Wednesday, April 23, 2014

Slipper animacule

Image taken from micro*scope

Paramecia were among the first ciliates to be seen through early microscopes in the late 17th century. The first description occurs in a letter by his contemporary Christiaan Huygens in 1678. Huygens was a prominent Dutch mathematician and scientist. Interestingly is not known for this particular discovery but more for his telescopic studies of the rings of Saturn and the discovery of its moon Titan which was honored by   the European Space Agency (ESA) by naming an atmospheric entry probe after him. The probe landed successfully on Titan in 2005. Huygens was clearly one of those all-round talents that were typical for scientists at that time, e.g. he also invented the pendulum clock

But back to the paramecia - in 1718, the French mathematics teacher Louis Joblot published a description and illustration of what he called "slipper animacule". In some countries this phrase still remains the common name for members of the genus Paramecium. The widely used German term "Pantoffeltierchen" literally means slipper animal. The name relates to the typically ovoid, elongate, foot- or cigar-shaped cell usually ranging from 50 to 300 micrometres in length. 

Paramecia are widespread in freshwater, brackish and marine environments, and are often very abundant in stagnant basins and ponds. Because some species are readily cultivated and easily induced to conjugate and divide, Paramecium has been widely used in classrooms and laboratories to study biological processes. I am fairly confident that many of my readers had an encounter with Paramecium at school or university. Consequently, some Paramecium species such as Paramecium aurelia or Paramecium caudatumare are among the best known protozoans. Other groups are almost unexplored and in most cases we know little about their DNA sequence variation which seems to be fundamental for the determination of boundaries between those species. Some studies have revealed the existence of reproductively isolated groups within Paramecium as well as other ciliates. Those were initially called syngens which stands for “generating together”. In some cases syngens were recognized as sibling cryptic species but in other cases the isolation between syngens is imperfect, thus not each syngen is equivalent to a true cryptic species. 

One of those less widely know species is Paramecium putrinum. It is one of the smallest members of the genus, a cosmopolitan, freshwater species, that prefers cold and moderate-climate regions. A neat little new study provides a closer look at this species and potential existence of cryptic variation within it:

Herein we present an assessment of molecular variation in 27 strains collected from widely separated populations by using two selected DNA fragments (ITS1-5.8S-ITS2-5′LSU rDNA and COI mtDNA). Both the trees and haplotype networks reconstructed for both genome fragments show that the studied strains of P. putrinum form five main haplogroups. The mean distance between the studied strains is p-distance = 0.007/0.068 (rDNA/COI) and exhibits similar variability as that between P. bursaria syngens. Based on these data, one could hypothesize that the clusters revealed in the present study may correspond to previously reported syngens and that there are at least five cryptic species within P. putrinum.

Tuesday, April 22, 2014

Are vouchers always necessary?

We live in times with a heightened sense of urgency to confirm the return of animals thought to be extinct, or to confirm the presence of newly discovered species. Global climate change and rapidly disappearing habitat is endangering species and we become increasingly concerned about the consequences of their disappearance. The standard approach in biology is to go out and collect specimens either to confirm that they do still exist in the wild or to discover new species. However, sometimes this field work may actually pose a risk to vulnerable animal populations already on the brink of extinction:

Cases such as the extinction of the great auk remind us what is at stake in taking animals from small and declining populations. The last wild great auk (Pinguinus impennis) was sighted in 1844 on Eldey Island, Iceland. Centuries of exploitation for food and feathers, and, to some degree, a changing climate, had stressed the species, but overzealous museum collectors also played a role in its extinction. As the bird's numbers dwindled in the 19th century, ornithologists and curators increasingly prized great auk skins and eggs, with museums and universities sending out collection parties to procure specimens. On Eldey, fishermen killed the final breeding pair of the flightless birds and sold them to a local chemist, who stuffed the specimens and preserved them in spirits. Their internal organs now reside at the Zoological Museum in Copenhagen.

The great auk's disappearance predates the rise of a robust societal ethic of conservation and the emergence of a scientific concern for global biodiversity decline in the late 20th century. Yet, there is still a strong and widespread impulse to procure specimens of rare or rediscovered species for scientific purposes.

