Otpornost na niske temperature i dehidrataciju kukuruznog plamenca (Ostrinia nubilalis Hb)- ćelijski i molekularni odgovori

Abstract

Kukuruzni plamenac, Ostrinia nubilalisHb, je insekt umerenog klimatskog područja koji niske zimske temperature preživljava ulaskom u dijapauzu u stadijumu gusenice. Tokom dijapauze gusenice postaju otporne na hladnoću i sposobne da prežive vanćelijsko zamrzavanje telesne tečnosti. Cilj ove teze je bio razumevanje mehanizama otpornosti na niske temperature gusenica O. nubilalis na ćelijskom i molekularnom nivou. Istraživanja su rađena na letnjim, nedijapauzirajućim gusenicama kao i zimskim gusenicama koje su bile izlagane uticaju niskih temperatura u različitim periodima dijapauze. Određivan je režim telesne vode, sadržaj poliola i šećera kao mogućih krioprotektora, kao i NMR metabolomički profili. Zbog pretpostavljene uloge atioksidativnog sistema zaštite (ADS) u procesima vezanim za otpornost na hladnoću određivane su aktivnosti određenih antioksidativnih enzima kao i produkcija H2O2i melanina. Rezultati ukazuju da je izlaganje niskim temperaturama kod dijapauzirajućih gusenica praćeno preraspodelom osmotski aktivne (OA) vode na nivou hemolimfe i masnog tela, pri čemu nivo OA opada u hemolimfi, a raste u masnom telu. Nivo osmotski neaktivne vode (OI) ostaje nepromenjen. Nivo glicerola je značajno veći kod dijapazirajućih u odnosu na nedijapauzirajuće gusenice, a njegov nivo se višestruko povećava pri izlaganju niskim temperaturama na sredini dijapauze ukazujući na njegov značaj kao glavnog krioprotektora u ovoj fazi dijapauze. Krajem dijapauze sa niskim temperaturama višestruko se povećava nivo trehaloze, glukoze i fruktoze, a tek neznatnoglicerola. NMR metabolomički profili su potvrdili značaj glicerola u otpornosti na hladnoću, ali i aminokiselina alanina i prolina. U nedijapuzirajućim i dijapauzirajućim gusenicama na 5oC registrovano je prisustvo putrescina, poznatog markera stresa, a njegova uloga, kao i moguća uloga njegovih daljih metabolita spermina i spermidina tek treba da se ispita. Odgovor ADS-a na niske temperature takođe je zavistan od perioda dijapauze. Na sredini dijapauze, na temperaturi od -3oC, a ne i na -16oC, dolazi do povećanja koncentracije H2O2 i signala melanin radikala, dok aktivnost katalaze i CuZnSOD, ali ne i MnSOD, opada. Kako je glavni izvor H2O2 mitohondrijalna MnSOD, ovaj enzim je verovatno uključen u regulaciju unutarćelijskog nivoa H2O2. Produkcija melanina verovatno je u funkciji odbrane organizma od oksidativnog stresa zbog uklanjanja intracelularnog H2O2. Predpostavljamo da je temperatura od -3oC ključna temperatura za pokretanje adaptivnih mehanizama za otpornost na hladnoću gusenica O.nubilalis. Promene u ADS sa izlaganjem niskim temperaturama nisu primećene na kraju dijapauze. Rezultati ove teze ukazuju da je dijapauza dinamično stanje podeljeno na faze, a sama otpornost na hladnoću dobroregulisana i usklađena sa ukupnim metaboličkim potrebama tokom dijapauze.

European corn borer, Ostrinia nubilalisHb, is insect species of the temperate zone that survives low winter temperatures as a diapausing larva. Diapausing larvae are cold hardy and tolerate the formation of extracellular ice crystals. The aim of this thesis was to investigate the cellular and molecular basis of O. nubilalis cold hardiness. Thus, our research was focused on the non-diapausing (ND) as well as the diapausing (D) larvae that were exposed to low temperatures during different diapausal phases. Body water management, content of polyols and sugars as well as metabolomic NMR profiles, were assessed. Because of the possible role of the antioxidant defence system (ADS) in insects’ cold hardiness, antioxidant enzymes activity as well as the content of H2O2 and melanin was measured. Results showed that the exposure of diapausing larvae to low temperatures provokes exchanges of osmotically active (OA) body water between hemolymph and fat body. With low temperatures, OA water was decreased in hemolymph, increased in fat body while the content of osmotically inactive (OI) water was constant. The level of glycerol was higher in diapausing compared to non-diapausing larvae, and showed 2-fold increase with the exposure to low temperatures in mid-diapause. This confirmed that glycerol is O.nubilalismajor cryoprotectant in mid-diapause cold hardiness. On the other hand, at the end of the diapause, the content of trehalose, glucose and fructose, except the glycerol, was substantially increased. NMR metabolic profiling confirmed the importance of glycerol in diapausal cold hardiness as well as implied the importance of alanine and proline. In addition to this, putrescine, a well-know stress marker, were detected in ND and D larvae at 5oC suggesting that its metabolites, namely sperimine and spermidine, can have potential role in diapause. The ADS response to low temperatures differs during diapause timescale. In mid-diapause, exposure of larvae to -3oC, but not -16oC, resulted in the elevation of H2O2 concentration and melanin radical signal. On the other hand, the activity of catalase and CuZnSOD, but not of MnSOD, was decreased. Since the mitochondrial MnSOD is the main source of H2O2, this enzyme probably regulates the intracellular level hydrogen peroxide. Melanin probably serves as a defence mechanism against oxidative stress while it reduces the level of intracellular H2O2. Temperature of -3oC is probably the ‘trigger temperature’ for mechanism in adaptation of larvae O.nubilalisto cold. There were no changes detected in ADS during exposure to cold in the end of diapausing period. The results of this thesis suggested that the O. nubilalis diapause is rather dynamic state divided into different phases and that the cold hardiness is highly regulated and in accordance with the current metabolic demands of each diapausal phase.