Friday, August 31, 2012

The Art of DNA Barcoding

Arts and DNA Barcoding? Yes, this combination works quite well.

Three years ago the renowned artist Josseph Rossano teamed up with Daniel Janzen, Paul Hebert, and the Ontario Genomics Institute (OGI), to engage the public around DNA Barcoding and how it helps to bring new appreciation to the life on our planet.

The resulting exhibit features mixed media sculptures of butterflies, fish and sea life abstractions by Rossano alongside large format photographs of Costa Rican butterflies and  caterpillars by Dan Janzen. Visitors with mobile devices are able to scan barcodes on each sculpture, which then links them to a series of web pages hosted by the OGI. 

As an artist, I strive to distill ideas, concepts, and reality into their bare essence.  My resulting minimalist sculptures, I hope, convey an emotion, ask a question, or direct the viewer on a path of introspection and investigation, as they explore man's impact on the environment.

My series "BOLD" is named for the acronym for the Barcode of Life Data Systems (BOLD) database. The subject of each specimen box is neither real nor is it an accurate representation of the creature it is designed to represent.  The subjects of these sculptures are a jeweled representation of reality that draw the viewer in for a closer inspection.  As the viewer shortens the distance between himself and the sculpture, the specimen becomes increasingly difficult to discern.  The viewer, now confronted with the frustration of being unable to make out exactly what is in the box, discovers the clear and legible text surrounding the specimen.

The sculptures were on display in a number of galleries in the United States and also traveled to the South Australian Museum in Adelaide on the occasion of the  4th International Barcode of Life Conference in November 2011. Some of you might have seen the exhibit there.

In case you haven't and you happen to be around San Diego between October 2012 and February 2013 I highly recommend you pay a visit to the San Diego National History Museum and don't forget your smartphone.

Thursday, August 30, 2012

I spy with my little eye

New successful eDNA experiments

Environmental DNA (eDNA) allows us to detect the presence of organisms without direct observation. Plants and animals shed cellular material in their surrounding environment, and this material can be collected and analyzed. Traces of DNA extracted from environmental samples can be used to determine if a target species has been in the vicinity of a sampling site. 

Generally there are two different approaches that utilize eDNA. The first is to design specific probes (e.g. TaqMan probes for real-time PRC assays) which are used to detect DNA of particular species in environmental samples. This method has great potential for invasive and endangered species detection and has been used e.g. to detect bullfrogs in French wetlands or Asian carp in the Great Lakes

The second approach uses Next-Generation Sequencing technology to gather sequence information from assemblages of specimens. This method has been called "environmental sequencing", environmental barcoding", or even "meta-barcoding". Name it as you please the idea remains the same: An environmental sample is filtered, homogenized, and all DNA is extracted from it. The DNA is amplified with a marker of your choice, e.g. the COI Barcode region, or more species- or group-specific markers or a combination of them. Subsequent massive parallel pyrosequencing will result in thousands of sequences that represent a genetic fingerprint of the ecosystem sampled. This snapshot is limited in its ability to identify all sequences in a given sample but the chance of comparing genetic diversity of different standardized samples at a single locus will help to understand and show community differences. This could include differences between regions, changes over time, or responses to environmental changes. Eventually changes could be monitored by looking at the full picture instead of relying on single representatives such as indicator species.  The challenge lies in the standardization of such datasets that would allow the observation of real differences and similarities in patterns.

Thomsen et al. 2012
Yesterday two papers of researchers from Denmark have been published in PLoS ONE describing the detection of macro-organisms through eDNA from marine water samples. One group was looking for fish and the other for mammals. This is the first time eDNA detection was successfully done with marine samples while it was already applied to terrestrial sediment samples, ancient cave sediments, ice cores, and freshwater samplesThe first group was able to identify 15 different fish species, including both species important for fisheries, as well as species rarely or never recorded by conventional monitoring programs. Interestingly they also detected 4 bird species in their sample among those the Red-throated loon (Gavia stellata) that passes through the sampling region only occasionally during migration. The authors were also able to prove that their eDNA method is as good as or mostly better than methods traditionally used for monitoring fish such as trawl and pots.

The second study tested the potential use of eDNA for genetic monitoring by using specific primers to detect the presence of  the harbor porpoise, Phocoena phocoena, both in a controlled environment and in natural locations. Results for the natural sites were compared to the current method of choice - detection of echolocation clicks by static acoustic monitoring devices. The genetic results were not as consistent as acoustic monitoring but the authors are confident that they will be able to change this by optimization of their protocol and the use of larger sample volumes.

