Wednesday, June 29, 2016

The demise of a stonefly

Much remains unknown about the genetic status and population connectivity of high-elevation and high-latitude freshwater invertebrates, which often persist near snow and ice masses that are disappearing due to climate change.

The four members of the stonefly genus Lednia are only known from high-elevation regions of western North America, e.g. Lednia tumana  is endemic to the Waterton-Glacier International Peace Park region of Montana, Alberta, and British Columbia. These species are adapted colder water temperatures as they occur in meltwater streams below glaciers, permanent snowfields, and alpine springs. Global climate change is expected to have dramatic effects on the structure and function of freshwater and the rise of ambient temperature has been correlated with increases in stream temperatures. Here we might have a good example of what might happen to a species that is adapted to colder temperatures. The region in where Lednia tumana is found used to be home to about 150 glaciers (around 1850), 125 of those have disappeared, and if we can believe the latest predictions based on current warming trends it will take less than 15 years for the remaining 25 to vanish as well. This will reduce the amount of suitable habitat for this stonefly by over 80% .

A group of US researchers now explored population genetic structure of this species over the last decade by using DNA barcodes and cytb sequence data. They collected nymphs and adults in 2010 and supplemented with those with older samples collected in 1997, 1998, and 2005. The results were quite sobering: The dominant haplotype was common in both time periods, while the second-most-common haplotype was found only in historic samples, having been lost in the interim. The 2010 populations also showed reduced gene and nucleotide diversity and increased genetic isolation. Our results imply small effective sizes, increased fragmentation, limited gene flow, and loss of genetic variation among contemporary L. tumana populations, which can lead to reduced adaptive capacity and increased extinction risk. This study reinforces concerns that ongoing glacier loss threatens the persistence of L. tumana, and provides baseline data and analysis of how future environmental change could impact populations of similar organisms.

Today all the newspapers and outlets are talking about Adélie penguins and the fact that habitat loss caused from warmer water and loss of sea ice could bring a 60% population decline of this penguin species by 2099. A new study shows that warming in Antarctica, once beneficial to the penguins, has reached a tipping point and is causing the sharp decline. However, as per usual nobody is talking about something as uninteresting as a stonefly and the fact that it is perhaps already too late to reverse the developments that will eventually lead to its extinction. 

Tuesday, June 28, 2016

Courses at Transmitting Science

Sharing knowledge between scientists is our objective. New methodologies need to be learned, and new ideas must be shared and debated in order to advance scientific knowledge.
Our courses cover a wide range of research methods and are aimed at postgraduate students, postdoctoral researchers and established academics from all over the world.

Transmitting Science started in 2012 to manage and offer courses and workshops on different topics related to Life Sciences. The organisation is based in Barcelona, Spain most of the course are held in the region although they also offered some in collaboration with colleagues in Argentina and Canada.

I find their portfolio striking and many courses offer up-to-date training in highly relevant analytical methodologies. Certainly worth a look for a lot of us.

Monday, June 27, 2016

5 Million barcodes

Did anybody notice? Perhaps not, as it happened with no celebratory noise or big announcements. A few days ago BOLD surpassed the 5 Million Barcode mark. The species count is very close to 550,000.

In the days of High-Throughput Sequencing it is not a big thing to upload Millions of sequences to one of the short read archives on the large genomic databases but hosting 5 Million DNA barcode records is a very different thing. These records come with a wealth of metadata, invaluable information that connects a short sequence to a specific specimen, a barcode voucher. 

By now everybody who uses DNA barcoding in one way or another should know that BOLD is the go-to site for DNA-based identification and barcode data management and analysis. Over the last 12 years it served as the central informatics platform for DNA barcoding and played a crucial role in assimilating and organizing data gathered by the international barcode research community. It is a very powerful workbench and analytics tool even more powerful now with more than 5 Million records under the hood. 

Behind every record stands a precious specimen, collected, databased, imaged and sequenced by researchers working on the vision of a bio-literate world. So, it is not only time to congratulate the BOLD team to this important milestone but also the large international barcoding community that has been tirelessly building this invaluable source of biodiversity knowledge. 

