Cold hardiness and dehydration of European corn Borer (Ostrinia nubilalis): molecularand cellmechanisme

Abstract

<p>Kukuruzni plamenac,&nbsp;<em> Ostrinia nubilalis</em>Hb, je insekt umerenog klimatskog područja koji niske&nbsp; zimske temperature preživljava ulaskom u dijapauzu u stadijumu gusenice. Tokom dijapauze gusenice postaju otporne na hladnoću i sposobne&nbsp; da prežive vanćelijsko zamrzavanje telesne tečnosti.<br />Cilj ove teze je bio razumevanje mehanizama otpornosti na niske temperature gusenica&nbsp; <em>O. nubilalis </em>na ćelijskom i molekularnom nivou.<br />Istraživanja su rađena na letnjim, nedijapauzirajućim gusenicama kao i zimskim gusenicama koje su&nbsp; bile izlagane uticaju niskih temperatura u različitim periodima dijapauze. Određivan je režim telesne vode, sadržaj poliola i &scaron;ećera kao mogućih krioprotektora, kao i NMR metabolomički profili. Zbog pretpostavljene uloge atioksidativnog sistema za&scaron;tite (ADS) u procesima vezanim za otpornost na<br />hladnoću određivane su aktivnosti određenih antioksidativnih enzima kao i produkcija H₂O₂i melanina.<br />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&nbsp; 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 vi&scaron;estruko povećava pri izlaganju niskim temperaturama na sredini dijapauze ukazujući na njegov značaj kao glavnog krioprotektora u ovoj fazi dijapauze. Krajem dijapauze sa<br />niskim temperaturama vi&scaron;estruko se povećava nivo trehaloze, glukoze i fruktoze, a tek neznatnoglicerola.<br />NMR metabolomički profili su potvrdili značaj glicerola u otpornosti na hladnoću, ali i<br />aminokiselina alanina i prolina. U nedijapuzirajućim i dijapauzirajućim gusenicama na 5^oC registrovano je prisustvo putrescina, poznatog markera stresa, a&nbsp; 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&nbsp; -3^oC, a ne i na&nbsp; -16^oC, dolazi do povećanja koncentracije H₂O_2&nbsp;i signala melanin radikala, dok aktivnost katalaze i CuZnSOD, ali ne i MnSOD, opada. Kako je glavni izvor H₂O₂mitohondrijalna MnSOD, ovaj enzim je verovatno uključen u regulaciju unutarćelijskog nivoa H₂O₂.<br />Produkcija melanina verovatno je u funkciji odbrane organizma od oksidativnog stresa zbog uklanjanja intracelularnog H₂O_2. Predpostavljamo da je temperatura od&nbsp; -3^oC ključna temperatura za pokretanje adaptivnih&nbsp; mehanizama za otpornost na hladnoću gusenica<em> O.nubilalis</em>. Promene u ADS sa izlaganjem niskim temperaturama nisu primećene na kraju dijapauze.<br />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.</p>

<p>&nbsp;European corn borer, <em>Ostrinia nubilalisHb</em>, 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.</p><p>The aim of this thesis was to investigate the cellular and molecular basis of&nbsp; <em>O. nubilalis </em>cold&nbsp;hardiness.</p><p>Thus, our research was focused on the non-diapausing (ND) as well as the diapausing (D) larvae that&nbsp;were exposed to low temperatures during different &nbsp;diapausal phases. Body water management,&nbsp;content of polyols and sugars as well as &nbsp;metabolomic NMR profiles, were assessed. Because of the&nbsp;possible role of the antioxidant defence system (ADS) in insects&rsquo; cold hardiness, antioxidant&nbsp;enzymes activity as well as the content of H₂O_2&nbsp;and melanin was measured.</p><p>Results showed that the exposure of diapausing larvae to low temperatures provokes exchanges of&nbsp;osmotically active (OA) body water between hemolymph and fat body. With low temperatures, OA&nbsp;water was decreased in hemolymph, increased in fat body while the content of osmotically inactive&nbsp;(OI) water was constant.</p><p>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&nbsp;O.nubilalismajor cryoprotectant in mid-diapause cold hardiness. On the other hand, at the end of&nbsp;the diapause, the content of trehalose, glucose and fructose, except the glycerol, was substantially&nbsp;increased.</p><p><br />NMR metabolic profiling confirmed the importance of glycerol in diapausal cold hardiness as well&nbsp;as implied the importance of alanine and proline. In addition to this, putrescine, a well-know stress&nbsp;marker, were detected in ND and D larvae at 5^oC suggesting that its metabolites, namely sperimine&nbsp;and spermidine, can have potential role in diapause.</p><p>The ADS response to low temperatures differs during diapause timescale. In mid-diapause,&nbsp;exposure of larvae to&nbsp; -3^oC,&nbsp; but not&nbsp; -16^oC, resulted in the elevation of H₂O_2&nbsp;concentration and&nbsp;melanin radical signal. On the other hand, the activity of catalase and CuZnSOD, but not of&nbsp;MnSOD, was decreased. Since the mitochondrial MnSOD is the main source of H₂O2, this enzyme&nbsp;probably regulates the intracellular level hydrogen peroxide. Melanin probably serves as a defence&nbsp;mechanism against oxidative stress while it reduces the level of intracellular H₂O₂. Temperature of -3^oC is probably the &lsquo;trigger temperature&rsquo; for mechanism in adaptation of larvae&nbsp; <em>O.nubilalisto </em>&nbsp;cold.&nbsp;There were no changes detected in ADS during exposure to cold in the end of diapausing period.</p><p>The results of this thesis suggested that the <em>O. nubilalis</em> diapause is rather dynamic state divided into&nbsp;different phases and that the cold hardiness is highly regulated and in accordance with the current&nbsp;metabolic demands of each diapausal phase.</p>