Amplifying the DNA

Once the separation of the samples and the extraction of their DNA are finished, it is time to amplify them, using the polymerase chain reaction (PCR). With this aim, we use a pair of primers capable of amplifying the chosen DNA fragment (in our case, a fragment of ribosomal RNA 18S, which is present in all eukaryotic organisms).

With this process, we obtain a mix of amplified DNA fragments, between 150 and 200 base pair long, representative of all the organisms present in each sample. Every fragment is also labeled with a "molecular barcode" which allows to identify the sample where it comes from. After checking the success of the amplification process on an agarose gel, we will send the samples to the ultrasequencing unit, where we will be able to read the sequences of the amplified DNA fragments.

Image of the agarose gel obtained with the DNA of the samples from Islas Cíes and Cabrera

Processing samples

We are now in the midle of sample processing. The work consists of separating the samples into three size fractions using sieves of 10, 1, and 0.06 mm. In this way each sample becomes three according to the organisms' size. Afterwards the DNA is extracted separately for each fraction using a special kit (PowerMax Soil Extraction Kit). The DNA is later amplified using universal primers for the target gene (we will begin using 18S).
As of today, the samples from one of the trips have been already sorted and we start the extractions... coming closer!

Sorting samples with a column of sieves

The three size fractions of a sample

First sampling in Cabrera done!

In September, 21^st to 26^th, we did the first sampling in the Cabrera Archipelago, in a joint trip with researchers from another National Parks research project, CORCLIM. Working together optimized costs and logistic needs, and it was an excellent occasion for interaction… and for fun!

The CORCLIM team with X Turon (photo Eneko Aspillaga)

Sampling was done in the Imperial, an islet in the SE of Cabrera. We could collect all the foreseen samples in spite of bad weather and rough seas some of the days. As an altered community we chose the shallow rocky bottoms occupied by the invasive seaweed Lophocladia lallemandii. The samples are currently being processed in the CEAB.

A sea of Lophocladia (photo Pol Capdevila)

We are in the news!

El Faro de Vigo dedicated two articles to Project Metabarpark last weekend! Here are the links:

CSIC biologists extract DNA from the rocks at Cíes Islands in order to identify every marine species

A CSI scratches the sea bottom at Rodas 

First sampling in the Parks done (Cíes Islands)!

Metabarpark Project has been launched! The first sampling trip to Cíes Islands (Galicia) was done in September, 8th to 11th. Thanks to the friendly collaboration of the Park staff and rangers, we could complete the work in a few days. We also enjoyed an exceptionally calm sea and high tides. We sampled both the Northern and Southern Islands, collecting material from rocky bottoms and Maërl communities. We also sampled sediment from the Lago dos Nenos.

The southern island of Cíes, known as San Martiño Island,

where some of the sampling has been carried out

We have returned with a full array of interesting samples to analyze! Now it's time to come down to the lab and put the lab-coats on...

Sampling at San Martiño Island

The Galician Atlantic Islands Maritime-Terrestrial National Park will pioneer in the application of new genetic techniques for marine biodiversity assessment

As part of a project funded by the Autonomous Organism of National Parks, and with the participation of scientists from the Centre for Advanced Studies of Blanes (CSIC), a study will be carried out on the biodiversity of the seabed of the Atlantic Islands NP using innovative genetic methods.

The project, beginning this year, aims at characterizing the species which inhabit these ecosystems, using the DNA found in the communities, the so-called environmental DNA. From this DNA, a number of indicator genes will be sequenced, which will allow to identify the present species. This method has been developed in recent years and its application to the study of marine seabed is virtually unprecedented, so the project will be a world premiere. 

The advantage of using the DNA present in the communities is the ability to detect both large and small-bodied organisms. Traditional biodiversity studies focus on species visible to the naked eye, which are the dominant biomass in communities. However, hundreds of species of small (up to a few tenths of a millimeter) animals, plants, fungi and protists present in the seabed are not analyzed. Over 90% of biodiversity is formed by these organisms and they are virtually unknown, due to the huge effort that would be needed to analyze these small species by conventional methods and the lack of specialists in these groups of organisms. However, the importance of these components of biodiversity is crucial, since they are involved in nutrient recycling, they lie at the base of the food chains and they are the first to respond to environmental stress or perturbations, before their effects are detectable in larger organisms.

With the new genetic techniques and the development of high-throughput sequencing devices in recent years, it is possible to make an exhaustive inventory of the present biodiversity. To achieve this, all the sequences of some particular genes present in the DNA of an environmental sample are obtained. These sequences serve as labels of the present species. Comparison of the sequences with those available in public databases allows assigning them to specific species or groups of organisms and therefore comprehensively characterize the present biodiversity. The advantages of this technique lie in its speed, its objectivity, and the fact that it does not rely on the availability of specialists in all groups. Moreover, the generated sequences are deposited in public databases and allow subsequent comparative studies.

The objectives of the project focus on analyzing seabed samples obtained from three types of communities: shallow rocky bottoms, deep rocky bottoms and gravel bottoms (maërl beds). At the same time, the researchers will study the effect of the presence of invasive seaweeds in the structure of communities. The study is to be performed in parallel in the Galician Atlantic Islands National Park and in the Archipelago of Cabrera National Park in the Balearic Islands, in order to have a comparison between similar communities in the Atlantic and Mediterranean areas.

The first samplings will be carried out in September and they are expected to be repeated seasonally for two years, in order to evaluate the seasonal and interannual variability. This study aims to achieve genetic characterization of biodiversity knowledge at a level that is not possible with traditional methods, to establish a database for future studies, and to be able to detect changes in the environment before they are evident in the communities.

The hidden biodiversity: hundreds of species of organisms present in seabed communities are invisible to the naked eye and must be studied using genetic techniques

The project begins!

The first months of the Metabarpark Project have been devoted to the setup of methods and protocols. We have been using samples from the Catalan and Balearic shores for the trials.

We have collaborated with Dr. Pierre Taberlet and his team at the Laboratory of Ecologie Alpine in Grenoble to apply methods developed for terrestrial soils to marine communities

We have amplified a fragment of the 18S rDNA using universal primers developed at LECA. The sequencing has been done using an Illumina MiSeq. We obtained >3 milion reads from 80 samples. The reads were processed using the OBITools pipeline, and ca. 1000 Molecular Taxonomic Units (MOTUs, our surrogate for species) were identified. Downstream analyses have revealed that the communities can be thoroughly inventoried, and that differences in composition (presence-absence) and in abundance (using read number as a proxy) can be detected between communities and localities using this method.

In a second trial we used a different extraction protocol (with lysis step) and combined amplification of DNA and amplification of cDNA obtained from RNA in the samples. We also tested an improved set of primers. The sequencing this time yielded 5 milion reads and 2700 MOTUs.
These trials allowed us to choose the best reagent kits and protocols for the study of the samples of the Parks. We are currently working in a set of primers to amplify the mitochondrial COI gene for comparison with the 18S nuclear gene, but we are having trouble in finding universal primers for sequence lengths adequate for Illumina sequencing. We will see if we succeed!