey and soil fauna prey, and stabilised herbivore vulnerability. Mechanistically, the vulnerability of herbivorous prey at crop ripening emerges from the combination of predator richness and trophic redundancy during this sampling round, rather than from carryover effects from previous crop stages. Synthesis and applications: Our results suggest that locally provided resource continuity through diversified cropping practices bolster biological pest regulation, thus underline the importance of lesser disturbance in arable ecosystems for the provision of ecosystem services. Enhanced predator species richness together with availability of alternative prey through the season underpins this enhanced pest regulation.

Increased ladybird predation and metabolism do not counterbalance increased field aphid population growth under experimental warming

Abstract Climate change may have diverse and complex impacts on species interactions, destabilizing food webs and ecosystem services. The effects of warming on the top‐down biological control of crop pests have been considerably less studied than bottom‐up effects through crop physiological changes. We studied the effect of a 2°C warming in the laboratory and in wheat fields on the predation and metabolism of Harmonia axyridis on wheat aphids using molecular gut content analysis. We also measured the effects of warming on the predation rate and functional response of H. axyridis on each aphid species in the laboratory, as well as on DNA degradation rate. Field densities of Sitobion avenae and Rhopalosiphum padi, the two most abundant wheat aphid species, were increased by 2 and 2.5 times, respectively, under experimental warming, but densities of H. axyridis were not. Field predation rate of H. axyridis on these two aphid was found to be about 25% lower under elevated temperature. This could have been due to faster prey digestion, since degradation of the preferred aphid species, Sitobion avenae, was 1.5 times faster under elevated temperature. However, the functional response of H. axyridis larvae on these two species was 1.5 times higher under warming over the range of prey densities tested (50–250 over 24 h). The total predation rate of H. axyridis larvae on a mixture of S. avenae, R. padi and Schizaphis graminum aphid prey was also increased by 1.4 times, but consumption of R. padi aphids was increased while that of S. graminum was decreased under warming. Overall, our results show that global warming could strongly increase pest outbreaks and destabilize biological pest control, which would likely result in accrued yield losses. Read the free Plain Language Summary for this article on the Journal blog.

Rethinking trophic interactions in agricultural landscapes through tracking secondary feeding

AbstractConservation biological control efforts depend on accurately estimating predator roles in crop fields, and knowledge of plant resources generalist predators utilize in agricultural landscapes. Generalist predators move among habitats to feed on insect prey and some predators feed on plants for non‐prey nutrients. Studying predation with molecular gut content analysis (MGCA), provides estimates of within field frequencies of predation on target pests and alternative prey. However, prey DNA takes time to move through the predator digestive system, so a portion of the observed predation likely occurs in adjacent crops or semi‐natural habitats. Therefore, we tested a strategy to estimate recent secondary feeding to help trace predation back to the source habitat. We selected the diamondback moth and three common predators with different mouthparts: Coccinella septempunctata, Geocoris punctipes, and Pardosa spiders, as model organisms for these proof‐of‐concept experiments. We estimated post‐feeding primary and secondary plant DNA detection time and compared it between these three predators using previously designed Lepidoptera primers and newly designed Brassica‐specific primers. Our results indicate secondary plant feeding detection largely depends on predator mouthparts. While secondary collard DNA detectability half‐life was 5.5 h and remained detectable for up to 30 h for the chewing predator, C. septempunctata, only 4% and 8.3% of G. punctipes and Pardosa spp. individuals, respectively, tested positive for collard DNA. However, more studies are required to confirm this mouthpart‐specific post‐feeding plant DNA detection time. Our feeding trials confirmed the possibility of primary and secondary plant feeding detection for chewing predators. Hence, when crops are not flowering (or anthesis), secondary plant feeding detection can be used to trace chewing predators to source habitats where they consumed sessile prey (e.g., nymphs and sessile adults). Such multitrophic linkage knowledge could unravel the landscape‐wise contribution of predators to pest control services.

Dietary history of click beetles and wireworms in the genus Limonius (Coleoptera: Elateridae) revealed by molecular gut content analysis.

