School Malaise Trap Program - Fall 2015

We were scrambling a little in the last weeks but here they are (and all the participating schools have them already) - the results of our School program fall run:

In 64 traps students collected 750 specimens on average for the two week period in September. Our collections group sorted 47,711 specimens and selected 16,798  to be barcoded. Our final dataset was made up of 15,043 DNA Barcodes (not all  worked and short barcodes were discarded). Using BOLDs BIN analysis we could  determine that the record number of 3,515 species were collected over the two week period of the program, 247 of which were brand new to BOLD. The map below shows all of these collection sites, which include elementary schools, secondary schools, and comparison sites (blue markers). You might notice that we also had some participants from the US. A shout out to our friends from some of the San Diego Libraries.

Our overall pie chart shows the typical species composition (largely grouped in orders) although this time the dipterans are not as predominant as they were in other years and runs. We caught almost as many different hymenopteran species. Mind you this is not a figure that corrects for abundance as flies clearly dominate each catch by their numbers.

Once more a great accomplishment by a large number of young citizen scientists. After three years still many surprises and new finds. Impressive.

Winter Bulletin is out

It's time for our Winter Issue of the Barcode Bulletin. For me that also means that I am about to head out for two weeks break. Consequently, posting will be rather light before we pick up speed again in early January.

Christmas greetings will follow in another post closer to crunch time. Meanwhile enjoy reading our jam-packed issue:

Citizen science to protect trees

Ablaze with colour in autumn and carpeted with bluebells in spring, native woodlands are one of Britain's most prized natural treasures. New research is showing that the public together with scientists can play a vital role in their protection. 

A study, carried out by scientists at Forest Research and Rothamsted Research in the UK, involved a survey of the Acute Oak Decline (AOD) disease which has affected oak trees across England and Wales. AOD reduces oak trees' ability to take up food and water, and has the potential to kill trees in 4-6 years. This threatens indigenous woodland and the many species of plants and animals that rely on oak trees for food and protection.

To make the survey even more far-reaching, the researchers also gathered sightings of AOD reported by concerned public volunteers. These 'citizen scientists' followed online instructions, and some even had training from Forest Research professionals on how to identify disease symptoms and to take non-destructive swab samples for experts to verify. The confirmed public sightings were compared with those of the scientific survey to determine how reliable they were.

This research provided a unique opportunity not only to map the known extent of AOD in the UK but to compare the results from historical and current records held by FR which were submitted by citizen scientists to that of data from a systematic and scientifically robust survey.

The results showed clear similarities between reports gathered from the scientific survey and those verified from the public volunteers. Both volunteers and scientists found AOD in central and southern England, whereas it was rare in Wales and in more northern and southwest areas of England. Help from the public can therefore play an important role in informing policy and management of woodland conservation, such as in helping scientists to predict which areas are at risk of AOD and other tree diseases, and preventing their spread.Consequently the researchers want to continue to involve citizen scientists in their work:

Observatree, an EU-funded citizen science project led by Forest Research which is training volunteers to identify and report tree pests and diseases. This approach can provide an efficient early warning system for pests and diseases, and our findings suggest volunteer detections can also be used to define the distribution of affected woodland.

If Nemo's kids can't find home

The transition from a pelagic to a benthic lifestyle is a crucial phase in the life cycle of many marine organisms. During early stages of development, dispersal of planktonic larvae may be mostly driven by currents; however, larvae progressively develop behavioural and physiological competencies that allow them to locate, orient towards and selectively settle in suitable benthic habitat. Competencies acquired during development include increased body size, development of functional fins and strong musculature, as well as sensory capabilities necessary for navigation and habitat selection. This set of adaptations means that settlement is far from a stochastic event.

Research has shown that acoustic cues help in the settlement of marine fishes and some invertebrates. Electrophysiological studies also revealed that the sense of hearing becomes more important throughout larval development and that at this particular life stage they are able to hear particular frequencies, an ability lost later in life. It is also known that some fish species have a very narrow window of competency for settlement. It seems likely that both findings are connected and that hearing guides little fish toward their future home.