Researchers at Arizona State University and Plymouth University in the United Kingdom want to change the way biologists think about the current state-of-the-art of collecting a voucher specimen for species description and often identification. They say using modern technologies can be just as effective in identifying an organism and will also avoid increasing the extinction risk for small and isolated populations. The researchers suggest using a combination of modern, non-lethal techniques to confirm a species' existence including high-resolution photography and audio recordings of sounds or mating calls. Also, using DNA sampling by taking swabs of the mouth or skin offer ways to identify an animal without taking a specimen from the field. Especially this suggestion has DNA Barcoding written all over it and I am convinced that the barcoding community will wholeheartedly support this request:

For this system to work, the DNA of relict populations and newly discovered species must be sequenced and the data made publicly available. This would, for example, make future population rediscoveries easier to document.

The discussion about replacing non-lethal identification techniques with less-invasive ones is part of a more complex issue. Balancing ecological impact against value of improved scientific understanding of threatened species for conservation is a touchy subject. However, I concur with the authors stating that a change of our standard practices for scientific description would have more advantages than we might think:

The multivariate description of a species that results from combining high-resolution photographs, sonograms (as appropriate), molecular samples, and other characteristics that do not require taking a specimen from the wild can be just as accurate as the collection of a voucher specimen without increasing the extinction risk. Clearly there remains a long-running debate over the appropriate standards for scientific description absent a voucher specimen. The benefits and costs of verification-driven specimen collection, however, should be more openly and systematically addressed by scientific societies, volunteer naturalist groups, and museums. Sharing of specimen information, including obligations to store genetic information from voucher specimens in widely accessible digital repositories, can also help to reduce the future need to collect animals from the wild.

h/t Claudia Kleint-Steinke

Thursday, April 17, 2014

Stream Bioassessment with DNA Barcodes

Bioassessments measure both the physical condition of a water body, and the integrity of the associated biological communities. Adding such physical and biological metrics to standard chemical and toxicological assessments provides a more comprehensive evaluation of the condition of a given body of water. Resident organisms can be better indicators of overall environmental health than measurements of individual stressors (such as toxic chemicals or other pollutants) or more general ecosystem attributes. 

This form of assessment provides information on the condition of a site based on the taxonomic composition and a priori knowledge about tolerances of some taxa to pollution or other stressors. However, this can be a roadblock as the use of coarse taxonomic resolution can obscure patterns in bioassessment metrics and hinder detection of biological impacts. Thus, fine-scale taxonomic resolution is desirable to maximize the diagnostic capability of assessment tools.

I am sure by now every regular reader knows where this is going. It is well-known that obtaining such detailed taxonomic data is challenging because identifications typically are done by using morphological characteristics. There are a lot of issues with that especially for standardized and repeated assessments. Limited taxonomic resources, cryptic species, small size, damaged specimens, and polymorphism just to name a few of those.

A group of US researchers now compared the ability of several commonly used bioassessment metrics calculated with data derived from morphology and from DNA Barcoding to detect differences in stream condition of 6 paired sites in southern California with relatively subtle impacts to habitat. Their paper has now been officially published in Freshwater Science which was previously titled Journal of the North American Benthological Society. 

The results of this study are very interesting as the authors focused more on the level of sensitivity a DNA barcoding approach provides and not so much on the question if it would work in general. They found increased metric sensitivity associated with barcoding was most pronounced at high-quality (i.e., relatively unimpacted) sites, which often have higher species richness and are inhabited by undescribed, cryptic, or regionally rare species. For example, 43% of the additional taxa identified through barcoding consisted of 1 or 2 individuals and occurred at only 1 stream. The presence or absence of rare species may be diagnostic of specific environmental changes, so the increased information provided by barcoding at taxon-rich sites allows finer-scale resolution of sources of stress and increases our ability to detect subtle changes in environmental quality.

The conclusions are fairly positive:
The DNA barcoding approach can improve existing BMI [benthic macroinvertebrate]-based bioassessment programs by enabling development of new or improved metrics based on taxonomic groups that currently are under-described and underused. Additional benefits include applications for quality control, taxonomic standardization, and improvement of taxonomic keys (Pilgrim et al. 2011, Sweeney et al. 2011). Barcoding probably will be used with increasing frequency to augment or support existing methods and to provide cost-effective improvement of taxonomic capacity.

