The sequencing of environmental DNA (eDNA) has emerged as important new tool to study present and past biodiversity allowing for the identification of multiple taxa from the genetic material shed by organisms into the environment. An approach for studying the eDNA is metabarcoding that has been widely applied to the study of aquatic ecosystems biodiversity, and to identify rare, endangered or invasive species. However, eDNA work in aquatic environments has been mostly focused on vertebrate species (84% on fish and amphibians) while invertebrates in coastal environments have received much less attention and the detection of aquatic insects using eDNA is weakly documented.
Our attention will focus on a peculiar aquatic environment, the marine rock-pools commonly found along the Mediterranean coasts. A few insect groups (chiefly Coleoptera and Diptera) are known to live in marine rock-pools, and among them, several species belong to the water beetle family Hydraenidae, genus Ochthebius Leach 1815. Most of the new hydraenid species belonging to the genus Ochthebius recently discovered from southern Europe were found in hypersaline coastal habitats, showing that these aquatic ecosystems is also a potential source of undetected biodiversity. Recent molecular and morphological studies strongly suggested the presence of a series of cryptic and thus far not formally recognized Ochthebius species around Mediterranean basin coasts and particularly along the coasts of Sicily.
Here, we intend to use eDNA as a tool for simultaneous identification of multiple rock-pools beetles species that may be gone undetected by traditional biomonitoring surveys that require observation and physical collection of whole organisms.
Aims and objectives
The general objective of our study is to document life in the peculiar aquatic environment of the hypersaline rock-pools by monitoring water beetles endemic fauna and potential cryptic species along the rocky shore of Sicily, Italy.
Specific objectives are:
- To design and validate taxon-specific primers for marine rock-pools beetles detection;
- To establish an eDNA based monitoring method to detect and resolve closely related species of marine rock-pools beetles;
To survey the distribution of endemic species and detect cryptic or unknown species of marine rock-pools beetles.
Expected results
Overall, we expect to establish an eDNA based methodology for the monitoring of water beetle in rock pools that represent a valid alternative to the traditional biomonitoring approach, thus avoiding bias in the species composition due to the difficulty of visual identification and recovery of the tiny individual even for experts.
Moreover, the analysis of molecular taxonomic units (MOTUs), rather than morphology-based species assignments, may uncover biodiversity signatures of rare and cryptic taxa that may be overlooked by conventional surveys based on the observation and physical collection of whole organisms, also due to similarity to resident taxa.
Direct outcomes expected from our study are:
o To compile a properly curated and comprehensive reference dataset with species-specific sequences of water beetles to support further molecular biomonitoring eDNA based studies.
o To identify suitable molecular markers that amplify short fragments, with conserved primer sites flanking variable regions specific to the target group species.
o To validated primers able to amplify all species of the target group without biases due to primers mismatches that favor amplification of certain taxa.
o To produce the first eDNA sequence datasets allowing for the detection of genetic signatures of resident beetle fauna including endemic, rare and cryptic members recovered from marine rock-pools of a Mediterranean coastal region, thus supporting the usefulness of the approach for detecting taxa present in the ecosystem.
o To demonstrate that marine hypersaline water found in rock-pools provide a source of detectable DNA from insects, despite the impact of salinity on the preservation and extraction of the eDNA.
o To find differences in water beetle species compositions recovered by eDNA from different sites sampled at a very narrow scale.