Judge a caterpillar by what they eat, not where they’re found

Judge a caterpillar by what they eat, not where they’re found

Judge a caterpillar by what they eat, not where they’re found

Gut content analysis of Peruvian caterpillars reveals new insights into host-plant relationships and the methods used to examine species interactions key to BIOSCAN

The original primary rainforest surrounding Panguana station, Peru, Dept Huánuco, western Amazonia.

PHOTO CREDIT: K. Wothe

Understanding species and their associations with each other and with the environment – a key aspect of synecological research – is of great importance. For example, data on insect-host plant relationships can aid investigations into food webs and extrapolations of global species numbers as well as inform forestry, agriculture, and conservation practices.

Supported by the Bavarian Ministry of Science (‘SNSB-Innovativ’), our recent pilot study examined the gut contents of Peruvian caterpillars demonstrating the potential for gathering large-scale data on species interactions when applying DNA barcoding and high-throughput sequencing technologies. We obtained 130 caterpillars (moth larvae) by canopy fogging at the Panguana research station, an area of tropical primary forest in western Amazonia. DNA barcode analysis resulted in 119 successfully sequenced larvae, more than half of which matched with moth reference sequences on BOLD. Surprisingly high biodiversity was uncovered from our modest sample – 92 BINs or species proxies. The trees from which caterpillars were collected were also identified, both by morphology and DNA barcoding.

Panguana research station, Peru, Dept Huánuco, western Amazonia, with the characteristic Lupuna tree (Ceiba pentandra, Malvaceae) in the background.

PHOTO CREDIT: J. Diller

Knowing the tree and larva identity is not enough to conclude a host-plant relationship, particularly in a dense tropical rainforest. Caterpillars may in fact be feeding on the epiphytes, lianas, lichens, algae, fungi, or mosses associated with trees (i.e., alternative feeding), and sometimes larvae may have been fogged down from neighbouring trees. To confirm a direct insect-host plant relationship, we partnered with the company Advanced Identification Methods (AIM) to design a high-throughput sequencing (HTS) protocol with plant markers (rbcL, psbA) that would enable the identification of plant matter from the gut contents of ten larvae. Results revealed only two matches between the fogged tree and larval gut content which suggests a rather high percentage of alternative feeding. In three cases, the gut content clearly indicated feeding on lianas and neighbouring trees. Interestingly, the analysis of four larvae resulted in the putative presence of Bryophyta, suggesting moss-feeding in Lepidoptera, a phenomenon rarely observed. Potential contamination (for example, through the diffusion of plant DNA into the alcohol of the bulk sample) has yet to be ruled out, work which is currently being validated in a subsequent project investigating the gut contents of an additional 190 larvae.

Automeris denticulata (Conte, 1906) (Saturniidae): Larva (left), selected from canopy fogging bulk samples of a Poulsenia (Moraceae) tree at the Panguana station, identified by its COI barcode; Adult (right), collected at the Panguana station.

PHOTO CREDIT: Mei-Yu Chen & Dr. R. Mörtter

Our approach of combined canopy fogging, DNA-based identification, and gut content analysis resulted in two key findings. First, a significant portion of both insect and plant taxa can be identified even in highly diverse, tropical regions – more than 97% to a family level and about 80% to a species or genus level. Secondly, we can successfully confirm or reject the hypothesis that caterpillars feed on the trees where they are collected by identifying their diets through an HTS protocol on gut contents. Importantly, the taxonomic resolution of animal and plant identifications will increase with further investments into DNA reference libraries. We recommend specimen de-contamination (e.g. by bleaching) and/or isolated storage of the target taxa rather than bulk storage to improve the reliability of gut content analysis.

Urania leilus (Linnaeus, 1758) (Uraniidae): Larva (left), selected from canopy fogging bulk samples of an Oxandra polyantha (Annonaceae) tree at the Panguana station, identified by its COI barcode; Adult (right).

PHOTO CREDIT: Mei-Yu Chen & Dr. J. Diller

The techniques employed in our pilot have immense potential for unveiling trophic interactions in tropical regions at a very large scale as they are fast and cost-effective. The latter is enabled, in part, by the availability of target specimens in the by-catch of other studies. For example, our efforts fogging 150 trees in a separate project assessing the biodiversity of ants have resulted in 1,200 lepidopteran larvae. Subsequent aspects of the workflow, from selecting the larvae from bulk samples, tissue sampling, photography, and databasing, required 10–20 minutes per larva and can be performed with relatively low expertise. The costs for subsequent lab work, i.e. identification of larvae and their gut contents, currently amount to 20–25 € per larva and these costs will soon drop considerably. In contrast, traditional approaches involving the searching and rearing of larvae, and the identification of hatched adults by experts is massively time and resource consuming.

