Using DNA barcodes in the fight against Malaria

Using DNA barcodes in the fight against Malaria

Using DNA barcodes in the fight against Malaria

Researchers in Ghana along with Target Malaria, the University of Oxford and the Centre for Biodiversity Genomics work to uncover the role of the Anopheles gambiae mosquito.

January 27, 2021

By Michelle Lynn D’Souza

Target Malaria aims to reduce malaria transmission by reducing the population of malaria-transmitting mosquitoes using genetic technologies. As part of their research, they want to know the ecological implications of reducing populations of the mosquito—the key vector for malaria in Africa.

Researchers are using DNA barcoding technologies to catalogue the insect community in Ghana. They will then use this catalogue or library to identify the insect species that are eaten by local birds, bats, and other insect-eating arthropods as well the host species in a mosquito bloodmeal, all through metabarcoding techniques that identify DNA fragments by matching sequences to the local DNA barcode library.

In the end, they will use these data to construct an ecological network that will quantitatively demonstrate how Anopheles gambiae is connected to the other members of the ecosystem.

These efforts are a demonstration of the power of DNA barcoding and its ability to reveal the nature and intensity of interactions among all species. This endeavour, to reveal species interactions to clarify their role in structing biological communities, is a key research theme of BIOSCAN, iBOL’s new seven-year, $180 million global research program that aims to revolutionize our understanding of biodiversity and our capacity to manage it.

For more information on Target malaria efforts in Ghana see: The important interactions behind the itch

For more information on BIOSCAN see:  BIOSCAN – Illuminating biodiversity and supporting sustainability

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The important interactions behind the itch

The important interactions behind the itch

The important interactions behind the itch

The potential consequences of reducing Anopheles gambiae mosquitoes to control Malaria

Ghanaian researchers sorting insect samples at the University of Ghana, Accra, Ghana.

IMAGE CREDIT: Michelle L. D’Souza; PHOTO CREDIT: Lema Concepts Africa

Written by

Talya D. Hackett

Talya D. Hackett

Department of Zoology, University of Oxford, Oxford 

Karen E. Logan

Karen E. Logan

Target Malaria, Dept Life Sciences, Imperial College London, UK

Michelle L. D'Souza

Michelle L. D'Souza

Centre for Biodiversity Genomics, University of Guelph, Guelph, CA

January 27, 2021

doi:10.21083/ibol.v11i1.6267

Only four months after China reported its first COVID-19 case to the World Health Organization (WHO) the virus had spread to every nation on the African continent. Despite being home to 17% of the world’s population, Africa currently accounts for just 2.5% of COVID-19 related deaths1. But the pandemic may well have caused many more to die, not from coronavirus, but from malaria.

The 2020 World Malaria Report warned that disruptions to malaria prevention and treatment caused by the coronavirus could see as many as 100,000 malaria-related deaths in Sub-Saharan Africa2. Similar effects were unfortunately experienced during the 2014-2015 outbreaks of Ebola in West Africa3. While most strategies being employed to control malaria have worked well, progress to reduce its incidence has stagnated. New strategies are needed to prevent the mortality rate from increasing further and to better prepare countries in the face of other unexpected pressures.

With the ambitious goal to create a world free of malaria, one not-for-profit research consortium–Target Malaria–is developing novel technologies using genetic modification to control the numbers of the malaria-transmitting mosquitoes.

The nature of malaria and the microbe responsible

Malaria is a disease that starts with a small single-celled parasite. This microorganism belongs to the genus Plasmodium, and of the four species that threaten humans, P. falciparum and P. vivax are the most common, and the former the most dangerous4.

Female mosquitoes alone spread malaria in nature. An infected mosquito injects a small number of parasites into its victim’s bloodstream while it feeds, and the parasites then travel to the liver where they multiply rapidly before infecting red blood cells. Flu-like symptoms begin when the parasites break out of the blood cells, one to four weeks after the bite.

Of the 229 million confirmed malaria cases worldwide in 2019, 94% occurred in Africa2. Even more devastating, of the 409,000 malaria-associated deaths, 84% occurred in children under the age of five.

While there are more than 3,500 species of mosquito worldwide and 837 in Africa, three very closely related species are responsible for most transmission of the disease: Anopheles gambiae, Anopheles coluzzii, and Anopheles arabiensis. These three species belong to the Anopheles gambiae complex which, if targeted, is likely to have the largest effect on the transmission of malaria. This species complex has tightly evolved with humans and is the key vector for malaria in sub-Saharan Africa.

Current vector control tools such as insecticides, bed nets, and drugs have been effective in reducing malaria cases but not in eradicating the disease. Target Malaria’s approach is meant to be complementary to the existing interventions by focusing on malaria control by mosquito control.

Researchers unify under Target Malaria

In 2003, Prof. Austin Burt published a seminal paper5 describing the principle of genetically modifying a population of mosquitoes for applications in the control of vector-borne diseases. Prof. Burt predicted that malaria-transmitting mosquitoes could potentially disappear in an area within two years when using these novel genetic tools. It was fortuitous that only a few years prior in Imperial College London, where Prof. Burt was working, a team lead by Prof. Andrea Crisanti had created the first reliable system for germline transformation of a malaria-transmitting mosquito6.

The two groups were brought together in 2005 with a grant given as part of the Grand Challenges in Global Health initiative and in a little over a decade the team’s scientific progress had resulted in a new mechanism for genetic control measures within An. gambiae7.