Wireworms, the larval stage of pest click beetle species (Coleoptera: Elateridae), are pests of many crops in North America including root vegetables and cereals. There is cause for concern amongst growers who are facing pressure from wireworms because there are a decreasing number of effective pesticides that can be used for wireworm management. Most research on pest elaterids has focused on the wireworm stage, which is the damage causing life stage. Recently, the focus in elaterid research has shifted to the adult click beetle stage, including identification of semiochemicals and development of effective traps. However, there is still a lot to be discovered about the basic biology of click beetles, including their feeding ecology. In an effort to understand the feeding ecology of click beetles, we investigated the presence of plant DNA in the digestive tracts of Limonius californicus (Mann.), L. canus (LeConte), and L. infuscatus (Mots.) beetles collected in 3 different locations within central Washington. To examine dietary histories of beetles and wireworms, specimens were collected from natural habitats and high-throughput sequencing of the plant genes trnF and ITS was used to identify their dietary history. Results revealed that click beetles do feed on a large variety of plants, which included a large quantity of brassicaceous plants commonly found in areas surrounding wireworm infested plots. The identification of the dietary history of the click beetles allowed us to infer their landscape-scale movements thus providing a means to better understand their behavior.

Testing the effectiveness of different wash protocols to remove body surface contaminants in invertebrate food web studies.

Molecular gut content analysis via diagnostic PCR or high-throughput sequencing (metabarcoding) of consumers allows unravelling of feeding interactions in a wide range of animals. This is of particular advantage for analyzing the diet of small invertebrates living in opaque habitats such as the soil. Due to their small body size, which complicates dissection, microarthropods are subjected to whole-body DNA extraction-step before their gut content is screened for DNA of their food. This poses the problem that body surface contaminants, such as fungal spores may be incorrectly identified as ingested food particles for fungivorous species. We investigated the effectiveness of ten methods for body surface decontamination in litter-dwelling oribatid mites using Steganacarus magnus as model species. Furthermore, we tested for potential adverse effects of the decontamination techniques on the molecular detection of ingested prey organisms. Prior to decontamination, oribatid mites were fed with an oversupply of nematodes (Plectus sp.) and postmortem contaminated with fungal spores (Chaetomium globosum). We used diagnostic PCR with primers specific for C. globosum and Plectus sp. to detect contaminants and prey, respectively. The results suggest that chlorine bleach (sodium hypochloride, NaClO, 5%) is most efficient in removing fungal surface contamination without significantly affecting the detection of prey DNA in the gut. Based on these results, we provide a standard protocol for efficient body surface decontamination allowing to trace the prey spectrum of microarthropods using molecular gut content analysis.

Who eats the yellowmargined leaf beetle? Field observations and genetic surveillance to identify local predators of a novel invasive pest

The yellowmargined leaf beetle, Microtheca ochroloma Stahl (Coleoptera: Chrysomelidae), invaded the Southeastern United States and rapidly emerged as a leading pest of crucifer crops, yet little is known about which native predators might effectively provide biological control. The objective of this research is to identify potential predators of this understudied invader through field observation and genetic surveillance. Over two years, arthropods were surveyed on a variety of susceptible Brassica cultivars and a shortlist of commonly associated predators was developed. In a survey of turnip plants in year 1, lady beetles were positively predicted by densities of M. ochroloma, suggesting a numerical response to densities of the invasive pest. Surveys at a different site in year 2 revealed that big-eyed bugs were also positively predicted by M. ochroloma, while pink-spotted lady beetles negatively associated, suggesting a pattern of suppression. Ants (primarily the red imported fire ant) and spiders were commonly observed predators on plants in both years, but neither associated with densities of M. ochroloma. No predators were observed consuming M. ochroloma eggs. Molecular gut-content analysis using specially generated M. ochroloma primers confirmed the observed predatory links and suggested a density-dependent tracking response from pink-spotted lady beetles and ground beetles also have density-dependent tracking re Table 3. Our results may enable farmers to bolster these predator populations with fine-tuned habitat management tools and better suppress the spreading pest.

The aphid Myzus persicae (Hemiptera: Aphididae) acquires chloroplast DNA during feeding on host plants.

