SPECIES DETECTION: ARTHROPODS
ARACHNIDS
Henschel J. R., Lubin Y. D. (1997): A test of habitat selection at two spatial scales in a sitand-wait predator: a web spider in the Namib desert dunes. Journal of Animal Ecology 66: 401-413.
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Distribution and web characteristics were examined for the dune-burrowing spider Seothyra henscheli in the Namib Desert to determine how these spiders relate to spatially heterogeneous habitat features that influence foraging costs and benefits, growth rate and survival. In particular, we set out to determine the relative importance of (i) habitat selection, (ii) site-dependent growth and survival, and (iii) restricted dispersal, to the occurrence of S. henscheli in different macro- and microhabitats. Spider webs were mapped along transects that crossed dune regions with dense vegetation hummocks, steep dune plinths and bare, gravelly interdune plains. Spiders were more abundant in the hummocks than in the interdunes and plinths. Translocation of spiders from the hummocks to the interdunes and plinths confirmed that spider density was correlated with habitat quality. In a comparison of web sites with dummy sites in terms of factors related to sand stability and food availability, it was found that, except for the avoidance of extreme conditions, web-site characteristics did not differ from dummy sites. Web geometry indicated that good sites were characterized by stable sand and abundant ants. However, many spiders occurred at poor sites when good sites were vacant nearby. It is concluded that S. henscheli do not actively choose web sites, but have restricted dispersal. Site-dependent growth and survival may explain the pattern of local abundance. Site tenacity may be a result of the spiders’ inability to predict site quality coupled with high costs of relocation. It is inferred that spiderlings tend to remain near their mother’s site, which has a previous record of success. Tolerance of variable conditions of dunes and the ability to sustain populations even at poor sites enable these spiders to occur in extreme desert conditions.
Nyffeler M., Bonte D. (2020): Where have all the spiders gone? Observations of a dramatic population density decline in the once very abundant garden spider, Araneus diadematus (Araneae: Araneidae), in the Swiss midland. Insects 11: 248.
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Aerial web-spinning spiders (including large orb-weavers), as a group, depend almost entirely on flying insects as a food source. The recent widespread loss of flying insects across large parts of western Europe, in terms of both diversity and biomass, can therefore be anticipated to have a drastic negative impact on the survival and abundance of this type of spider. To test the putative importance of such a hitherto neglected trophic cascade, a survey of population densities of the European garden spider Araneus diadematus – a large orb-weaving species – was conducted in the late summer of 2019 at twenty sites in the Swiss midland. The data from this survey were compared with published population densities for this species from the previous century. The study verified the above-mentioned hypothesis that this spider’s present-day overall mean population density has declined alarmingly to densities much lower than can be expected from normal population fluctuations (0.7% of the historical values). Review of other available records suggested that this pattern is widespread and not restricted to this region. In conclusion, the decline of this once so abundant spider in the Swiss midland is evidently revealing a bottom-up trophic cascade in response to the widespread loss of flying insect prey in recent decades.
CRUSTACEANS
Mächler E., Deiner K., Steinmann P., Altermatt F. (2014): Utility of environmental DNA for monitoring rare and indicator macroinvertebrate species. Freshwater Science 33: 1174-1183.
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Accurate knowledge of the distribution of rare, indicator, or invasive species is required for conservation and management decisions. However, species monitoring done with conventional methods may have limitations, such as being laborious in terms of cost and time, and often requires invasive sampling of specimens. Environmental DNA (eDNA) has been identified as a molecular tool that could overcome these limitations, particularly in aquatic systems. Detection of rare and invasive amphibians and fish in lake and river systems has been effective, but few studies have targeted macroinvertebrates in aquatic systems. We expanded eDNA techniques to a broad taxonomic array of macroinvertebrate species in river and lake systems. We were able to detect 5 of 6 species (Ancylus fluviatilis, Asellus aquaticus, Baetis buceratus, Crangonyx pseudogracilis, and Gammarus pulex) with an eDNA method in parallel to the conventional kicknet-sampling method commonly applied in aquatic habitats. Our eDNA method showed medium to very high consistency with the data from kicknet-sampling and was able to detect both indicator and nonnative macroinvertebrates. Furthermore, our primers detected target DNA in concentrations down to 10–5 ng/µL of total extracted tissue DNA in the absence of background eDNA in the reaction. We demonstrate that an eDNA surveillance method based on standard PCR can deliver biomonitoring data across a wide taxonomic range of macroinvertebrate species (Gastropoda, Isopoda, Ephemeroptera, and Amphipoda) in riverine habitats and may offer the possibility to deliver data on a more refined time scale than conventional methods when focusing on single or few target species. Such information based on nondestructive sampling may allow rapid management decisions and actions.
Forsström T., Vasemägi A. (2016): Can environmental DNA (eDNA) be used for detection and monitoring of introduced crab species in the Baltic Sea?. Marine Pollution Bulletin 109: 350-355.
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The need to detect and monitor introduced marine species has increased with the increasing number of marine invasions. To complement standard detection and monitoring techniques, new approaches using environmental DNA (eDNA) have recently been developed. However, most of the eDNA work has focused on vertebrate species in spatially limited freshwater habitats while benthic invertebrates in coastal environments have received much less attention. Here, we evaluated the suitability of the eDNA approach for detecting benthic, hard-shelled, crustacean mud crab species in a brackish water environment. We demonstrated for the first time that eDNA from an introduced mud crab Rhithropanopeus harrisii can be successfully amplified in aquarium water samples and detected in the brackish water environment. However, the detection rate was rather low. This suggests that in contrast to freshwater vertebrates, it may be more challenging to develop a highly sensitive eDNA method for detecting crustacean species in a marine environment.
Agersnap S., Larsen W. B., Knudsen S. W., Strand D., Thomsen P. F., Hesselsøe M., Mortensen P. B., Vrålstad T., Møller P. R. (2017): Monitoring of noble, signal and narrow-clawed crayfish using environmental DNA from freshwater samples. Plos One 12: e0179261.
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For several hundred years freshwater crayfish (Crustacea—Decapoda—Astacidea) have played an important ecological, cultural and culinary role in Scandinavia. However, many native populations of noble crayfish Astacus astacus have faced major declines during the last century, largely resulting from human assisted expansion of non-indigenous signal crayfish Pacifastacus leniusculus that carry and transmit the crayfish plague pathogen. In Denmark, also the non-indigenous narrow-clawed crayfish Astacus leptodactylus has expanded due to anthropogenic activities. Knowledge about crayfish distribution and early detection of non-indigenous and invasive species are crucial elements in successful conservation of indigenous crayfish. The use of environmental DNA (eDNA) extracted from water samples is a promising new tool for early and non-invasive detection of species in aquatic environments. In the present study, we have developed and tested quantitative PCR (qPCR) assays for species-specific detection and quantification of the three above mentioned crayfish species on the basis of mitochondrial cytochrome oxidase 1 (mtDNA-CO1), including separate assays for two clades of A. leptodactylus. The limit of detection (LOD) was experimentally established as 5 copies/PCR with two different approaches, and the limit of quantification (LOQ) were determined to 5 and 10 copies/PCR, respectively, depending on chosen approach. The assays detected crayfish in natural freshwater ecosystems with known populations of all three species, and show promising potentials for future monitoring of A. astacus, P. leniusculus and A. leptodactylus. However, the assays need further validation with data 1) comparing traditional and eDNA based estimates of abundance, and 2) representing a broader geographical range for the involved crayfish species.
Dunn N., Priestley V., Herraiz A., Arnold R., Savolainen V. (2017): Behavior and season affect crayfish detection and density inference using environmental DNA. Ecology and Evolution 7: 7777-7785.
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Although the presence/absence of aquatic invertebrates using environmental DNA (eDNA) has been established for several species, inferring population densities has remained problematic. The invasive American signal crayfish, Pacifastacus leniusculus (Dana), is the leading cause of decline in the UK’s only native crayfish species, Austropotamobius pallipes (Lereboullet). Methods to detect species at low abundances offer the opportunity for the early detection, and potential eradication, of P. leniusculus before population densities reach threatening levels in areas occupied by A. pallipes. Using a factorial experimental design with aquaria, we investigated the impacts of biomass, sex ratio, and fighting behavior on the amount of eDNA released by P. leniusculus, with the aim to infer density per aquarium depending on treatments. The amount of target eDNA in water samples from each aquarium was measured using the quantitative Polymerase Chain Reaction. We show that the presence of eggs significantly increases the concentration of crayfish eDNA per unit of mass, and that there is a significant relationship between eDNA concentration and biomass when females are egg‐bearing. However, the relationship between crayfish biomass and eDNA concentration is lost in aquaria without ovigerous females. Female‐specific tanks had significantly higher eDNA concentrations than male‐specific tanks, and the prevention of fighting did not impact the amount of eDNA in the water. These results indicate that detection and estimate of crayfish abundance using eDNA may be more effective while females are ovigerous. This information should guide further research for an accurate estimation of crayfish biomass in the field depending on the season. Our results indicate that detection and quantification of egg‐laying aquatic invertebrate species using eDNA could be most successful during periods when eggs are developing in the water. We recommend that practitioners consider the reproductive cycle of target species when attempting to study or detect aquatic species using eDNA in the field.
Mauvisseau Q., Coignet A., Delaunay C., Pinet F., Bouchon D., Souty-Grosset C. (2018): Environmental DNA as an efficient tool for detecting invasive crayfishes in freshwater ponds. Hydrobiologia 805: 163-175.
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Environmental DNA (eDNA) is a powerful method for assessing the presence and distribution of invasive aquatic species. We used this tool to detect and monitor several invasive crayfishes Procambarus clarkii, Orconectes limosus and Pacifastacus leniusculus present in, or likely to invade, the ponds of the Brenne Regional Natural Park. A previous study showed that the eDNA method was not very efficient in detecting P. clarkii. In the present study, we explored new improvements in the detection of invasive crayfish. We designed specific primers for each crayfish species, and set up an experimental mesocosm approach to confirm the specificity of the primers and the sampling protocol. We analysed samples taken from ponds in 2014 and 2015. We compared two qPCR protocols involving either SybrGreen or TaqMan assays. Using these same primers, we were able to detect crayfish eDNA with both assays during the mesocosm experiment. However, crayfish from field samples could only be detected by performing qPCR with a SybrGreen assay. We successfully monitored the presence of three invasive species of crayfish using eDNA. This method is a powerful tool for establishing the presence or absence of invasive species in various freshwater environments.