The initial group of researchers has now grown into a team of 180 project members, with collaborating research partners in Africa, Europe, and North America. As the work progressed, the project grew under its new brand–Target Malaria.

Target Malaria is working with five African partner sites.
IMAGE CREDIT: Michelle L. D’Souza

Today, Target Malaria is working with five African partner sites: Burkina Faso, Cape Verde, Ghana, Mali, and Uganda. This work is being headed by Dr. Abdoulaye Diabate at the Institut de Recherche en Sciences de la Santé (Research Institute on Health Sciences), Bobo-Dioulasso, Burkina Faso; Dr. Adilson de Pina at the Instituto Nacional da Saude (National Institute of Public Health, CCS-SIDA), Cape Verde; Dr. Fred Aboagye-Antwi at the University of Ghana, Accra, Ghana; Dr. Mamadou Coulibaly at the University of Bamako Malaria Research and Training Center, Bamako, Mali; and Dr. Jonathan Kayondo at the Uganda Virus Research Institute, Entebbe, Uganda.

Each of the African partners and their teams bring a different skillset to the collaboration, none being able to deliver all key elements independently. A relationship of co-development is fostered within the project with scientists working together across countries and with communities towards a common objective and vision–a world free of malaria.

The three key guiding pillars

Target Malaria’s work is structured around three key pillars: science, stakeholder engagement, and regulatory affairs. Each pillar is essential for the project’s success, supporting responsible research and development of genetic technologies, a commitment to engaging a wide variety of stakeholders, as well as ensuring compliance with all national regulations and laws. 

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Science

To date, Target Malaria has made significant scientific advancements on the path to developing a new tool for vector control for malaria. Researchers have demonstrated the proof of concept in creation of a transgenic sterile male An. gambiae8 strain, demonstration of the ability to modify a laboratory population of An. gambiae mosquitoes to be male biased9, suppression of a small cage population of laboratory reared An. gambiae mosquitoes10, creation of the first gene drive mosquitoes capable of suppressing a laboratory population of An. gambiae mosquitoes11, modelling the potential of genetic control of malaria mosquitoes12, modelling suppression of malaria vector using gene drive13, and the importation of the first genetically modified mosquitoes (a self-limiting sterile male line) into Burkina Faso for contained laboratory use in 2016 and regulatory approval for the subsequent release of the same self-limiting sterile male line in 2019.

Stakeholder engagement

The list of stakeholders is vast, from grass roots, those local communities where the project is working, through to local civil society organizations, regional governing bodies, and the appropriate governmental agencies, all in-country, as well as a range of interested parties outside of the African partner countries. Target Malaria is committed to ensuring that the stakeholders understand the research and long-term goals of the project enabling them to make an informed decision on whether to support the project’s efforts. Engagement also helps ensure that the research is welcomed and useful in the fight against malaria. Most importantly, Target Malaria will learn a lot from their stakeholders through the process.

Regulatory affairs

While an important aspect of Target Malaria’s strategy is to focus on the communities that might benefit from the technology and that are most concerned by the research activities, they also have an ongoing, transparent dialogue with other stakeholders at the national and international level. For example, the project is taking a phased approach to its development pathway in line with guidance from the WHO14.

Technology being developed

The goal is to develop modified mosquitoes that can pass on to their progeny a self-sustaining genetic change, a process aiming to reduce specific mosquito populations to break the malaria transmission cycle. To do so, Target Malaria is using gene drive, a phenomenon that occurs in nature and causes a selected trait to rapidly increase in frequency through a population via sexual reproduction over several generations. Gene drive works by increasing the likelihood–from the usual 50 per cent to greater than 95 per cent–that a modified gene will be inherited by its offspring. This means that over the course of several generations, a selected trait could become increasingly common within a specific species (depending on the specific area and how the animals move around within it).

Researchers are investigating the use of genes that produce enzymes that cut specific sequences of DNA. Called nucleases, these enzymes found in simple single celled organisms can copy themselves from one chromosome to another5. When introduced into the malaria mosquito, the nuclease works by identifying and cutting a selected site within essential genes targeted by researchers, rendering them functionless, such as reproductive genes. The subsequent effects depend on the nature and importance of the gene.

Target Malaria’s goal is to produce modified malaria mosquitoes that can pass these genes on to greater than 95 per cent of their offspring, so the modification is spread throughout the specific population relatively quickly and is effectively “self-sustaining”. This strategy is known as population suppression, and as the mosquitoes themselves do the work of spreading the modification, it makes the reduction of the malaria mosquito population relatively cost effective and simple to implement.

Current gene drive research is at an early stage, and so definitive decisions about gene drive-based tools are premature. Based on current progress, field releases of a gene drive-based tool are many years away. This gives scientists and stakeholders, specifically those from countries where gene drives might one day be employed, valuable time to consider the important questions of regulation, risk assessment, ethics, and engagement, and to prepare for assessing any application related to gene drive mosquitoes and their potential use as a tool for vector control for malaria.

An ecological approach spearheaded in Ghana

As the gene drive approach in development by Target Malaria will specifically target the An. gambiae complex to reduce its population, it is vital to ensure there are no undesirable consequences to the rest of the plant and animal communities. In Ghana, researchers are focusing on the ecological implications of the work; the role of the An. gambiae mosquito in the broader ecosystem. The ongoing research in Ghana aims to predict these potential effects.

 

Researchers setting off to sample the insect community around a Ghanaian village.

