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

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

The effect of low-dose spermidine supplementation on polyamine content and antioxidative defence mechanisms in honey bees

Summary. The honey bee, a widespread pollinator, contributes to the conservation of biodiversity. In recent decades, a trend of declining colony numbers has emerged. The unsustainable exploitation of the environment may be the cause of this phenomenon. One protective strategy of organisms is to strengthen their antioxidative capacity. A class of positively charged molecules, polyamines, plays important roles in various cellular processes. They exert a regulatory effect on gene expression, have antioxidative properties, and promote longevity in model organisms. The three main representatives are putrescine, spermidine and spermine. The aim of this study was to determine whether supplementation of bees with low-dose spermidine leads to an increased level of the mentioned polyamines and whether this could strengthen the antioxidative defence system. Two experimental groups were established: C group (control), fed with a 50% (w/v) sucrose solution, and S0.01 group, whose diet was supplemented with 0.01 mM spermidine. The experiment lasted for 10 and 17 days. A significant increase in putrescine, spermidine and spermine content was noted in the supplemented group after 17 days, compared to its control. These results show a positive impact of spermidine supplementation on maintaining polyamine levels throughout aging. FRAP and MDA biochemical assays were used for the assessment of oxidative status. FRAP assay showed increased antioxidative capacity in the S0.01 group. These results are in accordance with the results obtained from the MDA assay, which showed a decreased level of lipid peroxidation in the supplemented group, in comparison to the control. The potential practical outcome of this study could be the use of spermidine in beekeeping practice to promote overall honey bee health

Diapause induces remodelling of the fatty acid composition of membrane and storage lipids in overwintering larvae of Ostrinia nubilalis, Hubn. (Lepidoptera: Crambidae)

Seasonal changes in the FA composition of triacylglycerols and phospholipids prepared from the whole body of non-diapausing and diapausing fifth instar larvae of Ostrinia nubilalis, Hubn. (Lepidoptera: Crambidae) were determined to evaluate the role of these lipids in diapause. Substantial changes in the FA composition of triacylglycerols and phospholipids were triggered by diapause development. This led to a significant increase in the overall FA unsaturation (UFAs/SFAs ratio), attributable to an increase in the relative proportion of MUFAs and the concomitant decrease in PUFAs and SFAs. In triacylglycerols, the significant changes in FAs composition is the result of an increase in the relative proportions of MUFAs, palmitoleic acid (16:1n-7) and oleic acid (18:1n-9), and a concomitant reduction in composition of SFAs and PUFAs, mainly palmitic acid (16:0) and linoleic acid (18:2n-6), respectively. Changes in the composition of phospholipids were more subtle with FAs contributing to the overall increase of FA unsaturation. Differential scanning calorimetry (DSC) analysis revealed that the melt transition temperatures of total lipids prepared from whole larvae, primarily attributable to the triacylglycerol component, were significantly lower during the time course of diapause compared with non-diapause. These observations were correlated to the FA composition of triacylglycerols, most likely enabling them to remain functional during colder winter conditions. We conclude that O. nubilalis undergoes remodelling of FA profiles of both energy storage triacylglycerols and membrane phospholipids as an element of its overwintering physiology which may improve the ability to cold harden during diapause

Hypolipidemic and antioxidant effects of buckwheat leaf and flower mixture in hyperlipidemic rats

As a source of biologically active compounds, buckwheat has beneficial effects in nutrition due to its high content of flavonoids, particularly rutin. Aim of our study was to examine effects of buckwheat on plasma lipid status and phospholipids fatty acids composition, histological and parameters of oxidative stress in Wistar rats fed a high-fat diet. This study showed that buckwheat leaf and flower (BLF) mixture supplementation significantly reduce weight gain, plasma lipid concentrations and atherogenic index in rats fed a high-fat diet. Treatment of the high-fat group of animals with buckwheat significantly increased percentage of n-6 fatty acids as well as eicosapentaenoic acid (EPA) and decreased percentage of saturated fatty acids (SPA) and oleic acid. Buckwheat antioxidant effects diminished negative influence of high-fat diet in hyperlipidemic rats, while pathohistological analysis of liver confirmed changes after high-fat consumption. Our results showed hypolipidemic, antiatherogenic and antioxidative features of buckwheat leaf and flower mixture, and these parts of the plant with the highest rutin content could be beneficial in prevention and curing of hyperlipidemia

Antihyperlipidemic, antioxidant and weight-lowering effects of "Vitalplant"