Wu Q., Kawano K., Ishikawa T., Sakata M. K., Nakao R., Hiraiwa M. K., Tsuji S., Yamanaka H., Minamoto T. (2019): Habitat selection and migration of the common shrimp, Palaemon paucidens in Lake Biwa, Japan – An eDNA‐based study. Environmental DNA 1: 54-63.
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Palaemon paucidens has a large population and is an important food source for fish in Lake Biwa, Japan. They are abundant in shallow waters from spring to summer, after which most individuals migrate to offshore deep areas where they remain during autumn and winter. However, some individuals are nonmigratory, remaining in shallow waters over winter. It has been reported that P. paucidens individuals have declined in recent years; a better understanding of its seasonal distribution is needed to manage this species, and basic information on its seasonal distribution is indispensable. We tracked the environmental DNA (eDNA) distribution of P. paucidens in Lake Biwa over a year using a quantitative real-time polymerase chain reaction method. We collected water samples from offshore (both from the surface and from the benthic) and from shallow shore sites adjacent to the shorelines of the main lake and connecting freshwater lagoons. Offshore sampling took place in summer and winter, and shallow shore and lagoon sampling in all four seasons. During summer, eDNA concentrations were significantly higher in shallow and lagoon areas than offshore bottom sites. Conversely, during winter, eDNA concentrations were higher in offshore bottom sites, and relatively high and low eDNA concentrations in lagoons and shallow shore, respectively. These results most likely reflect the spatial and temporal distribution of this species in Lake Biwa. The eDNA concentrations peaked in early August at shallow shore sites in the main lake, with a significant decline in mid-October, while low eDNA concentrations were recorded at offshore bottom sites in late August. These results suggest that P. paucidens migrates from shallow waters to offshore bottom sites between early August and mid-October. These results provide important information for the management of this species.
Roux L. M. D., Giblot‐Ducray D., Bott N. J., Wiltshire K. H., Deveney M. R., Westfall K. M., Abbott C. L. (2020): Analytical validation and field testing of a specific qPCR assay for environmental DNA detection of invasive European green crab (Carcinus maenas). Environmental DNA 2: 309-320.
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Environmental DNA (eDNA) methods are providing tools for detecting invasive species in aquatic environments. Targeted qPCR assays applied to eDNA samples promise to overcome limitations of traditional methods, especially for early detection. The European green crab (Carcinus maenas) is considered one of the most successful invasive species globally due to the large range it has invaded and negative impacts on native species, marine habitats, and shellfish industries. We developed, laboratory‐validated, and field‐tested a specific qPCR assay for the detection of green crab from eDNA samples. We also show that the assay can detect green crab in bulk DNA extracted from plankton samples. Assay design, optimization, sensitivity, and specificity testing generally followed the validation pathway recommended by the World Organization for Animal Health for assays used to manage global aquatic animal health and infectious disease. Assay specificity was verified in silico and in vitro by laboratory testing 26 nontarget species, none of which showed potential for amplification. Assay sensitivity was appropriately high, with the limit of detection approaching two gene copies/μl. The assay was field‐tested on eDNA samples collected from filtered seawater at five sites on the Pacific coast of Canada known to harbor green crab based on historical monitoring data; green crab DNA was amplified from all sites. We also present early pilot field testing of the assay done on bulk DNA extracted from plankton samples from four sites from Australia, two sites with and two sites without reported records of green crab presence. Green crab was detected at both sites with known green crab records. Significant inhibition was recorded for some plankton samples but not for eDNA samples. This is the first qPCR assay for detection of European green crab, providing researchers and managers with a valuable new tool to aid early detection and ongoing monitoring.
Rusch J. C., Mojžišová M., Strand D. A., Svobodová J., Vrålstad T., Petrusek A. (2020): Simultaneous detection of native and invasive crayfish and Aphanomyces astaci from environmental DNA samples in a wide range of habitats in Central Europe. NeoBiota 58: 1-32.
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Crayfish of North American origin are amongst the most prominent high-impact invasive invertebrates in European freshwaters. They contribute to the decline of European native crayfish species by spreading the pathogen causing crayfish plague, the oomycete Aphanomyces astaci. In this study we validated the specificity of four quantitative PCR (qPCR) assays, either published or newly developed, usable for environmental DNA (eDNA) screening for widely distributed native and non-native crayfish present in Central Europe: Astacus astacus, Pacifastacus leniusculus, Faxonius limosus and Procambarus virginalis. We then conducted an eDNA monitoring survey of these crayfish as well as the crayfish plague pathogen in a wide variety of habitat types representative for Central and Western Europe. The specificity of qPCR assays was validated against an extensive collection of crayfish DNA isolates, containing most crayfish species documented from European waters. The three assays developed in this study were sufficiently species-specific, but the published assay for F. limosus displayed a weak cross-reaction with multiple other crayfish species of the family Cambaridae. In the field study, we infrequently detected eDNA of A. astaci together with the three non-native crayfish species under examination. We never detected eDNA from A. astaci together with native crayfish, but in a few locations eDNA from both native and non-native crayfish was captured, due either to passive transport of eDNA from upstream populations or co-existence in the absence of infected crayfish carriers of A. astaci. In the study, we evaluated a robust, easy-to-use and low-cost version of the eDNA sampling equipment, based mostly on items readily available in garden stores and hobby markets, for filtering relatively large (~5 l) water samples. It performed just as well as the far more expensive equipment industrially designed for eDNA water sampling, thus opening the possibility of collecting suitable eDNA samples to a wide range of stakeholders. Overall, our study confirms that eDNA-based screening for crayfish and their associated pathogen is a feasible alternative to traditional monitoring.
Trujillo‐Gonzalez A., Hinlo R., Godwin S., Barmuta L. A., Watson A., Turner P., Koch A., Gleeson D. (2021): Environmental DNA detection of the giant freshwater crayfish (Astacopsis gouldi). Environmental DNA 3: 950-958.
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The giant freshwater crayfish, Astacopsis gouldi Clark, 1936, is an endangered endemic freshwater species native to Northern Tasmania. Habitat loss, fishing pressure, and climate change have been identified as threatening processes. The Australian government approved a recovery plan for A. gouldi in 2017 that requires routine population surveys to assess the impact of ongoing threats and recovery actions. We developed a novel species-specific probe-based assay targeting a 219 bp fragment in the Cytochrome Oxidase 1 gene region to detect traces of A. gouldi DNA in environmental samples as a cost-effective, sensitive, and non-invasive surveillance method to assess the presence of this endangered species. We tested assay specificity against ten crayfish species commonly found in Tasmania within the Astacopsis, Cherax, Geocharax, Engaeus, and Ombrastacoides genera and determined assay sensitivity using tissue-derived genomic DNA and synthetic oligo standards designed for A. gouldi. We then tested water samples collected from aquaria and natural freshwater streams in Northern Tasmania with known occurrence of A. gouldi, as well as one site with no known A. gouldi occurrence. The probe-based assay designed in this study successfully detected A. gouldi DNA and eDNA with a 10 copies/µl limit of detection and showed no amplification of non-targeted co-existing crayfish species. We successfully detected the presence of A. gouldi eDNA in water samples from six sites with known occurrences of the species. There was no detection from the negative site. This study validates the use of eDNA-based detection of A. gouldi by real-time PCR as a non-invasive monitoring tool to assist field monitoring, assessment, and complement ongoing recovery actions to protect habitable ecosystems of A. gouldi.
Greenhalgh J. A., Collins R. A., Edgley D. E., Genner M. J., Hindle J., Jones G., Loughlin L., O’donnel M., Sweet M. J., Battarbee R.W. (2022): Environmental DNA‐based methods detect the invasion front of an advancing signal crayfish population. Environmental DNA 4: 596-607.
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Aquatic invasive species, such as the signal crayfish (Pacifastacus leniusculus), present a major threat to freshwater ecosystems. However, these species can be challenging to detect in recently invaded habitats. Environmental DNA (eDNA)–based methods are highly sensitive and capable of detecting just a few copies of target DNA from non-invasively collected samples. Therefore, they have considerable potential for broad-scale use in mapping and monitoring the spread of invasive species. In this study, we aimed to increase our understanding of the current distribution of signal crayfish in a headwater stream system in the United Kingdom (tributaries of the River Wharfe, Addingham, Yorkshire). Environmental DNA sampling, assessment of water chemistry variables, and conventional crayfish hand-searching were conducted across 19 study sites in five tributary streams. Using hand-searching, we detected signal crayfish at 26% of the sites (5/19 study sites). However, using eDNA-based methods, occupancy increased to 47% of study sites (9/19). Our sampling revealed previously unknown sites of crayfish occupancy, and using eDNA-based methods, we were able to define the geographical extent of the invasion front in each headwater stream sampled. This study highlights that eDNA-based methods are well-suited for detecting newly established signal crayfish populations in recently invaded habitats, even when the invasive species is at low abundance and, therefore, might otherwise be under-represented or undetected using conventional survey methods. Our study provides further evidence that headwater stream ecosystems are particularly vulnerable to signal crayfish invasion. However, their geomorphological features may make methods used to reduce or prevent invasive crayfish dispersal more effective than in other freshwater ecosystems.
Manfrin C., Zanetti M., Stanković D., Fattori U., Bertucci-Maresca V., Giulianini P. G., Pallavicini A. (2022): Detection of the endangered stone crayfish Austropotamobius torrentium (Schrank, 1803) and its congeneric A. pallipes in its last Italian biotope by eDNA analysis. Diversity 14: 205.