Providing reliable data on trophic interactions is one of the major goals of the BIOSCAN program, one that will be a powerful tool for investigating food webs, for determining the amplitude of alternative or multiple feeding sources, and for the study of phagism (monophagy versus polyphagy), thus gaining data for extrapolations of global species numbers. These data will also be particularly important for pest management in forestry, and agriculture, and for conservation purposes.

Overcoming the current lack of knowledge is a major challenge, particularly in ecoregions with megadiverse faunas and floras. Yet, its success is imperative for humanity considering the unprecedented biodiversity losses we currently face. In this context, the recently launched BIOSCAN with its focus on revealing species interactions will embolden an important plan for the international research community to come together in understanding nature and conserving it for a sustainable future.

 

Read the complete manuscript in PLoS ONE.

Written by

Axel Hausmann

Axel Hausmann

Juliane Diller

Juliane Diller

Amelie Höcherl

Amelie Höcherl

SNSB – Staatliche Naturwissenschaftliche Sammlungen Bayerns - Zoologische Staatssammlung München, Munich, Germany

May 6, 2020
https://doi.org/10.21083/ibol.v10i1.6133 

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Discovering ten new species of Paramyia Williston (Diptera: Milichiidae) in North America using DNA barcoding

Discovering ten new species of Paramyia Williston (Diptera: Milichiidae) in North America using DNA barcoding

Discovering ten new species of Paramyia Williston (Diptera: Milichiidae) in North America using DNA barcoding

Making the process of species identification more efficient by focusing morphological efforts using DNA-based tools

Flowering plant visited by Paramyia sp.
PHOTO CREDIT: Steve A. Marshall

The process of discovering and describing new species – the job of taxonomists – is time-consuming. To tackle the challenge, one must become an expert on a specific group in order to notice those rarities among the masses. This is without counting the added challenges of cryptic groups where the external morphology is of limited help as individuals often appear to belong to the same species despite being genetically distinct. In many cases, the taxonomist has to dissect hundreds of specimens to detect slight differences in their genitalia which are usually unique for each species. DNA barcoding can greatly assist any taxonomist by speeding up this laborious identification process, particularly with small flies like Paramyia Williston (Diptera: Milichiidae).

Flowering plant visited by Paramyia sp.

PHOTO CREDIT: Steve A. Marshall

Paramyia, a kleptoparasitic genus well represented worldwide, provides a perfect example of joining DNA barcoding and traditional taxonomy. Paramyia is a small genus, with under 30 described species, of tiny blackish flies, usually under 2mm long, with very similar external morphology. In the Nearctic, it was only represented by one species, P. nitens Loew. With that knowledge in mind, no particular attention was given to specimens collected in that geographic range. That is until multiple Barcode Index Numbers (BIN) were found on the Barcode of Life Data System (BOLD) under the same species name. This strongly indicated that multiple undescribed species may have been placed under one species – P. nitens. A closer look at their genitalia revealed this to be true, and so a revision of the genus was then needed.

Paramyia sp. displaying kleptoparasitism, that is, feeding on the captured prey (stink bug) of another predator (spider)

PHOTO CREDIT: Steve A. Marshall

As with any revision, I first acquired multiple loans from large museum collections to compare and study the many diverse and variable specimens from a specific geographic range, in this case, North America. Then, I studied the morphology of these specimens in-depth to detect variation between those grouped together based on their similarities (i.e, morphs) and dissected the genitalia to confirm if they were indeed new species. With a genus like Paramyia, most helpful characters to differentiate between the species are genitalic, which means that good dissection skills are essential. The skill needed to dissect the genitalia of such small flies is comparable to performing surgery on a baby’s tooth. Important to note, there are no morphology characters to split the females of most species apart.

This is where DNA barcoding comes in handy. I sequenced specimens from my different morphs, and then dissect males grouped in the same BIN to verify the correspondence between the BIN and the species concept. When the molecular and the morphological analysis align perfectly, females can get correctly associates with their male counterpart, which would have otherwise been impossible. Hence, the species description can be more complete and the sequences are available to be used by other researchers to correctly identified that group, e.g. in monitoring programs. I applied this process in the Nearctic revision of Paramyia and described 10 new species! Future revisions tackling the remaining geographic regions can build from this work.

Comparative morphology between the new species P. pseudonitens and P. brevikeraia with a body profile, frontal head and genitalia photos (top to bottom)

PHOTO CREDIT: Valerie Levesque-Beaudin

The taxonomic impediment coupled with the current rate of species extinction is making the job of the taxonomist increasingly more difficult and yet, there’s an urgent need to record species before they disappear. As this study demonstrates, by quickly sorting specimens based on morphology and sequencing representative of each group, the number of undescribed species can be assessed and the amount of dissection needed to make such a discovery can be managed. The focus can then be on the morphology and genitalia of the different BINs, hence speeding up the process of species identification.

For full details, please refer to the publication in Zootaxa.