PHOTO CREDIT: Michelle L. D’Souza

THE ECOLOGICAL ROLE OF MOSQUITO LARVAE IN AQUATIC ENVIRONMENTS

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WHAT WE KNOW, DON’T KNOW, AND THINK WE KNOW ABOUT THE PREDATORS OF MOSQUITOES

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POLLINATORS OR NECTAR THIEVES? THE ROLE OF MALARIA-TRANSMITTING MOSQUITOES IN POLLINATION

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ECOLOGICAL AND EPIDEMIOLOGICAL INSIGHTS FROM BLOOD MEALS

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While some aspects of An. gambiae ecology is well studied, research in Ghana will provide a more complete picture, specifically determining the interactions between An. gambiae and other mosquito species as well as predators, prey, and vertebrate hosts. In this sense, the research is based on a community ecology approach rather than looking at just mosquito ecology.

Researchers are sampling, as far as they are able, insects from the entire aerial communities across all habitats, not just where they expect to find high numbers of An. gambiae. Equally true for insectivores that might feed on mosquitoes or similar small aerial insects, they are taking fecal samples or stomach contents. In aquatic habitats, they are sampling from a range of water bodies and collecting representatives across all insect and insectivorous groups. And, because adult mosquitoes rely on flower nectar for food, they are sampling the pollinator community as well. Finally, they are looking at the community of biting flies and their vertebrate hosts by determining blood-meal interactions to better understand shared hosts and the potential for zoonotic disease transmission15. Importantly, all methods and target sample numbers have been cleared by independent ethics boards at both the University of Ghana and the University of Oxford to ensure there is no lasting impact on the community of plants and animals where the work takes place.

Insect samples collected around a Ghanaian village using Malaise traps (top) before being sorted and pinned (bottom) at the University of Ghana.
PHOTO CREDIT: Lema Concepts Africa

Using this broad community approach allows researchers not only to describe the role of An. gambiae in the ecosystem but also to predict how the rest of the ecological community would respond to An. gambiae reduction. For example, the data would allow them to determine which insects might face more pressure from predators if those that feed on mosquitoes shifted their feeding behaviour to replace An. gambiae in their diet and how this change might affect the rest of the food web. As there is no known animal or plant that relies solely on An. gambiae16, and food webs tend to rewire following minor perturbations17,18, it is predicted that there will not be any significant effects because of An. gambiae population reduction. Regardless, it is necessary to ensure this is the case and a community ecology approach will make this possible.

Some of the faces of the research team in Ghana. Dr. Fred Aboagye-Antwi (Ghanaian Principle Investigator, top left), Helen Selorm Wohoyie (Assistant Stakeholder Engagement and Communications Advisor, top center), Divine Dzokoto (Senior Stakeholder Engagement and Communications Advisor, top right) and Dr. Talya D. Hackett (project coordinator), Bernard Aiye Adams, Ezekiel Yaw Donkor, and Naa Na Afua Acquaah (laboratory technicians) (bottom, left to right).
PHOTO CREDIT: Lema Concepts Africa

Building a DNA barcode reference library for Ghana

The tools being used to construct the food web are mostly molecular-based, all requiring the creation of a DNA barcode library for insects in the area as the important first step. To this end, researchers in Ghana regularly collected terrestrial insects from villages in the southeast of Ghana for a year.

Once the library is established, they can then start looking at the feces and the gut contents of insectivores and use that library to match and identify prey DNA fragments using DNA metabarcoding.

Researchers are using a similar metabarcoding approach for the aquatic food web while for the pollination network they will use a combination of traditional observational methods and DNA metabarcoding of pollen from caught insects. Finally, for the blood meal analysis, they are metabarcoding the blood meals of fed mosquitoes and other biting flies to identify what has been bitten.

The ecological research in Ghana is a collaboration between Dr. Fred Aboagye-Antwi at the University of Ghana, Prof. Sir Charles Godfray and Prof. Owen Lewis at the University of Oxford, as well as an extensive team of postdoctoral researchers, Ph.D. students, and technicians at both institutions. Part of the UK team, postdoctoral researcher and project coordinator, Dr. Talya D. Hackett is organizing efforts between countries, including a collaboration with the Centre for Biodiversity Genomics (CBG) in Guelph, Canada, global leader in the field of DNA barcoding. Supported by Prof. Paul D. N. Hebert, the Director of CBG, and Dr. Michelle L. D’Souza, samples from Ghana have begun making their way to the large sequencing platforms housed at the CBG.

Dr. Talya D. Hackett (left) and Dr. Michelle L. D’Souza (right) discuss DNA barcode data compatibility across platforms EarthCape and BOLD Systems at the University of Ghana.
PHOTO CREDIT: Lema Concepts Africa

So far, about 3,000 insects have been processed and 530 BINs (species proxies) have been documented, about 70% of which are unique to the project. Efforts will ultimately barcode 100,000 specimens and fill a large gap in barcode data currently missing from West Africa.

Conclusions

Examining diets to determine species-specific interactions in a complex community food web is only possible at this large scale with molecular techniques, and only recently, because the costs of DNA barcoding and metabarcoding techniques have dropped. Even five years ago this sort of a project would not have been feasible.

Apart from building large, comprehensive food webs, these data can further inform our understanding of things like community structure and insect abundances across time and space, the dietary overlap of insectivorous species, and niche overlap of different mosquito species.

All data will be made publicly available. Ultimately, this project is creating a wealth of information, not just for Target Malaria’s research goals, but for the broader scientific community and for other people within Ghana and West Africa.