Bottom line is that half a litre of seawater can contain traces of local fish and whale faunas and combat traditional fishing methods - not bad.

Wednesday, August 29, 2012

High resolution melt

Shortly after the heated discussions about DNA Barcoding (Does it work? Is it science?...) abated people started to think about ways to apply it to existing challenges. Two parameters played a big role - cost and speed. Although prices for sequencing dropped over the last years a sequencing platform of any kind still represents quite an investment for a regular research lab, let alone the operating costs. The time it takes to obtain a sequence from a sample can be very short but this highly depends on equipment and workflows. Again, a small lab at a university usually doesn't provide results within a few hours. I am sure that readers with practical lab experience will also assent that it isn't easy to establish a new sequencing protocol in a lab and that it takes a while to have it run smoothly. 

So, what do you do when you need fast, robust and reliable identifications of a handful of species that are not easy to distinguish based on morphology? Ah yes, it needs to be cheap as well.

Elaine Fitzcharles a PhD student at the Scottish Oceans Institute, University of St Andrews is the most recent proponent of a method called High Resolution Melt (HRM). She was looking at a group of Antarctic fish, called Macrourus or rattails. These fish are the most common bycatch species in the toothfish industry and represent a very important prey item of toothfish. They were traditionally thought to consist of three species in the Southern Ocean: M. carinatus, M. holotrachys and M. whitsoni but a study using DNA Barcoding  found two morphotypes of M. whitsoni which differed from each other in their COI sequence, to the extent that they were considered to represent a fourth species. All of them are morphologically very similar and their taxonomic status has been confused, until recently, in part because of a paucity of comparative material. Some characters show overlap, and identification of species had been based on relatively few specimens, using a combination of characters including geographic and depth distributions. An ideal test case for any genetic technique and based on the results of earlier DNA Barcoding work Elaine Fitzcharles was able to develop a HRM protocol that represents a quick and cost effective method of genetic screening and an alternative to DNA sequencing between the four also genetically very similar species.

1.7 Million DNA Barcodes for some 160,000 species have been assembled so far. The study ends with the following paragraph and there isn't much I could add to it.

"HRM analysis can utilise this barcoding resource for primer design and provides a quick, efficient, low cost alternative to DNA sequencing for species identification screening that could increase sampling opportunities and possibilities. This has the potential to greatly expand the scope of research available for both conservation genetics and fisheries management. The simplicity of the technology required and the technique opens up the possibility of routine genetic screening at remote locations such as research stations and onboard ships."

Macrourus whitsoni (source NIWA)

Tuesday, August 28, 2012

A “monstrous and misshapen animal”

Cerataspis monstrosa, on the left, and Plesiopenaeus armatus, on the right. (Credit: Darryl L. Felder)

Have a look at these two animals. Would you think they belong to the same species? They actually do. On the left you see the larva and on the right the adult form. These two couldn't look more different and that's the reason why for over 180 years they have been considered two entirely different species. The larva was called Cerataspis monstrosa and the adult Plesiopenaeus armatus. Both were placed in very different crustacean groups although researchers over the years suggested affinities between C. monstrosa and paeneoid shrimps. However, a final placement was never made mainly due to the scarcity and extreme morphological uniqueness of the 'monster larva'.

Bracken-Grissom et al. 2012
That has changed. A few days ago researchers from Keith Crandall's lab (I believe it was at Brigham Young University at that time - Keith is now at George Washington University) published a paper with results that are currently going through the news.

The larva was only occasionally found in gut contents of fish and dolphins and a recent unexpected find of one individual in some mid-water collections provided the group with tissue suitable for DNA analysis. Based on the above mentioned affinities they also collected and sequenced reference material within the Paeneoida and more specifically one family, the Aristeidae. They sequenced five different gene regions and the resulting phylogenetic tree clearly shows that both species are actually one.

And did they use DNA Barcodes for this find? No - simply because COI failed to amplify in several attempts. That doesn't make this find less exciting. On the contrary, this study once more shows how useful modern DNA technology has become when more traditional methods (rearing in this case) can't be applied.

Monday, August 27, 2012

Shark fins

Over the course of last year a couple of US States and Cities in Canada have decided to ban shark fin products. Canada, which prohibits shark finning, has a heavily regulated shark fishing and trade industry but the import is much less regulated and therefore municipalities such as Toronto, Mississauga, and Nanaimo have started regional efforts to reduce the import. It is a slow process and many communities hesitate as there is opposition to such bans especially from business owners selling shark fin products. A few days ago in an attempt to test what species of sharks are actually sold activists from the Vancouver Animal Defense League tried to obtain shark fin soup samples for DNA testing from a Richmond restaurant owner. Activists and owner got into an argument that went through the press. This again fuels discussions about the pros and cons of such regional bans and their impact on the economy.

