Effects of consumer surface sterilization on diet DNA metabarcoding data of terrestrial invertebrates in natural environments and feeding trials

1. DNA metabarcoding is an emerging tool used to quantify diet in environments and consumer groups where traditional approaches are unviable, including small‐bodied invertebrate taxa. However, metabarcoding of small taxa often requires DNA extraction from full body parts (without dissection), and it is unclear whether surface contamination from body parts alters presumed diet presence or diversity.
2. We examined four different measures of diet (presence, rarefied read abundance, richness, and species composition) for a terrestrial invertebrate consumer (the spider Heteropoda venatoria) both collected in its natural environment and fed an offered diet item in contained feeding trials using DNA metabarcoding of full body parts (opisthosomas). We compared diet from consumer individuals surface sterilized to remove contaminants in 10% commercial bleach solution followed by deionized water with a set of unsterilized individuals.
3. We found that surface sterilization did not significantly alter any measure of diet for consumers in either a natural environment or feeding trials. The best‐fitting model predicting diet detection in feeding trial consumers included surface sterilization, but this term was not statistically significant (β = −2.3, p‐value = .07).
4. Our results suggest that surface contamination does not seem to be a significant concern in this DNA diet metabarcoding study for consumers in either a natural terrestrial environment or feeding trials. As the field of diet DNA metabarcoding continues to progress into new environmental contexts with various molecular approaches, we suggest ongoing context‐specific consideration of the possibility of surface contamination.

     

Invertebrate biodiversity in apple orchards: agrichemical sprays as explanatory variables for inter‐orchard community differences

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Redesign of PCR primers for mitochondrial cytochrome c oxidase subunit I for marine invertebrates and application in all‐taxa biotic surveys

DNA barcoding is a powerful tool for species detection, identification and discovery. Metazoan DNA barcoding is primarily based upon a specific region of the cytochrome c oxidase subunit I gene that is PCR amplified by primers HCO2198 and LCO1490 (‘Folmer primers’) designed by Folmer et al. (Molecular Marine Biology and Biotechnology, 3 , 1994, 294). Analysis of sequences published since 1994 has revealed mismatches in the Folmer primers to many metazoans. These sequences also show that an extremely high level of degeneracy would be necessary in updated Folmer primers to maintain broad taxonomic utility. In primers jgHCO2198 and jgLCO1490, we replaced most fully degenerated sites with inosine nucleotides that complement all four natural nucleotides and modified other sites to better match major marine invertebrate groups. The modified primers were used to amplify and sequence cytochrome c oxidase subunit I from 9105 specimens from Moorea, French Polynesia and San Francisco Bay, California, USA representing 23 phyla, 42 classes and 121 orders. The new primers, jgHCO2198 and jgLCO1490, are well suited for routine DNA barcoding, all‐taxon surveys and metazoan metagenomics.     

Relationships between management practices and ground‐active invertebrate biodiversity in New Zealand kiwifruit orchards

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Considerations for metabarcoding‐based port biological baseline surveys aimed at marine nonindigenous species monitoring and risk assessments

1. Monitoring introduction and spread of nonindigenous species via maritime transport and performing risk assessments require port biological baseline surveys. Yet, the comprehensiveness of these surveys is often compromised by the large number of habitats present in a port, the seasonal variability, and the time‐consuming morphological approach used for taxonomic identification. Metabarcoding represents a promising alternative for rapid comprehensive port biological baseline surveys, but its application in this context requires further assessments.
2. We applied metabarcoding (based on barcodes of the cytochrome c oxidase subunit I and of the 18S ribosomal RNA gene) to 192 port samples collected (a) from diverse habitats (water column—including environmental DNA and zooplankton, sediment, and fouling structures), (b) at different sites (from inner to outer estuary), and iii) during the four seasons of the year.
3. By comparing the biodiversity metrics derived from each sample group, we show that each sampling method resulted in a distinct community profile and that environmental DNA alone cannot substitute for organismal sampling, and that, although sampling at different seasons and locations resulted in higher observed biodiversity, operational results can be obtained by sampling selected locations and seasons.
4. By assessing the taxonomic composition of the samples, we show that metabarcoding data allowed the detection of previously recorded nonindigenous species as well as to reveal presence of new ones, even if in low abundance.
5. Synthesis and application. Our comprehensive assessment of metabarcoding for port biological baseline surveys sets the basics for cost‐effective, standardized, and comprehensive monitoring of nonindigenous species and for performing risk assessments in ports. This development will contribute to the implementation of the recently entered into force International Convention for the Control and Management of Ships' Ballast Water and Sediments.