Written by

Valerie Levesque-Beaudin

Valerie Levesque-Beaudin

Taxonomic Specialist – Diptera, Centre for Biodiversity Genomics

February 27, 2020
https://doi.org/10.21083/ibol.v10i1.6081

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Revealing the widespread adulteration of commercial herbal medicines and food supplements

Revealing the widespread adulteration of commercial herbal medicines and food supplements

Revealing the widespread adulteration of commercial herbal medicines and food supplements

DNA barcoding and metabarcoding have substantially contributed to the authentication of herbal products and they hold the key to validated, DNA-based diagnostics for ingredient identification
Commercial herbal products available for the consumers outside of the regulated/controlled marketing chains in an agri-food market in Romania.
PHOTO CREDIT: Paula Paraschiva Sosoi

The World Health Organization has estimated that up to 80% of the world’s population, especially in developing countries, rely on herbal medicinal products as a primary source of healthcare. Yet, these herbal products (HPs) – widely sold in food stores and supermarkets, as well as over the internet – are poorly regulated. By gathering and analyzing a dataset of a little less than 6,000 commercial HPs across 37 countries, this study found undeclared contaminant, substitute, and filler species, or none of the labelled species in more than a quarter of products thus highlighting the large-scale presence of herbal product adulteration across the globe.

Shelves with herbal products in a supermarket

PHOTO CREDIT: Mihael Cristin Ichim

Along with the tremendous increase in demand for both plant raw materials and processed HPs in the global marketplace, there has been growing scientific evidence of accidental contamination or intentional adulteration of commercial HPs. Irrespective of the authentication method used, the traditional pharmacopoeial (e.g., macroscopic and microscopic examination, chemical methods) or emerging DNA-based tools (e.g., DNA barcoding and metabarcoding), numerous studies have identified adulterants when the contents of commercial HPs were tested against their labelled ingredient species. But the scale of this phenomenon is unknown.

To understand whether herbal adulteration is a global phenomenon, I examined peer-reviewed scientific journals for studies on HP authentication that were based on detection methods using species-specific DNA sequences. By analyzing 5,957 commercial HPs, sold in 37 countries scattered across all continents, results revealed 27% of products contained undeclared contaminant, substitute, and filler species, or none of the labelled species. It is unclear whether these cases are accidental, or intentional and economically motivated.

Figure 1: The proportion of adulterated products (black) among continents. 

The proportion of adulterated products varied significantly among continents, as high as 79% in Australia and as low as 23% in Asia (Figure 1). Of the countries with at least 100 products successfully authenticated and reported, the highest percentage of adulterated commercial HPs was found in Brazil (68%), followed distantly by Taiwan (32%), India (31%), and others (Figure 2).

Figure 2: The proportion of adulterated products among countries with at least 100 products successfully authenticated and reported.

All the commercial herbal products examined in this study were authenticated with DNA-based methods. These gradually advanced from classical molecular methods (e.g., Restriction Fragment Length Polymorphism) in early 2000 to DNA barcoding and metabarcoding in more recent years. The latter DNA barcoding methods were used to authenticate the majority of the reported commercial HPs, illustrating their growing utility as a tool to quantify rates of adulteration.

DNA barcoding – which makes use of short, standardized regions of the genome as species ‘barcodes’ – is a targeted approach suitable for the authentication of raw plant materials and the testing of single-ingredient HPs. Metabarcoding – a combination of high-throughput sequencing and DNA barcoding – enables untargeted, simultaneous multi-taxa identification by using DNA from different origins that can be extracted from more complex mixtures.

Besides the indisputable analytical advantages brought to herbal authentication, DNA barcoding and metabarcoding also have limitations. These are mainly derived from their high sensitivity to any amplifiable DNA extracted from the herbal product. Air-borne biological particles, such as pollen grains from neighbouring plants, can be deposited on the target plant and, when harvested, can be amplified, identified, and reported as adulterants of the finished herbal products rather than an accidental contamination. The ever-increasing analytical sensitivity of high-throughput DNA sequencing likely means that the proportion of adulterated HPs is expected to significantly increase.

It is important that the sensitivity of DNA-based authentication methods be optimized and effectively used to manage contaminated and adulterated HPs as they pose a significant risk for both the environment and human health. For example, often among the adulterants detected in commercial HPs are plant and animal species protected by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). More relevant to human health, HPs are often used in combination with conventional drugs. As HPs contain many pharmacologically active ingredients, the risk of adverse drug reactions – due to herb-drug interactions – increases when unknown, unlabeled plant ingredients are present in adulterated herbs taken with prescribed medications. Chronic, acute, and sometimes even lethal adverse health effects have been reported after the use of adulterated herbal products.

An accurate estimation of the adulteration of the HPs commercially available in the global marketplace is of paramount importance to all involved in their value chain, from growers, collectors, and producers, to pharmacists and medical practitioners, and finally, to the consumer.

For full details, please refer to the publication in Frontiers in Pharmacology.