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The stone crayfish, Austropotamobius torrentium, is a European freshwater crayfish. Although this species is relatively widespread throughout the continent, it is undergoing significant declines throughout its range. However, as the decline rates have not been quantified in detail, this species is classified as data deficient by the IUCN Red List of Threatened Species. The present study describes the development and validation of two species-specific assays based on hydrolysis probe chemistry for the detection of A. torrentium and A. pallipes environmental DNA (eDNA) in water samples collected in the Julian Alps of Italy (Friuli Venezia Giulia). The eDNA-based method was applied to 14 sites within the Danubian Slizza basin, known to be inhabited by A. torrentium, but with insufficient information on their distribution. In addition, one station in the Tagliamento River basin was sampled to test the performance of the A. pallipes probe. The presence of A. torrentium is confirmed at 6 out of 15 sites. At four of these sites, A. torrentium is detected for the first time. In contrast, the presence of A. torrentium was not detected at two sites already known to harbour the species. Finally, the presence of A. pallipes was confirmed in the station belonging to the Tagliamento basin. The methodology described, which allows the distinction between the two species, paves the way for the parallel detection of the stone crayfish and the white-clawed crayfish (A. pallipes) through eDNA analysis.
Westfall K. M., Therriault T. W., Abbott C. L. (2022): Targeted next‐generation sequencing of environmental DNA improves detection of invasive European green crab (Carcinus maenas). Environmental DNA 4: 440-452.
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In the northeast Pacific Ocean, there is high interest in developing eDNA-based survey methods to aid management of invasive populations of European green crab (Carcinus maenas). Expected benefits are improved sensitivity for early detection of secondary spread and to assess the outcome of eradication efforts. A new eDNA-based approach we term “targeted next-generation sequencing (tNGS)” is introduced here and shown to improve detection relative to qPCR at sites with lower green crab CPUE values measured by trapping. DNA standards (gBlock) with starting molecule copies that were 10 to 100 times lower than the qPCR limit of detection returned significant numbers of sequencing reads, which in our field assessments translated to a 7%–10% increase in detection probability from tNGS relative to qPCR at sites with lower CPUE. We also found the number of sequencing reads from tNGS was significantly correlated with green crab CPUE whereas Ct values from qPCR were not. When sources of variation were partitioned for each assay, we found the difference between mean within-site and mean between-site variation was much larger and had non-overlapping confidence intervals for tNGS relative to qPCR, suggesting the former may offer more power for detecting spatial variation in eDNA availability. Results presented here indicate this approach is suitable for species of known low abundances where a positive detection has high economic or environmental consequences, or for labs doing eDNA surveys for whom NGS-based workflows are operationally more efficient than qPCR given its upward scalability. Any species with an existing qPCR assay can be easily tested with a tNGS assay using the approach presented here. We conclude with a discussion on the fitness for purpose of tNGS vs. qPCR for various applications and on how to best apply molecular surveys in management programs.
INSECTS
Tóth F., Árpás K., Szekeres D., Kádár F., Szentkirályi F., Szénási Á., Kiss J. (2004): Spider web survey or whole plant visual sampling? Impact assessment of Bt corn on non-target predatory insects with two concurrent methods. Environmental Biosafety Research 3: 225-231.
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Impact assessment studies rely on relevant sampling methods. Correct evaluation of methods can be done by their concurrent use in the same sampling site and period. Collecting webs of Theridion impressum L. Koch (Araneae: Theridiidae) may serve as an arthropod sampling method: empty cuticles of prey items remain attached to the back side of the leaves with adhesive silk. Our study aimed to compare the applicability of the two methods concurrently (spider web survey and whole plant visual sampling) in a risk assessment study. The spider web survey recorded more predatory insect families than the whole plant visual sampling. Both methods were able to detect significant differences in the quantity of predatory insects in Bt vs. isogenic plots, but not in the same taxa (Nabidae: spider-web, 2001, Bt > Iso; Coccinellidae: plant sampling, 2001, Iso > Bt; Welsh-test, P < 0.05); thus, they could not confirm each other. The lack of confirmation can be explained by differences in the sensitivity and selectivity of the two methods. A web survey of T. impressum has the practical advantage that although we concentrate only on the one species during field sampling, we gain additional information on a wide range of foliage-dwelling arthropods. Due to several biological uncertainties, interpretation and explanation of the results remain problematic. Thus, additional research – based on in-situ observation and recording of T. impressum-prey interactions – is necessary before we could propose web survey method as a complementary tool in ecological impact assessment
Lee H. Y., Oh S. Y., Jang Y. (2012): Morphometrics of the final instar exuviae of five cicada species occurring in urban areas of central Korea. Journal of Asia-Pacific Entomology 15: 627-630.
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Cicadas produce loud calling songs for mate attraction that can be a nuisance to city dwellers in Korea. Exuviae of final instars can be used to estimate population density. We investigated the morphological characteristics of final instar exuviae for five cicada species that are abundant in central Korea: Cryptotympana atrata, Hyalessa fuscata, Graptopsaltria nigrofuscata, Meimuna opalifera, and Meimuna mongolica. The characters analyzed were pro-mesonotum length, pronotum width, pronotum length, head width, abdominal circumference, body length, wing length, and femoral tooth angle. The results of a general linear model showed that species and sex were significant for morphological characters, and the result of post hoc analyses revealed that the mean values of most of the morphological characters measured were significantly different among the five cicada species. Three groups, Cryptotympana atrata, H. fuscata/G. nigrofuscata, and M. opalifera/M. mongolica, were distinctively identified based on most morphological characters. The distributions of femoral tooth angles did not overlap between H. fuscata and G. nigrofuscata, and the distributions of abdominal circumference did not overlap between M. opalifera and M. mongolica. Thus, the exuviae of all five cicada species could be easily distinguished based on morphological characters. We also provide a morphological key for the exuviae of all five cicada species in Korea.
Richter A., Weinhold D., Robertson G., Young M., Edwards T., Hnatiuk S., Osborne W. (2013): More than an empty case: a non invasive technique for monitoring the Australian critically endangered golden sun moth, Synemon plana (Lepidoptera: Castniidae). Journal of Insect Conservation 17: 529-536.
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Monitoring programs for butterflies and moths are focused on the adult stage, rarely considering other life stages. Transect-based counts of adults and searches for empty pupal cases have been suggested as standard monitoring protocols for the critically endangered golden sun moth Synemon plana in Australia. To date, surveys and monitoring have focused only on counts of adults. However, undertaking such counts is constrained by the short adult life of the species (1–2 days), and the fact that prevailing weather conditions can seriously influence detectability. We tested whether empty pupal cases of S. plana can be used to supplement the monitoring of adults and whether this technique can be undertaken by citizen scientists. Volunteers from Canberra (Australia) collected 650 pupal cases from 11 grassland areas. The cases were found in native grasslands and in grassland comprised entirely of the exotic Chilean needle grass (Nassella neesiana). Pupal cases of S. plana were found to be durable, with most persisting in the field for greater than 3 weeks after first sighting, and exhibited a male biased sex ratio. This study demonstrates that detection of empty pupal cases provides a potential additional tool to monitor S. plana that is not dependent on the restrictions of prevailing weather conditions and time of day, and can be undertaken by citizen scientists.
Yang C., Wang X., Miller J. A., de Blécourt M., Ji Y., Yang C., Harrison R. D., Douglas W. Y. (2014): Using metabarcoding to ask if easily collected soil and leaf-litter samples can be used as a general biodiversity indicator. Ecological Indicators 46: 379-389.
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The targeted sequencing of taxonomically informative genetic markers, sometimes known as metabarcoding, allows eukaryote biodiversity to be measured rapidly, cheaply, comprehensively, repeatedly, and verifiably. Metabarcoding helps to remove the taxonomic impediment, which refers to the great logistical difficulties of describing and identifying species, and thus promises to improve our ability to detect and respond to changes in the natural environment. Now, sampling has become a rate-limiting step in biodiversity measurement, and in an effort to reduce turnaround time, we use arthropod samples from southern China and Vietnam to ask whether soil, leaf litter, and aboveground samples provide similar ecological information. A soil or leaf-litter sample can be collected in minutes, whereas an aboveground sample, such as from Malaise traps or canopy fogging, can require days to set up and run, during which time they are subject to theft, damage, and deliberate contamination. Here we show that while the taxonomic compositions of soil and leaf-litter samples are very different from aboveground samples, both types of samples provide similar ecological information, in terms of ranking sites by species richness and differentiating sites by beta diversity. In fact, leaf-litter samples appear to be as or more powerful than Malaise-trap and canopy-fogging samples at detecting habitat differences. We propose that metabarcoded leaf-litter and soil samples be widely tested as a candidate method for rapid environmental monitoring in terrestrial ecosystems.
Jeliazkov A., Bas Y., Kerbiriou C., Julien J. F., Penone C., Le Viol I. (2016): Large-scale semi-automated acoustic monitoring allows to detect temporal decline of bush-crickets. Global Ecology and Conservation 6: 208-218.
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Monitoring biodiversity over large spatial and temporal scales is crucial to assess the impact of global changes and environmental mitigation measures. However, large-scale monitoring of invertebrates remains poorly developed despite the importance of these organisms in ecosystem functioning. The development of new recording techniques and new methods of automatic species recognition based on sound detection and easily applicable within a citizen-science framework, offers interesting possibilities. However, the value of such protocols has not been tested for the study of temporal trends on a large spatial scale. We used an acoustic region-wide citizen-monitoring program of Orthoptera, conducted along roads, to assess the relevance of automatic species recognition methods to detect temporal trends while taking into account spatial and seasonal patterns of two Orthoptera species activity (Tettigonia viridissima Linnaeus, 1758, and Ruspolia nitidula Scopoli, 1786) at a large scale. Additionally, we tested the effect of climate and land-use variables on spatio-temporal abundance patterns using generalized linear mixed models. We found negative temporal trends for the two species across the survey period (2006–2012). The spatial variations were largely explained by the geoclimatic conditions and, to a lesser extent, by land use (negative effects of urbanization). The temporal variations were highly correlated to the climatic conditions of the year, and of the previous year (nonlinear effect of temperature, precipitation). To our knowledge, this paper describes the first successful attempt to calculate large-scale temporal trends of insect populations on the basis of an automatic identification process of acoustic data. We argue that acoustic monitoring along roads, coupled with the automatic recognition of species sounds, offers several advantages for assessing Orthoptera biodiversity response to global changes and environmental measures.