     

DNA metabarcoding and the cytochrome c oxidase subunit I marker: not a perfect match

DNA metabarcoding enables efficient characterization of species composition in environmental DNA or bulk biodiversity samples, and this approach is making significant and unique contributions in the field of ecology. In metabarcoding of animals, the cytochrome c oxidase subunit I (COI) gene is frequently used as the marker of choice because no other genetic region can be found in taxonomically verified databases with sequences covering so many taxa. However, the accuracy of metabarcoding datasets is dependent on recovery of the targeted taxa using conserved amplification primers. We argue that COI does not contain suitably conserved regions for most amplicon-based metabarcoding applications. Marker selection deserves increased scrutiny and available marker choices should be broadened in order to maximize potential in this exciting field of research.     

Characterization of wild bee communities in apple and blueberry orchards

1. Wild bees provide invaluable ecosystem services in agricultural landscapes such as pollination. However, in recent decades, pollinator biodiversity, especially in wild bees, is declining on a global scale, with potentially far‐reaching consequences for crop production. Thus, there is an urgent need to determine whether wild bees are present in agricultural systems, such as fruit orchards.
2. In the present study, we examined the wild bee fauna at species and community levels during the period of bee activity (May to August) in apple and high‐bush blueberry orchards in New England.
3. Bee communities are crop‐specific and dominated by very few species, which fluctuate according to crop and season. The blueberry associated bee fauna was more diverse. In apple, communities were phylogenetically clustered at the genus level and dominated by solitary ground nesting bees within the genus Andrena. Species fluctuated widely in presence and abundance throughout the season, leading to differences in community composition and functional trait structure.
4. The results obtained in the present study show that apple and blueberry harbour a distinct and diverse bee fauna that performs vital pollination services in orchards. Our results provide essential baseline data for wild bees in blueberry and apple orchards and this can be used to improve management and conservation strategies for wild bee preservation in these crops.

     

Nesting activity of cavity-nesting bees and wasps is lower in small-scale apple orchards compared to nearby semi-natural habitats

1. Commercially reared cavity-nesting bees have been studied mainly in large, intensively managed orchards. However, knowledge on wild cavity-nesting bee and wasp communities and their potential limitations in smaller orchards remain insufficient.
2. We compared the colonization rate of trapnests, nesting success, parasitism and response to flower resources of cavity-nesting bees and wasps between apple orchards and nearby semi-natural habitats (SNHs).
3. Trapnests were placed in orchards and neighbouring SNHs. Colonization dynamics were studied and herbaceous flower resources were estimated. Furthermore, nest and brood cell quantity, number of alive offspring and nest parasitism rate were assessed.
4. We found a higher colonization rate in the SNHs than in the orchards. Both bees and wasps made more nests, completed more brood cells and had a higher number of alive offspring in the SNHs. The number of bee nests in the orchards showed a positive correlation with the species richness of the flowering plants. The nest parasitism of wasps was higher in the SNHs.
5. Apple orchards in the studied small-scale system were generally less colonized by cavity-nesting hymenopterans than nearby SNHs that can be important reservoirs of these ecosystem service provider hymenopterans. Our results highlight the importance of diverse flowering herbaceous vegetation in the understory that increased the number of bee nests in orchards and that could have a positive effect on the nesting activity of the bee species active in summer. Therefore, management practices that support flowering plant species in the understory vegetation are highly recommended in such orchards.

     

Targeted and passive environmental DNA approaches outperform established methods for detection of quagga mussels,Dreissena rostriformis bugensis in flowing water

1. The early detection of invasive non‐native species (INNS) is important for informing management actions. Established monitoring methods require the collection or observation of specimens, which is unlikely at the beginning of an invasion when densities are likely to be low. Environmental DNA (eDNA) analysis is a highly promising technique for the detection of INNS—particularly during the early stages of an invasion.
2. Here, we compared the use of traditional kick‐net sampling with two eDNA approaches (targeted detection using both conventional and quantitative PCR and passive detection via metabarcoding with conserved primers) for detection of quagga mussel, Dreissena rostriformis bugensis, a high priority INNS, along a density gradient on the River Wraysbury, UK.
3. All three molecular tools outperformed traditional sampling in terms of detection. Conventional PCR and qPCR both had 100% detection rate in all samples and outperformed metabarcoding when the target species was at low densities. Additionally, quagga mussel DNA copy number (qPCR) and relative read count (metabarcoding) were significantly influenced by both mussel density and distance from source population, with distance being the most significant predictor.
4. Synthesis and application. All three molecular approaches were more sensitive than traditional kick‐net sampling for the detection of the quagga mussel in flowing water, and both qPCR and metabarcoding enabled estimates of relative abundance. Targeted approaches were more sensitive than metabarcoding, but metabarcoding has the advantage of providing information on the wider community and consequently the impacts of INNS.