A new study published today provides some more proof for the assumption that the interpretation of normal ocean sound cues helps juvenile fish to find an appropriate home. However, tests conducted as part of the study also showed that fish were completely confused under the levels of CO2 predicted to be found in oceans by the end of the century as a result from Ocean acidification:

Here we show that larvae of a catadromous fish species (barramundi, Lates calcarifer) were attracted towards sounds from settlement habitat during a surprisingly short ontogenetic window of approximately 3 days. Yet, this auditory preference was reversed in larvae reared under end-of-century levels of elevated CO2, such that larvae are repelled from cues of settlement habitat. These future conditions also reduced the swimming speeds and heightened the anxiety levels of barramundi. Unexpectedly, an acceleration of development and onset of metamorphosis caused by elevated CO2 were not accompanied by the earlier onset of attraction towards habitat sounds. This mismatch between ontogenetic development and the timing of orientation behaviour may reduce the ability of larvae to locate habitat or lead to settlement in unsuitable habitats. 

There is a nice little video that explains all this in simpler terms:

Combining Public Health Education and Research

Chagas disease is caused by a parasitic protozoan (Trypanosoma cruzi) and transmitted via the so called kissing bugs (Triatominae) Due to the success of community based surveillance and collection in Central and South America, researchers from Texas A & M University set up a citizen science program to gain insight into the distribution and infection prevalence of the vector insects in Texas.

The researchers used printed pamphlets, phone communication, an educational website with a dedicated email address and local news station announcements to provide public information about the program. Citizens were encouraged to submit triatomine insect specimens. Submitters were informed of the risk of Chagas disease and cautioned not to touch the insects with bare hands. Each submission was required to include the date, time and location of capture and whether the insect was alive or dead.

Each insect was identified to species, measured, sexed and dissected. DNA from the insect gut was extracted and tested for the presence of the parasite. Any insects reported to have fed on humans were sent to the state health department for further processing. Submitters were notified of the species and infection status of their sample. An interactive map of the location of all submissions is published on the project's website.

Over 4000 emails were received from the public over the course of the 2 years, and 1980 kissing bug samples comprising seven species submitted. Of the submitted insects, 25% were found in dog kennels, 19% in patios or porches and 11% inside homes. In total, 375 citizens submitted samples, the majority submitting only one insect. Citizens submitting >20 samples were most likely to have found them in their dogs sleeping quarters. Tests for Trypanosoma cruzi were carried out on 694 of the insects and 63% were found to be infected. The geographic distribution of the insects was similar to previous documentation spanning 8 decades, suggesting that citizen collections are a valid way to document insect distribution in future.

Using public citizens for this kind of initiative means that insects will only be found in places citizens have visited. This does, however, create a unique sample of the insects likely to be encountered by the public, which is a useful sub-set for studies of disease vectors. Using DNA-based identification of samples also negates observer error which can be a risk of citizen identification. Finally the set up and running of the program strengthens relationships between researchers, health departments, disease control centers, clinical vets and the public, and provides a resource of public information on a local illness.

Ballast water

Maritime transport is considered one of the most important ways that native species are moved between marine regions. The trip can be especially successful if these species latch on to the vessel's anchors or chains, or even if they travel in the ship's ballast water tanks. Each year, between 2.2 and 12 billion tons of water are transported around the oceans of the world in these ballast water tanks which also serve as a means of transport for about 7,000 species per day. In a survey colleagues found more than 1,000 taxa of living specimens in ballast tanks of vessels arriving in European ports.

A golden rule for successful invaders is ‘the more tolerant are the more dangerous'. Therefore, migrants that survive long cross-latitudinal voyages within ballast tanks should be of particular concern as potential invaders. Identifying such species is crucial for conducting reliable risk analyses, preventing expansions and developing efficient control methods. However, many species transported in ballast water as eggs or larvae are often very difficult to identify.

In order to identify which organisms are most capable of tolerating non-native waters and are thus the most invasive, a team of researchers from Spain and Lithuania applied metabarcoding of the environmental DNA present in 70 m3 of ballast water of the scientific research vessel Polarstern. The tanks were filled in the North Sea and the vessel traveled from Bremerhaven (Germany) and Cape Town (South Africa). Over the long travel distance the ballast water was subjected to extreme temperature variations in addition to anoxic conditions. 

