Individuazione di micropredatori di ricci di mare formanti barren tramite l'impiego di marcatori molecolari

Responsabili di progetto

Roberto De Michele, Vesna Macic

Accordo

MONTENEGRO - MoS-not in force - Ministero della Scienza del Montenegro

Bando

CNR/MoS 2015-2016

Dipartimento

Scienze bio-agroalimentari

Area tematica

Scienze bio-agroalimentari

Stato del progetto

Rinnovo

Proposta di ricerca

The project aims to unravel the mechanisms of population control of Mediterranean sea urchin at the settlement stage, due to predation by small invertebrates (micropredators).
Rocky reefs can shift from forest, a state dominated by erect algae with high biodiversity, to barren, an impoverished state dominated by encrusting algae. Sea urchins, abundant in barrens, are usually held responsible for the maintenance of this state. Predation by large fish can revert the barren state to forest by controlling sea urchin populations. However, the persistence of a community state sometimes seems to be independent from the presence of such large predators, suggesting the existence of other unknown mechanisms ensuring their stability.
Processes occurring at the immediate post-settling stage could represent an important level of control of sea urchin populations. Since the supply of urchin larvae and the induction of settlement seem to occur independently of the community state (e.g., forest vs. barren) and of conspecific abundance, population density is necessarily determined in large part by post-settlement mortality. In particular, some authors suggest this to be the actual bottleneck for sea urchin populations. Among the causes of mortality of early benthic invertebrate settlers, predation is likely to be the most important. Small and relatively inconspicuous predators may present a similar or even greater impact than large and abundant predators and microfauna is known to be abundant in algal forests.
It may be relatively easy to observe, identify and quantify the prey of a large predator, such as a lion, which kills just one or two prey every few days. It is much more difficult, however, to determine the prey of a small predatory beetle or mite, hunting at night under a closed canopy of vegetation. Any attempt to make observation of such an arthropod easier (e.g. clearance of vegetation, use of extra light) will result in disturbance of the system, invalidating any quantitative data on predation that may be recorded. Even if one were fortunate enough to observe a predator attacking a prey under such circumstances there would be no way of knowing whether this particular interaction was a common or rare event. Most laboratory simulations (other than, perhaps, within an elaborate ecotron) are unable to recreate the vegetation structure and microclimates that exist in the field. If such environmental conditions are altered then the ability of the predator to find and capture its various prey will be affected as will, in turn, prey choice and functional responses. It is also difficult to recreate, and then maintain, the prey range available in the field, while thigmotactic responses to boundaries (by both predators and prey) will always be a problem in cages and arenas.
To overcome these difficulties a range of techniques have been developed for identifying prey consumed by predators directly in the field. Most involve the collection of predators (particularly invertebrates) from the field followed by postmortem analysis of their gut contents. The advantage of this approach is that, prior to collection, the system under study is not disturbed in any way and should thus provide direct information on prey choices in any ecosystem. Unfortunately, most invertebrate predators are basically fluid feeders (e.g. spiders, hemipteran bugs) consuming no identifiable remains. Even those predators that can ingest all parts of their prey may either eat many prey that provide no or few recognizable remains (such as the eggs of many insects or invertebrates such as slugs) or may simply choose to avoid consuming hard, indigestible remains. Alternatively, digestive processes render many samples visually unrecognizable. Any analyses based upon indigestible remains are thus likely to provide biased results and may miss many trophic interactions.
For these reasons a range of techniques have been developed for identifying prey remains at the molecular level, whether they be prey-specific protein sites or DNA sequences. Polymerase chain reaction (PCR)-based techniques for detecting prey remains in the guts, faeces and regurgitates of predators are being developed to study complex trophic interactions in the field.
Detecting degraded, semidigested DNA is not always easy. Major areas of difficulty seem to be lack of sensitivity, short post-ingestion detection periods and cross-amplification problems. Two factors affect the successful amplification of prey DNA from the guts of invertebrate predators and from faecal material: the amount of target DNA present in the sample and the quantity of that DNA remaining after storage of the predator, dissection (if applicable), then extraction and purification of the DNA. Optimization of each of these steps can enhance the success and consistency of any molecular analysis of predation.
Both single and multiple-copy DNA regions from both nuclear and mitochondrial genomes have been used for molecular detection of predation. After early attempts using nuclear DNA regions, including randomly amplified polymorphic DNA-derived sequence characterized amplified region (SCAR) markers, internal transcribed spacer region 1 (ITS-1) and á esterase genes, the majority of recent studies have used mitochondrial DNA (mtDNA) genes as the source of their target-specific primers. There are two main reasons for this. First, hundreds or thousands of copies of the mitochondrial genome may be present within each invertebrate cell, greatly increasing sensitivity and hence the probability that prey DNA can be amplified from a predator's gut. Second, there are many published sets of 'universal' primers available for the amplification of mtDNA genes, facilitating the rapid screening of suitable regions from both predator and prey species. From these sequences, prey-specific primers can be designed.

Obiettivi della ricerca

The project wi use modern molecular techniques to amplify trace DNA remains in the guts of small invertebrate, in order to identify potential micropredators of sea urchins.
DNA analysis is a recent advancement in the study of predator-prey interaction and, to date, has never been used for sea urchins.
Objectives of the present project are:

1) Design of specific and group primers for DNA from the two most common Mediterranean se urchin species from rocky reefs, Paracentrotus lividus and Arbacia lixula. This task will be facilitated by the presence of a wide collection of expressed sequence tags (ESTs) in public databases, eg. www.ncbi.nlm.nih.gov. For P. lividus, the complete genome sequence is already known and available. We will design primers based on both mitochondrial (cycloygenase I, ITS) and nuclear (bindin, P16, P19) DNA, known to be highly polymorphic among species

2) Identification of potential micropredators of sea urchin by amplification of total DNA with the specific primers designed in objective 1. PCR techniques will overcome the challenges of handling small invertebrates, stored in ethanol for weeks or months.

Ultimo aggiornamento: 14/12/2024