Identification of Chattonella species (Raphidophyceae) through molecular methods on fixed samples from Santa Giusta Lagoon (Sardinia, Italy).

The genus Chattonella Biecheler (Raphidophyceae) has a worldwide distribution and includes harmful species, which cause extensive kills of farmed and wild fishes, with very important ecological damage and economic losses. Only two species are currently taxonomically accepted as members of the genus: C. marina (Subrahmanyan) Hara & Chihara and Chattonella subsalsa Biecheler. These two species show very similar cell morphology, with some characters that overlap, such as the length and the width of the cell. Therefore, their certain identification can be obtained only through the ultrastructure and genetics. The mechanism of toxic action is known better for C. marina but, even if a number of different killing pathways have been described, the debate is still ongoing. Potential deleterious effects of C. subsalsa are less known and only few studies indicate the production of toxic substances (e.g. brevitoxins, peroxide radicals). Chattonella spp. blooms associated with fish-kill events were observed in Sardinian lagoons since the middle of nineties. The first observation occurred in Santa Giusta Lagoon (Gulf of Oristano), during a massive fish mortality case, between the end of July and August 1994. The presence of Chattonella spp. was well documented also in 1998, 1999 and 2010, during further harmful blooms always in Santa Giusta Lagoon, which belongs to the LTER-Italy network. The presence of C. subsalsa was established in samples collected in Santa Giusta Lagoon in 2005 through molecular methods, when kill-fish events were absent. The main objective of this work is to investigate on which species were present during the fish-kill events in Santa Giusta Lagoon using fixed samples (with Lugol's iodine or formalin) collected at the time of the events. The analysis is based on the method developed by Connel (2002), which allows a rapid identification of Raphidophyceae using three-primer PCR amplification of nuclear internal transcribed spacer (ITS) regions. The internal transcribed spacer region of the nuclear rRNA gene was used to discriminate among the marine raphidophyte species, so it’s a useful support especially when cells cannot be identified morphologically and fixation methods have destroyed cellular RNA content. Our study is a first tentative to apply the method to natural samples in which only one or both the investigated Chattonella species may be present.