Thursday, June 23, 2016

What's inside a rumen?

Roe deer fawn (Capreolus capreolus)
Many central questions in ecology focus on the use and selection of food by individuals, populations or species. Particularly, in large herbivores the applications of such knowledge of the diet is wide ranging. For example, management related questions and the consequences for commercially valuable crops and trees are dependent on knowledge about diets. Most studies of wild ungulate diets use traditional techniques such as macroscopic ruminal, or histological fecal analysis. However these techniques are hampered with inherent methodological problems that involve inaccurate determination of fragmented plant material or simply missing the very small fragments.

This likely introduces bias related to digestibility. Some food items are digested faster than other components and often the latter remain the only identifiable bits for macroscopical ruminal analysis. Quantitative comparisons of diet components based on such analyses will only be of limited used for interpretations of relative importance of food items. In a new studies colleagues compared "metabarcoding" to macroscopic identifications of rumen contents in two species of wild free-ranging ungulates. I set the term metabarcoding in quotation marks simply because the authors did not use a standard plant barcode marker but rather the trnL (UAA) intron. The locus has been used successfully in the past but it is not a DNA barcode.

The authors used Ion Torrent PGM system to analyze diet composition based on rumen content from two large herbivores living in sympatry, roe deer (Capreolus capreolus) and fallow deer (Dama dama), and compared it to a classical macroscopic method to determine food items.

Overall we found a greater number of identified taxa when using DNA. This may reflect the fact that food material stays in the rumen for only a few hours before it is ruminated and later passed along to the stomach. Thus, after rumination some plant species might become unidentifiable macroscopically, while they still are detectable using DNA. Thus, using DNA methods may also increase the window of time for detection of some food items. 

Overall, the high-throughput sequencing data did prove to be more sensitive and taxonomically more reliable. More taxa could be identified and it showed a positive correlation with the amount of biomass identified traditionally, suggesting that the number of DNA sequences obtained may be used as an indicator for how much biomass was consumed.

Wednesday, June 22, 2016

Plant virus detection

Plant viruses in the genus Begomovirus, family Geminiviridae often cause substantial crop losses. These viruses have been emerging in many locations throughout the tropics and subtropics. Like many plant viruses, they are often not recognized by plant diagnostic clinics due in large part to the lack of rapid and cost effective assays. 

When a plant gets infected with a pathogen, it often shows very general symptoms. An accurate diagnosis is essential to knowing which disease one is looking at. However, a number of different pathogens can cause the same symptoms and there are about 1,600 plant viruses out there. BEcause viruses can't be cultured, plant pathology labs use PCR and RT-PCR based tests to detect a part of the virus. Although used regularly in research these tests can be time-consuming and expensive. As a consequence diagnostic laboratories don't test for viruses, and the diseases go unmanaged or managed incorrectly, which is expensive for the grower. 

In a newly published study UF/IFAS scientists examined several ways to detect DNA of begomoviruses. These viruses have emerged over the last 30 years to become plant pathogens that threaten crop production in tropical and sub-tropical regions globally. The researchers found that recombinase polymerase amplification identified the cause of a disease faster and cheaper than commonly used assays.  RPA relies on the extension of primers induced by recombination proteins. These and DNA binding proteins bind the primers and scan for their target. The primers recombine with the target, and a mesophilic polymerase (Bacillus subtilis DNA polymerase I) extend the 3’ end of the invading primer using the opposite strand as a template. As opposed to PCR these reactions can be done at low temperatures (25 °C to 42 °C), with amplification in as short as 15 min which means that there won't be a need for thermocyclers.

The colleagues conclude: RPA could prove useful for the cost effective detection of plant viruses by plant diagnostic clinics. It can be performed in one hour or less with a reagent cost similar to that of PCR but with a lower labor cost, and with an acceptable level of sensitivity and specificity.

Perhaps something to look into not only for plant virus detection.

