Publication: Azadırachtın’ın galleria mellonella larvalarının bağışıklık sistemine etkisi
Abstract
ÖZETAZADIRACHTIN’IN Galleria mellonella LARVALARININ BAĞIŞIKLIK SİSTEMİNE ETKİSİNeem ağacı tohumlarından elde edilen Azadirachtin (AZA)’nin böceklerde toksik etkileri olduğu bilinmektedir. AZA’nın kimyasal insektisitlere oranla çevresel ve ekonomik zararları daha azdır. Ayrıca memeliler üzerinde toksik etkilerinin olmaması AZA için bir kullanım tercihi oluşturmaktadır.Çalışmamızda AZA’nın böcek hemositleri üzerindeki etkisi incelenmiştir. Çalışmada kullanılan büyük bal mumu güvesi Galleria mellonella, bilimsel araştırmalar için iyi bir model organizma olması ve laboratuvar koşullarında kolay ve hızlı yetiştirilmesi sebebiyle tercih edilmiştir. Deneylerde son evre larvalar (0.16-0.17 g) kullanıldı. Dört farklı dozda AZA (0.5, 1.0, 1.5, 2.0 μg/ larva) böceklere force-feeding yöntemi ile uygulandı. Larvalar force-feeding öncesi 3 saat aç bırakıldı. Force-feeding uygulamasından 48 saat sonra böcekler incelemeye alındı. Bir grup larvaya enkapsülasyon tepkimesi için Sephadex A-25 cam boncuk enjekte edildi ve bu işlemden sonraki 4 ve 24 saatte gösterdikleri enkapsülasyon ve melanizasyon tepkimeleri gözlendi. Diğer bir grup larvanın hemolenfleri alındı. Boyama işleminden sonra hemositlerin gösterdiği apoptoz ve nekroz tepkimeleri, mitotik indeks ve mikronukleus oluşumları incelendi.AZA’ya bağlı olarak tüm dozlarda canlı hücre sayısında azalma tespit edildi. AZA dozlarına bağlı olarak geç apoptoz ve nekroz verilerinde artış gözlendi. Nekrotik hücre sayısında 0.5 ve 1.0 μg/ larva AZA uygulanan gruplarda önemli bir artış belirlendi. 0.5 μg/ larva dozu 0.5 μg/ larva AZA dozunun mitotik hücre sayısında önemli bir artışa sebep olduğu tespit edildi. Mikronukleus oluşumunun 1.5 ve 2.0 μg/ larva AZA dozlarında arttığı belirlendi. 0.5, 1.0 ve 1.5 μg/ larva AZA dozlarında 4 saatlik enkapsülasyon yanıtında artış bulunurken 24 saatlik enkapsülasyon yanıtında fark tespit edilmedi. 4 saatlik melanizasyon yanıtında tüm AZA dozlarında ve etanol uygulanan grupta artış gözlendi. AZA uygulamasına bağlı olarak 24 saatlik melanizasyon yanıtında azalma gözlendi. Sonuç olarak AZA, G. mellonella larvalarının bağışıklık sistemini etkilemiş ve genotoksik hasara sebep olmuştur.SAYFATEŞEKKÜR iİÇİNDEKİLER iiÖZET vABSTRACT viSEMBOLLER viiKISALTMALAR viiiŞEKİL LİSTESİ xTABLO LİSTESİ xiii1.GİRİŞ 11.1.Amaç 1 1.2.Azadirachtin’in Özellikleri ve Toksik Etkileri 21.3.Azadirachtin ile İlgili Yapılan Çalışmalar 61.4.Böceklerde Bağışıklık Sistemi ve Hemositler 91.5.Böceklerde Bağışıklık Sistemi ve Hemositler ile İlgili Yapılan Çalışmalar 181.6.Böcek Hemositlerinde Apoptoz Mekanizması 21 1.7.Böcek Hemositlerinde Apoptozla İlgili Yapılan Çalışmalar 231.8.Galleria mellonella L. 1758 (Büyük Balmumu Güvesi)’nin Biyolojisi 252.MATERYAL VE YÖNTEM292.1.G. mellonella Kültürünün Oluşturulması ve Devamının Sağlanması 292.2.Azadirachtin’in Force-Feeding Yöntemi ile Böceklere Uygulanması 302.3.Apoptotik İndeks, Mitotik İndeks ve Mikronukleus Durumu 322.4.Enkapsülasyon ve Melanizasyon 342.5.İstatiksel Analiz 353.BULGULAR VE TARTIŞMA363.1.AZA’nın İmmün Sisteme Etkisi 363.2. Farklı Dozlardaki AZA’nın G. mellonella Larvalarının Hemositlerinde Apoptotik İndeks, Mitotik İndeks ve Mikronukleus Oluşumuna Etkisi 363.2.1. Farklı Dozlardaki AZA’nın G. mellonella Larvalarının Hemositlerinde Apoptotik İndekse Etkisi 36 3.2.2. Farklı Dozlardaki AZA’nın G. mellonella Larvalarının Hemositlerinde Mitotik İndekse Etkisi 403.2.3. Farklı Dozlardaki AZA’nın G. mellonella Larvalarının Hemositlerinde Mikronukleus Oluşumuna Etkisi 41 3.3.Farklı Dozlardaki AZA’nın G. mellonella Larvalarının Hemositlerinde Enkapsülasyon ve Melanizasyon Davranışı 43 3.3.1. Farklı Dozlardaki AZA’nın G. mellonella Larvalarının Hemositlerinde Enkapsülasyona Etkisi 433.3.2. Farklı Dozlardaki AZA’nın G. mellonella Larvalarının Hemositlerinde Melanizasyona Etkisi 484.SONUÇLAR 55KAYNAKLAR 57 ÖZGEÇMİŞ 68