Doi H., Katano I., Sakata Y., Souma R., Kosuge T., Nagano M., Ikeda K., Yano K., Tojo K. (2017): Detection of an endangered aquatic heteropteran using environmental DNA in a wetland ecosystem. Royal Society Open Science 4: 170568.
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The use of environmental DNA (eDNA) has recently been employed to evaluate the distribution of various aquatic macroorganisms. Although this technique has been applied to a broad range of taxa, from vertebrates to invertebrates, its application is limited for aquatic insects such as aquatic heteropterans. Nepa hoffmanni (Heteroptera: Nepidae) is a small (approx. 23 mm) aquatic heteropteran that inhabits wetlands, can be difficult to capture and is endangered in Japan. The molecular tool eDNA was used to evaluate the species distribution of N. hoffmanni in comparison to that determined using hand-capturing methods in two regions of Japan. The eDNA of N. hoffmanni was detected at nearly all sites (10 eDNA-detected sites out of 14 sites), including sites where N. hoffmanni was not captured by hand (five eDNA-detected sites out of six captured sites). Thus, this species-specific eDNA technique can be applied to detect small, sparsely distributed heteropterans in wetland ecosystems. In conclusion, eDNA could be a valuable technique for the detection of aquatic insects inhabiting wetland habitats, and could make a significant contribution to providing distribution data necessary to species conservation.
Mukundarajan H., Hol F. J. H., Castillo E. A., Newby C., Prakash M. (2017): Using mobile phones as acoustic sensors for high-throughput mosquito surveillance. eLife 6: e27854.
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The direct monitoring of mosquito populations in field settings is a crucial input for shaping appropriate and timely control measures for mosquito-borne diseases. Here, we demonstrate that commercially available mobile phones are a powerful tool for acoustically mapping mosquito species distributions worldwide. We show that even low-cost mobile phones with very basic functionality are capable of sensitively acquiring acoustic data on species-specific mosquito wingbeat sounds, while simultaneously recording the time and location of the human-mosquito encounter. We survey a wide range of medically important mosquito species, to quantitatively demonstrate how acoustic recordings supported by spatio-temporal metadata enable rapid, non-invasive species identification. As proof-of-concept, we carry out field demonstrations where minimally-trained users map local mosquitoes using their personal phones. Thus, we establish a new paradigm for mosquito surveillance that takes advantage of the existing global mobile network infrastructure, to enable continuous and large-scale data acquisition in resource-constrained areas.
Newson S. E., Bas Y., Murray A., Gillings S. (2017): Potential for coupling the monitoring of bush‐crickets with established large‐scale acoustic monitoring of bats. Methods in Ecology and Evolution 8: 1051-1062.
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Monitoring biodiversity over large spatial and temporal scales is crucial for assessing the impact of global changes and environmental mitigation measures. However, large-scale monitoring of invertebrates remains poorly developed despite the importance of these organisms in ecosystem functioning. Exciting possibilities applicable to professional and citizen science are offered by new recording techniques and methods of semi-automated species recognition based on sound detection. Static broad-spectrum detectors deployed to record throughout whole nights have been recommended for standardised acoustic monitoring of bats, but they have the potential to also collect acoustic data for other species groups. Large-scale deployment of such systems is only viable when combined with robust automated species identification algorithms. Here we examine the potential of such a system for detecting, identifying and monitoring bush-crickets (Orthoptera of the family Tettigoniidae). We use incidental sound recordings generated by an extensive citizen science bat survey and recordings from intensive site surveys to test a semi-automated step-wise method with a classifier for assigning species identities. We assess species’ diel activity patterns to make recommendations for survey timing and interpretation of existing nocturnal data sets and consider the feasibility of determining site occupancy. Of six species of bush-crickets, the species classifier achieved over 85% accuracy for three, speckled bush-cricket, dark bush-cricket and Roesel’s bush-cricket. It should be possible to automatically scan recordings for these species with minimal manual validation. Further refinement of the classifier is required for the three remaining species, in particular for the acoustically similar short-winged conehead and long-winged conehead. Diel activity patterns are species specific and it may be necessary to adjust the hours over which the detectors record to increase detection of key species, but this must be weighed against the costs in terms of increased memory and battery use and equipment security during daytime. We conclude that with logistical support and centralised semi-automated species identification it is now possible for the public to contribute to large-scale acoustic monitoring of Orthoptera while recording bats. Further innovation of sound classifier algorithms is needed and would be aided by improved reference sound libraries from multiple locations spanning species’ ranges.
Hill A. P., Prince P., Piña Covarrubias E., Doncaster C. P., Snaddon J. L., Rogers A. (2018): AudioMoth: Evaluation of a smart open acoustic device for monitoring biodiversity and the environment. Methods in Ecology and Evolution 9: 1199-1211.
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The cost, usability and power efficiency of available wildlife monitoring equipment currently inhibits full ground-level coverage of many natural systems. Developments over the last decade in technology, open science, and the sharing economy promise to bring global access to more versatile and more affordable monitoring tools, to improve coverage for conservation researchers and managers. Here we describe the development and proof-of-concept of a low-cost, small-sized and low-energy acoustic detector: “AudioMoth.” The device is open-source and programmable, with diverse applications for recording animal calls or human activity at sample rates of up to 384 kHz. We briefly outline two ongoing real-world case studies of large-scale, long-term monitoring for biodiversity and exploitation of natural resources. These studies demonstrate the potential for AudioMoth to enable a substantial shift away from passive continuous recording by individual devices, towards smart detection by networks of devices flooding large and inaccessible ecosystems. The case studies demonstrate one of the smart capabilities of AudioMoth, to trigger event logging on the basis of classification algorithms that identify specific acoustic events. An algorithm to trigger recordings of the New Forest cicada (Cicadetta montana) demonstrates the potential for AudioMoth to vastly improve the spatial and temporal coverage of surveys for the presence of cryptic animals. An algorithm for logging gunshot events has potential to identify a shotgun blast in tropical rainforest at distances of up to 500 m, extending to 1 km with continuous recording. AudioMoth is more energy efficient than currently available passive acoustic monitoring devices, giving it considerably greater portability and longevity in the field with smaller batteries. At a build cost of ∼US$43 per unit, AudioMoth has potential for varied applications in large-scale, long-term acoustic surveys. With continuing developments in smart, energy-efficient algorithms and diminishing component costs, we are approaching the milestone of local communities being able to afford to remotely monitor their own natural resources.
Valentin R. E., Fonseca D. M., Nielsen A. L., Leskey T. C., Lockwood J. L. (2018): Early detection of invasive exotic insect infestations using eDNA from crop surfaces. Frontiers in Ecology and the Environment 16: 265-270.
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The number of exotic species invasions has increased over recent decades, as have the ecological harm and economic burdens they impose. Rapid-response eradication of nascent exotic populations is a viable approach to minimizing damage, but implementation is limited by the difficulty of detecting such species during the early stages of infestation due to their small numbers. The use of environmental DNA (eDNA) has helped address this issue in aquatic ecosystems, but to the best of our knowledge has not been trialed for surveillance of exotic species in terrestrial systems. Using a high-resolution, real-time (quantitative) polymerase chain reaction assay, we developed a highly efficient protocol to survey agricultural fields for the invasive non-native brown marmorated stink bug (BMSB; Halyomorpha halys). We compared results using eDNA to those for conventional monitoring traps and documented substantially higher sensitivity and detection effectiveness. Our methodology is transferable to situations in which the DNA of terrestrial target species can be accumulated into a single substrate, suggesting that eDNA-based approaches could transform our ability to detect exotic insects in non-aquatic settings.
Kawakita S., Ichikawa K. (2019): Automated classification of bees and hornet using acoustic analysis of their flight sounds. Apidologie 50: 71-79.
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To investigate how to accurately identify bee species using their sounds, we conducted acoustic analysis to identify three pollinating bee species (Apis mellifera, Bombus ardens, Tetralonia nipponensis) and a hornet (Vespa simillima xanthoptera) by their flight sounds. Sounds of the insects and their environment (background noises and birdsong) were recorded in the field. The use of fundamental frequency and mel-frequency cepstral coefficients to describe feature values of the sounds, and supported vector machines to classify the sounds, correctly distinguished sound samples from environmental sounds with high recalls and precision (0.96–1.00). At the species level, our approach could classify the insect species with relatively high recalls and precisions (0.7–1.0). The flight sounds of V.s. xanthoptera, in particular, were perfectly identified (precision and recall 1.0). Our results suggest that insect flight sounds are potentially useful for detecting bees and quantifying their activity.
Mauvisseau Q., Davy-Bowker J., Bulling M., Brys R., Neyrinck S., Troth C., Sweet M. (2019): Combining ddPCR and environmental DNA to improve detection capabilities of a critically endangered freshwater invertebrate. Scientific Reports 9: 14064.
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Isogenus nubecula is a critically endangered Plecoptera species. Considered extinct in the UK, I. nubecula was recently rediscovered (in one location of the River Dee, Wales), after 22 years of absence. In a similar way to many other species of Perlodidae, I. nubecula could be utilised as a bio-indicator, for assessing water quality and health status of a given freshwater system. However, conventional monitoring of invertebrates via kick-sampling, is invasive and expensive (time consuming). Further, such methods require a high level of taxonomic expertise. Here, we compared the traditional kick-sampling method with the use of eDNA detection using qPCR and ddPCR-analyses. In spring 2018, we sampled eDNA from twelve locations on the River Dee. I. nubecula was detected using kick-sampling in five of these locations, three locations using both eDNA detection and kick-sampling and one location using eDNA detection alone – resulting in a total of six known and distinct populations of this critically endangered species. Interestingly, despite the eDNA assay being validated in vitro and in silico, and results indicating high sensitivity, qPCR analysis of the eDNA samples proved to be ineffective. In contrast, ddPCR analyses resulted in a clear detection of I. nubecula at four locations suggesting that inhibition most likely explains the large discrepancy between the obtained qPCR and ddPCR results. It is therefore important to explore inhibition effects on any new eDNA assay. We also highlight that ddPCR may well be the best option for the detection of aquatic organisms which are either rare or likely to shed low levels of eDNA into their environment.