Aphids (Hemiptera: Aphididae) extract nutrients from host plant phloem via stylets that facilitate salivation and sap uptake. When navigating to the phloem, aphids periodically puncture nonvascular cells and sample cell contents, but rarely cause significant cell damage. As a result, aphids are considered "stealthy" feeders. In contrast, insects that do cause damage, such as chewing herbivores, will take up host cell contents-including DNA-into their guts. Researchers can use molecular barcoding methods to identify recent host use patterns of chewing herbivores. This information is valuable for both pest management and basic ecological studies. Because of their stealthy feeding style, it was assumed that host plant DNA could not be recovered from aphids and other Sternorrhyncha. However, several recent studies document host plant DNA uptake by psyllids, which feed in a similar manner to aphids. Therefore, we hypothesized that aphids may also acquire DNA from host plants. Since aphids puncture and sample cytosol contents from cells, we predicted that aphids would be most likely to acquire DNA from chloroplasts. To test this, we performed host feeding and host transfer experiments with Myzus persicae (Sulzer), then used PCR to recover and sequence a region between the trnT and trnF genes from acquired chloroplast DNA. We found that M. persicae readily acquires chloroplast DNA, even prior to phloem contact, and that fragment sizes sufficient for host plant identification can be recovered. Our work suggests that molecular gut content analysis is a viable tool for studying aphid-host interactions.

Low Incidence of Avian Predation on the Brown Marmorated Stink Bug, Halyomorpha halys (Hemiptera: Pentatomidae), in Southeastern Orchard Systems.

In many agroecosystems, brown marmorated stink bugs (Halyomorpha halys) (Hemiptera: Pentatomidae) are polyphagous pests that cause significant economic losses to numerous crops every year. Insectivorous birds may provide a means of sustainable predation of invasive pests, such as H. halys. In forest margins surrounding peach, pecan, and interplanted peach-pecan orchards, we monitored H. halys populations with pheromone-baited traps, mist-netted birds, and collected avian fecal samples for molecular gut content analysis. We screened 257 fecal samples from 19 bird species for the presence of H. halys DNA to determine whether birds provide the biological control of this pest. Overall, we found evidence that four birds from three species consumed H. halys, including Northern cardinal (Cardinalis cardinalisis), Tufted titmouse (Baeolophus bicolor), and Carolina wren (Thryothorus ludovicianus). Halyomorpha halys captured in traps increased over time but did not vary by orchard type. Although incidence of predation was low, this may be an underestimate as a result of our current avian fecal sampling methodology. Because birds are members of the broader food web, future studies are needed to understand avian ecosystem services, especially in terms of pest control, including H. halys and other pest species.

Estimating predation rates from molecular gut content analysis.

Several methods have been published to estimate per capita predation rates from molecular gut content analysis relying on intuitive understanding of predation, but none have been formally derived. We provide a theoretical framework for estimating predation rates to identify an accurate method and lay bare its assumptions. Per capita predation can be estimated by multiplying the prey decay rate and the prey quantity in the predators. This assumes that variation in per capita predation rate is approximately normally distributed, prey decay occurs exponentially, and predation is in steady state. We described several ways to estimate steady state predation, including using only qualitative presence-absence data to estimate the decay rate and in addition, we provided a method for estimating per capita predation rate when predation is not in steady state. We used previously published data on aphid consumption by a ladybird beetle in a feeding trial to calculate the predation rate and compare published methods with this theoretically derived method. The estimated predation rate (3.29 ± 0.27 aphids/h) using our derived method was not significantly different from the actual predation rate, 3.11 aphids/h. In contrast, previously published methods were less accurate, underestimating the predation rate (0.33 ± 0.02 to 1.66 ± 0.8 aphids/h) or overestimating it (3.64 ± 0.30 aphids/h). In summary, we provide methods to estimate predation rates even when variation in predation rates is not exactly normally distributed and not in steady state and demonstrate that the prey decay rate, and not the prey detection period, is required.