     

Towards routine DNA metabarcoding of macroinvertebrates using bulk samples for freshwater bioassessment: Effects of debris and storage conditions on the recovery of target taxa

1. Macroinvertebrates are commonly sampled for bioassessment of freshwater ecosystems. However, current bioassessment protocols involve laborious sorting of the animals from the debris (sample matrix) and morphological identification, where species level identifications are often difficult. DNA metabarcoding has the potential to improve bioassessment by reducing the time taken to process samples and improve the accuracy and speed of macroinvertebrate species identification.
2. In this study, we evaluated DNA metabarcoding of macroinvertebrate samples, which include macroinvertebrates and the debris collected in the sample nets, to test if bulk, unsorted samples can be used to assess macroinvertebrate diversity. First, we tested if the sample matrix prevented the detection of six target macroinvertebrate taxa when DNA metabarcoding. Second, we tested if sample storage influenced the detection of the same six target macroinvertebrates. We also explored different levels of replication at the sample, sub-sample, and polymerase chain reaction levels and compared the overall macroinvertebrate families detected using DNA metabarcoding to those identified morphologically.
3. We found that the presence of the sample matrix did not interfere with or inhibit the detection of the six target macroinvertebrate taxa. Furthermore, we found that the various sample storage methods did not affect target macroinvertebrate detection. The reliability of detection of the target macroinvertebrates improved as hierarchical levels of replication were combined. We found strong overlap between the detection of overall macroinvertebrate family diversity when comparing DNA metabarcoding to morphological identification.
4. Extracting DNA from the bulk macroinvertebrate samples that included the sample matrix and using this for DNA metabarcoding could improve bioassessment by removing the need for laborious sorting of samples. Furthermore, DNA metabarcoding detection of the six target taxa was not dependent on sample storage of up to 1 year in 95% ethanol, at room temperature or after heating. DNA metabarcoding had the advantage of identifying macroinvertebrate species, but good DNA barcode libraries are needed for widespread species identifications. Further investigation should focus on including multiple samples with different macroinvertebrate composition and densities to refine and standardise bulk sample processing protocols, and on building comprehensive DNA barcode libraries for aquatic macroinvertebrates.

     

A from‐benchtop‐to‐desktop workflow for validating HTS data and for taxonomic identification in diet metabarcoding studies

The main objective of this work was to develop and validate a robust and reliable “from‐benchtop‐to‐desktop” metabarcoding workflow to investigate the diet of invertebrate‐eaters. We applied our workflow to faecal DNA samples of an invertebrate‐eating fish species. A fragment of the cytochrome c oxidase I (COI) gene was amplified by combining two minibarcoding primer sets to maximize the taxonomic coverage. Amplicons were sequenced by an Illumina MiSeq platform. We developed a filtering approach based on a series of nonarbitrary thresholds established from control samples and from molecular replicates to address the elimination of cross‐contamination, PCR/sequencing errors and mistagging artefacts. This resulted in a conservative and informative metabarcoding data set. We developed a taxonomic assignment procedure that combines different approaches and that allowed the identification of ~75% of invertebrate COI variants to the species level. Moreover, based on the diversity of the variants, we introduced a semiquantitative statistic in our diet study, the minimum number of individuals, which is based on the number of distinct variants in each sample. The metabarcoding approach described in this article may guide future diet studies that aim to produce robust data sets associated with a fine and accurate identification of prey items.     

Effective mosquito and arbovirus surveillance using metabarcoding

Effective vector and arbovirus surveillance requires timely and accurate screening techniques that can be easily upscaled. Next‐generation sequencing (NGS) is a high‐throughput technology that has the potential to modernize vector surveillance. When combined with DNA barcoding, it is termed ‘metabarcoding.’ The aim of our study was to establish a metabarcoding protocol to characterize pools of mosquitoes and screen them for virus. Pools contained 100 morphologically identified individuals, including one Ross River virus (RRV) infected mosquito, with three species present at different proportions: 1, 5, 94%. Nucleic acid extracted from both crude homogenate and supernatant was used to amplify a 269‐bp section of the mitochondrial cytochrome c oxidase subunit I (COI) locus. Additionally, a 67‐bp region of the RRV E2 gene was amplified from synthesized cDNA to screen for RRV. Amplicon sequencing was performed using an Illumina MiSeq, and bioinformatic analysis was performed using a DNA barcode database of Victorian mosquitoes. Metabarcoding successfully detected all mosquito species and RRV in every positive sample tested. The limits of species detection were also examined by screening a pool of 1000 individuals, successfully identifying the species and RRV from a single mosquito. The primers used for amplification, number of PCR cycles and total number of individuals present all have effects on the quantification of species in mixed bulk samples. Based on the results, a number of recommendations for future metabarcoding studies are presented. Overall, metabarcoding shows great promise for providing a new alternative approach to screening large insect surveillance trap catches.     