Organisms that were alive upon entry into the tank in Bremenhaven could have been subjected to conditions of stress likely resulting in their death, thus meaning that the number of DNA molecules within the tank would decrease over the course of a trip. However, this is not what happened to a mudsnail (Peringia ulvae). The researchers found that the number sequences of a particular haplotype increased during the journey.

Although this is not conclusive evidence confirming that the little mollusk is alive but it certainly confirms the resistance of its DNA to adverse conditions. Up until now there has not been any evidence of the presence of this small snail outside of its natural habitat, although some studies have indeed described its ability to tolerate diverse ecological conditions.

The results of this study indicate the high likelihood of species survival in ballast water or ballast sediments on cross-regional voyages. It can therefore be used for the species-specific risk assessments required by the Ballast Water Management Convention and for prioritizing species of greatest management concern.

Citizen Science in Malaysia

Over the past 50 years, Southeast Asia has suffered the greatest losses of biodiversity of any tropical region in the world. Malaysia is a biodiversity hotspot in the heart of Southeast Asia with roughly the same number of mammal species, three times the number of butterfly species, but only 4% of the land area of Australia. Consequently, in Malaysia, there is an urgent need for biodiversity monitoring and also public engagement with wildlife to raise awareness of biodiversity loss. Citizen science is “on the rise” globally and can make valuable contributions to long-term biodiversity monitoring, but perhaps more importantly, involving the general public in science projects can raise public awareness and promote engagement. Butterflies are often the focus of citizen science projects due to their charisma and familiarity and are particularly valuable “ambassadors” of biodiversity conservation for public outreach.

The Peninsular Malaysia Butterfly Count is a great example for citizen science involving school children. We were following their work through Facebook already for a while and it is wonderful that all this work and its results now made it into the scientific literature through an article just published in Pensofts Biodiversity Data Journal.

Congrats to John Wilson and his team - keep up the good work. Here's their summary:

1) The level of participation in the first Peninsular Malaysia Butterfly Count was encouraging, but reaching and engaging rural communities remains a challenge.

2) The non-lethal DNA barcoding approach for species identification worked effectively, however, protocols could be improved to limit the number of returned samples which could not be identified. The family-level identification guide could use some improvement but provides an important educational tool for the participants.

3) The sampled butterflies revealed that widely distributed, cosmopolitan species, often recently arrived to the peninsula or with documented "invasive" potential, dominate the habitats sampled by the participants. Data from the first Butterfly Count helps establish a baseline from which we can monitor changes in butterfly communities in Peninsular Malaysia.

Complementary and alternative medicines

Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds.

A new study just published in Nature Scientific Reports does just that (including metabarcoding) and the results are - perhaps not surprising - pretty alarming. Researchers of Curtin University, Murdoch University and the University of Adelaide found that 90 % of 26 widely available medicines (in Australia) tested were not fit for human consumption. Half contained illegal substances, including toxic metals, prescription medications, stimulants and animal DNA, none of which were listed on the product's label.

Arsenic, cadmium and lead were found in some of the Chinese medicine. One of the herbal concoctions contained over 10 times the recommended daily limit for arsenic exposure. Another contained strychnine, which is used as a rat poison and at lower levels as a performance-enhancing drug. The researchers also found one of the herbal medicines for sale had trace amounts of snow leopard DNA in it. In addition they found DNA from pit vipers, frogs and trace amounts of cat and dog DNA. Whether the animal products were primary ingredients or the result of a poor manufacturing processes is yet to be determined.

The researchers were especially concerned about contamination with undisclosed pharmaceuticals. Over-the-counter drugs like paracetamol and ibuprofen were found but also steroids, blood thinner warfarin and even sildenafil, the active ingredient in Viagra.

Here the summary of the paper:

This study presents genetic, toxicological, and heavy metal data that should be of serious concern to regulatory agencies, medical professionals and the public who choose to adopt TCM as a treatment option. Of the 26 TCMs investigated, all but two can be classified as non-compliant on the grounds of DNA, toxicology and heavy metals, or a combination thereof. In total, 92% were deemed non-compliant with some medicines posing a serious health risk. The multi-tiered approach outlined in this study provides a much-needed auditing toolkit that should swiftly form the basis of best-practice pharmacovigilance across the CAM  [complementary and alternative medicines] sector.