Secondary predation by omnivores: Cereal aphid consumption bears no risk of misinterpretation in DNA‐based diet analysis

AbstractMany omnivorous arthropods act as important pest control agents in agroecosystems. Assessing their prey choices helps to better understand their biocontrol potential in different environmental settings. To this end, multi‐target approaches of molecular gut content analysis allow studying their prey choices. Yet, DNA‐based analysis of trophic interactions is challenged by the question of whether the detected food DNA was ingested primarily as direct prey, or secondarily via the gut content of a consumed animal. Experimental data are needed to understand the potential bias derived through secondary predation. Here we address this issue for a phloem‐feeding aphid consumed by omnivorous carabids. In feeding experiments, Pseudoophonus rufipes beetles were offered grain aphids Sitobion avenae as primary prey, which had fed on barley seedlings. Gut content samples of carabids collected after 0, 3, 9, 12, 24, 32 or 64 h of digestion time and whole‐body aphids were screened for the presence of aphid and plant DNA. Samples positive for plant DNA were subsequently tested with Poaceae‐specific primers. None of the 7.8% gut content samples that tested positive for general plant DNA amplified with the Poaceae primers. Moreover, none of the whole‐body extracted aphids tested positive for plant DNA, neither with general nor with family‐specific primers. We, therefore, conclude that the plant DNA detections in our experiment can be ascribed to remnants of plant material in the guts of the field‐collected carabids. Our findings demonstrate that these phloem‐feeding aphids are an unlikely cause of false trophic link assignment because no secondarily consumed plant DNA, derived either from aphid gut content or host‐plant environmental DNA on aphid bodies, could be detected in carabid gut content samples.

An experimental framework for quantifying the degree of intraguild predation in omnivorous food webs in the field

AbstractIntraguild predation (IGP) is common in natural and human‐managed systems and plays a critical role in food web dynamics. Although studies have documented the occurrence of IGP across a wide range of predator taxa, quantitative understanding regarding the degree/intensity of IGP remains lacking. I propose an experimental framework combining controlled feeding trials and stable isotope analysis to quantify the degree of IGP in an omnivorous food web in the field consisting of a top predator, a mesopredator, and a shared prey. The degree of IGP is defined as the proportion (in number) of mesopredators consumed in the total diet (shared prey + mesopredator) of top predators. Feeding trials along with stable isotope analysis are used to construct a standard curve of the relationship between the diet composition of top predator and its nitrogen isotope signatures. The nitrogen isotope signatures of field‐collected top predator individuals are then analyzed and interpolated to the standard curve to estimate the degree of IGP in the field. The proposed framework leverages the strengths of different experimental approaches to study trophic interactions, providing a practical tool for quantifying IGP in a more accurate (controlled feeding trials and standard IGP curve) and realistic (stable isotope analysis of field samples) fashion. The current framework can be further extended to food webs involving more complex interactions (e.g., multiple shared prey) and complemented with other approaches (e.g., molecular gut content analysis) to capture a more complete picture of IGP dynamics in the field.

Association of Two Bactericera Species (Hemiptera: Triozidae) With Native Lycium spp. (Solanales: Solanaceae) in the Potato Growing Regions of the Rio Grande Valley of Texas.

Bactericera cockerelli (Šulc) (Hemiptera: Triozidae) is a vector of 'Candidatus Liberibacter solanacearum' (Lso), the pathogen that causes potato zebra chip. Zebra chip incidence varies regionally, perhaps because of geographic differences in species of noncrop hosts available to the vector and in susceptibility of those hosts to Lso. Native and introduced species of Lycium (Solanales: Solanaceae) are important noncrop hosts of B. cockerelli in some regions of North America. Susceptibility of native Lycium species to Lso is uncertain. We investigated the use of two native species of Lycium by B. cockerelli in South Texas and tested whether they are susceptible to Lso. Bactericera cockerelli adults and nymphs were collected frequently from L. berlandieri Dunal and L. carolinianum Walter. Greenhouse assays confirmed that B. cockerelli develops on both species and showed that Lso infects L. carolinianum. Molecular gut content analysis provided evidence that B. cockerelli adults disperse between potato and Lycium. These results demonstrate that L. berlandieri and L. carolinianum are likely noncrop sources of potato-colonizing B. cockerelli in South Texas and that L. carolinianum is a potential source of Lso-infected psyllids. We also routinely collected the congeneric psyllid, Bactericera dorsalis (Crawford), from both Lycium species. These records are the first for this psyllid in Texas. Bactericera dorsalis completed development on both native Lycium species, albeit with high rates of mortality on L. berlandieri. B. dorsalis acquired and transmitted Lso on L. carolinianum under greenhouse conditions but did not transmit Lso to potato. These results document a previously unknown vector of Lso.

