The otter is very difficult to study. It has nocturnal habits, and it's extremely elusive: direct observation is infeasible. Moreover, individual identification would be impossible without capturing and marking the animals, but this option, in addition to be technically complex and potentially harmful, is complicated by the regulations on protected species.
The only alternative is to use non-invasive genetic techniques (NGS).
Otters are used to mark their territory on rocks emerging along the river courses leaving faeces (known as spraints) and special jellies. These marking signs can be used to retrieve traces of DNA of the individual who dropped them off.
The first difficulty comes from the rain and frequent variations in the river flow which could wash away the marking signs. Furthermore, DNA degrades fast if left to the elements (that's why checks are to be performed daily, early in the morning, soon after the otters' activities overnight).
Finally, another problem comes from the tiny quantities of otters' DNA in the spraints compared to preys' DNA... and the jellies, which contain better quality DNA, are far rarer.
In the picture above you can read:
As soon as they are collected, the samples are stored in specific solutions to prevent DNA degradation and avoid contamination. Finally, they are sent to the ISPRA laboratories in Ozzano dell'Emilia to be analysed.
To identify individuals from DNA fragments we analysed the microsatellites loci. These are small portions of repeated DNA, highly variable among individuals: each locus may present different configurations but each individual has his own arrangement. Here below the six LUT in blue are the ones commonly used to identify European otters.
We encountered an early problem: the Sangro otters are strictly related, hence the similarity among the genotypes rendered virtually impossible a certain identification using just the six LUT loci.
We had to add seven more microsatellites loci (the OT, shown in red here below) previously used only for Asian otters.
Using all the 13 microsatellites loci the probability of false identification dropped to 0.2% (weren't the otters so strictly related, 6 loci would have been enough to reach a 0.1% probability).
Due to the mentioned difficulties in sampling storage and DNA degradation, we were able to genotype only 67 on 191 collected samples (about one third). Despite everything, we were able to identify 14 individuals living along the Sangro River: eight males, four females, and 2 of uncertain sex.
An interesting angle emerged from the research is the overlay among territories of animals of the same sex. It's possible that this behaviour is mostly related to individuals belonging to the same family cluster and the marking signs being more "social communication" than "actual territorial marks".
Genetic analysis allow us to determine some kinship among the identified otters. Sadly, there are enough holes in the data that we cannot yet understand the actual relationships (e.g. who's the parent and who's the child).
Even so, we were able to sort the 14 individuals in five groups as depicted in the picture below (in which the two undetermined otters were assigned the more probable female sex) spread along the 64 Km as in the previous page.