Tuesday, June 21, 2016

Phlebotomine sand fly identification using HTS

Lutzomyia longipalpis
Phlebotomine sand flies are hematophagous dipterans of primary medical importance. They
represent the only proven vectors of leishmaniasis worldwide and are involved in the transmission of various other pathogens. Studying the ecology of sand flies is crucial to understand the epidemiology of leishmaniasis and further control this disease. A major limitation in this regard is that traditional morphological-based methods for sand fly species identifications are time-consuming and require taxonomic expertise. DNA metabarcoding holds great promise in overcoming this issue by allowing the identification of multiple species from a single bulk sample. 

According to the WHO about 12 million people worldwide are currently infected with Leishmania. Each year about 20000 - 50000 people die from the disease and 2 million new cases are reported annually. About 200 million people live in areas where the disease is common. The disease is transmitted by the bite of infected female phlebotomine sandflies which can transmit the protozoan.

The sandfly genus Lutzomyia genus currently includes 477 described species, among which 56 are proven or suspected vectors of Leishmania. A new study of colleagues from France and French Guiana now assessed the reliability of mitochondrial 16S rRNA sequences using paired-end sequencing on a MiSeq platform. They call their approach metabarcoding but I beg to differ as 16S is not a barcode marker for animals. Not sure what to call it and I also disagree with their assessment that  the standard COI barcode is however not well suited for metabarcoding applications. There are a number of good studies that show that this claim is rather premature. The authors actually suggest the construction of a further reference library for 16S rRNA. 

Nevertheless, the study as such is very good. It is based on a newly constructed reference database for 40 species found in French Guiana. The colleagues successfully experimented with sand flies mixtures of known content and with field samples caught in a 1 ha forest plot in French Guiana. Given the serious basis for the study every progress in species identification should first and foremost be considered a very positive development and disagreement on markers is rather a question of efficiency and community standards but one that should be discussed further.

Thursday, June 16, 2016

BID funding for sub-Saharan Africa announced

In 2015 the European Union announced funding for the multi-year, €3.9 million program Biodiversity Information for Development (BID), led by GBIF. Its aim is to increase the amount of biodiversity information available from the so-called APC (African, Caribbean and Pacific region) nations. The first call for proposals from sub-Saharan Africa, brought in 143 initial concept notes for projects coordinated from 34 African countries. The GBIF Secretariat selected 23 projects with a total funding of €1 million and announced them earlier this week.

The selected projects range across not only diverse geographies but also many thematic areas. The proposed collaborations will seek to build institutional capacity, to mobilize data on Red Listed species and targeted groups of species, and to digitize and share data from museum collections, parks and protected areas. Others harvest species data from activities focused on human health, food security and the reduction of emissions from deforestation and forest degradation.

Here they are:

Wednesday, June 15, 2016

Post-Doctoral Fellowship University of Minho

Expressions of interest for applications to FCT fellowships:
Post-Doctoral Fellowships in marine metabarcoding
Centre for Molecular and Environmental Biology (CBMA),
Department of Biology, University of Minho, Braga, PORTUGAL

Research Project:
We have been recently granted a 3–year research project titled: “NEXT-SEA: Next generation monitoring of coastal ecosystems in a scenario of global change” (Structured–Project R&D&I Norte-45-2015-02). We are seeking for individuals interested in preparing applications for a Post-Doctoral Fellowship in the scope of Next-Sea, to submit to the 2016 fellowships call from Fundação para a Ciência e Tecnologia (FCT), which is open from June 15 to July 15, 2016

Tasks and profile of the Post-Doc Fellow (PDF):
We seek for fellows holding a Doctorate degree for no longer than 6 years, with a relevant CV and a good publication record. Candidates should have preferably a background in marine ecology and/or molecular ecology, although other closely related backgrounds may be considered. Experience in sampling design and fieldwork, and in molecular analyses and bioinformatics are highly valued. Candidates must also be fluent in English.

The fellow will be hosted by the CBMA, a research unit graded Excellent (FCT 2014), located at the Department of Biology, University of Minho. We offer the opportunity to engage in a cutting-edge multidisciplinary project and to work in the new research institute IB-S (Institute for Science and Innovation for Bio-sustainability). The project will also provide opportunities for international partnerships, and will involve travelling within the country and abroad.

