Nematodes and genetically-modified crops

Once in a while one comes across applications of DNA Barcoding you would never thought of in the first place although they perfectly make sense. It is nice to see that so many creative researchers are picking up the method and using it for questions they are interested in.

This post looks at the potential of DNA Barcoding for ecological risk assessment of genetically-modified crops. Dutch researchers from the National Institute for Public Health and the Environment were interested in crop induced changes in nematode assemblages in agricultural soils. Nematodes constitute a very diverse group within the soil fauna, feeding on microbes, invertebrates (including other nematodes) and plant roots. Such an intimate feeding relationship with all major players in agro-ecosystems implies that any effect genetically modified crop might have will likely be reflected in the nematode community. Better knowledge of nematode assemblage compositions could be used to extrapolate impacts on soil systems.

Nematodes in a sample

The classical methods to characterize nematode communities is based on morphological identification using light microscopy. However, this method is both very time consuming (on average several hours for 150 individuals per sample) and the resolution depends on the level of taxonomic expertise. For me they are all white to translucent small inconspicuous worms.

The overall goal of the study was to implement DNA Barcoding into a long-term monitoring network for soil impact of pest management products and the use of genetically-modified crops. Three parts are supposed to serve as baseline for future application. A first part represents the control and looks at seasonal fluctuations of nematode communities as a measure for natural variability over time. The second part focuses on the effects of current pest management (bio-fumigation using Brassica juncea) on nematodes, while the last part investigates the effects of genetically-modified crops (pathogen-resistant potatoes and amylase-potatoes) on soil nematode community structure.

The DNA-based approach to identification allowed for the analysis of nematode communities without microscopic pre-selection because it was based on a considerably broad (2,400 taxa) genetic database that covers the majority of terrestrial and freshwater nematode taxa. For the Dutch researchers one of the main advantages of using molecular methods is the time saving aspect as it allows for more intense and frequent sampling schedules.

A whitefly puzzle solved

Bemisia tabaci or maybe not

The silverleaf whitefly (Bemisia tabaci, which is also referred to as Silverleaf whitefly strain B) is one of several whiteflies that are currently important agricultural pests.Their nymphs use parts of their mouth to stab into the plant and consume the plant’s juices. The honeydew they leave behind can induce the growth of sooty molds, which can then reduce the plants ability to absorb light. This results in less growth, lower yield, and poor quality plants.It is thought that the United States alone has suffered crop and ornamental plant damages in excess of $1 billion through this pest.

For 100 years, outbreaks were sporadic and relatively small, but this changed in the mid-1980s with widespread outbreaks occurring across the south western USA. This was odd because the whitefly was well known across the region as a minor pest yet here it was destroying large amounts of crops.  Although there were no morphological differences, molecular and biological data indicated that the outbreak pest was a different species. Using this information, it was proposed that rather than one species, Bemisia tabaci was composed of at least 28 different morphologically indistinguishable species, all separated by at least 3.5% divergence in their DNA Barcode. To date 34 species have been delimited using the same metrics.

These findings are supported by mating compatibility studies. These have shown that crosses between individuals identified as different cryptic species are reproductively isolated to the point that in most cases copulation does not occur and where it does, the resulting progeny are either sterile or reproductively inferior to their parents.

There was only one question left: Who is the the real Bemisia tabaci originally described in 1889?

A group of researchers has now solved this mystery. They were able to sequence a 496 bp DNA Barcode fragment belonging to a single whitefly taken from the original 1889 collection by Panayiotis Gennadius who first described the species. He had travelled to Greece to identify a small fly-like pest that was devastating tobacco crops there. It is clear now that it wasn't this species that invaded the US but rather a species that was provisionally called "Mediterranean". This species has now begun its own global journey of invasion spreading from its Mediterranean home range to at least 10 different countries in Europe.

Bigfoot does not exist!

It is a myth as much as the yeti, the unicorn, mermaids, giant octopus that attack ships and drag them to the bottom of the ocean, the Bermuda triangle, aliens at area 51, little green aliens on Mars, Santa Claus (sorry kids), the Easter bunny and so on. I could probably fill the entire blog post with mystical creatures and suspicious encounters of similar credibility. 

Why would I do that? Because some really bad science is making the headlines again.

