Vrednotenje toksičnosti kompleksov egerolizinov in proteinov z domeno MACPF za žitnega strgača in kapusovega bolhača : magistrsko delo

Pavšič, Ana (author), Razinger, Jaka (mentor), Gunde-Cimerman, Nina (member of the commission for defense), Turk, Tom (member of the commission for defense), Sepčić, Kristina (comentor)

Potreba po iskanju novih bioinsekticidov je vedno večja. V tej magistrski nalogi smo testirali proteine egerolizinske družine iz bukovega ostrigarja, ki v kombinaciji s pleurotolizinom B, proteinskim partnerjem z domeno MACPF (ang. membrane-attack domain/perforin) deluje insekticidno na koloradskega in koruznega hrošča, ki spadata v družino lepencev (Chrysomelidae). Insekticidno delovanje je posledica vezave egerolizinskega partnerja na membranski sfingolipidni receptor ceramid fosfoetanolamin (CPE), ki je specifičen za nevretenčarje. Da bi preverili, ali so kompleksi na osnovi egerolizinov toksični tudi za druge hrošče iz družine lepencev, smo komplekse egerolizin/pleurotolizin B testirali še na dveh rastlinskih škodljivcih iste družine: na žitnem strgaču (Oulema melanopus) in na kapusovem bolhaču (Phyllotreta atra). Žuželke smo nabrali na netretiranih poljih in nastavili poskus tako, da smo z egerolizini in egerolizinskimi kompleksi obdelali liste, s katerimi so se žuželke prehranjevale. Naši testi so pokazali občutljivost žitnega strgača na izpostavitev proteinom, medtem ko osebki kapusovega bolhača za to niso bili dovzetni. Z analizo lipidnih ekstraktov žuželk smo preverili prisotnost egerolizinskega receptorja CPE pri žitnem strgaču in kapusovem bolhaču kakor tudi pri drugih rastlinskih škodljivcih (marmorirana smrdljivka, plodova vinska mušica, velika žitna uš, koruzni hrošč in koloradski hrošč). Prisotnost CPE smo dokazali v vseh vzorcih, a v različnih koncentracijah. Najvišjo koncentracijo CPE ima plodova vinska mušica, najmanjšo pa velika žitna uš. Primerjava lipidomskih analiz in testov toksičnosti je pokazala, da koncentracija CPE v membranah nima vloge pri delovanju egerolizinov in egerolizinskih kompleksov na žuželke.

• Language: Slovenian
• Keywords: bioinsekticid, ceramid fosfoetanolamin, družina lepencev, egerolizini, kapusov bolhač, Oulema melanopus, Phyllotreta atra, protein z domeno MACPF, testiranje toksičnosti, žitni strgač
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Place of publishing: Ljubljana
• Publisher: [A. Pavšič]
• Year: 2021
• Number of pages: IV f., 81 str.
• PID: 20.500.12556/RUL-131376
• UDC: 577.2(043.2)
• COBISS.SI-ID: 78160643
• Publication date in RUL: 26.09.2021

Secondary language

• Language: English
• Title: Assessment of toxicity of aegerolysin and MACPF-domain protein complexes against cereal leaf beetle and cabbage flea beetle : magistrski študij - 2. stopnja

Abstract

The need to find new bioinsecticides is continuously increasing. In this MSc thesis, we tested aegerolysin proteins from oyster mushrooms that had been found to exert insecticidal effects against two plant pests that belong to the Chrysomelidae family: the Colorado potato beetle and the Western corn rootworm. These aegerolysins bind to insect-specific membrane sphingolipid receptor ceramide phosphoethanolamine (CPE) and form transmembrane pores in insect cell membranes in concert with their protein partner pleurotolysin B, which bears a membrane-attack complex/perforin (MACPF) domain. In order to check whether these aegerolysin-based protein complexes are toxic also against other beetles from the Chrysomelidae family, we assessed their toxicity against the cereal leaf beetle (Oulema melanopus) and against the cabbage flea beetle (Phyllotreta atra). The insects were collected on non-treated fields and fed with leaves treated with aegerolysins and aegerolysin/pleurotolysin B mixtures. Our tests showed the sensitivity of cereal leaf beetle to these treatments, while cabbage flea beetles were not susceptible. We also analysed the presence of CPE in lipid extracts from cereal leaf beetle and cabbage flea beetle, as well as from other plant pests (brown marmorated stink bug, Drosophila suzukii, English Grain Aphid, Colorado potato beetle and Western corn rootworm). The presence of CPE was confirmed in all pests’ extracts, but in different concentrations. The highest CPE amount was detected in D. suzukii, and the lowest in English Grain Aphid. The comparison of lipidomic data and toxicity tests showed no correlation between the CPE membrane content and the toxic activity of aegerolysin-based complexes on tested insects.

• Keywords: bioinsecticide, cabbage flea beetle, ceramide phosphoethanolamine, cereal leaf beetle, Chrysomelidae family, aegerolysins, Oulema melanopus, Phyllotreta atra, MACPF protein, toxicity testings