Applications and inquiries:
Candidates should send a single Word or PDF file by email until the 20th of June containing: 1) a short motivation letter 2) detailed CV with list of publications and 3) indicate two researchers that may provide references. Foreign researchers with suitable profile are welcome to apply.

CBMA, Departamento de Biologia, Universidade do Minho, 4710-057 Braga,

Friday, June 10, 2016

DNA barcoding to prepare for the invasion

Puccinia psidii or better known as myrtle or guava rust is a fungus with a wide and expanding host range within the tree family Myrtaceae, with over 440 host species currently known, 250 of those were reported from Australia only. It is native to Brazil but since the late 1970s it has spread into continental US, most of the Hawaiian Islands, Japan, Australia, China, South Africa, and Indonesia. 

Unlike other plant pathogens, rust usually affects healthy plants. An infection may cause deformities such as growth retardation, witches brooms, stem canker, or formation of galls. Plants with severe rust infection may appear stunted and discoloured. Rusts are considered one of the most harmful pathogens to agriculture, horticulture and forestry, and they are very hard to treat.

New Zealand has been afforded the opportunity to prepare for a myrtle rust invasion. The Ministry for Primary Industries' (MPI) High Risk Site Surveillance programme is tasked with providing early detections of unwanted plant pests of tree species present in New Zealand, and the programme has been put on a myrtle rust alert. The identification of species of Myrtaceae by morphological means can be time consuming and often requires a combination of characters, which may not all be present at a particular time. The more rapidly species of Myrtaceae can be identified, the more rapidly surveying for myrtle rust and a response to an incursion can take place. Accurate surveillance of infected hosts will enable MPI to consider appropriate biosecurity measures in order to protect threatened plant species and vegetation communities and to reduce the risk of spread to new areas by workers and visitors.

In an attempt to prepare for a potential but also likely incursion to New Zealand a team of researchers has started to assemble a DNA barcode reference library for more than 100 species of Myrtaceae occurring in New Zealand. The team used the internal transcribed spacer (ITS), the external transcribed spacer (ETS) and the barcode standard marker matK. New Zealand has only 24 native species of Myrtaceae but a large number (386) of non-native cultivated Eucalyptus species that are very susceptible to this pathogen.

The Myrtaceae DNA barcode database will enable rapid identification of large numbers of myrtaceous specimens in the event of a myrtle rust incursion. This will help MPI to accurately survey the spread of the disease and thereby enable timely decision making and consideration of biosecurity measures in order to protect threatened plant species and reduce the risk of spread to new areas. 

Thursday, June 9, 2016

Evolution of real glowfish

Bioluminescence, the production and emission of light from a living organism, is a fascinating phenomenon that is documented in over 700 genera of metazoans across the tree of life, with the vast majority living in the ocean. Among vertebrates, bioluminescence has evolved in cartilaginous (Chondrichthyes) and ray-finned fishes (Actinopterygii), and it is not observed in any lobe-finned fishes or tetrapods.

Bioluminescence is  a form of chemiluminescence produced in living organisms. The substrate luciferin reacts with oxygen and the enzyme luciferase and the reaction releases a photon:
A new study aimed to determine the number of independent evolutionary origins of bioluminescence in ray-finned fishes. The authors discovered that bioluminescence has evolved many more times than previously hypothesized across fishes and the tree of life. Actually,  the colleagues showed that it has evolved independently 27 times in 14 major fish clades on of which includes the most abundant vertrebrate on Earth, the bristlemouth, numbering in the hundreds of trillions to quadrillions. 

There is a variety of ways bony fish can deploy bioluminescence, such as leveraging bioluminescent bacteria, channeling light though fiber-optic-like systems, or using specialized light-producing organs.  This shows how important bioluminescence is to vertebrate fish that life in the world's deep seas.

You have this whole habitat where everything that's not living at the top or bottom of the ocean or along the edges - nearly every vertebrate living in the open water - around 80 percent of those fish species are bioluminescent. So this tells us bioluminescence is almost a requirement for fishes to be successful.