Written by

Mihael Cristin Ichim

Mihael Cristin Ichim

“Stejarul” Research Centre for Biological Sciences, National Institute of Research and Development for Biological Sciences, Piatra Neamt, Romania

February 10, 2020
https://doi.org/10.21083/ibol.v10i1.5928

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Assessing herbarium material with novel molecular techniques reveals a wealth of new data from old treasure troves

Assessing herbarium material with novel molecular techniques reveals a wealth of new data from old treasure troves

Assessing herbarium material with novel molecular techniques reveals a wealth of new data from old treasure troves

Using large-scale genome skimming to build a resilient resource for the future
Hamersley Range, Pilbara, Western Australia

PHOTO CREDIT: Stephen van Leeuwen

Herbaria are valuable sources of extensive curated plant material that are important reference specimens for plant identification. These plant materials are now also accessible to genetic studies because of advances in high-throughput, next-generation sequencing (NGS) methods.

In our study, we conducted a large-scale applied assessment of one such NGS approach – genome skimming – and its ability to recover plastid and ribosomal genome sequences from a broad taxonomic sampling of herbarium material for the Western Australian flora. We sequenced 672 samples covering 21 families, 142 genera, and 530 named and proposed named species, and explored the impact of sample age, DNA concentration and quality, read depth and fragment length on plastid assembly error.

We demonstrate that herbaria are a valuable source of plant material for building a comprehensive DNA sequence database which serves various applications from modernizing plant surveys to improving the resolution of plant phylogenies.

Gastrolobium grandiflorum, Pilbara, Western Australia

PHOTO CREDIT: Stephen van Leeuwen

Genome skimming1 was effective at producing genomic information at large scale. Substantial sequence information on the chloroplast genome was obtained from 96.1% of samples, and complete or near-complete sequences of the nuclear ribosomal RNA gene repeat were obtained from 93.3% of samples.

Eucalyptus kingsmillii, Pilbara, Western Australia

PHOTO CREDIT: Stephen van Leeuwen

Grevillea wickhamii, Pilbara, Western Australia

PHOTO CREDIT: Stephen van Leeuwen

We extracted sequences for plastid markers rbcL and matK – the core DNA barcode regions – from 96.4% and 93.3% of samples, respectively. Read quality and DNA fragment length had significant effects on sequencing outcomes and error correction of reads proved essential. Assembly problems were specific to certain taxa with low GC and high repeat content (e.g. Goodenia, Scaevola, Cyperus, Bulbostylis, Fimbristylis), suggesting the influence of biological rather than technical factors. The structure of related genomes was needed to guide the assembly of repeats that exceeded the read length. DNA-based matching proved highly effective and showed that the efficacy for species identification declined in the following order: total chloroplast DNA >> ribosomal DNA > matK >> rbcL.

Ptilotus rotundifolius, Pilbara, Western Australia

PHOTO CREDIT: Stephen van Leeuwen

Our success is important as it demonstrates that herbaria can be used as a source of plant material for building a comprehensive DNA sequence database. These data form the basis of development of a molecular identification system for the Western Australian flora. This will enable identification of specimens throughout the year (e.g., non-flowering times) and for hard-to-identify species (e.g., those with constrained or reduced morphological characters) or for specimens where only fragments of non-diagnostic material are available. The availability of this technology will modernize plant surveys by reducing constraints on survey effort through moderating sampling timing restrictions and seasonal effects, as well as enabling rapid verifiable identification. It will also have practical applications in a wide range of ecological contexts using eDNA metabarcoding, such as gut and scat analysis of animals to determine dietary preferences of threatened species and livestock, and checking the integrity of seed collections for seed banking and use in land restoration/revegetation programs. Other potential uses of extensive plastid sequence data, beyond species identification, include improving the resolution of plant phylogenies and studies on the evolution of plastid genome function, including understanding adaptive changes.

References:

1. Straub S, Parks M, Weitemier K, Fishbein M, Cronn R, Liston A (2012) Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics. American Journal of Botany 99(2), 349-364. https://dx.doi.org/10.3732/ajb.1100335

For full details, please refer to the publication in BMC Plant Methods.

Written by

Paul Nevill

Paul Nevill

Curtin University, School of Molecular and Life Sciences, ARC Centre for Mine Site Restoration

& Trace and Environmental DNA (TrEnD) Lab, Perth, Western Australia

February 4, 2020
https://doi.org/10.21083/ibol.v10i1.5934 

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Revolutionizing fish monitoring in Alpine rivers

Revolutionizing fish monitoring in Alpine rivers

Revolutionizing fish monitoring in Alpine rivers

Environmental DNA provides high resolution data on Alpine fish populations and facilitates their management.
The river Inn (Kufstein, Tyrol, Austria) as an example of epipotamal habitats under anthropogenic influence.

