The research carried out in recent years, and exposed in this Thesis, concerns a basic study on the entomopathogenic nematodes (EPN) of Sicily. The biodiversity and distribution of EPNs in the Region was investigated, by sampling thoughout 26 different sites located in 8 of the 9 Sicilian provinces. 290 samples were collected, in which a total of 45 isolates were found, 34 belonging to steinernematids and 11 to heterorabditids. Five species have been identified: Steinernema feltiae Filipjev, 1934; Steinernema kraussei (Steiner, 1923) Travassos, 1927; Steinernema apuliae Triggiani, Mráček & Reid, 2004; a so far unclassified Steinernema sp., possibly a new species belonging to the “Steinernema feltiae group”; Heterorhabditis bacteriophora Poinar, 1976. Samplings were carried out in different environments, owning distinct ecological characteristics, and confirmed the wide presence of EPNs in Sicily, both in terms of species and isolates, more in natural environments than in anthropized ones, allowing to highlight the preference of specific habitats for some species. The isolates were characterized from a molecular point of view by sequencing the ITS locus, both for their specific identification and to investigate their phylogenetic relationships with conspecific isolates coming from other geographical areas. This analysis helped to clarify the taxonomic position for almost all the isolates found, except for three for which a clear specific identification was not achieved. Different phylogenetic reconstruction approaches agree on placing this taxon within the Feltiae group and on the possibility that they represent a new species for science. The analysis of ITS sequences has also highlighted, in the case of S. feltiae isolates, the occurrence of microevolutionary phenomena able to determine peculiar haplotypes in local EPNs populations. In another series of experiments, some traits of the biology of these species, of their pathogenic capabilities against Galleria mellonella, as well as and some aspects of their life cycle, were studied. The results obtained confirmed how some species best show their pathogenic activity at different temperatures and highlighted consistent levels of pathogenicity in all studied isolates. These data, together with those concerning habitat preferences, could allow for a targeted use of autochthonous isolates instead of commercial formulations in biological control, with obvious benefits for local biodiversity. The isolate of S. apuliae showed an anomalous life cycle, with different juvenile and adult stages capable of completing the cycle outside the insect, unlike what happens in the normal course of the EPNs cycle. These observations raise interesting questions about the ongoing evolutionary processes, regarding the complex nematode-bacterium-insect symbiosis. Finally, some preliminary data relating to EPN endosymbionts were acquired. They were isolated from each EPN strain, identified by growing on selective plates, both chromogenic and antimicrobial, and stored in 20% glycerol at -80°C. For all the EPNs found it was possible to isolate the respective symbionts. Tests were also carried out to verify their resistance to the antibiotic Tetracycline. The data obtained reinforce the discussed link between these bacteria and host nematodes and their synergistic action as entomopathogens.
La ricerca svolta in questi anni, ed esposta in questo elaborato, attiene a uno studio di base sui nematodi entomopatogeni (EPN) della Sicilia. È stata indagata la diversità e la distribuzione degli EPN nella Regione, con campionamenti in 8 delle 9 province siciliane e in 26 siti differenti, per un totale di 290 campioni raccolti, nei quali sono stati rinvenuti complessivamente 45 isolati (34 steinernematidi e 11 heterorabditidi). Le specie identificate sono cinque: Steinernema feltiae Filipjev, 1934; S. kraussei (Steiner, 1923) Travassos, 1927; S. apuliae Triggiani, Mráček & Reid, 2004; Steinernema sp., probabile nuova specie, appartenente allo “Steinernema feltiae group” ed Heterorhabditis bacteriophora Poinar, 1976. I campionamenti sono stati effettuati in ambienti differenti per caratteristiche ecologiche ed evidenziano l’ampia presenza degli EPN in Sicilia, sia in termini di specie che di isolati, maggiormente negli ambienti naturali rispetto a quelli antropizzati, mettendo in luce per alcune specie la preferenza di specifici habitat. Gli isolati sono stati caratterizzati dal punto di vista molecolare attraverso lo studio del locus ITS, sia per la loro identificazione specifica, sia per indagarne i rapporti filogenetici con isolati conspecifici e provenienti da altre aree geografiche. Quest’analisi ha permesso di chiarire la posizione tassonomica della quasi totalità degli EPN rinvenuti, a esclusione di tre isolati per i quali non si è giunti a una identificazione specifica certa. Le ricostruzioni filogenetiche convergono sulla loro collocazione all’interno del Feltiae group e sulla possibilità che essi rappresentino una specie nuova per la scienza. L’analisi delle sequenze ITS ha inoltre messo in evidenza, come nel caso degli isolati di S. feltiae, l’occorrenza di fenomeni di microevoluzione in grado di determinare aplotipi peculiari nelle popolazioni locali di EPN. Sono stati studiati, inoltre, alcuni tratti della biologia