A dubious scientist claims to have sequenced Bigfoot DNA. Dr. Melba Ketchum from Nacogdoches (her company) claims:

“Our study has sequenced 20 whole mitochondrial genomes and utilized next generation sequencing to obtain 3 whole nuclear genomes from purported Sasquatch samples. The genome sequencing shows that Sasquatch mtDNA is identical to modern Homo sapiens, but Sasquatch nuDNA is a novel, unknown hominin related to Homo sapiens and other primate species. Our data indicate that the North American Sasquatch is a hybrid species, the result of males of an unknown hominin species crossing with female Homo sapiens."

First and foremost, these results are nowhere published and Ketchum is not sharing them not even more details on the methods used. No reputable scientist makes a press release before a paper has been published. If true any big journal would take the story and insist on doing the PR using their own machinery. Obviously this didn't happen which makes these 'results' not more reliable than any of the shaky videos, blurry images or dubious witness accounts that are called evidence for the existence of Bigfoot.

Good science is shared with the scientific community to allow colleagues a critical view at results and the conclusions. Currently we are left with a pretty much meaningless press release. 

Dr. Ketchum, if you want anyone to believe your findings release them! Until that happens I and hopefully anybody else with some common sense sees Bigfoot as just another hoax. Unfortunately, these 'news' are currently all over the place and many outlets take the outrageous claim for granted. Bye bye critical thinking. Dear press - you shouldn't buy everything just because it says DNA on the label. The fact that these researchers claim they used DNA analyses doesn't mean a thing. The new hominid nuclear DNA might as well be chimeric and the human mtDNA from a researcher that sneezed on the samples. Most importantly, where did the samples come from? I hardly dare ask, but is there a voucher specimen?

That leaves us with two unproven claims. (1) There is such a being like Bigfoot, and (2) the hybrid between an unknown hominid and a human was actually fertile and evolved into a species that survived the last 15,000 years in North America.

I don't buy it!

PeerJ

When I recently did my little number crunching exercise I was also looking at the percentage of DNA Barcoding papers that have been published in open access journals. Among the major ones was of course PLoSONE but there were also a few others that have papers available without subscription fees. However, it only amounts to a total of 12% of the publications that are available to everyone. In turn that means that about 88% of all DNA Barcoding papers are not available to every researcher on the planet. I find that very frustrating as I thought that especially in this new field there is more inclusiveness!

In 2006 Marguerite Holloway published an article in Conservation in Practice in which she tried to capture the innovative thinking behind the at that time very new method called DNA Barcoding. The title was "Democratizing Taxonomy" referring to Dan Janzen and Winnie Hallwachs who believe passionately that greater access to knowledge and technology will transform the future of conservation. I've heard this term from Dan in various of his talks and I am always reminded of it when it comes to publishing as researchers do not live in a free publishing world. This is especially true for all of us in early phases of our careers where an insufficient measure (impact factor) is dictating where to publish. The result is that many researchers, maybe even the majority, have no access to a lot of what we've published over the last 10 years just because they or their institution can't afford the often ridiculously high subscription fees. That's a shame!

The more happy I am to announce the birth of a new journal with a new business model of open access publishing. PeerJ is a new multidisciplinary open access journal that announced today the first formal call for papers. Unique among academic publishers, PeerJ provides authors with low cost lifetime memberships giving them the rights to publish their papers freely thereafter. A basic membership plan is only $99 that would give an author the right to publish once a year for the rest of their life. That seems reasonable and compared to some of the usual fees of other open access journals it is very cheap. Access to the articles is of course free!

Agarwood barcoding

Aquilaria crassna

Aquilaria crassna is a tree species that has been of great ethnobotanical importance to people throughout the Greater Mekong region and beyond. Its heartwood and resin are highly valued commodities that have been transported along long-established trade routes for thousands of years. However, this species has now become Critically Endangered, owing to over-exploitation.

The wood from Aquilaria crassna (Agarwood) contains aromatic resin, known as ‘gaharu’. This is produced by the tree in response to injury if the production of callus tissue is inhibited. It acts as a chemical barrier to attack by insects and fungi. However, under natural conditions gaharu is not produced by trees at all. This resin is often used in temples as perfume wood. The wood can also be distilled to yield a valuable essential oil, which is widely used in Chinese and Southern Asian medicine and also in the Middle East for making perfumes and cosmetics. 

Currently the majority of the wood comes from wild populations and there is now a very real danger this species may become extinct if wild harvesting continues at the current rate. To conserve this species, it is vital that this plant becomes more widely grown in cultivation, to reduce the pressure on the few wild populations that remain. Cultivation requires high quality seed and especially in Vietnam there is concern that a range of hybrid species or other members of the genus could lead to production loss and disappearance of the original species. Proper species identification starts with the seeds used and Vietnamese researchers have now tested if DNA Barcoding could help. Not surprisingly it worked quite well and one of the official plant markers, rbcL along with ITS was tested most effective.