Thomsen P. F., Sigsgaard E. E. (2019): Environmental DNA metabarcoding of wild flowers reveals diverse communities of terrestrial arthropods. Ecology and Evolution 9: 1665-1679.
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Terrestrial arthropods comprise the most species‐rich communities on Earth, and grassland flowers provide resources for hundreds of thousands of arthropod species. Diverse grassland ecosystems worldwide are threatened by various types of environmental change, which has led to decline in arthropod diversity. At the same time, monitoring grassland arthropod diversity is time‐consuming and strictly dependent on declining taxonomic expertise. Environmental DNA (eDNA) metabarcoding of complex samples has demonstrated that information on species compositions can be efficiently and non‐invasively obtained. Here, we test the potential of wild flowers as a novel source of arthropod eDNA. We performed eDNA metabarcoding of flowers from several different plant species using two sets of generic primers, targeting the mitochondrial genes 16S rRNA and COI. Our results show that terrestrial arthropod species leave traces of DNA on the flowers that they interact with. We obtained eDNA from at least 135 arthropod species in 67 families and 14 orders, together representing diverse ecological groups including pollinators, parasitoids, gall inducers, predators, and phytophagous species. Arthropod communities clustered together according to plant species. Our data also indicate that this experiment was not exhaustive, and that an even higher arthropod richness could be obtained using this eDNA approach. Overall, our results demonstrate that it is possible to obtain information on diverse communities of insects and other terrestrial arthropods from eDNA metabarcoding of wild flowers. This novel source of eDNA represents a vast potential for addressing fundamental research questions in ecology, obtaining data on cryptic and unknown species of plant‐associated arthropods, as well as applied research on pest management or conservation of endangered species such as wild pollinators.
Tiusanen M., Huotari T., Hebert P. D., Andersson T., Asmus A., Bêty J., Davis E., Gale J., Hardwick B., Hik D., Körner C., Lanctot R. B., Loonen M. J. J. E., Partanen R., Reischke K., Saalfeld S. T., Senez-Gagnon F., Smith P. A:, Sulavik J., Syvanpera I., Urbanowicz C., Williams S., Woodard P., Zaika Y., Roslin T. (2019): Flower‐visitor communities of an arcto‐alpine plant – Global patterns in species richness, phylogenetic diversity and ecological functioning. Molecular Ecology 28: 318-335.
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Pollination is an ecosystem function of global importance. Yet, who visits the flower of specific plants, how the composition of these visitors varies in space and time and how such variation translates into pollination services are hard to establish. The use of DNA barcodes allows us to address ecological patterns involving thousands of taxa that are difficult to identify. To clarify the regional variation in the visitor community of a widespread flower resource, we compared the composition of the arthropod community visiting species in the genus Dryas (mountain avens, family Rosaceae), throughout Arctic and high-alpine areas. At each of 15 sites, we sampled Dryas visitors with 100 sticky flower mimics and identified specimens to Barcode Index Numbers (BINs) using a partial sequence of the mitochondrial COI gene. As a measure of ecosystem functioning, we quantified variation in the seed set of Dryas. To test for an association between phylogenetic and functional diversity, we characterized the structure of local visitor communities with both taxonomic and phylogenetic descriptors. In total, we detected 1,360 different BINs, dominated by Diptera and Hymenoptera. The richness of visitors at each site appeared to be driven by local temperature and precipitation. Phylogeographic structure seemed reflective of geological history and mirrored trans-Arctic patterns detected in plants. Seed set success varied widely among sites, with little variation attributable to pollinator species richness. This pattern suggests idiosyncratic associations, with function dominated by few and potentially different taxa at each site. Taken together, our findings illustrate the role of post-glacial history in the assembly of flower-visitor communities in the Arctic and offer insights for understanding how diversity translates into ecosystem functioning.
Brydegaard M., Jansson S., Malmqvist E., Mlacha Y. P., Gebru A., Okumu F., Killeen G. F., Kirkeby C. (2020): Lidar reveals activity anomaly of malaria vectors during pan-African eclipse. Science Advances 6: eaay5487.
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Yearly, a quarter billion people are infected and a half a million killed by the mosquito-borne disease malaria. Lack of real-time observational tools for continuously assessing the unperturbed mosquito flight activity in situ limits progress toward improved vector control. We deployed a high-resolution entomological lidar to monitor a half-kilometer static transect adjacent to a Tanzanian village. We evaluated one-third million insect observations during five nights, four days, and one annular solar eclipse. We demonstrate in situ lidar classification of several insect families and their sexes based on their modulation signatures. We were able to compare the fine-scale spatiotemporal activity patterns of malaria vectors during ordinary days and an eclipse to disentangle phototactic activity patterns from the circadian mechanism. We observed an increased insect activity during the eclipse attributable to mosquitoes. These unprecedented findings demonstrate how lidar-based monitoring of distinct mosquito activities could advance our understanding of vector ecology.
Kudoh A., Minamoto T., Yamamoto S. (2020): Detection of herbivory: eDNA detection from feeding marks on leaves. Environmental DNA 2: 627-634.
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Many techniques have been developed to investigate the interactions between plants and herbivorous insects in natural environments and are generally used to determine either (a) which plant species are eaten by a specific herbivorous insect or (b) which herbivorous insect species are herbivores of a specific plant. The former problem is usually addressed by the direct observation of feeding and microscopic observation of gut contents and excrements, as well as the application of DNA‐barcoding techniques. However, the latter problem has typically been addressed using time‐consuming methods, such as direct observation and rearing. Therefore, more efficient techniques are needed for identifying and quantifying the interactions of plants with herbivorous insects. The present study demonstrates that the environmental DNA (eDNA) of herbivorous insects can be recovered from leaves with external foliage feeding marks. Mitochondrial DNA fragments of herbivorous insects were detected from insect‐exposed leaves using primer sets that amplified the DNA of target species. The amplification rate of the herbivorous insect DNA was positively associated with the rim length of feeding marks, which suggests that most of the insect DNA came from the feeding marks. Additionally, we showed that this method has the potential to detect eDNA from field‐collected leaves. This time‐efficient approach will contribute to the detection of plant–insect herbivore interactions.
Nagarajan R. P., Goodbla A., Graves E., Baerwald M., Holyoak M., Schreier A. (2020): Non-invasive genetic monitoring for the threatened valley elderberry longhorn beetle. Plos One 15: e0227333.
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The valley elderberry longhorn beetle (VELB), Desmocerus californicus dimorphus (Coleoptera: Cerambycidae), is a federally threatened subspecies endemic to the Central Valley of California. The VELB range partially overlaps with that of its morphologically similar sister taxon, the California elderberry longhorn beetle (CELB), Desmocerus californicus californicus (Coleoptera: Cerambycidae). Current surveying methods are limited to visual identification of larval exit holes in the VELB/CELB host plant, elderberry (Sambucus spp.), into which larvae bore and excavate feeding galleries. Unbiased genetic approaches could provide a much-needed complementary approach that has more precision than relying on visual inspection of exit holes. In this study we developed a DNA sequencing-based method for indirect detection of VELB/CELB from frass (insect fecal matter), which can be easily and non-invasively collected from exit holes. Frass samples were collected from 37 locations and the 12S and 16S mitochondrial genes were partially sequenced using nested PCR amplification. Three frass-derived sequences showed 100% sequence identity to VELB/CELB barcode references from museum specimens sequenced for this study. Database queries of frass-derived sequences also revealed high similarity to common occupants of old VELB feeding galleries, including earwigs, flies, and other beetles. Overall, this non-invasive approach is a first step towards a genetic assay that could augment existing VELB monitoring and accurately discriminate between VELB, CELB, and other insects. Furthermore, a phylogenetic analysis of 12S and 16S data from museum specimens revealed evidence for the existence of a previously unrecognized, genetically distinct CELB subpopulation in southern California.
Valentin R. E., Fonseca D. M., Gable S., Kyle K. E., Hamilton G. C., Nielsen A. L., Lockwood J. L. (2020): Moving eDNA surveys onto land: Strategies for active eDNA aggregation to detect invasive forest insects. Molecular Ecology Resources 20: 746–755.
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The use of environmental DNA (eDNA) surveys to monitor terrestrial species has been relatively limited, with successful implementations still confined to sampling DNA from natural or artificial water bodies and soil. Sampling water for eDNA depends on proximity to or availability of water, whereas eDNA from soil is limited in its spatial scale due to the large quantities necessary for processing and difficulty in doing so. These challenges limit the widespread use of eDNA in several systems, such as surveying forests for invasive insects. We developed two new eDNA aggregation approaches that overcome the challenges of above‐ground terrestrial sampling and eliminate the dependency on creating or utilizing pre‐existing water bodies to conduct eDNA sampling. The first, “spray aggregation,” uses spray action to remove eDNA from surface substrates and was developed for shrubs and other understorey vegetation, while the second, “tree rolling,” uses physical transfer via a roller to remove eDNA from the surface of tree trunks and large branches. We tested these approaches by surveying for spotted lanternfly, Lycorma delicatula, a recent invasive pest of northeastern USA that is considered a significant ecological and economic threat to forests and agriculture. We found that our terrestrial eDNA surveys matched visual surveys, but also detected L. delicatula presence ahead of visual surveys, indicating increased sensitivity of terrestrial eDNA surveys over currently used methodology. The terrestrial eDNA approaches we describe can be adapted for use in surveying a variety of forest insects and represent a novel strategy for surveying terrestrial biodiversity.
Allen M. C., Nielsen A. L., Peterson D. L., Lockwood J. L. (2021): Terrestrial eDNA survey outperforms conventional approach for detecting an invasive pest insect within an agricultural ecosystem. Environmental DNA 3: 1102-1112.