Potential challenges and solutions to meet them are also discussed:
However, full integration of barcode data in routine bioassessment will be challenging. First, a robust barcode reference library must be developed and vouchered. Standard handling and quality-control procedures must be developed to reduce risk of loss of samples because of contamination or DNA degradation (as happened for 1 of the sites in our study). Improved primers are needed for certain taxonomic groups to minimize bias caused by differential amplification. More research is needed on the effect of short-sequence reads on conclusions about taxonomic resolution.

Overall this paper is a very good read. It is clear, concise, and provides an objective assessment of a new approach to freshwater bioassessments.

Wednesday, April 16, 2014

Hydrological niche segregation

Mountain Fynbos
Fynbos is a natural shrubland or heathland vegetation occurring in a small belt of the Western Cape of South Africa, mainly in areas with a Mediterranean climate. Fynbos is known for its exceptional degree of biodiversity and endemism. As this floral community has the capacity to regenerate after fire it provides an opportunity to study the genesis of a variety of ecological phenomena such as hydrological niche segregation.

Species in plant communities normally separate along fine-scale hydrological gradients. Different plant species settle in different ecological niches based on the availability of water in the soil. One open question is at which stage of a plant's life history this segregation actually happens. one hypothesis is that it starts at the seedling stage because it is the most vulnerable as it is most prone to drought, competition, herbivory and disease. A group of researchers from the UK and Switzerland put this hypothesis to the test with a soil translocation experiment performed in the fynbos in South Africa, after a fire and before seed germination had started:

Following wildfires at two field sites where we had previously mapped the vegetation and monitored the hydrology, seeds were moved experimentally in >2500 intact soil cores up and down soil-moisture gradients to test the hypothesis that hydrological niche segregation is established during the seedling phase of the life cycle. Seedling numbers and growth were then monitored and they were identified using DNA Barcoding, the first use of this technology for an experiment of this kind.

The study focused on endemic species in the family Restionaceae because it is species-rich, ubiquitous, contains many keystone species and most species have been sequenced for the matK gene which is one of the plant DNA Barcode markers for plants.

According to the results of the study seedling growth on hydrological gradients in the field is affected by soil moisture status and by root competition. This means that hydrological niche segregation could indeed potentially originate in the seedling stage. In particular below-ground competition seems to be decisive in determining a species' hydrological niche. Fynbos species, as in other fire-prone plant communities, divide between those that regenerate from seed and those that resprout. The resprouters were probably the chief source of below-ground competition for seedlings in our experiment

Tuesday, April 15, 2014


Regular readers of my blog might have noticed that I have a weak spot for the DIY movement especially in biology and biotechnology. I try to follow the news in this sector and secured them a spot in the DNA Barcoding Bulletin that we produce quarterly. That being said, I was surprised that I didn't find out about the BioCoder newsletter that is published by O’Reilly.

O'Reilly is an american media company that mostly publishes books on computer technology topics. Their distinctive brand features a woodcut of an animal on many of their book covers. If you don't know what I am talking about I suggest you type in "O’Reilly covers" in a Google Image search. The animal illustrations are quite beautiful. I have a few of their books in my shelf and my favorite is a book on BLAST showing a coelacanth on the cover (see image below).

So what is BioCoder? Here is what their website has to say about that:
We’re at the start of a revolution that will transform our lives as radically as the computer revolution of the 70s. The biological revolution will touch every aspect of our lives: food and health, certainly, but also art, recreation, law, business, and much more.

BioCoder is the newsletter of that revolution. It’s about biology as it moves from research labs into startup incubators, hacker spaces, and even homes. It’s about a very old programming language that we’re just beginning to understand, and that’s written in a code made up of organic chemicals. It’s the product of a sharing community of scientists that stretches from grade school to post docs and university faculty.

The new spring issue contains an article about DNA Barcoding of fungi coming from a DIY lab in Victoria here in Canada. The organism choice clearly tells me that DIY people are up for challenges and not necessarily aiming for the low hanging fruits. The article is pretty interesting also from a technical standpoint and there is a part II following in the next issue. 

Great new resource. Well, not that new. This was their third issue. So, it is new to me but I am sure not news to the DIY biohack community.