These efforts are a demonstration of the power of DNA barcoding and its ability to reveal the nature and intensity of interactions among all species. This endeavour, to reveal species interactions to clarify their role in structuring biological communities, is a key research theme of BIOSCAN, iBOL’s new seven-year, $180 million global research program that aims to revolutionize our understanding of biodiversity and our capacity to manage it.

If you would like to know more about Target Malaria, go to www.targetmalaria.org

References:

1. World Health Organization (2020) WHO Coronavirus Disease (COVID-19) Dashboard. Accessible at: https://covid19.who.int/

2. World Health Organization (2020) World malaria report. Accessible at: https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2020

3. Wang J et al. (2020) Preparedness is essential for malaria-endemic regions during the COVID-19 pandemic. The Lancet 395(10230), 1094–1096. doi: 10.1016/s0140-6736(20)30561-4

4. Crutcher JM, Hoffman SL. Malaria. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 83.

5. Burt A (2003) Site-specific selfish genes as tools for the control and genetic engineering of natural populations. R. Soc. London. Ser. B Biol. Sci. 270:921–928. doi: 10.1098/rspb.2002.2319

6. Catteruccia F, Nolan T, Loukeris TG, Blass C, Savakis C, Kafatos FC, Crisanti A (2000) Stable germline transformation of the malaria mosquito Anopheles stephensi. Nature 405(6789):959-962. doi: 10.1038/35016096.

7. Windbichler N, Menichelli M, Papathanos PA, Thyme SB, Li H, Ulge UY, Hovde BT, Baker D, Monnat RJ Jr, Burt A, Crisanti A. (2011) A synthetic homing endonuclease-based gene drive system in the human malaria mosquito. Nature. 473(7346):212-215. doi: 10.1038/nature09937.

8. Windbichler N, Papathanos PA, Catteruccia F, Ranson H, Burt A, Crisanti A (2007) Homing endonuclease mediated gene targeting in Anopheles gambiae cells and embryos. Nucleic Acids Res. 35(17):5922-5933. doi:10.1093/nar/gkm632

9. Galizi R, Doyle LA, Menichelli M, Bernardini F, Deredec A, Burt A, et al. (2014) A synthetic sex ratio distortion system for the control of the human malaria mosquito. Commun., 5: 1–8.

10. Hammond AM, Kyrou K, Bruttini M, North A, Galizi R, Karlsson X, et al. (2017). The creation and selection of mutations resistant to a gene drive over multiple generations in the malaria mosquito. PLOS Genet. 13:e1007039.

11. Kyrou K, Hammond AM, Galizi R, Kranjc N, Burt A, Beaghton AK, et al. (2018) A CRISPR–Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes. Biotechnol. 36:1062–1071

12. North AR, Burt A & Godfray HCJ (2019) Modelling the potential of genetic control of malaria mosquitoes at national scale. BMC Biol. 17: 26.

13. North AR, Burt A & Godfray HCJ (2020) Modelling the suppression of a malaria vector using a CRISPR-Cas9 gene drive to reduce female fertility. BMC Biol. 18:98.

14. World Health Organization (2014) Guidance framework for testing of genetically modified mosquitoes. Accessible at: https://www.who.int/tdr/publications/year/2014/en/

15. Bellekom B, Hackett TD & Lewis OT (2021) A Network Perspective on the Vectoring of Human Disease. Trends Parasitol.

16. Collins CM, Bonds JAS, Quinlan MM & Mumford JD (2018) Effects of the removal or reduction in density of the malaria mosquito, Anopheles gambiae s.l., on interacting predators and competitors in local ecosystems. Vet. Entomol. 33(1):1–15. doi: 10.1111/mve.12327

17. Timóteo S, Ramos JA, Vaughan IP & Memmott J (2016) High resilience of seed dispersal webs highlighted by the experimental removal of the dominant disperser. Biol. 26:910–915. doi: 0.1016/j.cub.2016.01.046

18. Bartley TJ, McCann KS, Bieg C, Cazelles K, Granados M, Guzzo MM, et al. (2019) Food web rewiring in a changing world. Ecol. Evol. 3: 345–354. doi: 10.1038/s41559-018-0772-3

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ABOL BioBlitz: DNA barcoding safeguards taxonomic knowledge

ABOL BioBlitz: DNA barcoding safeguards taxonomic knowledge

ABOL BioBlitz: DNA barcoding safeguards taxonomic knowledge

The Austrian Barcode of Life (ABOL) initiative uses DNA barcoding to safeguard and make publicly accessible rare knowledge on biodiversity generated in the course of local BioBlitz events
Local fisher presents catch of the day from lake Millstatt PHOTO CREDIT: Susanne Glatz-Jorde, Biosphere Reserve Nockberge

A BioBlitz is an event organized to survey the biodiversity of a designated area as fast and as comprehensively as possible1. Along with the general public, taxonomic experts from various fields, whether laymen or academics, swarm a location to record every identifiable species, usually within 24 hours.

The primary goal of these events is to increase bioliteracy—awareness of biodiversity and its importance for healthy, functioning ecosystems. We believe increased bioliteracy could transform societal perceptions of nature and, ultimately, humanity’s relationship with other species. Consequently, we deem it a prerequisite in our arsenal to abate the dramatic loss of biodiversity2. Additionally, the BioBlitz approach is becoming increasingly important for conservation efforts as it generates high-quality biodiversity data while simultaneously enhancing research capacity3.

In 2019, the Austrian Barcode of Life Initiative (ABOL) successfully introduced a new extended BioBlitz format to Austria—the ABOL BioBlitz—which combines existing collecting events with DNA barcoding. The organisms acquired and identified by experts during BioBlitz events are subsequently DNA barcoded. Importantly, specimens are photographed and stored in a scientific collection along with their associated metadata to be fully compliant with DNA barcoding standards for reference sequences.