DNA High-Throughput Sequencing for Arthropod Gut Content Analysis to Evaluate Effectiveness and Safety of Biological Control Agents.

The search for effective biological control agents without harmful non-target effects has been constrained by the use of impractical (field direct observation) or imprecise (cage experiments) methods. While advances in the DNA sequencing methods, more specifically the development of high-throughput sequencing (HTS), have been quickly incorporated in biodiversity surveys, they have been slow to be adopted to determine arthropod prey range, predation rate and food web structure, and critical information to evaluate the effectiveness and safety of a biological control agent candidate. The lack of knowledge on how HTS methods could be applied by ecological entomologists constitutes part of the problem, although the lack of expertise and the high cost of the analysis also are important limiting factors. In this review, we describe how the latest HTS methods of metabarcoding and Lazaro, a method to identify prey by mapping unassembled shotgun reads, can serve biological control research, showing both their power and limitations. We explain how they work to determine prey range and also how their data can be used to estimate predation rates and subsequently be translated into food webs of natural enemy and prey populations helping to elucidate their role in the community. We present a brief history of prey detection through molecular gut content analysis and also the attempts to develop a more precise formula to estimate predation rates, a problem that still remains. We focused on arthropods in agricultural ecosystems, but most of what is covered here can be applied to natural systems and non-arthropod biological control candidates as well.

Molecular gut-content analysis in phytoseiid mites

Molecular gut content analysis has been used widely to elucidate predator-prey interaction both in natural and agricultural ecosystems. The two-spotted spider mites, Tetranychus urticae (Acari: Tetranychidae), is one of the most important pest species worldwide, mainly due to their acaricide resistance. In greenhouses, commercially available biological control agents (BCAs), such as Phytoseiiulus persimilis and Neoseiulus californicus (Acari: Phytoseiidae), have been used. However, in open fields like orchards, conservation biological control (CBC) is desirable rather than augmentation, because natural populations of phytoseiid mites, which can potentially control spider mites, are distributed. There can be several species of phytoseiid mites feeding on spider mites in the same habitat, and therefore it is important to know which species can control spider mites effectively. To this end, Hinomoto (2017) elucidated the effectiveness of molecular gut-content analysis in the predatory mite, N. californicus, and showed that ordinary agarose gel electrophoresis of PCR products could not detect any bands, but fluorescent-labeled primers successfully detected the gut contents of phytoseiid mites and confirmed they were derived from spider mite eggs. Furthermore, he also found that newly deposited spider mite eggs (less than 3 hr of oviposition) consumed by the predatory mites could not be detected even if fluorescent-labeled primers were used. To apply this technique in the fields, this should be carefully taken in the consideration, although it is unknown whether old or new eggs of spider mites are preferred. Regardless of this phenomenon, a combination of barcoding of phytoseiid mite species and molecular cut-content analysis will reveal real predator-prey food web in fields.

New perspectives on soil animal trophic ecology through the lens of C and N stable isotope ratios of oribatid mites

Knowledge of the trophic ecology of soil animals is important for understanding their high alpha diversity as well as their functional role in soil food webs and systems. In the last 20 years, the analysis of natural variations in stable isotope ratios (15N/14N, 13C/12C) has revolutionized our view on soil animal trophic ecology. Here, we review the state of the art of the trophic ecology of a highly abundant and diverse soil animal taxon, oribatid mites (Oribatida), investigated by stable isotope analyses. The review is based on 25 papers reporting stable isotope data of 292 oribatid mite taxa from 30 different sites. Four main findings emerged. (1) Oribatid mites cluster into six trophic groups, i.e. moss feeders, lichen feeders, primary decomposers, fungal feeders/secondary decomposers, predators/scavengers and marine algal feeders, plus one additional group, which incorporates CaCO3 in their cuticle for defence but still belongs to the fungal feeders/secondary decomposers group. (2) Of the 292 species studied 43.7% were classified as fungal feeders/secondary decomposers, 27.0% as primary decomposers and 15.7% as predators/scavengers, only few species include CaCO3 into their skeleton (6.1%), feed on lichens (4.9%), mosses (2.1%) or marine algae (0.7%). (3) In about one-third of the species studied the trophic niche was constant or varied little between sites or habitats, but in two-thirds of the species their trophic niche varied between habitats, with some species even shifting trophic levels, indicating trophic plasticity. (4) When aggregated at higher taxonomic level oribatid mite species clustered in only three instead of six trophic groups. This indicates that species within the same high level taxon often belong to different trophic groups, for example because feeding habits evolved convergently. Therefore, to accurately reflect the trophic ecology of oribatid mites their stable isotope signatures need to be analysed at species level. However, stable isotope analyses also have limitations, e.g. feeding on bacteria and fungi cannot be separated, and the same is true for feeding on ectomycorrhizal and arbuscular mycorrhizal fungi. Other methods such as fatty acid, amino acid and molecular gut content analyses as well as microbiome analyses may complement stable isotope studies and resolve oribatid mite trophic niche differentiation at a higher resolution. This will contribute to a better understanding of the local coexistence of large numbers of species in soil. Finally, we provide perspectives on how to integrate microarthropods into soil food webs using stable isotope and other methods allowing deeper insight into their trophic structure.