BACKGROUND: "VITALPLANT" medicinal plant mixture, aimed at body weight regulation and metabolism enhancement was formulated in order to be incorporated as an ingredient in functional food formulations on the basis of known biological activity of the alder buckthorn bark, peppermint, caraway and parsley. METHODOLOGY: The body weight-lowering potential, hypolipidemic effects and antioxidant activity of "Vitalplant" mixture were examined on five groups of male Wistar rats on different food regimes for 14 weeks. RESULTS: "Vitalplant" mixture supplementation caused an increase in food intake, but induced a decrease in body weight gain. The lipid status and atherogenic index (AI) were significantly improved in "Vitalplant" mixture supplemented hyperlipidemic (HF) groups in comparison to HF group. "Vitalplant" mixture was able to influence the activity of antioxidant enzymes and to protect erythrocytes of the rats fed the HF diet from oxidative damage, which was indicated by significantly lower malondialdehyde (MDA) levels in "Vitalplant"- HF groups in comparison to the HF group. CONCLUSION: "Vitalplant" mixture seemed to be beneficial for the reduction of body weight and improvement of antioxidant status of the erythrocytes, and its antihyperlipidemic property was highly active for enhancing the profile of plasma lipids in rats

Echocardiography-based Left Ventricle Cardiac Hypertrophy Simulations

Clinical scenarios can be evaluated using numerical modeling of the cardiac cycle prior to experimental or clinical application. Changes in wall thickness, displacement fields, and general cardiac function are all affected by hypertrophy. In our study, we calculated the effects of eccentric and concentric hypertrophy and monitored changes in ventricular thickness and shape. Concentric hypertrophy results in thicker walls, while eccentric hypertrophy results in thinner walls. Passive stresses were calculated using recently established material modals based on Holzapfel’s work. Our modeling approach is based on composite shell finite elements, allowing easier and more efficient modeling compared to traditional 3D finite elements. A left ventricular model was constructed using echocardiographic images. Our modeling technology is based on accurate patient-specific geometries and realistic constitutive curves, so it can be used as the basis for real-world applications. Our model can be used to test medical hypotheses about the development of hypertrophy in healthy and diseased hearts under the influence of different conditions and factors.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Effect of Cold Acclimation on Selected Metabolic Enzymes During Diapause in The European Corn Borer Ostrinia nubilalis Hbn

The European corn borer, Ostrinia nubilalis Hbn., is a pest Lepidopteran species whose larvae overwinter by entering diapause, gradually becoming cold-hardy. To investigate metabolic changes during cold hardening, activities of four metabolic enzymes – citrate synthase (CS), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in whole-body homogenates of pupae, non-diapausing and diapausing larvae acclimated to 5 °C, −3 °C and −16 °C. The highest CS activity was detected in non-diapausing larvae, reflecting active development, while the highest in vitro LDH activity was recorded in diapausing larvae at temperatures close to 0 °C, evidencing a metabolic switch towards anaerobic metabolism. However, in-gel LDH activity showed that production of pyruvate from lactate is triggered by sub-zero temperatures. The activities of both aminotransferases were highest in non-diapausing larvae. Our findings suggest that during diapause and cold hardening the aminotransferases catalyse production of L-alanine, an important cryoprotectant, and L-aspartate, which is closely tied to both transamination reactions and Krebs cycle. The results of this study indicate that, during diapause, the activity of metabolic enzymes is synchronized with exogenous factors, such as temperatures close to 0 °C. These findings support the notion that diapause is metabolically plastic and vibrant, rather than simply a passive, resting state

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

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

Laboratory bioassays on the response of honey bee (Apis mellifera L.) glutathione S-transferase and acetylcholinesterase to the oral exposure to copper, cadmium, and lead.

In the present study, the influence of cadmium, copper, and lead on two enzymes often used as biomarkers in toxicological analysis was investigated. Bees were fed with 1 M sucrose solution containing 10-fold serial dilutions of CuCl2 (1000 mg L-1, 100 mg L-1, and 10 mg L-1), CdCl2 (0.1 mg L-1, 0.01 mg L-1, and 0.001 mg L-1), or PbCl2 (10 mg L-1, 1 mg L-1, and 0.1 mg L-1) during 48 h. Our results showed that the total glutathione S-transferase activity was not changed under the influence of cadmium and lead, and it was decreased with the highest concentration of copper. The level of gene expression of the three analyzed classes of glutathione S-transferase was significantly increased with increasing concentrations of copper and cadmium. Lead did not cause significant changes in glutathione S-transferase activity and gene expression, while it showed biphasic effect on acetylcholinesterase activity: lower concentration of lead, 0.1 mg L-1 inhibited and higher dose, 10 mg L-1 induced acetylcholinesterase activity in honey bees. Furthermore, our results showed a significant decrease of the acetylcholinesterase activity in honey bees treated with 0.001 and 0.01 mg L-1 CdCl2. Our results indicate the influence of cadmium, copper, and lead on GST and AChE in the honey bees. These results form the basis for future research on the impact of metallic trace element pollution on honey bees