The Great Lakes

Locations of the ‘Days of Biodiversity’ 2019 in Austria. The logos represent the organizing institutions.

Base map from d-maps.com

Although running for the first time, the ABOL BioBlitz efforts were very well received and highly successful. From 2000 individuals comprising 1400 taxa, approximately 1500 DNA barcodes were obtained and these data are now available on BOLD.

The number of individuals per higher taxon provided for DNA barcode analysis in the course of the ABOL BioBlitzes 2019. Sites indicated by colours.

Images from ClipArt ETC

In 2019, ABOL joined six ‘Days of Biodiversity’ with ABOL BioBlitzes in five different federal states of Austria. These events covered very different habitat types, from an organic farmstead in Upper Austria, a valley in the mountainous area in Tyrol, to annually organized events in protected areas like National Park Hohe Tauern or the Biosphere Reserves of Nockberge and Wienerwald. It is important to note that these events are only possible with the generosity and cooperation of the institutional hosts to whom we wish to extend our deepest thanks.

In total 54 taxonomic experts joined our efforts at these events. They not only provided us with samples from their collected material, along with the respective metadata and photos, but they also prepared reference individuals and assured their appropriate storage in public collections. After receiving samples from the experts, the ABOL team transferred tissue into microplates and shipped them to the Canadian Centre for DNA Barcoding (CCDB) in Canada for barcode analysis. We thank all the experts for their incredible efforts as well as staff at the CCDB for their obligingness and cooperation.

Day of Nature in the Biosphere Reserve Nockberge

The 4th Day of Nature named ‘Shores and mountain slopes of Lake Millstatt in Carinthia’ in the Biosphere Reserve Nockberge contributed substantially to the species inventory of the Carinthian part of the Biosphere Reserve. 45 taxonomic experts recorded 1166 species, some rare while others new records for the Biosphere Reserve Nockberge and for Carinthia. The event was organized by the team at the Biosphere Reserve, together with two ecological agencies, Ökoteam and E.C.O.

IMAGE: Local fisher presents catch of the day from lake Millstatt
CREDIT: Susanne Glatz-Jorde, Biosphere Reserve Nockberge

Day of Biodiversity in Upper Austria

The Day of Biodiversity in Upper Austria, organized by the Naturschutzbund Oberösterreich and the Biologiezentrum of the Upper Austrian State museums, took place around the Mühlbergerhof, an organic farmstead covering around 20 ha of species-rich grassland, pastures, and deciduous forest. In addition to the species inventory, excursions with different topics (e.g. moths and bats, plants, fungi and lichens, insects) were offered to the public.

IMAGE: Members of the ABOL coordination team examine collected insects from Mühlbergerhof
CREDIT: Heidi Kurz, Naturschutzbund OÖ

Day of Biodiversity in the Biosphere Reserve Wienerwald

The annual Day of Biodiversity in the Biosphere Reserve Wienerwald took place in 2019 in Pressbaum, Lower Austria. Within 24 hours, 1151 species were recorded. This data forms a valuable basis for scientific research and nature conservation activities in the Biosphere Reserve. Excursions led by experts explored water insects, plants, and birds. More than 30 stands provided information on various topics for adults and children. The festival of biodiversity was completed with regional food, kid’s programs and an open-air concert.

IMAGE: ABOL information stand at the festival of biodiversity in Pressbaum
CREDIT: Michaela Sonnleitner

Day of Biodiversity in Vienna
The Day of Biodiversity in Vienna was the 1st transnational event of its kind within the Interreg project CITY NATURE jointly organized by the Vienna municipality (MA22), the University of Natural Resources & Life Sciences (BOKU), and Bratislava, Slovakia. The collecting event around the BOKU area was complemented by excursions exploring birds, plants, insects & mammals.

IMAGE: Group photo from the closing event with project partner from Bratislava at the Day of Biodiversity in Vienna CREDIT: Barbara Reinwein, MA22

Day of Biodiversity in Tyrol

During the Day of Biodiversity in Tyrol, the Brandenberg Valley attracted many experts and visitors with its diverse natural habitat. A highlight of the event was the demonstration of different types of light traps catching night-active insects. Experts held lectures on butterflies, which fascinated children and adults. Arctia matronula, a rare and locally distributed Noctuidae was among the observed species. The days of biodiversity in Tyrol are organized by the Tyrolean State Museums, the University of Innsbruck and the State of Tyrol.

IMAGE: Expert demonstrates light traps and explains nocturnal butterflies
CREDIT: Michaela Sonnleitner

Day of Biodiversity in the National Park Hohe Tauern

The motto of the 13th Day of Biodiversity in the National Park Hohe Tauern was Summit of biodiversity at the foot of the Großglockner (the highest mountain in Austria). More than 60 experts investigated the species inventory of the Gössnitztal, an elongated alpine valley up to more than 2000 m and the surroundings of Heiligenblut in Carinthia. In addition to plants, insects, birds etc., bats were observed in different altitudes and their call sequences recorded. The event was perfectly organized by the Team of the National Park.

IMAGE: Group photo from the Day of Biodiversity in the National Park Hohe Tauern in Heiligenblut with Großglockner in the background.
CREDIT: Manfred Schmucker

Due to the current Coronavirus pandemic, some of the 2020 ‘Days of Biodiversity’ were cancelled or postponed to 2021, while some will take place but with great care. For the future, we hope to be able to increase the number of participants and, ultimately, the data collected.