PHOTO CREDIT: Bettina Thalinger

Many fish communities in Alpine rivers are impacted by anthropogenically altered riverbeds, making effective biomonitoring critical for the long-term sustainability and health of river systems. Traditional surveys require fish to be temporarily captured, an approach that can only be done outside of protected periods and at low water flow. A new method of detecting fish DNA in the environment has the potential to revolutionize monitoring efforts. Our study applied environmental DNA (eDNA) analyses in dynamic Alpine habitats to successfully detect species even at low population densities and to obtain information on the whole fish species community at a regional scale.

The river Inn upstream of a hydropower plant in Tyrol, Austria.

PHOTO CREDIT: Bettina Thalinger

Hydropower plants, transverse structures, and artificially straightened riverbeds are just some examples of how Alpine rivers and the landscapes around them are being altered for the benefit of neighbouring communities. These changes exert negative influences on fish species and make population management challenging. For instance, straightened river segments lack areas with low flow rates, which can be crucial for spawning and refuge seeking during the annual spring and summer high waters. Hence, detailed knowledge on the species present and how they are distributed along rivers is essential for their management, which also needs to incorporate sport fishing and artificial stocking in many places.
Traditionally, fish populations in Alpine rivers are assessed via electrofishing, but this method can only be applied during short periods outside of spawning times and when water levels and turbidity are low enough to effectively see and capture fish. Additionally, the considerable time and people necessary for electrofishing campaigns means only a small number of locations can be assessed, which are then taken as a representation for the whole river.
Water sampling for eDNA analysis in the river Rissbach (Hinterriß, Tyrol, Austria).

PHOTO CREDIT: Bettina Thalinger

Environmental DNA (eDNA) – genetic material obtained directly from environmental samples (soil, sediment, water, etc.) without any obvious signs of biological source material1 – has the potential to revolutionize fish monitoring in rivers as it enables the cost-effective analysis of multiple water samples from different locations within river networks. It allows us to easily pinpoint the upstream distribution limits of species and the changes in their longitudinal distribution. Although Alpine rivers are characterized by a comparably low species diversity, low population densities coupled with high water flow fluctuations represent a significant challenge for the broad-scale application of eDNA-based fish monitoring. Our team, scientists based at the University of Innsbruck and freshwater ecologists from the ARGE Limnologie, is examining what is needed for successful eDNA-based fish detections in Alpine rivers through the Austrian Research Promotion Agency (FFG) project “eDNA-AlpFish”.

First, our team defined detection limits for distinct flow situations in the summer and winter months. We were able to confirm the high sensitivity of our species-specific eDNA amplification approach by detecting signals from fish at low numbers and at a considerable downstream distance and water flow. For example, we detected signals stemming from ~50 individuals (200 g) of Eurasian minnow (Phoxinus phoxinus) at a downstream distance of 550 m and at a discharge of 0.25 m³/s. Under similar circumstances, a detection via electrofishing is uncertain as usually a shorter river stretch is sampled and not all present fish individuals can be caught.

High water flow during summer due to snowmelt (top) in comparison to low winter flow (bottom). In both situations, fish were placed in cages in the river Melach (Tyrol, Austria) for downstream eDNA detection.

PHOTO CREDIT: Bettina Thalinger

In the next step, we evaluated the performance of these molecular approaches in direct comparison to electrofishing by sampling water from an Alpine river and its tributaries in the epirhithral zone. Whilst water samples from the whole river network could be obtained in a day, quantitative electrofishing was only possible at three sites within this timeframe. Additionally, the molecular analysis enabled the detection of bullhead (Cottus gobio) upstream of the point where low population levels made catches via electrofishing impossible.

Samples were also taken at epipotamal locations along the river Inn where the fish community is more diverse, and they were analysed with DNA metabarcoding. This approach permits the simultaneous assessment of the whole fish community and led to the detection of all the species known to occur in these waters. Additionally, we also obtained eDNA signals stemming from the surrounding tributaries in these samples.

The river Inn (Kufstein, Tyrol, Austria) as an example of epipotamal habitats under anthropogenic influence.

PHOTO CREDIT: Bettina Thalinger

The results of the eDNA-AlpFish project will inform sampling strategy and data interpretation of future projects and, overall, highlight the massive potential of eDNA for large-scale fish monitoring in Alpine rivers.