ABSTRACTEFFECT OF AZADIRACHTIN ON IMMUNE SYSTEM OF Galleria mellonella LARVAEAzadirachtin (AZA) obtained from Neem tree seeds is known to have toxic effects on insects. Environmental and economic harms of AZA are less than chemical insecticides. In addition, it is preferred because it has no toxic effect on mammals.In our study, the effects of AZA on insect hemocytes were investigated. The Greater Wax Moth, Galleria mellonella used in the study was preferred because of being an excellent model organism for scientific research and easy and fast growing in laboratory conditions. Last instar larvae (0.16-0.17 g) were used for the experiments. Four different doses of AZA (0.5, 1.0, 1.5, 2.0 µg/ larva) were applied to larvae via force-feeding method. Larvae were starved for 3 h before feeding. Larvae were investigated 48 hours after force-fed. Sephadex A-25 glass beads were injected into a group of larvae for encapsulation reactions, and encapsulation and melanization reactions were determined at 4 and 24 hours after this procedure. The hemolymph was taken from another group of larvae. The apoptosis and necrosis reactions, micronucleus and mitotic index of the hemocytes were investigated in preparations prepared with hemolymph after dyeing. The number of viable cells decreased in all doses due to AZA. There was an increase in late apotosis and necrosis data depending on the doses of AZA. There has been a significant increase in the number of necrotic cells in 0.5 and 1.0 µg/ larvae of AZA applied groups. 0.5 µg/ larvae AZA caused a significant increase in the number of mitotic cells. The significant increase of micronucleus formation was found in the groups applied to 1.5 and 2.0 µg/ larvae doses. 0.5, 1.0 and 1.5 µg/ larva doses caused an increase in 4 hours encapsulation response, but did not have effect at 24 hours encapsulation response. There was increase at 4 hours melanization response in all AZA doses and groups of ethanol. Depending on AZA application, a decrease was observed at 24 hours melanization response.As a result of this study, AZA affected the immune system of G. mellonella larvae and caused genotoxic damages.Table of ContentsAcknowledgements iTable of Contents iiSummary vAbstract viSymbols viiAbbreviations viiiList of Figures xList of Tables xiii1.Introduction 11.1.Purpose 11.2.Properties and Toxic Effects of Azadirachtin 2 1.3.Studies on Azadirachtin 6 1.4.Immune System in Insects and Hemocytes 9 1.5.Studies on Immune System in Insects and Hemocytes 18 1.6.Mechanism of Apoptosis in Insect Hemocytes 21 1.7.Studies Related to Apoptosis in Insect Hemocytes 23 1.8.Biology of Galleria mellonella L. 1758 (Greater Wax Moth) 25 2.Materials and Methods 29 2.1.Creation and Continuation of G. mellonella Culture 292.2.Application of Azadirachtin to Insects by Force-Feeding Method 302.3. Apoptotic Index, Mitotic Index and Micronucleus Reactions 322.4. Encapsulation and Melanization 342.5. Statistical Analysis 353.Discovery and Discussion 363.1. The Effect of AZA on the Immune System 363.2. Effect of AZA in Different Doses on Apoptotic Index, Mitotic Index and Micronucleus Formation in Hemocytes of G. mellonella Larvae 36 3.2.1. Apoptotic Index Effect in Hemocytes of G. mellonella Larvae of AZA in Different Dose 363.2.2. Mitotic index effect of hematocytes of G. mellonella larvae of AZA in different doses 403.2.3. Effect of AZA in Different Doses on the Formation of Micronucleus in Hemocytes of Larger G. mellonella 413.3. Encapsulation and Melanism Behavior in the Hemocytes of G. mellonella Larvae of AZA in Different Dose 433.3.1. Effect of AZA in Different Dose on Encapsulation in Hemocytes of Larval Larvae of G. mellonella 433.3.2. Effect of AZA in Different Doses on Melanization in Hemocytes of Larvae of G. mellonella 48Results 55References 57Resume 68