The concerted actions of the ABOL team at the ‘Days of Biodiversity’ significantly enhance the value and reach of these local BioBlitz events. For example, our efforts contribute to the completion of DNA barcode reference databases, important resources for society in the long term. They also support taxonomic research by providing the genetic resources important to the morphological determination of species. Additionally, we significantly promote these events amongst the public, raise awareness of the importance of biodiversity as well as build and strengthen bonds within the biodiversity community by enabling a process of mutual learning between cross-disciplinary experts.

As events this past year have shown, combining a BioBlitz with DNA barcoding is a very successful way to obtain both taxonomic and barcode data as well as integrate experts, especially for national DNA barcoding initiatives, which are not always fully funded, as is the case in Austria.

These events also foster a greater appreciation of biodiversity and ecosystems amongst the public and, therefore, are a very successful approach to increase bioliteracy. The currently ongoing biodiversity crisis implies rapidly growing importance of biodiversity data. As taxonomic expertise is increasingly rare in the academic environment, it is becoming more dependent on nature enthusiasts. This knowledge of biodiversity has always received too little attention, although a substantial part of it is held outside of academic institutions. Therefore, we wish to emphasize, that increased appreciation of nature should go hand in hand with that of private taxonomic expertise. ABOL BioBlitzes take all this into account.

References:

1. Baker G.M., Duncan N., Gostomski T., Horner M.A., Manski D. (2014). The bioblitz: Good science, good outreach, good fun. Park Science 31(1): 39–45.
2. IPBES (2019). Global assessment report on biodiversity and ecosystem services of the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services. E. S. Brondizio, J. Settele, S. Díaz and H. T. Ngo (eds.) Bonn, IPBES secretariat.
3. Parker S.S., Pauly G.B., Moore J., Fraga N.S., Knapp J.J., Principe Z., Brown B.V., Randall J.M., Cohen B.S. Wake T.A. (2018). Adapting the bioblitz to meet conservation needs. Conservation Biology 32(5): 1007–1019. https://doi.org/10.1111/cobi.13103

Written by

Michaela Sonnleitner

Michaela Sonnleitner

Sabine Schoder

Sabine Schoder

Oliver Macek

Oliver Macek

Nikolaus U. Szucsich

Nikolaus U. Szucsich

Natural History Museum Vienna, Austria

August 24, 2020

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The butterfly effect: geographic patterns of DNA barcode variation in subtropical Lepidoptera

The butterfly effect: geographic patterns of DNA barcode variation in subtropical Lepidoptera

The butterfly effect: geographic patterns of DNA barcode variation in subtropical Lepidoptera

Forest dynamics, spatial distribution patterns, and sampling scale are associated with mitochondrial DNA variation in Argentinian butterflies

Andean forests in northwestern Argentina.
PHOTO CREDIT: Ezequiel Núñez Bustos

Argentina harbours more than 1,200 species of butterflies, most of them found in two biodiversity hotspots and priority areas for conservation: the Atlantic Forest and the Andean forests1.

Figure 1: Sampling localities in northwestern Argentina (NWA, black squares) and northeastern Argentina (NEA, white triangles)2. The Atlantic Forest (dark blue) extends along the Brazilian coast and reaches its southernmost portion in NEA, while the Central Andean forests (red) descend from southern Peru and reach NWA. The distribution of eight other ecoregions indicated.

Despite their current isolation (Figure 1), these two areas have been cyclically and transiently connected in the past, promoting the interchange of flora and fauna, and resulting in a pattern of disjunctly co-distributed taxa. While the historical relationship between these allopatric forests and its evolutionary effects on shared fauna has been the subject of recent (and ongoing) research, studies have been concentrated mostly on vertebrates.

This study explores the butterflies of the Atlantic Forest and the Andean forests providing new insights into both the diversification patterns in southern South America and the impact of increasing the geographic and taxonomic scale of sampling on DNA barcoding performance in the region.

Atlantic Forest in northeastern Argentina. PHOTO CREDIT: Ezequiel Núñez Bustos

In 2017, we assembled and analyzed a DNA barcode reference library for 417 species from northeastern Argentina (NEA)2, focusing on the Atlantic Forest and covering around one-third of the butterfly fauna of the country. To expand the geographic and taxonomic distribution of this library, we generated DNA barcodes for 213 butterfly species from northwestern Argentina (NWA) with a focus on the Andean forests.

We then used these libraries to examine three themes, outlined below.

1.The effectiveness of DNA barcodes for species discrimination and identification

 

The mean intraspecific distance for the butterflies of NWA was 0.29%, while mean interspecific distance among congeneric species was 7.24% (Figure 2). More importantly, mean distance to the nearest neighbour (7.56%) was nearly 13 times larger than the mean distance to the furthest conspecific (0.60%), resulting in a distinct barcode gap for all but two species represented by two or more individuals (Figure 2).

Genetic distance or sequence variation in the COI sequences within and between species was estimated using the Kimura-2-parameter (K2P) model of nucleotide substitution

Substitution models describe the process of genetic variation through fixed mutations, constituting the foundation of evolutionary analysis at the molecular level.

Arenas M (2015) Trends in substitution models of molecular evolution. Frontiers in Genetics 6(319). 