The study also shows that once an evolutionary line of fish developed the ability to produce light, it tended soon thereafter to branch into many new species. The process is not fully understood and will require further study but there are some ideas:

Our exploration of the macroevolutionary patterns of bioluminescent lineages indicates that the present day diversity of some inshore and deep-sea bioluminescent fish lineages that use bioluminescence for communication, feeding, and reproduction exhibit exceptional species richness given clade age. We show that exceptional species richness occurs particularly in deep-sea fishes with intrinsic bioluminescent systems and both shallow water and deep-sea lineages with luminescent systems used for communication.

Tuesday, June 7, 2016

Pristine landscapes are perhaps long gone

The exhibition of increasingly intensive and complex niche construction behaviors through time is a key feature of human evolution, culminating in the advanced capacity for ecosystem engineering exhibited by Homo sapiens. A crucial outcome of such behaviors has been the dramatic reshaping of the global biosphere, a transformation whose early origins are increasingly apparent from cumulative archaeological and paleoecological datasets.

A new study is taking a new look at how the world's landscapes have been shaped by repeated human activity over many thousands of years. An exhaustive review of archaeological data from the last 30 years  reveals a pattern of significant, long-term, human influence on the distribution of species across all of the earth's major occupied continents and islands. It draws on new datasets using ancient DNA, stable isotopes, and microfossils, as well as the application of new statistical and computational methods.

The authors argue that it is wrong to assume that societies before the Industrial Revolution had little effect on the environment or diversity of species. They show that many living species of plants, trees and animals that thrive today are those that were favoured by our ancestors. Furthermore, large-scale extinctions started thousands of years ago due to over-hunting or change of land use by humans

One of the take home messages of the study is that pristine landscapes likely do not exist anywhere in the world today and, in most cases, have not existed for at least several thousand years. Consequently, the colleagues conclude that in light of their findings and other evidence of long-term anthropogenic change, we need to be more pragmatic in our conservation efforts rather than aiming for perhaps impossible natural states. I am sure that view will spark further discussions about what constitutes a natural state and and how far back in time we want to go to determine our efforts for restoration, conservation, invasive species eradication and so on. The main message of the paper is nicely summarized by the lead author:

Cumulative archaeological data clearly demonstrates that humans are more than capable of reshaping and dramatically transforming ecosystems. Now the question is what kind of ecosystems we will create for the future. Will they support the wellbeing of our own and other species or will they provide a context for further large-scale extinctions and irreversible climate change?

Monday, June 6, 2016

GBIF/GGBN survey

I received this from GBIF and I like to encourage all readers to do the survey. It actually takes less than 20 min of your time:

The Global Biodiversity Information Facility (GBIF) and the Global Genome Biodiversity Network (GGBN) are seeking expert opinions on how to link morphological and molecular biodiversity data, i.e. physical specimens, derived DNA, sequences, observations and other evidence of species' presence and abundance in space and time. This survey targets data users and providers of specimen and sequence information.

We know your time is extremely valuable, this survey should take about 25 minutes. We are particularly eager to receive early responses from those who are able to complete the survey by 15 June 2016, as we plan to report on preliminary results during workshop 9 of the SPNHC/GGBN conference, Documentation of environmental samples and eDNA on 20 June 2016.

To take the survey, please follow this link.

We encourage you to share the survey with your contacts and networks, especially with users and holders of molecular biodiversity data. The survey will run until 15 September 2016, and the final results will be reported at TDWG 2016 Conference in Costa Rica. If you have any questions about this survey, please contact Dmitry Schigel, Thanks in advance for your help.

Gabriele Dröge (GGBN), Siro Masinde, and Dmitry Schigel (GBIF).

Oh no! Researchers think DNA barcoding is dead.

A new week and a new paper I need to comment on. This one comes from one of DNA barcoding's old critics, Malte Ebach, and he and his co-authors take on Big Data and of course DNA barcoding although the latter it is not necessarily Big Data science yet. Actually the barcoding critique is pretty much out of context and as per usual incorrect: 

DNA barcoding has had minor impact in taxonomy, with some benefit for phylogeneticists, ecologists and conservationists; it has largely been superseded by technological advances, namely Next Generation Sequencing (NGS), in which whole genomes can now be routinely sequenced.