References:

1. Thomsen, P. F., & Willerslev, E. (2015). Environmental DNA – An emerging tool in conservation for monitoring past and present biodiversity. Biological Conservation. doi: 10.1016/j.biocon.2014.11.019

Written by

Bettina Thalinger

Bettina Thalinger

Department of Ecology, University of Innsbruck, Austria

Centre for Biodiversity Genomics, University of Guelph, Canada
Yannick Pütz

Yannick Pütz

Department of Ecology, University of Innsbruck, Austria

Dominik Kirschner

Dominik Kirschner

Department of Ecology, University of Innsbruck, Austria

Christian Moritz

Christian Moritz

ARGE Limnologie GesmbH, Innsbruck, Austria

Richard Schwarzenberger

Richard Schwarzenberger

ARGE Limnologie GesmbH, Innsbruck, Austria

Josef Wanzenböck

Josef Wanzenböck

Research Department for Limnology, Mondsee, University of Innsbruck, Austria

Michael Traugott

Michael Traugott

Department of Ecology, University of Innsbruck, Austria

November 26, 2019
https://doi.org/10.21083/ibol.v9i1.5798

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Understanding the impacts of widespread forest die-offs across France, Germany, and China

Understanding the impacts of widespread forest die-offs across France, Germany, and China

Understanding the impacts of widespread forest die-offs across France, Germany, and China

Metabarcoding increases the taxonomic resolution and geographic scale at which researchers can assess the impacts of climate change on insect communities in forests

Entomologists studying tree dieback of Picea abies in Bavarian National Park.

PHOTO CREDIT: Heiner M. Elsner

Forests throughout the world are suffering from an increase in the frequency and severity of droughts as well as pathogen and insect infestations. These climate-exacerbated factors are leading to tree diebacks—the progressive death of tree branches—and subsequent large-scale forest die-offs, the effects of which are not well understood. Preliminary results from our study indicate that while the number of insect species along forest die-off gradients might not be affected, their composition is changing.

CLIMTREE is an international project funded by the Belmont Forum that assesses the impact of climate-induced forest die-off on invertebrate biodiversity in highland forests in France, Germany, and China. This project aims to better understand how tree mortality and associated changes in forest composition affect biodiversity and ecosystem functions.

Silver fir (Abies alba) dieback in the French eastern Pyrenees

PHOTO CREDIT: Carlos Lopez-Vaamonde

Our study measures changes in the insect communities along dieback and salvage-logging gradients of silver fir (Abies alba) forests in the French eastern and central Pyrenees, Norway, spruce (Picea abies) forests in the Bavarian Forest National Park, Germany, and Yunnan pine (Pinus yunnanensis) forests in Yunnan province, China. We examined patterns of variation in the species diversity of flying insects collected by Malaise traps. As these passive traps can collect a large number of specimens, we analyzed samples in bulk using DNA metabarcoding. This approach uses DNA barcode reference libraries to identify species from a mixed sample using high-throughput technologies that effectively provide large amounts of DNA sequence data.

Members of CLIMTREE assessing the level of decline of silver fir stands in Pays de Sault (French eastern Pyrenees)

PHOTO CREDIT: Carlos Lopez-Vaamonde

We also examined saproxylic beetles, a key functional group used as bioindicators in forest assessments of dead-wood availability. We collected these beetles using flight interception traps, and we also sampled specimens from natural history collections to build a DNA barcode reference library for the French saproxylic beetle fauna as a resource for future investigations.

The preliminary results from the 56 Malaise traps deployed in the French eastern and central Pyrenees have revealed more than 3,500 OTUs (Operational Taxonomic Units, a proxy for species) belonging to 18 insect orders, with considerable change in the compositions of the species detected along the dieback gradient as well as across a 4-month period. However, results to date do not suggest significant declines in species richness over the dieback gradient.

Rosalia alpina (Cerambycidae) is one of the 2,663 species of saproxylic beetles known to occur in France

PHOTO CREDIT: Carlos Lopez-Vaamonde

Polytrap flight interception trap

PHOTO CREDIT: Carl Moliard

Of the 55,571 saproxylic beetles collected by the flight interception traps, about 70% were morphologically identified to one of 284 species. Similar to the flying insects collected by Malaise traps, the diversity of beetles along dieback gradients did not decline. We are now trying to use a non-destructive metabarcoding approach that involves processing the collection media to determine whether we can uncover a comparable number of species with a morphology-based sorting approach. If results are similar, we will have a strong case for using this technique as a time-efficient alternative for biomonitoring moving forward.

Overall, there is an urgent need to obtain detailed baseline data on insect communities to quantify the impacts of climate change. By taking advantage of DNA metabarcoding approaches, our study is assessing biodiversity patterns at scales previously impossible and providing the data essential for evaluating future changes in insect communities. Our workflows are simple to use and provide an affordable, reliable, and repeatable method of assessing insect diversity in forests at a large geographical scale.