Figure 2: Frequency histogram of COI sequence distances within species (orange) and among congeneric species (blue) of butterflies in NWA. The inset graph shows the barcode gap analysis for species represented by two or more COI sequences, where each dot represents a specimen. Red dots correspond to individuals with a maximum intraspecific distance higher than the distance to the nearest heterospecific. The vertical dashed line shows the 95th percentile of all intraspecific distances (2.02%), while the horizontal line corresponds to the lower 5% of all congeneric distances (3.36%).

Consistently, sequence-based specimen identification simulations showed that this library is extremely effective in the identification of the butterflies of NWA, exceeding a 98% success rate regardless of the identification criteria implemented.

We then used different clustering algorithms to assess the presence of cryptic species. Overall, these methods generated between 1.4–9.9% more Molecular Operational Taxonomic Units (MOTUs) than the number of reference species, suggesting that the butterfly diversity of NWA might be higher than currently recognized.

Figure 3: Taxonomic coverage of the complete DNA barcode reference library for the butterflies of Argentina. Dark shading indicates the proportion of species covered within each family based on the total known for the country.

Merging the NWA and NEA databases resulted in a DNA barcode reference library for nearly 500 butterfly species, covering ~40% of the butterfly fauna of Argentina (Figure 3) and representing 549 barcode clusters (BINs) on BOLD (170 of which are new to the platform).

2.The impact of increasing the spatial and taxonomic coverage on DNA barcoding performance

When we compared the two reference libraries, we found that the barcode gap was significantly narrower in the NEA than in the NWA library (Figure 4). This is most likely associated with the higher geographic and taxonomic coverage of the former, since expanding the spatial scale of sampling is expected to not only increase intraspecific variation as a result of isolation by distance but also reduce interspecific divergences as more closely related species appear.

Figure 4: Maximum intraspecific distance (blue) and minimum interspecific distance (red) for the three datasets. Note the different scales.

When we tried to identify specimens from NWA by using the reference library of NEA, a considerably high proportion of individuals representing shared species between these regions could not be identified or resulted in an ambiguous identification, even when we allowed a maximum intraspecific distance of as high as 2% in the identification procedure. This was due to the existence of deep intraspecific divergences between conspecifics from northeastern and northwestern Argentina, two regions separated on average by more than 1,000 km.

At the same time, however, we observed that the effect of increasing the geographic (and taxonomic) scale was more profound on the minimum interspecific distances than on the maximum intraspecific distances. Therefore, it is possible that butterfly species in NEA are also naturally more variable than in NWA based on our current sampling. While specimens from NWA came almost exclusively from the montane forest on the east slope of the Andes, the sampling in NEA covered not only larger geographic distances but also a more heterogeneous landscape, characterized by the existence of different ecoregions (Figure 1) and physical barriers such as river, specifically the Paraná-Paraguay River axis. Regardless, our results show that both large geographic distances and increased taxonomic coverage can affect DNA barcoding identification performance, especially when using a local library to identify the fauna from another distant region.

As expected, the maximum intraspecific distance was significantly higher and minimum interspecific distance was significantly lower in the complete database (NEA + NWA) than within the NWA and NEA libraries alone (Figure 4). However, the logical and anticipated reduction in the barcode gap did not have, in this case, a significant impact on the identification performance of DNA barcodes, which were able to correctly identify ~99% of the individuals. This reflects the importance of increasing the spatial and taxonomic coverage of DNA barcode libraries to improve identification success, and of considering the use of a local database to identify regional fauna when a more comprehensive COI database is not available.

Doxocopa cyane burmeisteri
Doxocopa cyane burmeisteri
Parides erithalion erlaces

Parides erithalion erlaces

Pteronymia ozia tanampaya

Pteronymia ozia tanampaya

Butterfly species from the Andean forests. PHOTO CREDIT: Ezequiel Núñez Bustos

3.Geographic patterns of intraspecific variation across Argentina

A total of 135 butterfly species are shared between the databases of NEA and NWA. Mean intraspecific distance for these species was significantly higher between regions (1.02%) than within them (NEA, mean 0.35%; NWA, mean 0.33%), especially for a subset of 43 species that showed particularly deeper distance (mean 2.43%) between NEA and NWA.

We then focused only on the 85 species that are present in both the Atlantic Forest and the Andean forests (Figure 5), 27 of which have a disjunct distribution between forests, being absent from intermediate ecoregions, while the remaining 57 have a continuous range across northern Argentina.

Figure 5: Proportion of shared species between NEA and NWA that occur in both forests. The spatial distribution pattern (disjunct vs continuous) and the percentage of species with a deep intraspecific divergence between forest populations indicated.

We found that mean intraspecific distance between forest populations was significantly higher for the disjunctly distributed species (1.65%) than for species with continuous ranges (0.78%), showing that spatial distribution patterns have an influence on the level of intraspecific variation. Moreover, the proportion of species showing the deep divergence between populations from the Atlantic Forest and the Andean forests was notably higher among species with fragmented distributions (nearly 50%) than for species with continuous ranges (less than 30%) (Figure 5).

Lastly, based on standard molecular rates and COI sequence divergence, all diversification events between forest populations were dated to the last two million years, a time period when the currently isolated Atlantic Forest and Andean forests experienced multiple transient connections across the open vegetation corridor, a diagonal of more open and drier savanna-like environments (Caatinga, Cerrado and Chaco) that isolates the Atlantic Forest from the Andean forests (and the adjacent Amazonia) (Figure 1). These past connections were promoted mainly by Pleistocene climatic changes and habitat shifts.