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Recent methodological advances permit surveys for terrestrial insects from the direct collection of environmental DNA (eDNA) deposited on vegetation or other surfaces. However, in contrast to well-studied aquatic applications, little is known about how detection rates for such terrestrial eDNA-based surveys compare with conventional survey methods. Lycorma delicatula, the spotted lanternfly, is an emerging invasive insect in eastern North America, and a significant ecological and economic pest of forested and agricultural systems, especially grapes. During fall 2019, we conducted two rounds of paired eDNA and visual surveys for spotted lanternflies within 48 plots at 12 vineyards in New Jersey, USA. We compared detection probabilities within a multimethod occupancy modeling framework and used the results to extrapolate and inform survey design. The probability of detecting spotted lanternflies given presence in a plot was over two times higher for eDNA (84%) versus visual surveys (36%). In mid-September, lanternfly eDNA was detected at five plots in three vineyards, while visual surveys revealed only a single individual in one plot. In early October, after dispersal of lanternflies into vineyards, lanternfly eDNA was detected in 12 plots within six vineyards compared with visual detections in six plots in two vineyards. Extrapolations based on detection and local-scale occupancy rates indicate that only five and 12 plots would have been needed to positively detect lanternfly presence with 95% confidence using eDNA in contrast to 14 and 29 plots with visual surveys alone, respective to survey rounds. Log-linear models revealed that visual counts of lanternflies were positively related to eDNA concentrations (R2 = 71%). We provide some of the first quantitative evidence to support the enhanced sensitivity of terrestrial eDNA approaches compared with conventional methods. Such methods can augment efforts to combat invasive species through improved ability to delimit invasion fronts, identify satellite populations, and confirm local eradications.
Camila L., Tony D., Alice V., Pauline J., Pierre J. R. (2021): A novel trap design for non-lethal monitoring of dung beetles using eDNA metabarcoding. Journal of Insect Conservation 25:629–642.
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The current biodiversity crisis calls for rapid and wide-ranging surveys to assess living organisms. However, some taxa are more elusive than others, making monitoring challenging. This is the case for soil invertebrates, but new molecular technologies such as eDNA metabarcoding could help to alleviate this problem. In this study, we evaluated the feasibility of using an eDNA approach to survey dung beetles, adapting existing monitoring methods for surveying dung fauna to enable eDNA collection in a non-destructive way. The main design idea is to capture species secretions and excretions from a serum-soaked nonwoven compress in a baited non-destructive trap. While the attractiveness of the device to dung beetles and the sampling protocol would benefit from further development, eDNA allowed the identification of more than 68% of trapped species and an identification of relative abundance match rate of 79%. The results of the study demonstrate the effectiveness of eDNA-based detection tools for the monitoring of dung beetles compared to standard surveying and identification techniques. Moreover, the adapted collecting device developed for the study could be used for similar surveys of other terrestrial invertebrates or even re-adapted. Ultimately, we hope this study encourages more non-invasive studies of insects by enabling others to utilize these emerging, non-destructive molecular techniques and therefore foster wide insect monitorings and conservation programs.
Droissart V., Azandi L., Onguene E. R., Savignac M., Smith T. B., Deblauwe V. (2021): PICT: A low‐cost, modular, open‐source camera trap system to study plant–insect interactions. Methods in Ecology and Evolution 12: 1389-1396.
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Commercial camera traps (CTs) commonly used in wildlife studies have several technical limitations that restrict their scope of application. They are not easily customizable, unit prices sharply increase with image quality and importantly, they are not designed to record the activity of ectotherms such as insects. Those developed for the study of plant–insect interactions are yet to be widely adopted as they rely on expensive and heavy equipment. We developed PICT (plant–insect interactions camera trap), an inexpensive (<100 USD) do-it-yourself CT system based on a Raspberry Pi Zero computer designed to continuously film animal activity. The system is particularly well suited for the study of pollination, insect behaviour and predator–prey interactions. The focus distance can be manually adjusted to under 5 cm. In low light conditions, a near-infrared light automatically illuminates the subject. Frame rate, resolution and video compression levels can be set by the user. The system can be remotely controlled using either a smartphone, tablet or laptop via the onboard Wi-Fi. PICT can record up to 72-hr day and night videos at >720p resolution with a 110-Wh power bank (30,000 mAh). Its ultra-portable (<1 kg) waterproof design and modular architecture is practical in diverse field settings. We provide an illustrated technical guide detailing the steps involved in building and operating a PICT and for video post-processing. We successfully field-tested PICT in a Central African rainforest in two contrasting research settings: an insect pollinator survey in the canopy of the African ebony Diospyros crassiflora and the observation of rare pollination events of an epiphytic orchid Cyrtorchis letouzeyi. PICT overcomes many of the limitations commonly associated with CT systems designed to monitor ectotherms. Increased portability and image quality at lower costs allow for large-scale deployment and the acquisition of novel insights into the reproductive biology of plants and their interactions with difficult to observe animals.
Kirkeby C., Rydhmer K., Cook S. M., Strand A., Torrance M. T., Swain J. L., Prangsma J., Johnen A., Jensen M., Brydegaard M., Græsbøll K. (2021): Advances in automatic identification of flying insects using optical sensors and machine learning. Scientific Reports 11: 1555.
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Worldwide, farmers use insecticides to prevent crop damage caused by insect pests, while they also rely on insect pollinators to enhance crop yield and other insect as natural enemies of pests. In order to target pesticides to pests only, farmers must know exactly where and when pests and beneficial insects are present in the field. A promising solution to this problem could be optical sensors combined with machine learning. We obtained around 10,000 records of flying insects found in oilseed rape (Brassica napus) crops, using an optical remote sensor and evaluated three different classification methods for the obtained signals, reaching over 80% accuracy. We demonstrate that it is possible to classify insects in flight, making it possible to optimize the application of insecticides in space and time. This will enable a technological leap in precision agriculture, where focus on prudent and environmentally-sensitive use of pesticides is a top priority.
Pumkaeo P., Takahashi J., Iwahashi H. (2021): Detection and monitoring of insect traces in bioaerosols. PeerJ 9: e10862.
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Studies on bioaerosols have primarily focused on their chemical and biological compositions and their impact on public health and the ecosystem. However, most bioaerosol studies have only focused on viruses, bacteria, fungi, and pollen. To assess the diversity and composition of airborne insect material in particulate matter (PM) for the first time, we attempted to detect DNA traces of insect origin in dust samples collected over a two-year period. These samples were systematically collected at one-month intervals and categorized into two groups, PM2.5 and PM10, based on the aerodynamic diameter of the aerosol particles. Cytochrome-c oxidase I (COI) was the barcoding region used to identify the origins of the extracted DNA. The airborne insect community in these samples was analyzed using the Illumina MiSeq platform. The most abundant insect sequences belonged to the order Hemiptera (true bugs), whereas order Diptera were also detected in both PM2.5 and PM10 samples. Additionally, we inferred the presence of particulates of insect origin, such as brochosomes and integument particles, using scanning electron microscopy (SEM). This provided additional confirmation of the molecular results. In this study, we demonstrated the benefits of detection and monitoring of insect information in bioaerosols for understanding the source and composition. Our results suggest that the PM2.5 and PM10 groups are rich in insect diversity. Lastly, the development of databases can improve the identification accuracy of the analytical results.
Sigsgaard E. E., Olsen K., Hansen M. D., Hansen O. L. P., Høye T. T., Svenning J. C., Thomsen P. F. (2021): Environmental DNA metabarcoding of cow dung reveals taxonomic and functional diversity of invertebrate assemblages. Molecular Ecology 30: 3374-3389.
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Insects and other terrestrial invertebrates are declining in species richness and abundance. This includes the invertebrates associated with herbivore dung, which have been negatively affected by grazing abandonment and the progressive loss of large herbivores since the Late Pleistocene. Importantly, traditional monitoring of these invertebrates is time-consuming and requires considerable taxonomic expertise, which is becoming increasingly scarce. In this study, we investigated the potential of environmental DNA (eDNA) metabarcoding of cow dung samples for biomonitoring of dung-associated invertebrates. From eight cowpats we recovered eDNA from 12 orders, 29 families, and at least 54 species of invertebrates (mostly insects), representing several functional groups. Furthermore, species compositions differed between the three sampled habitats of dry grassland, meadow, and forest. These differences were in accordance with the species’ ecology; for instance, several species known to be associated with humid conditions or lower temperatures were found only in the forest habitat. We discuss potential caveats of the method, as well as directions for future study and perspectives for implementation in research and monitoring.
Sinka M. E., Zilli D., Li Y., Kiskin I., Msaky D., Kihonda J., Mkandawile G., Kaindoa E., Killeen G., Zanga J., Metelo E., Kirkham D., Mansiangi P., Herreros-Moya E., Rafique W., Chareonviriyaphap T., Tisgratog R., Wang L., Chan H., Gutteridge B., Portwood H., Roberts S., Willis K. J. (2021): HumBug–an acoustic mosquito monitoring tool for use on budget smartphones. Methods in Ecology and Evolution 12: 1848-1859.
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Mosquito surveys are time-consuming, expensive and can provide a biased spatial sample of occurrence data—the data often representing the location of the surveys, not the occurrence of the mosquitoes. We present the HumBug project, an acoustic system that can turn any Android smartphone into a mosquito sensor. Our sensor has the potential to significantly increase the quantity of mosquito occurrence data as well as access locations that are more difficult to survey by traditional means. We describe our database of wild-captured mosquito fight tone audio data and outline our mosquito detection algorithms that these data train. We also present our MozzWear App, designed to work on budget smartphones, which, together with our HumBug Net (an adapted traditional bednet), facilitates data collection and allows the user to record and directly upload mosquito flight tones from any dwelling with a bednet in the field. Our HumBug system has the potential to vastly increase our understanding of the distribution of mosquito species in space and time and greatly improve surveys needed to assess the success or failure of ongoing vector control measures. At a time when the WHO reports a plateauing in the decade-long decline in malaria mortality rates, this new technological solution for surveying mosquito vectors will provide a timely new resource.
van Gent-Pelzer M., Cornelissen B. (2021): Detection of small hive beetle: frass as a source of DNA. Journal of Apicultural Research 60: 683-685.
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Current diagnostic techniques for the detection of the small hive beetle (SHB), Aethina tumida are limitedly available and not cost effective. More sensitive pragmatic methods are preferred for early detection. To improve diagnostics, we focused on sampling techniques for SHB frass, as an indicator for SHB presence in a honey bee colony. In this study, we successfully tested a novel approach of employing swab sample collection of frass for real-time PCR detection of SHB.