Written by

Lucas Sire

Lucas Sire

Institut de Recherche sur la Biologie de l’Insecte, Tours, France

Paul Schmidt Yañez

Paul Schmidt Yañez

Leibniz Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin, Germany

Rodolphe Rougerie

Rodolphe Rougerie

Muséum National d’Histoire Naturelle, Paris, France

Christophe Bouget

Christophe Bouget

IRSTEA, Nogent-sur-Vernisson, France

Laurent Larrieu

Laurent Larrieu

Dynafor INRA & CRPF Occitanie, Toulouse, France

Douglas W. Yu

Douglas W. Yu

Kunming Institute of Zoology, Kunming, China

Michael T. Monaghan

Michael T. Monaghan

Leibniz Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin, Germany

Institut Für Biologie, Freie Universität Berlin, Germany

Jörg Müller

Jörg Müller

Bavarian Forest National Park, Grafenau, Germany

Elisabeth A. Herniou

Elisabeth A. Herniou

Institut de Recherche sur la Biologie de l’Insecte, Tours, France

Carlos Lopez-Vaamonde

Carlos Lopez-Vaamonde

Institut de Recherche sur la Biologie de l’Insecte, Tours, France

INRA, Orléans, France

October 28, 2019
https://doi.org/10.21083/ibol.v9i1.5726

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Insects don’t talk, but new DNA-based technologies are helping to tell their stories

Insects don’t talk, but new DNA-based technologies are helping to tell their stories

Insects don’t talk, but new DNA-based technologies are helping to tell their stories

Researchers unlock clues about vertebrate diversity by examining arthropod samples
Malaise trap deployed in Carajás National Forest in Brazil. PHOTO CREDIT: Christina Lynggaard

The study of arthropod diversity often uses primers solely targeting arthropod DNA, ignoring those originating from non-arthropod sources. Yet, some arthropods (e.g. sand flies, blowflies, mosquitoes) feed on vertebrates and vertebrate remains. By specifically targeting these vertebrate-feeding arthropods, invertebrate derived DNA (iDNA) can be used to understand vertebrate diversity as well. Our study has found that by using samples collected for arthropod diversity research, we can tap into a rich information source of vertebrate diversity.

Arthropods are highly abundant and provide vital ecosystem services such as pollination, decomposition of organic matter, and they serve as a food source for other animals. Because of this, hundreds to thousands of arthropod samples are collected globally every year for monitoring programs, conservation efforts, and ecosystem assessments.

However, identifying arthropod species based on physical characteristics is difficult, requiring specific expertise and a significant amount of time. Due to this, molecular analyses, such as DNA metabarcoding, are being used to identify the arthropods present in bulk samples, speeding-up the otherwise time-demanding identification process by screening the contents of hundreds of samples simultaneously.

Bulk arthropod sample

PHOTO CREDIT: Christina Lynggaard

In this study, bulk arthropod samples were collected using pitfall and Malaise traps deployed at the Carajás National Forest in Brazil and in the Udzungwa Mountains in Tanzania. Our aim was to study the arthropod diversity in these areas by using metabarcoding primers targeting arthropod DNA. Nevertheless, we conducted an additional analysis targeting vertebrate DNA remains in the bulk samples by using metabarcoding vertebrate primers.

We were able to detect vertebrate DNA in 19 per cent of our 265 analyzed samples, identifying more than 30 vertebrate taxa including mammals, amphibians and birds. For example, we found South American tapir (Tapirus terrestris), chirinda screeching frog (Arthroleptis xenodactyloides), velvety free-tailed bat (Molossus molossus), Zanzibar bushbaby (Paragalago zanzibaricus) and honeyguide greenbul (Baeopogon indicator). The presence of some species was further confirmed through visual observations during the sample collections.

Some cases represent the first time those vertebrate species have been detected in the area. This could indicate that we are detecting vertebrates that live in different areas to where the arthropods were collected or that with this method it is possible to detect local animals that are difficult to observe.

Tapir footprint found in one of the Brazilian study sites during bulk arthropod sample collection confirmed the presence of some of the detected vertebrate taxa PHOTO CREDIT: Christina Lynggaard

Bushbaby (Paragalago zanzibaricus) found in the Udzungwa Mountains in the Tanzanian study site during bulk arthropod sample collection confirmed the presence of some of the detected vertebrate taxa

PHOTO CREDIT: mnielsenphotography

The sensitivity of new DNA-based technologies is changing the way we explore and understand the relationships between species. Approaches like DNA metabarcoding can not only tell us about the different types of insects in our environment, but also what they eat and where they have been. Our study is the first to not target a specific invertebrate group to detect vertebrate DNA but instead to use bulk arthropod samples. We encourage others to consider bulk samples during environmental monitoring not only as a source for arthropod diversity information, but one for vertebrate diversity as well.

This study is published in the eDNA Journal.