Sharks are fascinating but they have been in trouble long before Spielberg's Jaws (which didn't help to make things better). Many humans fear them although there is little evidence indicating that sharks are really very dangerous. Sharks have much more to fear from humans than humans have to fear from sharks.

Most sharks are active predators and eat primarily fish, although the great white shark will also prey upon seals, sea lions and other marine mammals. Some sharks eat bottom-dwelling animals such as crabs, and others scavenge for dead animals. Some sharks such as the whale shark feed rather passively by swimming with their mouth open to collect plankton and small fish. Sharks are high on the ocean's food chain. They typically eat thousands of smaller fish during the course of their lives. As predators, they conserve energy by eating the slow, weak or sick fish in a school. They are, therefore, important to the ecological balance of the marine environment in that they eliminate the genetically weaker animals in a population as well as keeping population sizes in balance. 

From the currently known 30,000 fish species some 1100 are sharks of which 1000 are living in marine waters and 100 occur in freshwater. An alarming 40% of the recorded sharks species at the IUCN are listed as threatened and endangered and for 35% of them sufficient data are missing while only 10 are currently listed on the Convention on International Trade in Endangered Species of Wild  Fauna and Flora (CITES).

Sharks, and their relatives the rays and chimaeras, provide about 1% of world fisheries landings reported to FAO, or some 700,000 to 800,000 tons; perhaps 70-100 million animals. About 60% of these are sharks, around 40% skates and rays. These figures are undoubtedly an underestimate, because many countries under-report their catches and ignore bycatch, discards and recreational fisheries. Actual catches may be twice that reported. Also, there is worldwide concern that the increased demand for shark fins will have a devastating impact on shark populations and stocks. Shark fins are a highly prized commodity in many Asian cultures. Shark fins infer financial status and are traditionally used in soups served at important occasions such as weddings. The increase in middle-class wealth in many shark fin consumer countries has significantly increased the demand for such products; the largest market is Hong Kong. The 2005 FAO estimate of world exports of dried shark fins was US$ 220 million.

 There is strong evidence that populations of sharks and rays have declined considerably in recent times.  This decline is generally attributed to increasing fishing pressure, the effects of which are accentuated by the generally slow growth, late maturity and low reproductive output of most sharks and rays. Other major threats to sharks include habitat alteration, damage and loss from coastal developments, pollution, and the impacts of fisheries on the seabed and food species.

Most shark fisheries around the world are virtually unmonitored and completely unmanaged. However, it is widely recognized that the need for introducing shark fisheries management is now very urgent and that a precautionary approach to the management is essential. One of the major problems associated with monitoring and enforcement is the likelihood of misidentification. This can arise both because some shark and ray species are difficult to distinguish morphologically, and because of ethnic differences in understanding or interpreting common names. Also, little is known of the identity of species in confiscated shark fin catches and processed shark meat, but in recent years genetic approaches have been deployed to resolve this issues. Way back in 2002 Mahmood Shivji and colleagues proposed a multiplex primer method to identify a few shark species from body parts and in 2009 we published a study about  the successful DNA Barcoding of shark fins confiscated in Australia. Over the years researchers have assembled DNA reference Barcodes for 51% of all elasmobranch species (

Unfortunately this work largely went unnoticed and several attempts to obtain funds for a global collaborative project to assemble a barcode library for the remaining species were unsuccessful. The figure above and some text in this blog are part of some of my own unsuccessful grant proposals. They either didn't fit the portfolio of some funding agencies at that time or simply weren't attractive enough. This might have changed though. A collaborative study of the Stony Brook University in New York, the Field Museum in Chicago and Pew Environment Group in Washington, D.C. barcoded samples of shark fin soup in 14 U.S. cities and their results went through the press also triggering the events described at the beginning. They were able to detect the presence of shark in just 32 of 51 bowls of soup. Of those, six could not be linked to a particular species, but the rest were identified. Those identified were all threatened (one endangered) or vulnerable, according to the IUCN Red List. 

The real issue is not the cruel practice of shark finning as such. No doubt it is bad and absolutely unnecessary. However, a ban of shark fins can only be the beginning. Sharks and rays need far more protection from humans as they are also fished for other food items, cosmetics, and pharmaceuticals, the latter leading to products of highly questionable benefit. It is a multimillion dollar business to the expense of these mighty ocean creatures.