ABSTRACTEFFECT OF AZADIRACHTIN ON IMMUNE SYSTEM OF Galleria mellonella LARVAEAzadirachtin (AZA) obtained from Neem tree seeds is known to have toxic effects on insects. Environmental and economic harms of AZA are less than chemical insecticides. In addition, it is preferred because it has no toxic effect on mammals.In our study, the effects of AZA on insect hemocytes were investigated. The Greater Wax Moth, Galleria mellonella used in the study was preferred because of being an excellent model organism for scientific research and easy and fast growing in laboratory conditions. Last instar larvae (0.16-0.17 g) were used for the experiments. Four different doses of AZA (0.5, 1.0, 1.5, 2.0 µg/ larva) were applied to larvae via force-feeding method. Larvae were starved for 3 h before feeding. Larvae were investigated 48 hours after force-fed. Sephadex A-25 glass beads were injected into a group of larvae for encapsulation reactions, and encapsulation and melanization reactions were determined at 4 and 24 hours after this procedure. The hemolymph was taken from another group of larvae. The apoptosis and necrosis reactions, micronucleus and mitotic index of the hemocytes were investigated in preparations prepared with hemolymph after dyeing. The number of viable cells decreased in all doses due to AZA. There was an increase in late apotosis and necrosis data depending on the doses of AZA. There has been a significant increase in the number of necrotic cells in 0.5 and 1.0 µg/ larvae of AZA applied groups. 0.5 µg/ larvae AZA caused a significant increase in the number of mitotic cells. The significant increase of micronucleus formation was found in the groups applied to 1.5 and 2.0 µg/ larvae doses. 0.5, 1.0 and 1.5 µg/ larva doses caused an increase in 4 hours encapsulation response, but did not have effect at 24 hours encapsulation response. There was increase at 4 hours melanization response in all AZA doses and groups of ethanol. Depending on AZA application, a decrease was observed at 24 hours melanization response.As a result of this study, AZA affected the immune system of G. mellonella larvae and caused genotoxic damages.Table of ContentsAcknowledgements iTable of Contents iiSummary vAbstract viSymbols viiAbbreviations viiiList of Figures xList of Tables xiii1.Introduction 11.1.Purpose 11.2.Properties and Toxic Effects of Azadirachtin 2 1.3.Studies on Azadirachtin 6 1.4.Immune System in Insects and Hemocytes 9 1.5.Studies on Immune System in Insects and Hemocytes 18 1.6.Mechanism of Apoptosis in Insect Hemocytes 21 1.7.Studies Related to Apoptosis in Insect Hemocytes 23 1.8.Biology of Galleria mellonella L. 1758 (Greater Wax Moth) 25 2.Materials and Methods 29 2.1.Creation and Continuation of G. mellonella Culture 292.2.Application of Azadirachtin to Insects by Force-Feeding Method 302.3. Apoptotic Index, Mitotic Index and Micronucleus Reactions 322.4. Encapsulation and Melanization 342.5. Statistical Analysis 353.Discovery and Discussion 363.1. The Effect of AZA on the Immune System 363.2. Effect of AZA in Different Doses on Apoptotic Index, Mitotic Index and Micronucleus Formation in Hemocytes of G. mellonella Larvae 36 3.2.1. Apoptotic Index Effect in Hemocytes of G. mellonella Larvae of AZA in Different Dose 363.2.2. Mitotic index effect of hematocytes of G. mellonella larvae of AZA in different doses 403.2.3. Effect of AZA in Different Doses on the Formation of Micronucleus in Hemocytes of Larger G. mellonella 413.3. Encapsulation and Melanism Behavior in the Hemocytes of G. mellonella Larvae of AZA in Different Dose 433.3.1. Effect of AZA in Different Dose on Encapsulation in Hemocytes of Larval Larvae of G. mellonella 433.3.2. Effect of AZA in Different Doses on Melanization in Hemocytes of Larvae of G. mellonella 48Results 55References 57Resume 68