Molecular diet analysis in mussels and other metazoan filter feeders and an assessment of their utility as natural eDNA samplers.

Molecular gut content analysis is a popular tool to study food web interactions and has recently been suggested as an alternative source for DNA-based biomonitoring. However, the overabundant consumer's DNA often outcompetes that of its diet during PCR. Lineage-specific primers are an efficient means to reduce consumer amplification while retaining broad specificity for dietary taxa. Here, we designed an amplicon sequencing assay to monitor the eukaryotic diet of mussels and other metazoan filter feeders and explore the utility of mussels as natural eDNA samplers to monitor planktonic communities. We designed several lineage-specific rDNA primers with broad taxonomic suitability for eukaryotes. The primers were tested using DNA extracts of different limnic and marine mussel species and the results compared to eDNA water samples collected next to the mussel colonies. In addition, we analysed several 25-year time series samples of mussels from German rivers. Our primer sets efficiently prevent the amplification of mussels and other metazoans. The recovered DNA reflects a broad dietary preference across the eukaryotic tree of life and considerable taxonomic overlap with filtered water samples. We also show the utility of a reversed version of our primers, which prevents amplification of nonmetazoan taxa from complex eukaryote community samples, by enriching fauna associated with the marine brown algae Fucus vesiculosus. Our protocol will enable large-scale dietary analysis in metazoan filter feeders, facilitate aquatic food web analysis and allow surveying of aquacultures for pathogens. Moreover, we show that mussels and other aquatic filter feeders can serve as complementary DNA source for biomonitoring.

Flavonoid-producing tomato plants have a direct negative effect on the zoophytophagous biological control agent Orius sauteri.

Orius sauteri (Poppius) (Hemiptera: Anthocoridae) is often used for biological control of small arthropod pests in greenhouse vegetable production systems in Asia. In addition to feeding on arthropod prey, O. sauteri consumes small quantities of plant material. Previous studies demonstrated that tomato plant chemistry confers antixenosis resistance to phloem-feeding whiteflies, but the potential nontarget effects of phytochemicals on the beneficial predator O. sauteri are unknown. Comparison of O. sauteri confined to near-isogenic lines (NILs) of tomatoes producing high levels of flavonoids (NIL-purple hypocotyl; resistant to whiteflies) and low levels of flavonoids (NIL-green hypocotyl; susceptible to whiteflies) revealed that O. sauteri had reduced oviposition, nymphal survival, and development on resistant plants, even if they were also provided with prey that did not feed on the host plant. Moreover, O. sauteri showed a significant ovipositional preference in choice assays, laying significantly more eggs on susceptible than on resistant plants. Molecular gut content analysis using the specific chloroplast trnL gene from tomato confirmed that adult and immature O. sauteri feed on both resistant and susceptible genotypes, and feeding behavior assays revealed that resistance did not affect plant feeding or prey acceptance by O. sauteri adults. These results demonstrate a direct negative effect of phytochemicals on a nontarget beneficial species and indicate that resistance mediated by phytochemicals can affect organisms that do not solely feed on phloem sap. The results also indicate that the mode of action and the potential ecological effects of phytochemical-mediated resistance are broader than previously recognized.