Lots of marine species actually unprotected

The increase in the number MPAs in recent years is encouraging, but most of this increase has come from a few very large MPAs. Those very large MPAs provide important value, but they can be misleading in thinking that biodiversity is being well protected because of them. Species all around the planet need protection, not just those in some locations. 

The first comprehensive assessment of protected areas coverage on marine life says that more than 17,000 marine species worldwide remain largely unprotected

The authors of the study looked at the ranges of some 17,348 species of marine life, including whales, sharks rays and fish, and found that 97.4 percent have less than 10 percent of their range represented in marine protected areas. Nations with the largest number of "gap species" or species whose range lie entirely outside of protected areas include the U.S., Canada, and Brazil. 

Despite these dismal and somewhat shameful results, the authors say the study underscores opportunities to achieve goals set by the Convention on Biological Diversity to protect 10 percent of marine biodiversity by 2020. For example, the majority of species that were considered very poorly represented (less than two percent of their range found in marine protected areas) are found in exclusive economic zones. This suggests an important role for particular nations (see above) to better protect biodiversity.

As most marine biodiversity remains extremely poorly represented, the task of implementing an effective network of MPAs is urgent. Achieving this goal is imperative for not just for nature but for humanity, as millions of people depend on marine biodiversity for important and valuable services.

Combat fish food fraud

Over the years I have presented several studies that showed ongoing food fraud in the seafood trade and how DNA Barcoding helped to unravel these cases. Now a large multi-species survey of fish labeling accuracy indicates a marked reduction of seafood mislabeling in supermarkets, markets and fishmongers in the EU.

Scientists in six European countries tracked samples of the mostly commonly consumed fish, including cod, tuna, hake and plaice, after a series of studies going back 5 years had shown mislabeling in up to 40% of cases.

Of the 1 563 DNA sequence samples examined, just 77 (4.9%) proved to be mislabeled. Most commonly mislabeled was anchovy (15.5%), hake (11.1%) and tuna (6.8%). By contrast only 3.5% of cod and 3% of haddock was  wrongly labeled. 

The study argues that this positive trend is due to a combination of transnational legislation, governance and public outreach, which has forced new regulation and self-regulation, and it contrasts the European 'turn-around' with the experience of the United States, where improvements appear more sluggish.

Genetic identification methods have progressively exposed the inadequacies of the seafood supply chain, raising awareness among the public, and serving as a warning to industry that malpractice will be detected. This evidence indicates we are now on the road to greater transparency, which should help the management of exploited stocks worldwide, but further standardised studies on a greater range of food provision channels, such as restaurants and auctions, are warranted, in order to have a complete understanding of the current state of the trade.

Topical Issue on DNA Barcoding of Fishes

A new special issue dedicated to FishBOL work has been published in the journal DNA Barcodes. Quite a few articles on recent research of the global fish barcoding community. Have a look:

Cods for sale. Do we know what we are buying? 

DNA barcoding increases resolution and changes structure in Canadian boreal shield lake food webs 

DNA Barcoding, species delineation and taxonomy: a historical perspective 

Extending DNA barcoding coverage for Lake Whitefish (Coregonus clupeaformis) across the three major basins of Lake Huron 

An exploration of sufficient sampling effort to describe intraspecific DNA barcode haplotype diversity: examples from the ray-finned fishes (Chordata: Actinopterygii) 

Community engagement in seafood identification using DNA barcoding reveals market substitution in Canadian seafood 

Standards for Conducting a DNA Barcoding Market Survey: Minimum Information and Best Practices 

Status of DNA Barcoding Coverage for the Tropical Western Atlantic Shorefishes and Reef Fishes 

A case study of the forensic application of DNA Barcoding to sharkfin identification in the Mexican Pacific 

Molecular identification of Leporinus from the south portion of South America 

Species delimitation in Neoplecostomus (Siluriformes: Loricariidae) using morphologic and genetic approaches 

Use of DNA barcode in the identification of fish species from Ribeira de Iguape Basin and coastal rivers from São Paulo State (Brazil)

Knodus moenkhausii (Characiformes: Characidae): one fish species, three hydrographic basinsa natural or anthropogenic phenomenon? 