Campbell C. D., Gleeson D. M., Furlan E. M., Muirhead K. A., Caron V. (2022): Detection of a cryptic terrestrial insect using novel eDNA collection techniques. Environmental DNA 4: 820-829.
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Sensitive detection techniques are key to inform management practices for invasive and pest species by identifying the pest species’ distribution or identifying the presence of biological control agents that may negate the need for chemical control. Environmental DNA (eDNA) can be detected to infer the presence of rare, cryptic, and invasive species. This study provides a method that expands the application of eDNA techniques within terrestrial systems. Our study is focused on a cryptic biological control agent of the pointed snail Cochlicella acuta, the fly parasitoid Sarcophaga villeneuveana, which remains within the snail shell for most of its lifecycle, making it difficult to detect. Three sites were sampled using traditional surveys and three types of eDNA sampling 1/ crushing live snails, 2/ water washing live snails, old snail shells or vegetation, and 3/ vacuuming live snails or vegetation. An assay was developed to amplify a 129 bp fragment of S. villeneuveana, with positive detections recorded in crushed snail samples, water washed live snail, water washed vegetation, and laboratory vacuumed vegetation. Presence of the endoparasitoid was validated by traditional survey techniques conducted at the same time, indicating that all techniques tested are comparable for detection of the parasitoid. The new technique which only requires a small vegetation sample from the field post-harvest and a portable vacuum has great potential to be applied to other insects and environments, particularly cryptic species, pests, and biological control agents in crops.
Kirtane, A., Dietschler, N. J., Bittner, T. D., Lefebvre, M. B., Celis, S., O’Connor K., Havill N., Whitmore M. C. (2022): Sensitive environmental DNA (eDNA) methods to detect hemlock woolly adelgid and its biological control predators Leucotaraxis silver flies and a Laricobius beetle. Environmental DNA 4: 1136-1149.
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Environmental DNA (eDNA) analysis can be a powerful tool for the early detection of invasive organisms. However, research on terrestrial eDNA detection from foliage surfaces has been limited. In this study, we developed methods to capture and detect eDNA using qPCR from an invasive forest pest, hemlock woolly adelgid (Adelges tsugae), and three of its biological control predators Leucotaraxis piniperda, Leucotaraxis argenticollis, and Laricobius nigrinus. We designed four highly efficient qPCR assays with a low limit of detection (1–10 copies/reaction). The assay targeting A. tsugae was species-specific. The assays targeting Le. piniperda, and Le. argenticollis were biotype-specific in addition to being species-specific demonstrating applications of eDNA analysis beyond species-level detection. The La. nigrinus assay also detected DNA from closely related and hybridizing Laricobius rubidus. The eDNA methods were evaluated against traditional detection methods. We collected foliage samples from three strata (bottom, middle, and top) of eastern hemlock trees to detect the presence of A. tsugae. The detection of the biological control predators was evaluated using western hemlock foliage samples collected from the predators’ native range in western Washington. The eDNA methods had significantly higher positive detection rates (2.8–4.5 times) than conventional methods of all target species. The strata of sampling were not significant in determining the presence of A. tsugae infestation. The eDNA concentration positively correlated with the observed density for all species. This study demonstrates the efficacy of eDNA analysis as a more sensitive tool for early detection of A. tsugae and to track the establishment of its biological control predators.
Krehenwinkel H., Weber S., Broekmann R., Melcher A., Hans J., Wolf R., Hochkirch A., Kennedy S. R., Koschorreck J., Künzel S., Müller C. (2022): Environmental DNA from archived leaves reveals widespread temporal turnover and biotic homogenization in forest arthropod communities. eLife 11: e78521.
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A major limitation of current reports on insect declines is the lack of standardized, long-term, and taxonomically broad time series. Here, we demonstrate the utility of environmental DNA from archived leaf material to characterize plant-associated arthropod communities. We base our work on several multi-decadal leaf time series from tree canopies in four land use types, which were sampled as part of a long-term environmental monitoring program across Germany. Using these highly standardized and well-preserved samples, we analyze temporal changes in communities of several thousand arthropod species belonging to 23 orders using metabarcoding and quantitative PCR. Our data do not support widespread declines of α-diversity or genetic variation within sites. Instead, we find a gradual community turnover, which results in temporal and spatial biotic homogenization, across all land use types and all arthropod orders. Our results suggest that insect decline is more complex than mere α-diversity loss, but can be driven by β-diversity decay across space and time.
Krehenwinkel H., Weber S., Künzel S., Kennedy S. R. (2022): The bug in a teacup – monitoring arthropod–plant associations with environmental DNA from dried plant material. Biology Letters 18: 20220091.
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Environmental DNA analysis (eDNA) has revolutionized the field of biomonitoring in the past years. Various sources have been shown to contain eDNA of diverse organisms, for example, water, soil, gut content and plant surfaces. Here we show that dried plant material is a highly promising source for arthropod community eDNA. We designed a metabarcoding assay to enrich diverse arthropod communities while preventing amplification of plant DNA. Using this assay, we analysed various commercially produced teas and herbs. These samples recovered ecologically and taxonomically diverse arthropod communities, a total of over a thousand species in more than 20 orders, many of them specific to their host plant and its geographical origin. Atypically for eDNA, arthropod DNA in dried plants shows very high temporal stability, opening up plant archives as a source for historical arthropod eDNA. Considering these results, dried plant material appears excellently suited as a novel tool to monitor arthropods and arthropod–plant interactions, detect agricultural pests and identify the geographical origin of imported plant material. The simplicity of our approach and the ability to detect highly diverse arthropod communities from all over the world in tea bags also highlights its utility for outreach purposes and to raise awareness about biodiversity.
Oettel J., Braun M., Hoch G., Connell J., Gschwantner T., Lapin K., Schöttl S., Windisch-Ettenauer K., Essl F., Gossner M. M. (2022): Rapid assessment of feeding traces enables detection of drivers of saproxylic insects across spatial scales. Ecological Indicators 145: 109742.
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Knowledge of habitat requirements of saproxylic insects and their response to habitat changes is critical for assessing the ecological impacts of forest management. Several studies have demonstrated a positive relationship of tree-species richness, deadwood volume, or structural diversity with saproxylic species diversity, while the relationship with the abundance of potential pest species have often been negative. A better understanding of which factors drive saproxylic insects’ occurrence is therefore essential for deriving urgently needed thresholds for key habitat conditions. We tested a rapid assessment method applicable at large scale based on recorded feeding galleries and boreholes assessed during the Austrian National Forest Inventory to investigate the drivers and habitat thresholds of different saproxylic insect families; i.e. Buprestidae, Cerambycidae, Curculionidae, Siricidae, at multiple spatial scales; i.e. at the object, forest stand and landscape level. We modelled the relative abundance of all insects and these families considering nineteen explanatory variables using ordinal logistic regression models. Key habitat characteristics were identified using recursive partitioning. Our results revealed complex interactions among influencing factors at different spatial scales. We showed that deadwood volume was of surprisingly little importance. Instead, individual tree characteristics were of major importance, demonstrating the value of resource quality and variability. The abundance of all saproxylic insect families increased with advancing decomposition, on trees taller than 18 m, and above a living stand volume of 41 m3ha−1. Aiming to guide forest management, not only forest type-specific, but tree species-specific deadwood management is needed, taking into account site-specific conditions, including temperature and precipitation. For assessing temporal trends in insect colonization and habitat dynamics as well as the effects of forest management, we propose a continuous monitoring of insect traces, including living but weakened trees. This will allow for further thresholds that are urgently needed for maintaining biological diversity in forest ecosystems in the face of climate change.
Roger F., Ghanavi H. R., Danielsson N., Wahlberg N., Löndahl J., Pettersson L. B., Andersson G. K., Boke Olén N., Clough Y. (2022): Airborne environmental DNA metabarcoding for the monitoring of terrestrial insects – A proof of concept from the field. Environmental DNA 4: 790-807.
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Biodiversity is in decline due to human-induced pressures on ecosystems around the world. To be able to counteract this alarming trend, it is paramount to closely monitor biodiversity at global scales. Because this is practically impossible with traditional methods, the last decade has seen a strong push for new solutions. In aquatic ecosystems, the monitoring of species from environmental DNA (eDNA) has emerged as one of the most powerful tools at our disposal, but in terrestrial ecosystems, the power of eDNA for monitoring has so far been hampered by the local scale of the samples. In this study, we report the successful detection of insects from airborne eDNA from samples taken in the field. We compare our results to two traditional insect monitoring methods (1) light traps for moth monitoring and (2) transect walks for the monitoring of butterflies and wild bees. Airborne eDNA metabarcoding revealed DNA from six classes of arthropods, and twelve order of insects—including representatives from the four largest orders: Diptera (flies), Lepidoptera (butterflies and moths), Coleoptera (beetles), and Hymenoptera (bees, wasps, and ants). We did not detect all species observed using traditional methods and suggest further directions for the development of airborne eDNA metabarcoding. We also recovered DNA from nine species of vertebrates, including frogs, birds, and mammals as well as from 12 other phyla. Airborne eDNA has the potential to become a powerful tool for terrestrial biodiversity monitoring, with many impactful applications including the monitoring of pests, invasive, or endangered species and disease vectors.
Wang J., Bu Y. (2022): Internet of Things‐based smart insect monitoring system using a deep neural network. IET Networks 11: 245-256.
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According to recent studies, insects make up the vast majority of all animal species on the planet, and their numbers are rapidly dwindling. This occurrence has been recorded by a variety of insect taxa and geographic locales, but there is insufficient data to identify its true extent. Most monitoring methods are ineffective and time-consuming, making insect population analysis difficult. Computer vision and deep learning advancements, on the other hand, may soon give new answers to this global challenge. Using cameras and other sensors, entomologists can gather data continuously and non-invasively at any time of day or season. To capture specimens’ physical appearance, automated imaging can be utilised in the laboratory. This data can be used to build a deep learning model to predict the quantity, biomass, and variety of insects. Additionally, deep learning models can measure the variability of phenotypic features, behaviour, and interactions. We may thank current advances in deep learning and computer vision for the immediate demand for more cost-effective insect and invertebrate monitoring systems. Insect monitoring using sensors is demonstrated. Here, we show how deep-learning algorithms might extract ecological information from massive data sets, and we explore the challenges ahead. There are four areas that will help us achieve our goal: There is a need for verification of taxonomic identification using pictures; sufficient training data; public reference databases; and ways for integrating molecular and deep learning technology.