Monday, April 14, 2014

When pharmaceuticals become too effective

Sepsid fly
The veterinary pharmaceutical ivermectin has been used for more than thirty years all over the world to combat parasites like roundworms, lice and mites in livestock and pets. The active ingredient belongs to the chemical group of avermectins, which generally disrupt cell transport. However, when ivermectin is used in high dosage excess quantities are excreted in the faeces of treated animals which also harms dung-degrading beneficial insects like dung beetles and dung flies. This has a profound impact on the the functioning of surrounding ecosystems. In extreme cases the dung is not decomposed and the pasture is destroyed.

Since 2000 public regulators in many countries therefore mandate standardized safety tests for the use of avermectin derivatives. A research team consisting of scientists from the University of Zurich and an ecotoxicology company in Germany, has now shown that the currently used safety tests are not able to sufficiently prevent environmental damage. Even closely related dung organisms react with varying degrees of sensitivity to the same veterinary pharmaceutical.

The group examined 23 species of sepsid flies that typically live in cow dung. It turns out that individual species vary by a factor of 500 in their sensitivity to ivermectin. Standardized safety tests typically performed in toxicology laboratories today are based on single, arbitrarily selected dung organisms. This poses the considerable risk that more sensitive species will continue to be harmed by ivermectin and that important ecosystem functions will suffer long-term damage as a consequence. In order to prevent this, safety tests should be extended to include a representative selection of all dung-degrading organisms, if not the entire community:

We close by reiterating that sepsid flies are very well suited as test organisms for any toxic residues in the dung of livestock or other large vertebrates, due to their ease and speed of rearing and handling. While the choice of a particular species will be crucial because species vary strongly in sensitivity, use of several local species can offset the arbitrariness of choice to some degree, rendering overall representative results. Sepsids as ecotoxicological test organisms could be particularly useful and economical in the tropics, where high-tech laboratory equipment is often not available.

By including more species in the tests costs for the authorization process would increase especially because all relevant organisms would need to be properly identified. For that reason the authors suggest to include DNA Barcoding in the test protocol as its inclusion would represent a rather modest increase in costs.

Friday, April 11, 2014

A different take on Escargot

Gastropod shells and bodies extracted after microwaving
And today for something completely different. Let's start with a description of the problem:
Extracting DNA from gastropods presents particular difficulties due to the capacity of the living animal to retract into the shell, resulting in poor penetration of the ethanol into the tissues. Because the shell is essential to establish the link between sequences and traditional taxonomic identity, cracking the shell to facilitate fixation is not ideal. 

This sounds very familiar to me. While working on my masters project I had to remove tissue from coiled shells of a number terrestrial gastropods and some of those specimens were quite small and delicate. Most of the time I was working with a dissecting probe which tip I had bent to be able to reach the fully retracted animal. A very tedious and not always successful method to retrieve a tiny tissue sample for DNA analysis. Over the years a variety of methods to retrieve tissue without damaging the shell have been developed but for the most part they are suffering from the same problem. Due to the fact that they all take a fair bit of time they are not useful for large scale surveys or expeditions.

In a new paper a group of French researchers present an alternative method for the easy, efficient and nondestructive tissue removal from shells. It involves the use of a regular microwave oven. The use of microwaves in molecular biology is actually not unknown and has been applied in the extraction of DNA from viruses, bacteria, soil micro-organisms, and animal tissue. The colleagues placed the living gastropods in a microwave oven in which the electromagnetic radiation very quickly heats both the animal and the water trapped inside the shell, which results in the separation of the muscles that anchor the animal to the shell. If done properly, the body can be removed intact from the shell and the shell voucher is undamaged as well. The authors conducted comparative tests to find out if microwaving the snail tissue will have any effect on DNA extraction or subsequent PCRs. They couldn't find any difference in DNA quantity or quality.

The method was then implemented on a large scale during expeditions, resulting in higher percentage of DNA extraction success. The microwaves are also effective for quickly and easily removing other molluscs from their shells, that is, bivalves and scaphopods. Workflows implementing the microwave technique show a three- to fivefold increase in productivity compared with other methods.

That seems to be worth the effort. I wish we had thought of that 12 years ago.