Probably it needs to reemphasized that DNA Barcoding cannot identify hybrids (oh well, I probably should say mostly, but that's a different story...) but it can help to distinguish between seeds of different Aquilaria species obtained in the wild and destined to be used in cultivation.

Helpful weevils

Eurasian milfoil (Myriophyllum spicatum)

Eurasian milfoil (Myriophyllum spicatum), a submerged aquatic plant that grows in still or slow-moving water, is one of the worst aquatic plant pests in North America. Like native aquatic milfoils, it has feather-like underwater leaves and emergent flower spikes. Usually leaf shape and size can be used to distinguish it from other milfoil species. However, Eurasian milfoil is a variable species, often making it difficult to identify without chemical or DNA analysis. It has been known to crowd out native plants and create dense mats that interfere with recreational activity. Eurasian milfoil can grow from broken off stems which increases the rate in which the plant can spread and grow. 

One of the many lakes that are heavily invested with this invasive plant is Christina Lake in British Columbia, Canada. In the lake milfoil is currently controlled by having dive teams who manually extract the plant from June until October. Those teams work seven days a week with one overlapping day just to keep the nasty aquatic weed at bay. 

Milfoil weevil (Euhrychiopsis lecontei)

Obviously it would be much easier and cost-effective to develop biological pest control strategies and there are a few species that have been discussed and used. The water veneer moth (Acentria ephemerella), feeds upon and damages this water milfoil. It has been used as biocontrol but carefully, as it lacks host specificity and attacks other plant species, including natives. In addition it is also an introduced species. Another method for biocontrol is grass carp, (Ctenopharyngodon idella), is sometimes released into affected areas, since these fish primarily feed on aquatic plants and have proven effective at controlling the spread. However, the carp prefers native species before turning against Eurasian milfoil and more imporantly it has become a serious pest in North American lakes as well. That leaves us with the milfoil weevil (Euhrychiopsis lecontei), a native species that specifically loves to eat the new growth on the pest thereby stunting the plant’s growth and propagation.  that a weevil that lives in the region is actually this native species, making it possible to rear and release more of the bug. In the case of Christina Lake it needed DNA tests to confirm that the right kind of weevil naturally occurs in the lake. This makes it possible to rear and release more of the bug as it will be easier to get permission to introduce more of them to help control the ever growing milfoil. Regional authorities now hope that a pilot project can be started as early as coming summer. Eventually they would like to see a business created that will raise weevils to release not only into Christina Lake, but other lakes around the province to naturally control the alien species of Eurasian Milfoil.

Barcoding types

When describing a new species, taxonomists provide us with a species name and designate a type specimen to which this name will be permanently attached. In other words, a type is an example that serves to anchor the defining features of a particular taxon. 

DNA Barcodes can be used both to attribute species names to a given specimen and to flag genetic clusters for which no name is available. In an ideal situation this can be achieved by sequencing type specimens. However, the majority of the type specimens that are in our Natural History Museums is fairly old and not necessarily stored under DNA-friendly conditions. Many are conserved in formalin which destroys DNA over time. Others, like many insects, are pinned and boxed. From time to time researchers were able to extract DNA from such specimens but mostly they focused on considerably younger specimens (collected and described more recently) or addressed a very small number of samples as it is a more  laborious process.

Eois isographata (credit discoverlife.org)

In a new study researchers from Austria and Germany report successful sequencing of DNA Barcodes from 96 historical specimens (92 type specimens +4 non-types) of the moth genus Eois (in the 79 to 157 year age range). They used six primer combinations (recovering between 109 and 130 bp each) to assemble a full-length barcode sequence for each specimen. And indeed they were able to generate sequences with an average length of ~500 bp. Furthermore, they developed a non-destructive DNA extraction which means that the often very valuable type is not destroyed in the sense that no additional damage compared to conventional preparation of genitalia slides is inflicted on the specimen. Genitalia slides are usually necessary for morphology-based identification and description. This is still possible after the extraction procedure.

What strikes me is the fact that they were still able to retrieve enough DNA to apply the primer sets they had designed to come up with DNA Barcodes of reasonable length albeit they had to admit that the work is considerably more expensive than 'regular' DNA Barcoding. Nevertheless, a big step forward to anchor another defining feature to some types. Only this time it is one far more objective than many morphological characters out there.