Were do I start? Well, DNA barcoding ain't dead and it had a big impact on taxonomy and still has. Actually, I think it is fair to say, that it became a regular tool in many taxonomist's toolbox. After reading the above statement I did a meta-analysis of the DNA barcoding literature searching for species descriptions that involved barcoding as diagnostic tool. I knew I would find some but I was surprised that I was looking at some 500 papers over the last decade. That is anything but a minor impact. 

Ebach also completely ignores other societal benefits of DNA barcoding, e.g. food authentication in his futile attempt to downplay a method he never liked. However, the best is his claim that new technology has superseded barcoding. Nope, that's not true. Actually both barcoding and high-throughput sequencing (HTS) complement each over very well and metabarcoding was probably DNA barcoding's most successful spin-off. Many colleagues around the world are already using HTS (that acronym will soon supersede NGS) in conjunction with organismal barcode reference libraries. 

All this doesn't sound like DNA barcoding's swan song to me.

However, this wasn't even the main story of the paper which is rather a diatribe on the use of Big Data in biology. I will not attempt to criticize the paper from a informatics standpoint. This should be done by an expert but it speaks volumes that Ebach's examples for what he refers to as "Big Data hubris" are 6 and 14 years old which in computer science are ages. Even worse, one is an opinion piece taken from Wired, and another one from the New York Times which today would trigger a lot of questions with respect to an almost homophobic connotation.

There is nothing wrong with cautioning users of the vast amounts of internet information sources, and Ebach actually nicely illustrates the issues of taking information provided by search engines such as Google Scholar at face value. Our new age of communication with ubiquitous information requires new critical thinking skills especially when it comes to the interpretation of such results but to me that is not an issue of the use of Big Data in science as proper research always questions results and correlations. It is actually our job as scientists to question them as much as possible and only if it endures such scrutiny a new scientific finding should be considered likely. And it is our job as educators to teach the new generation of students how to critically evaluate information and analytical results. So, if the intention was to write a piece that should caution us I am afraid that this publication failed to do so and the choice to include DNA barcoding can be at best considered mission-drift or at worst missionary.

Friday, June 3, 2016

New issue of the Barcode Bulletin

Here it is the newest issue of our Barcode Bulletin with the official announcement of the next International Barcode of Life Conference. If you can't wait - their website has a cool counter.

Enjoy reading!

Thursday, June 2, 2016

Metabarcoding for honey authenticity

Let's start today's post with a question: Do you know what melissopalynology is? Perhaps not everyone can answer this question with a clear yes, of course. In short melissopalynology is the study of pollen contained in honey in particular to determine the source of the pollen. Not only is this used to determine the food sources and habitat range of bee colonies but also to combat fraud and mislabeling of honey. There are large value differences between honey derived from particular plants. Some monofloral honey can be much more valuable than honey derived from a mixture of plant species. Also, in the light of current threats to bee health it is crucial to determine if there is sufficient foraging habitat available as it is known that pollen diversity and quantity affect disease tolerance and longevity in bees.

Traditionally honey bee collected pollen is identified through morphological features revealed with light microscopy. However, this requires considerable skill and experience and in some plant families (Campanulaceae, Lamiaceae, Poaceae, Rosaceae) this is hampered by either a lack of characteristic features or high levels of morphological variation. It comes to no surprise that colleagues are looking into DNA-based alternatives such as metabarcoding. DNA barcoding of pollen collected by bees has been successfully done before but that pollen was directly sampled from the animals. 

Researchers from the UK compared DNA metabarcoding utilizing rbcL and 454 pyrosequencing with microscopic analysis for nine honey samples provided by domestic beekeepers in Wales and England (UK). Wales was the first nation in the world to have barcoded all of its native flowering plants and conifers, enabling the identification of any plant species in Wales from the tiniest fragment of leaf, seed or pollen grain. In total 98% of the Welsh native flora (1143 species) has DNA barcodes for rbcL and 90% for rbcL and matK. Such a reference library provides perfect conditions for a metabarcoding approach. I am happy to report that Wales is not alone anymore as DNA barcoding  of Canada's flowering plants is nearly completed as well.