Relationships between wild bees,hoverflies and pollination success in apple orchards with different landscape contexts

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基于STICS模型的黄土高原苹果园生态系统服务评估

苹果园在确保最大生产力的同时应适当考虑环境和自然资源,如何权衡其生态系统服务是苹果园可持续发展面临的重要问题之一。利用大田试验和STICS模型相结合的方法研究农业管理措施和气候对黄土高原苹果园生态系统服务的影响,并在此基础上对黄土高原南部半湿润区和北部半干旱区土壤氮可利用性、气候调节、水循环调节和果实生产四种苹果园生态系统服务进行了系统的评估。结果表明:(1)STICS模型均能较好模拟白水和子洲试验果园的产量、单果重、土壤含水量和蒸散发等生态系统服务指标;(2)除固碳与产量、单果重及果树相关指标是协同关系以外,其他生态系统服务之间都是权衡关系;(3)对于各管理措施下的果园平均服务标准值,秸秆覆盖(0.67)>地布覆盖(0.52)>清耕(0.30),充分灌溉(0.56)>轻度亏缺灌溉(0.44)>重度亏缺灌溉(0.30),其中秸秆覆盖和充分灌溉的果园服务概况最佳,地布覆盖和轻度亏缺灌溉的果园服务概况较相似且最平衡;(4)地布覆盖和轻度亏缺灌溉管理措施缓和权衡作用的效果优于其他管理措施。综上所述,STICS模型能够较好的量化果园生态系统服务概况,农业管理措施是果园生态系统服务强有力的驱动因子。     

Biosurveillance for invasive insect pest species using an environmental DNA metabarcoding approach and a high salt trap collection fluid

1. With the increase in global trade and warming patterns, the movement, introduction, and establishment of non‐native insect species has increased. A rapid and effective early detection biosurveillance program to identify species of concern is needed to reduce future impacts and costs associated with introduced non‐native species. One of the challenges facing insect surveillance trapping methods is the sheer volume of individual specimens in the collections. Although molecular identification methods are improving, they currently have limitations (e.g., destructive processing of specimens) and a protocol addressing these limitations can support regulatory applications that need morphological evidence to corroborate molecular data.
2. The novel protocol presented here uses a metabarcoding approach to amplify environmental DNA from a saturated salt solution trap fluid, which retains trap specimens for downstream morphological identifications. The use of a saturated salt solution to preserve specimens in traps addresses issues with the high evaporation rate of ethanol in traps, and public safety concerns with other fluid preservation options with unattended traps in public settings.
3. Using a metabarcoding approach, a 407‐nucleotide segment of the cytochrome c oxidase subunit 1 (COI) animal barcode region was successfully amplified from Lindgren funnel trap collection fluids. These traps were placed in forested areas to survey for wood‐boring beetles of regulatory concern. Our results displayed successful amplification of target taxa, including the molecular identification of the Japanese Beetle Popillia japonica, a species regulated in Canada. A second species, Anisandrus maiche, recently introduced to North America, was identified in every trap. The genus Lymantria, which contains numerous species of concern to North American woodlands, was also detected. Also, there were six other species identified of interest due to their potential impacts on native and crop flora and fauna.
4. Our results show how this protocol can be used as an efficient method for the surveillance of insects using a trap with a saturated salt solution and eDNA metabarcoding to detect species of regulatory concern.

     

Bio-ecology of false codling moth,Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) within citrus orchards in Kenya and Tanzania

1. False codling moth, Thaumatotibia leucotreta is among the key constraints of the agricultural industry. Little is known about the population dynamics and genetic diversity of this pest in East Africa.
2. The spatial–temporal population dynamics and genetic diversity of false codling moth were evaluated in citrus orchards in Kenya and Tanzania between May 2017 and August 2018 covering two fruiting seasons. The diversity of false codling moth sampled in these orchards and from solanaceous hosts, as well as from specimens received from Uganda, Sudan, and South Africa were assayed using the mitochondrial cytochrome oxidase I gene.
3. A similar spatial–temporal pattern of false codling moth was found in both Kenya and Tanzania, with the most male moths in August 2017 and 2018. In Tanzania, the number of male moths caught at high and mid altitudes did not differ but were significantly higher than those caught at low altitude.
4. A relatively low false codling moth genetic diversity was recorded at the sites where sampling was done in the respective countries as well as between specimens sampled from different host plants.
5. The low false codling moth genetic diversity determined in this trial can be exploited in the management of the pest in the studied countries.