Catonephele numilia neogermanica

Catonephele numilia neogermanica

Callicore hydaspes

Callicore hydaspes

Doxocopa agathina vacuna

Doxocopa agathina vacuna

Butterfly species from the Atlantic Forest.
PHOTO CREDIT: Ezequiel Núñez Bustos

Conclusions

Our study has not only expanded the DNA barcode reference library for the butterflies of Argentina, but it also constitutes, to our knowledge, the first multi-species assessment of the historical relationship between the currently isolated Atlantic Forest and Andean forests using butterfly species as model organisms.

Importantly, our research supports the fact that, even in the era of genomic data, large-scale analyses of mitochondrial DNA variation are still extremely useful for evolutionary studies, as they unveil spatial diversification patterns and highlight cases that deserve further investigation.

ACKNOWLEDGEMENTS:

We thank our colleagues from the Museo Argentino de Ciencias Naturales and the staff at the Centre for Biodiversity Genomics (CBG) for their help during different stages of this ongoing investigation. We also thank Michelle D’Souza for her helpful comments and suggestions that improved this contribution. This project is supported by Richard Lounsbery Foundation, the CBG, the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), the Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación, Fundación Williams, Fundación Bosques Nativos Argentinos and Fundación Temaiken. For granting the permits and transit guides, we thank the Offices of Fauna of the Argentinian provinces in which fieldwork was conducted, the Administración de Parques Nacionales, and the Ministerio de Ambiente y Desarrollo Sostenible from Argentina.

References:

1. Klimaitis J, Núñez Bustos E, Klimaitis C, Güller R (2018) Mariposas-Butterflies-Argentina. Guía de Identificación-Identification Guide. Vazquez Mazzini Editores. Buenos Aires. pp. 327.

2. Lavinia P, Núñez Bustos E, Kopuchian C, Lijtmaer D, García N, Hebert P, Tubaro P (2017) Barcoding the butterflies of southern South America: Species delimitation efficacy, cryptic diversity and geographic patterns of divergence. PLOS ONE 12(10), e0186845. https://dx.doi.org/10.1371/journal.pone.0186845

Written by

Natalí Attiná

Natalí Attiná

Ezequiel Núñez Bustos

Ezequiel Núñez Bustos

Darío A. Lijtmaer

Darío A. Lijtmaer

Pablo L. Tubaro

Pablo L. Tubaro

Pablo D. Lavinia

Pablo D. Lavinia

Museo Argentino de Ciencias Naturales “Bernardino Rivadavia” (MACN–CONICET)

July 31, 2020

doi: 10.21083/ibol.v10i1.6256  

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Hunting for a water mite neotype in southern Norway

Hunting for a water mite neotype in southern Norway

Hunting for a water mite neotype in southern Norway

Scientists rediscover lost specimens of water mite in Norway 120 years after they were first described

A stream near the church of Vanse at Lista in southern Norwaythe type locality of Lebertia porosa Thor, 1900.

PHOTO CREDIT: Torbjørn Ekrem

Did you know that scientists can assess natural water quality by monitoring the diversity of aquatic invertebrates? Freshwater insect and arachnid populations are often important indicators of environmental change. This is evident in particularly species-rich groups, such as water mites and biting or non-biting midges, which have great potential for monitoring water quality. The problem is only that they are too difficult and time consuming to identify in routine water quality assessments. This hurdle can be overcome with DNA metabarcoding, but only if a good reference barcode library is available.

Elisabeth Stur of the Norwegian University of Science and Technology (NTNU) University Museum, along with her team, have been doing summer fieldwork for the Water Mites and Midges in southern Norway (Water M&M) project. One of the many goals for this year’s fieldwork was not only to contribute to the reference barcode library, but also to sample the type locality of the water mite Lebertia porosa, described 120 years ago by Sig Thor, a Norwegian priest and acarologist.

The Great Lakes
Phaenopsectra flavipes (Diptera: Chironomidae) with water mite larvae attached. PHOTO CREDIT: Aina Mærk Aspaas, NTNU University Museum

Barcode data indicate that there are at least six cryptic genetic lineages within this species, but it is unknown which of these applies to the nominal species. Since the original type material is lost, re-sampling L. porosa from its type locality is important in designating a neotype that most likely belongs to the species described by Thor in 1900. This way, researchers can stabilize the definition of the L. porosa species name, such that potential new species could be described. This species delineation is part of a MSc. project by Valentina Tyukosova at NTNU: Integrative taxonomy and species delimitation in the Lebertia porosa species complex (Acari, Parasitengona: Hydrachnidia).

The type locality of L. porosa was vaguely described in Thor’s original publication as a “stream near the church of Vanse”. After studying maps of the surrounding area, researchers learned that this church still stands, and were able to locate two nearby streams.

Now they wondered, would these streams still be in good condition 120 years later? As the team of researchers approached what they thought might be the stream in June 2020, they were pleased to see running, clear water under the bridge. Next mystery: could the streams hold a population of L. porosa 120 years after first collection? They found that yes, the waters could, and the water mite populations were bountiful!

The Great Lakes DNA Barcoding Project team

Water mites from the type locality of Lebertia porosa Thor, 1900.
PHOTO CREDIT: Torbjørn Ekrem

Stur and her team are now looking forward to getting these critters under the compound microscope. Using DNA analysis, they hope to identify which barcode clusters they match with, potentially revealing the nominal species of L. porosa. We’re sure that Sig Thor would be thrilled to learn that his identified species is still thriving, 120 years later.

Written by

Katherine Perry

Katherine Perry

Centre for Biodiversity Genomics, Guelph, ON, Canada

July 24, 2020

doi: 10.21083/ibol.v10i1.6243

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