di questi animali, valutandone le capacità patogene nei confronti di Galleria mellonella, e alcuni aspetti del ciclo vitale. I risultati ottenuti hanno confermato come alcune specie esplichino al meglio la loro attività patogena a temperature differenti e hanno evidenziato buoni livelli di patogenicità in tutti gli isolati oggetto dello studio. Questi dati, insieme a quello legato alle preferenze di habitat, possono consentire un più mirato utilizzo degli isolati autoctoni, in sostituzione dei formulati commerciali, nel controllo biologico di insetti nocivi, con evidenti ricadute a vantaggio della biodiversità locale. L’isolato appartenente alla specie S. apuliae ha mostrato un ciclo vitale anomalo, con diversi stadi giovanili e adulti in grado di completare il ciclo all'esterno dell'insetto, a differenza di quanto accade nel normale svolgimento del ciclo degli EPN. Queste osservazioni pongono interessanti interrogativi sui processi evolutivi, tuttora in corso, riguardanti la complessa simbiosi nematode-batterio-insetto. Infine, sono stati acquisiti alcuni dati preliminari relativi ai batteri simbionti. Questi sono stati isolati e identificati fenotipicamente su piastre selettive, sia cromogeniche che antimicrobiche, e conservati in glicerolo a -80 °C. Per tutti gli EPN rinvenuti è stato possibile isolare i rispettivi simbionti, sui quali sono stati anche effettuati test per verificarne la resistenza all’antibiotico Tetraciclina. I dati ottenuti rafforzano il discusso legame tra questi batteri e i nematodi ospiti e la loro azione sinergica come entomopatogeni.
Biodiversità dei nematodi entomopatogeni in Sicilia: caratterizzazione molecolare, biologia e batteri simbionti / Leone, Diego. - (2024 Mar 01).
Biodiversità dei nematodi entomopatogeni in Sicilia: caratterizzazione molecolare, biologia e batteri simbionti
LEONE, DIEGO
2024-03-01
Abstract
The research carried out in recent years, and exposed in this Thesis, concerns a basic study on the entomopathogenic nematodes (EPN) of Sicily. The biodiversity and distribution of EPNs in the Region was investigated, by sampling thoughout 26 different sites located in 8 of the 9 Sicilian provinces. 290 samples were collected, in which a total of 45 isolates were found, 34 belonging to steinernematids and 11 to heterorabditids. Five species have been identified: Steinernema feltiae Filipjev, 1934; Steinernema kraussei (Steiner, 1923) Travassos, 1927; Steinernema apuliae Triggiani, Mráček & Reid, 2004; a so far unclassified Steinernema sp., possibly a new species belonging to the “Steinernema feltiae group”; Heterorhabditis bacteriophora Poinar, 1976. Samplings were carried out in different environments, owning distinct ecological characteristics, and confirmed the wide presence of EPNs in Sicily, both in terms of species and isolates, more in natural environments than in anthropized ones, allowing to highlight the preference of specific habitats for some species. The isolates were characterized from a molecular point of view by sequencing the ITS locus, both for their specific identification and to investigate their phylogenetic relationships with conspecific isolates coming from other geographical areas. This analysis helped to clarify the taxonomic position for almost all the isolates found, except for three for which a clear specific identification was not achieved. Different phylogenetic reconstruction approaches agree on placing this taxon within the Feltiae group and on the possibility that they represent a new species for science. The analysis of ITS sequences has also highlighted, in the case of S. feltiae isolates, the occurrence of microevolutionary phenomena able to determine peculiar haplotypes in local EPNs populations. In another series of experiments, some traits of the biology of these species, of their pathogenic capabilities against Galleria mellonella, as well as and some aspects of their life cycle, were studied. The results obtained confirmed how some species best show their pathogenic activity at different temperatures and highlighted consistent levels of pathogenicity in all studied isolates. These data, together with those concerning habitat preferences, could allow for a targeted use of autochthonous isolates instead of commercial formulations in biological control, with obvious benefits for local biodiversity. The isolate of S. apuliae showed an anomalous life cycle, with different juvenile and adult stages capable of completing the cycle outside the insect, unlike what happens in the normal course of the EPNs cycle. These observations raise interesting questions about the ongoing evolutionary processes, regarding the complex nematode-bacterium-insect symbiosis. Finally, some preliminary data relating to EPN endosymbionts were acquired. They were isolated from each EPN strain, identified by growing on selective plates, both chromogenic and antimicrobial, and stored in 20% glycerol at -80°C. For all the EPNs found it was possible to isolate the respective symbionts. Tests were also carried out to verify their resistance to the antibiotic Tetracycline. The data obtained reinforce the discussed link between these bacteria and host nematodes and their synergistic action as entomopathogens.File | Dimensione | Formato | |
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