     

Plant–soil feedbacks of forest understorey plants transplanted in nonlocal soils along a latitudinal gradient

• Climate change is driving movements of many plants beyond, as well as within, their current distributional ranges. Even migrant plants moving within their current range may experience different plant–soil feedbacks (PSF) because of divergent nonlocal biotic soil conditions. Yet, our understanding to what extent soil biotic conditions can affect the performance of within‐range migrant plants is still very limited.
• We assessed the emergence and growth of migrant forest herbs (Milium effusum and Stachys sylvatica) using soils and seeds collected along a 1,700 km latitudinal gradient across Europe. Soil biota were manipulated through four soil treatments, i.e. unsterilized control soil (PSF_US), sterilized soil (PSF_S), sterilized soil inoculated with unsterilized home soil (PSF_S+HI) and sterilized soil inoculated with unsterilized foreign soil (PSF_S+FI, expected to occur when both plants and soil biota track climate change).
• Compared to PSF_S, PSF_US had negative effects on the growth but not emergence of both species, while PSF_S+FI only affected S. sylvatica across all seed provenances. When considering seed origin, seedling emergence and growth responses to nonlocal soils depended on soil biotic conditions. Specifically, the home–away distance effect on seedling emergence differed between the four treatments, and significant responses to chemistry either disappeared (M. effusum) or changed (S. sylvatica) from PSF_US to PSF_S.
• Soil biota emerge as an important driver of the estimated plant migration success. Our results of the effects of soil microorganisms on plant establishment provide relevant information for predictions of the distribution and dynamics of plant species in a changing climate.

     

Conserved primers for DNA barcoding historical and modern samples from New Zealand and Antarctic birds

Our ability to DNA barcode the birds of the world is based on the effective amplification and sequencing of a 648 base pair (bp) region of the mitochondrial cytochrome c oxidase (COI or cox1) gene. For many geographic regions the large numbers of vouchered specimens necessary for the construction of a DNA barcoding database have already been collected and are available in museums and other institutions. However, many of these specimens are old (>20 years) and are stored as either fixed study skins or dried skeletons. DNA extracted from such historical samples is typically degraded and, generally, only short DNA fragments can be recovered from such specimens making the recovery of the barcoding region as a single fragment difficult. We report two sets of conserved primers that allow the amplification of the entire DNA barcoding region in either three or five overlapping fragments. These primer sets allow the recovery of DNA barcodes from valuable historical specimens that in many cases are unique in that they are unable or unlikely to be collected again. We also report three new primers that in combination allow the effective amplification from modern samples of the entire DNA barcoding region as a single DNA fragment for 17 orders of Southern Hemisphere birds.     

Molecular‐ and pollen‐based vegetation analysis in lake sediments from central Scandinavia

Plant and animal biodiversity can be studied by obtaining DNA directly from the environment. This new approach in combination with the use of generic barcoding primers (metabarcoding) has been suggested as complementary or alternative to traditional biodiversity monitoring in ancient soil sediments. However, the extent to which metabarcoding truly reflects plant composition remains unclear, as does its power to identify species with no pollen or macrofossil evidence. Here, we compared pollen‐based and metabarcoding approaches to explore the Holocene plant composition around two lakes in central Scandinavia. At one site, we also compared barcoding results with those obtained in earlier studies with species‐specific primers. The pollen analyses revealed a larger number of taxa (46), of which the majority (78%) was not identified by metabarcoding. The metabarcoding identified 14 taxa (MTUs), but allowed identification to a lower taxonomical level. The combined analyses identified 52 taxa. The barcoding primers may favour amplification of certain taxa, as they did not detect taxa previously identified with species‐specific primers. Taphonomy and selectiveness of the primers are likely the major factors influencing these results. We conclude that metabarcoding from lake sediments provides a complementary, but not an alternative, tool to pollen analysis for investigating past flora. In the absence of other fossil evidence, metabarcoding gives a local and important signal from the vegetation, but the resulting assemblages show limited capacity to detect all taxa, regardless of their abundance around the lake. We suggest that metabarcoding is followed by pollen analysis and the use of species‐specific primers to provide the most comprehensive signal from the environment.