Development of a novel molecular approach for the rapid assessment of the biodiversity changes of benthic nematodes assemblages during habitat recovery of Zostera noltii seagrass beds
Benthic nematode assemblages are widely regarded as an ideal group to assess the potential ecological effects of natural and anthropogenic disturbance in aquatic ecosystems. Their ability to reflect changes of the environmental conditions make them informative to assess estuarine and marine biological integrity.
Novel, rapid high-resolution assessment of field samples of nematodes is urgently needed to gain an insight of nematode diversity at a functional level to use nematode community response as a tool in marine ecological research. Molecular methods offer a potentially efficient alternative but the sequencing of individual marine nematodes (barcoding) as well as community analyses has been limited, due the restrictions of validating morphology-based surveys.
The purpose of this project is to establish the methodological foundations to allow the development and testing a tool for rapid assessment of the estuarine/marine nematode responses to environmental changes, based on specific nematode assemblages (core assemblage) of Mira estuary (SW, Portugal).
Seagrass meadows are declining worldwide at an unprecedented rate, suggesting a global crisis with important consequences in the diversity of the associated benthic communities, although the causes of collapses are poorly understood. During 2008, Zostera noltii seagrass beds of the Mira estuary suffered a total collapse, though since 2009 slight and intermittent recovery has been observed. This context creates an almost unique opportunity to follow the ecological processes associated with the natural recovery of this key habitat and its associated benthic communities. A monitoring programme have been develop by members of the project team to analyse the function and dynamic response of nematodes assemblages to natural recovery of seagrass beds applying traditional morphological methods for analysis.
Molecular techniques have been successfully applied to monitor soil environmental condition using the nematodes. A molecular profiling system, using directed terminal-restriction fragment length polymorphism (dt-RFLP), has been designed and tested by a member of the project team that characterizes nematode assemblages by relative abundance of feeding guilds.
The main aim of this project is to undertake a combined morphological and molecular analysis of estuarine nematodes assemblages to establish the potential to provide a tool for fast assessment of the biodiversity changes within habitat recovery of Zostera noltii seagrass beds and provide preliminary validation of dT-RFLP as a high-throughput tool for assessing the recovery of the system. As part of this aim we will also develop a database of sequences related to individuals identified at a species level that will form the basis of new taxonomic reference system.
The following methodology will be achieved the goal in 4 major tasks:
Task 1- Collection of samples, extraction and preservation of nematode assemblages:
Task 2- Linking morphological identification with bar-code sequencing of individual nematodes:
Tasks 3- Theoretical testing of dT-RFLP approach for estuarine nematode assemblages.
Task 4- dT-RFLP- Molecular approach for rapid assessment of estuarine nematodes biodiversity changes.
The combined experience of the project team will allow the generation of a candidate tool for rapid assessment of nematode biodiversity and provide depth, currently lacking, to worldwide databases of marine nem
Objectives, activities and expected/achieved results
In summary this will project will :
1) Further analyse nematode morphology-based biodiversity surveys to build morphological vouchers suitable for molecular analyses.
2) Extract DNA from individuals identified at a species level, amplify target genes and sequence to provide a barcode library for key species within the system.
3) Generate assemblage sequence libraries from key samples to ensure capture of all sequence types and understand intra-species variation.
4) Use these molecular libraries to assess, and adjust if necessary, the existing dT-RFLP strategy designed to separate nematode taxa at order/family level.
5) Apply the dT-RFLP profiling molecular system as a tool to assess the changes in assemblage composition across degradation gradients.