A record of the Porbeagle, Lamna nasus, in coastal waters of Buenos Aires (Argentina) confirmed by DNA barcoding 

DNA barcoding Indonesian freshwater fishes: challenges and prospects 

Fishing for barcodes in the Torrent: from COI to complete mitogenomes on NGS platforms 

Adding nuclear rhodopsin data where mitochondrial COI indicates discrepancies – can this marker help to explain conflicts in cyprinids? 

Quantitative metabarcoding

It is a bit of a holy grail for metabarcoding and a dream of many: Using it not only to determine species identities but also for quantifying relative species abundances. It is widely accepted that metabarcoding has its limits and is biased both biologically and technically. For example, chimeric sequences, contaminants and clustering algorithms can bias even the most basic outputs of DNA metabarcoding studies such as species richness. This becomes very problematic if one tries to infer abundance from the proportions of species DNA. Attempts have been made to extrapolate differences in mass or abundance of species through differences in sequence read abundance but biases have been repeatedly reported.

Previous attempts to control biasing factors in DNA metabarcoding studies have primarily focused on correcting for a single source of bias, or altering protocol steps that are known to introduce bias...An alternative approach to correcting for individual biases is to create control materials for target organisms, which when sequenced alongside environmental samples can be used to create correction factors that account for multiple sources of bias simultaneously.

A new study that just showed up in the accepted article section of Molecular Ecology Resources went the latter route and used sequencing of 50/50 mixtures (target species/control species) to establish relative correction factors (RCF) that account for multiple sources of bias and are applicable to field studies.

The colleagues focused on a rather small model system containing a few fish and a small prey library for Pacific harbour seals. However, they also applied the prey library derived correction factors to some wild seal scat samples to determine the impact of the correction method on a real world scenario. Their results show that the 50/50 RCF approach represents an effective tool for evaluating and correcting biases for the model chosen and likely for other studies as well. The authors also clearly state that their new method will not solve the problem for all possible scenarios and that there will be lots of cases where it is simply unrealistic to expect accurate estimates of species proportion based on DNA sequence read abundances.

However, in study systems focused on a limited number of species which have conserved barcode priming regions, 50/50 RCFs offer potential to improve proportional estimates by accounting for multiple sources of bias. The 50/50 RCF approach will be particularly useful when biases to sequence read abundance are substantial and the resulting species correction factor magnitudes are large. Even when it is not possible to generate a complete tissue library, a 50/50 RCF library consisting of a subset of key species could be used to screen for large species-specific biases and aid in the interpretation of sequencing results.

We're crowdfunding!

Dear Colleagues,   Today is Giving Tuesday, the global day of giving, and we are excited to announce that this year the School Malaise Trap Program will be hosting a crowdfunding campaign through the University of Guelph!    Over the past three years, the program has become an outstanding success through the constant dedication of educators, students, and supporters of the Biodiversity Institute of Ontario. Through our generous funders we have been able to reach nearly 15,000 students and over 250 schools across Canada! Furthermore, the scientific findings associated with the program are quite impressive. Over 6,500 arthropod species have been collected by participants, with 1041 of those species being new to BOLD.   As resources diminish, we are asking for support from generous donors like you so that we may keep the program alive! This year we are hoping to raise at least $10000 in order to keep the program free for participants (average cost = $1000 per participant). For more information about our program costs and our goals, please click here.   Every little bit helps! If each person who receives this email donates $15.43, we would be able to reach our goal TODAY! We ask that you kindly consider donating as well as spreading the word by forwarding this email or sharing it via social media.    To donate, please click here. All donations are eligible for a tax receipt, and you can feel good about continuing to bring modern biodiversity science to students and educators across Canada. We thank you in advance for your contribution to keep this valuable educational opportunity alive!     Sincerely, The School Malaise Trap Program Team In the past three years, over250 schools and almost15,000 students have contributed 68,000 specimens, representing6,500 different species, to BOLD (BIO’s online barcode reference library). 1,041 of these species were NEW to BOLD, having never been DNA barcoded before! Learn More about the School Malaise Trap Program