Gamonal Gomez N., Sørensen D. H., Chua P. Y. S., Sigsgaard L. (2023): Assessing flower‐visiting arthropod diversity in apple orchards through metabarcoding of environmental DNA from flowers and visual census. Environmental DNA 5: 117-131.
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Arthropods are essential in maintaining healthy and productive agricultural ecosystems. Agricultural crops such as apples are typically pollinated by domesticated honey bees, but wild bees and other arthropod flower visitors also contribute to pollination. Flower visitors can also be natural enemies of crop-pests or herbivores. Biodiversity is under pressure and knowledge of wildflower visitors is an important tool in designing orchards that can support high functional biodiversity. In our study, we assessed the diversity of arthropod flower visitors in four Danish apple orchards using both molecular and nonmolecular techniques to study arthropod communities in agricultural ecosystems. Arthropod DNA collected from apple flowers was analyzed using a DNA metabarcoding approach using the mitochondrial COI marker, while arthropod pollinators were recorded through visual assessment surveys. These complementary techniques resulted in a total of 19 arthropod taxa detected. Nonbee arthropods constituted a large proportion of arthropods detected by both methods (84%, 16 taxa). Metabarcoding detected 12 taxa and had 83% species resolution. Visual census recovered flower visiting groups to the order level (Coleoptera, Diptera, Hymenoptera and Lepidoptera) but not species level and also provided relative abundance data, which is not possible with molecular methods. We demonstrated that by utilizing both molecular and nonmolecular techniques to assess arthropod communities, we are able to obtain a broader overview of the arthropod fauna present. The methodology used and the outcome of this study can be used to inform and tailor suitable arthropod-pest management practices in orchards to increase crop yield and maintain healthy agricultural systems.
Harper L. R., Niemiller M. L., Benito J. B., Paddock L. E., Knittle E., Molano‐Flores B., Davis M. A. (2023): BeeDNA: Microfluidic environmental DNA metabarcoding as a tool for connecting plant and pollinator communities. Environmental DNA 5: 191-211.
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Pollinators are declining globally, and this loss can reduce plant reproduction, erode critical ecosystem services and resilience, and drive economic losses. Monitoring pollinator biodiversity trends is essential for adaptive conservation and management, but conventional surveys are often costly, time-consuming, and requires considerable taxonomic expertise. Environmental DNA (eDNA) metabarcoding surveys are booming due to their rapidity, nondestructiveness, and cost efficiency. Microfluidic technology allows multiple primer sets from different markers to be used in eDNA metabarcoding for more comprehensive inventories, minimizing associated primer bias. We evaluated microfluidic eDNA metabarcoding for pollinator community monitoring in both controlled greenhouse and natural field settings. Using a variety of sampling, preservation, and extraction methods, we assessed pollinator communities with a number of markers using microfluidic metabarcoding. In greenhouse experiments, microfluidic eDNA metabarcoding detected the target bumblebee in two of four focal flower species as well as greenhouse insects in all focal flower species. In the field, numerous common regional arthropods, including some directly observed, were detected. Pollinator detection was maximized using whole flower heads preserved in ATL buffer and extracted with a modified Qiagen® DNeasy protocol for amplification with COI primers. eDNA surveillance could enhance pollinator assessment by detecting protected and endangered species and being more applicable to remote, inaccessible locations, whilst reducing survey time, effort, and expense. Microfluidic eDNA metabarcoding requires optimization to address remaining efficacy concerns, but this approach shows potential in revealing complex networks underpinning critical ecosystem functions and services, enabling more accurate assessments of ecosystem resilience.
Hutchins P. R., Giersch J. J., Sepulveda A. J., Muhlfeld C. C. (2023): Validation of a species-specific probe-based qPCR assay for the threatened meltwater stonefly, Lednia tumana, in environmental samples. Conservation Genetics Resources 15: 105-107.
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A probe-based quantitative real-time PCR assay was developed to detect meltwater stonefly (Lednia tumana) environmental (e)DNA in water samples. The limits of detection and quantification, respectively, were 12.1 and 58.4 gene copies for calibration standards and these values were similarly low in a relevant environmental sample matrix (8.6 and 174.2, respectively). The assay’s utility was demonstrated in situ on water samples with concomitant manual invertebrate surveys from a wide range of alpine streams across L. tumana’s native range.
Johnson M. D., Katz A. D., Davis M. A., Tetzlaff S., Edlund D., Tomczyk S., Molano-Flores B., Wilder T., Sperry J. H. (2023): Environmental DNA metabarcoding from flowers reveals arthropod pollinators, plant pests, parasites, and potential predator–prey interactions while revealing more arthropod diversity than camera traps. Environmental DNA 5: 551-569.
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Arthropods can strongly impact ecosystems through pollination, herbivory, predation, and parasitism. As such, characterizing arthropod biodiversity is vital to understanding ecosystem health, functions, and services. Emerging environmental DNA (eDNA) methods targeting trace arthropod eDNA left behind on flowers have the potential to track arthropod biodiversity and interactions. The goal of this study was to determine the extent to which eDNA metabarcoding can identify plant-arthropod and arthropod-arthropod interactions and assess eDNA metabarcoding compared to conventional sampling. We deployed camera traps to document arthropod activity on specific flowers, sampled eDNA from those same flowers, then performed a metabarcoding analysis that targets a partial fragment of the cytochrome c oxidase subunit I gene (COI) to determine all arthropod eDNA present. We found that our eDNA metabarcoding analysis detected small arthropod pollinators, plant pests, and parasites, and shed light on potential predator–prey interactions while detecting 55 species compared to just 21 species from conventional camera trapping. The camera trapping survey, however, detected larger, more conspicuous nectarivores more successfully. We also explored the ecology of residual arthropod eDNA, finding that rainfall had a significant negative effect on the ability to detect residual arthropod eDNA. Preliminary evidence also indicates flower species may impact the amount of arthropod eDNA that can be detected. We found that eDNA metabarcoding can provide clues to potential predator–prey interactions on flowers and highlights the potential insights that can be gained from future eDNA metabarcoding studies. We show that eDNA metabarcoding is a valuable tool for not only detecting pollinator communities but for revealing potential interactions among plants, pollinators, pests, parasites, and predators. Future research should focus on how to improve the detection of large pollinators/nectivores and studying the ecology of residual arthropod eDNA to further explore this method’s utility.
Macher T. H., Schütz R., Hörren T., Beermann A. J., Leese F. (2023): It’s raining species: Rainwash eDNA metabarcoding as a minimally invasive method to assess tree canopy invertebrate diversity. Environmental DNA 5: 3-11.
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Forest canopies are highly diverse ecosystems, but despite several decades of intense research, there remain substantial gaps in our knowledge of their biodiversity and ecological interactions. One fundamental challenge in canopy research is the limited accessibility of the ecosystem. Consequently, previous studies have relied on the application of either highly invasive methods such as chemical knockdown, or on time-consuming and expensive setups such as canopy walkways or cranes. Therefore, time- and cost-efficient, ideally minimally invasive yet comprehensive applications are required to help close this knowledge gap. High-throughput metabarcoding of environmental DNA (eDNA) collected from water, soil, or air provides a minimally invasive method for biodiversity assessment, yet its potential for canopy biodiversity monitoring has not been explored. Herein, we conducted metabarcoding of eDNA washed off the canopy via rainwater to explore its potential for biodiversity monitoring and ecological research. We placed four 1 m2 rain samplers beneath the canopies of four different trees (beech, oak, larch, and pine) prior to a major rain event, filtered eDNA from the collected rainwater, and performed cytochrome c oxidase subunit I (COI) gene metabarcoding to profile the invertebrate community. Additionally, we collected and identified all specimens present in the rainwater to assess if eDNA only came from specimens physically present in the rainwater. We detected 50 invertebrate species by eDNA metabarcoding, of which 43 were not physically present in the water sample, thus likely representing true canopy biodiversity signals. Furthermore, we observed distinct species occurrence patterns corresponding to the four trees, suggesting that ecological patterns such as host specificity can potentially be assessed using the method. In conclusion, our study provides a proof of principle that rainwash eDNA metabarcoding offers a minimally invasive and comprehensive method for biodiversity monitoring in tree canopies.
MYRIAPODS
Seeber J., Rief A., Seeber G. U., Meyer E., Traugott M. (2010): Molecular identification of detritivorous soil invertebrates from their faecal pellets. Soil Biology and Biochemistry 42: 1263-1267.
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Faecal pellets of macro-decomposers form an important component of upper soil layers. It is, however, not yet specifically known which species’ faecal pellets are important in the process of humus and soil formation. Here we present a novel approach, based on the detection of short DNA fragments, to species-specifically assign faecal pellets to their detritivorous producers. Using diagnostic polymerase chain reaction (PCR), DNA of the two millipede species Cylindroiulus fulviceps and C. meinerti, abundant macro-decomposers on European alpine pastureland, could be detected in their faecal pellets. Although in both species detection was possible up to 5 days post-defecation (no longer intervals were tested), detection rates were significantly higher in C. fulviceps (81.3%) than in Cylindroiulus meinerti (60.9%) in repetitive PCRs. Employing a binomial mixture model, the probability that the producer’s DNA was present in a faecal sample which tested negative in five subsequent PCRs was estimated as 22.0% for C. fulviceps and 41.9% for C. meinerti, suggesting to adopt a flexible stopping rule for assaying this type of samples. This proof-of-concept study demonstrates that the molecular assignment of faecal pellets to their macro-invertebrate producers is possible, offering a new approach to examine the role of decomposer faecal pellets in soil formation and functioning as well as to complement molecular profiling of faunal communities.