Analysis of internal transcribed specers (ITS) regions in Steinernema feltiae sicilain strains

Extended abstract: Biodiversity of entomopathogenic nematodes (EPN) in Italy is particularly high, with a greater number of species (12 species) than in other European and Mediterranean countries (Tarasco et al., 2009). Sicily is the Italian region with the largest number of species, with 6 reported species (Tarasco et. al., 2015). In the last two decades, investigations on EPN's biodiversity in Sicily and the neighboring islands led to isolate more than 50 different strains, belonging to the genera Heterorhabditis and Steinernema (Clausi et al., 2010 a; 2010 b; Tarasco et al., 2010; Leone et al., 2011) and including the new species for science, Steinernema vulcanicum, found only on Etna Volcano (Clausi et al., 2011). Furthermore, some intraspecific differences were found in morphological, molecular, and pathogenic characterization of local strains (Leone et al., 2014; Clausi et al., 2014; Clausi et al., 2020). For this reason, further investigations have been made in order to study more extensively the variability of Sicilian EPN. During the course of the present study, we have found 22 new isolates belonging to Steinernema genus; 16 among them were Steinernema feltiae, the most common species in the island, found in more than 70 % of the samples. The aim of the work was to investigate the intraspecific genetic diversity of new Sicilian strains of S. feltiae by the analysis of ITS rDNA sequences. Fourteen new strains of S. feltiae were included in this study. Samples were collected between November 2019 and October 2021 from different localities and habitats throughout the island. Nematodes were obtained by the technique of Galleria mellonella bait (Bedding and Akhurst, 1975). IJs were collected in modified White traps (Kaya and Stock, 1997) and stored at 4-8 °C in polyethylene bags filled with wet polyurethane sponges. DNA was extracted from a single female or several juveniles. The ITS region was amplified by PCR performed using KAPA Taq HotStart PCR Kit (Roche ® ) and the primers proposed by Vrain et al., 1992. PCR reaction products were run in 1 % agarose gel and the bands excised for DNA extraction with GFX PCR DNA and Gel band purification Kit (Cytiva™). Purified DNA was directly sequenced by the Eurofins Genomics (Ebersberg, Germany) sequencing services. In addition, sequences with high similarity with respect to S. feltiae were searched in NCBI GenBank using BLAST algorithm (Altschul et al., 1990). ClustalW (Thompson et al., 1994) was used to generate the alignments under default values for gap opening and gap extension penalties and MEGA software (version 11.0.11; Tamura, Stecher, and Kumar 2021) was used to calculate phylogenetic relationships between sequences. A total of 74 sequences of S. feltiae were analyzed, including those from Sicilian and Italian strains as well as other countries. By analyzing multiple alignment of the chosen sequences, we have found a number of nucleotide substitutions in Sicilian/Italian strains never discovered in strains from other countries, except for a strain from Switzerland. In the course of the study, a peculiarity of haplotypes of S. feltiae in Sicilian/Italian strains was detected/revealed by the presence of a synapomorphy shared by Sicilian/Italian strains and the Swiss strain of S. feltiae. Extensive sampling throughout Italy is necessary to find out if other minor haplotypes of S. feltiae are present. Its analysis and comparison with the isolates from neighboring countries will provide the wider picture of the distribution of this most sampled Steinernema species.