Written by

Christina Lynggaard

Christina Lynggaard

The GLOBE Institute, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark

Martin Nielsen

Martin Nielsen

The GLOBE Institute, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark

Luisa Santos-Bay

Luisa Santos-Bay

The GLOBE Institute, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark

NTNU University Museum, Norwegian University of Science and Technology (NTNU), Trondheim, Norway
Markus Gastauer

Markus Gastauer

Instituto Tecnológico Vale, Belém, Pará, Brazil

Guilherme Oliveira

Guilherme Oliveira

Instituto Tecnológico Vale, Belém, Pará, Brazil

Kristine Bohmann

Kristine Bohmann

The GLOBE Institute, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark

University of East Anglia, Norwich Research Park, Norwich, UK

October 17, 2019
https://doi.org/10.21083/ibol.v9i1.5727

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The celebrities of the microcosmos aren’t always easy to find: detecting tardigrades in environmental DNA

The celebrities of the microcosmos aren’t always easy to find: detecting tardigrades in environmental DNA

The celebrities of the microcosmos aren’t always easy to find: detecting tardigrades in environmental DNA

The hidden diversity of tardigrades is being uncovered in Norwegian forests using DNA barcoding and metabarcoding

Scanning electron microscopy image of Diploechiniscus oihonnae

PHOTO CREDIT: Lasse Topstad

Found across every continent on Earth, to now potentially living on our moon, tardigrades are some of the most resilient microorganisms we know of. But despite our fascination with these microscopic water bears, there is still much to discover. Our study is exploring the applicability of using environmental DNA to facilitate the examination of tardigrade diversity.

The popular narrative that tardigrades can withstand anything – from -272 degrees Celsius to as high as 150 degrees Celsius, 6,000 times the atmospheric pressure, extreme radiation, and vacuum – has earned them celebrity status of the microcosmos. However, tardigrades are more than just superstars. They constitute their own phylum of life, ranked at the same taxonomic level as arthropods (insects and spiders), and currently hold around 1,270 described species. Many of these species fulfill ecologically important roles related to the breakdown of organic material in the soil. Other species are found in freshwater streams, sediments, mosses, lichens, and leaf-litter, occurring in most ecosystems throughout the world. As with other tiny taxa, telling tardigrade species apart can be challenging. Confident identifications of many species depend on the presence of both adult specimens and eggs. Additionally, tardigrade taxonomy is traditionally based on a limited set of morphological traits. This has resulted in several complex species groups, comprising morphologically inseparable, but genetically distinct species.

The claws of one of the species in the Macrobiotus hufelandi group. These species are often inseparable based on morphology, but clearly distinct species based on the COI gene.

PHOTO CREDIT: Lasse Topstad

DNA barcodes offer a solution to these impediments by generating unique genetic characteristics for each of these species. In recent years, there has been an increase in the use of molecular tools on tardigrades, but currently, only a small portion of the known species have barcodes deposited in public databases. Such reference sequences are essential if tardigrades are to be included in large-scale biomonitoring methods such as metabarcoding of environmental DNA (eDNA). Our study is the first to compare the applicability of eDNA-based metabarcoding of tardigrade diversity with morphologically identified communities.

Collection of lichen samples during fieldwork in Southern Norway

PHOTO CREDIT: Torbjørn Ekrem

We extracted tardigrades and eggs from samples of moss, lichens, and leaf-litter and identified them using morphology. The 3,788 recorded tardigrade specimens and eggs were identified as 40 morphologically distinct species, of which 24 were successfully sequenced for the gene cytochrome c oxidase I (COI). These were represented by 151 successfully sequenced individuals. Interestingly, the barcodes revealed 32 genetically distinct linages among the 24 morpho-species, showing high levels of hidden diversity.

Figure 1. Overlap in species recovery by the different methods.

Next, we extracted eDNA from the same environmental samples and sequenced two fragments of the COI marker and one fragment of the 18S marker using the Illumina MiSeq next-generation sequencing platform. This method recovered 57 species of tardigrades compared to the 40 species detected by conventional methods. Mostly, the two methods identified the same species (Figure 1), yet, metabarcoding detected cryptic species elusive to morphological identification. This indicates that metabarcoding of eDNA successfully captures tardigrade diversity.

However, the credibility of such records needs to be evaluated thoroughly. While the COI marker distinguishes well between tardigrade species, the 18S marker might not be as useful as there is not sufficient sequence variation between species (a so-called barcode gap). Furthermore, the 18S marker detected Acutuncus antarcticus in two of the samples, a species endemic to Antarctica. This species is likely not found in Norway and highlights the danger of blindly trusting marker-based identifications without carefully evaluating taxonomic assignments and possibilities of contamination.

Our findings were dependent on our barcode reference library of locally sampled species and the use of multiple markers. As only a small portion of tardigrade species are deposited with reference sequences in public databases, both the COI and 18S markers are limited in their ability to detect species of tardigrades as most sequences will go unmatched. We demonstrate that metabarcoding is applicable for large-scale biomonitoring of tardigrades, but highlight the need for better reference libraries for tardigrade species.

Aknowledgements:

This research is part of a Master thesis at the NTNU University Museum and the project ‘Tardigrades in Norwegian Forests’ funded by the Norwegian Taxonomy Initiative and NorBOL. Special thanks to Roberto Guidetti at University of Modena and Reggio Emilia for his supervision during my stay in Italy.

Written by

Lasse Topstad

Lasse Topstad

Norwegian University of Science and Technology University Museum, Department of Natural History

September 18, 2019
https://doi.org/10.21083/ibol.v9i1.5722

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