The study results: There was 92% correspondence for the plant taxa that had an abundance of over 20%. However, the level of similarity when all taxa were compared was lower, ranging from 22–45%, and there was little correspondence between the relative abundance of taxa found using the two techniques. DNA metabarcoding provided much greater repeatability, with a 64% taxa match compared to 28% with melissopalynology. DNA metabarcoding has the advantage over melissopalynology in that it does not require a high level of taxonomic expertise, a greater sample size can be screened and it provides greater resolution for some plant families. However, it does not provide a quantitative approach and pollen present in low levels are less likely to be detected.

It is true that at this point metabarcoding is not suitable if one wants to determine pollen quantity and it a known fact that it fails to detect the rare species of a sample. There is some debate around those issues with some people claiming that especially PCR-based metabarcoding assessments can only provide presence-absence metrics while others think that this depends on the HTS platform used.

Anyway, I couldn't agree more with their final conclusion - DNA metabarcoding can be easily and widely used to investigate floral visitation in honey bees and can be adapted for use with other insects. It provides a starting point for investigating how we can better provide for the insects that we rely upon for pollination. I might add that the study clearly shows that we are capable to determine food sources of honey bees through analyzing pollen and other plant remnants in honey for food authentication and safety especially in countries where a large portion of the floral biodiversity has been barcoded.

Wednesday, June 1, 2016

Barcoding with microfluidic dynamic arrays

The family of tephritid fruit flies is very large and contains about 5000 described species. Many species are of major economic importance in agriculture. Some have negative effects, some positive. Various species cause extensive damage to fruit and other plant crops. The genus Bactrocera is globally known for its destructive impact on agriculture. The high morphological similarity between many tephritid species makes identification notoriously difficult and species boundaries are hotly debated.

About 350  species are  considered  economically  important and most of them belong  to  five genera:  Anastrepha, Bactrocera, Ceratitis, Dacus, and Rhagoletis.

China  is  a  globally  significant  importer  of  fresh  produce;  hence,  fruit  flies  are  of  particular concern.  The  very  large  trade  of  fresh  commodities  into  China,  the  country’s  range  of climatic conditions  which  make  it  suitable  for  most  exotic  fruit  fly  species,  and  the  economic and  social importance  of  its  domestic  fresh-commodity  production,  means  that  exotic  pest  fruit flies  pose  a continual and high profile threat to the country. Intercept information from Chinese ports in the last ten years show that fruit flies have been intercepted from baggage and cargo on a staggering 19000 occasions, with each interception consisting of one or more individuals.

Due to the increasing number and diversity of intercepted fruit flies, a high-throughput method combined  with  high  accuracy, high speed, and low cost  has  become  a  necessity  not  only  for China,  but  all  major  trading  countries. A group of Chinese researchers developed a promising approach to this challenge. They designed species-specific  qPCR  primer  and  probe  combinations for  27  economically  important  tephritidae species of the six genera mentioned above. The probes were designed using some 1000 DNA barcodes of fruit fly species available on BOLD. Each primer pair was tested through qPCR. In a follow-up step the colleagues developed a standardized reaction system for detecting all target species based on a microfluidic dynamic  array,  and  also  applied  the method  to  identify  unknown  immature  samples  from  port interceptions and field monitoring.

They were quite successful and were able to properly detect all 27 species in a rather short time (7.5h), using  only  0.2μl  of  reaction  system  in  each  reaction  chamber. All intercepted specimens were correctly identified which they confirmed by rearing and morphological identification of adults.

Microfluidic dynamic arrays have been used in a number of novel applications, such as medical diagnosis, gene expression, genotyping, and GMO analysis but it has not been applied for  species detection  in  plant  quarantine  and  invasion  biology,  where  it  offers  great  potential  for high-throughput and species diversity screening. Available chips can perform as many as 9000 reactions in a single run which makes high-throughput screening a realistic prospect.