الفهرس 
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Abstract
SUMMARY
The present work was undertaken to study certain aspects which might affect the phenomenon of resistance or tolerance of the pink bollworm (Pectinophora gossypiella Sannd.) field populations collected from some Governorates in Egypt to certain conventional insecticides, comparing with ore and nanoparticles formulation of natural compounds. Moreover, chitosan biopolymers and it’s nanoparticles was also evaluated.
The discriminating dose was using in monitoring the resistance of field population to tested insecticides and compounds. Comparative investigation of the rate of development of selected resistance strains of pink bollworm to each of organophosphorus and Pyrethroids compounds was carried out. The enzymatic activity which have a role in resistance to insecticide was investigated in certain field population. The study included also the identification and detection of genetic diversity of pink bollworm field population. Also the insecticide-resistance gene expression using Real-Time (PCR) was evaluated in certain population. The following results were obtained:
5.1. Efficiency of certain conventional toxicants and some naturally compounds and their nanoparticler against the 1st and 4th instar larvae of the pink bollworm, Pectinophora gossypiella, laboratory strain:
5.1.1. The main criteria of the toxicity line representing the biological activity of the investigated compounds against the 1st and 4th instar larvae of p. gossypiella lab- strain i.e. The LC50, LC90 values, toxicity index, relative potency levels conformed each other revealed that Lambada-cyhalothrin Pyrethroid (Lambada star) was the most effective one than the other investigated compounds; Chlorpyrifos (OP) (Pyriban A) and naturally inorganic ore Silica and Aluminum oxide, and biopolymer Chitosan traditional and it’s nano-particles formulation.
5.1.2. The nanoparticle formulations of natural inorganic ore compounds were proved to be highly toxic to both instar larvae of P. gossypieilla lab – strain, than each of traditional natural ore products. Also the nano Chitosan was more efficacy than traditional form of Chitosan.
5.1.3. It is an interestingly note that nanoparticles of naturally inorganic ore Aluminum oxide (Aluminum) and biopolymers Chitosan proved to be better than or equal organophosphorus Pyriban A against 1st instar larvae of pink bollworm.
5.1.4. These naturally nanoparticles materials did not gave the same efficacy in the field application as lab – treatment due to the behavior of pest infestation and factors affecting the molecules delivery to target pest larvae in field.
5.2. Monitoring of insecticidal resistance percentage in certain field population of pink bollworm by using discriminating concentration technique:
5.2.1. It could be concluded here that the monitoring of percent resistance in field population of P. gossypiella by using discriminating concentration (LC99) of certain compounds, revealed that the resistance (%) was varied from Governorate to another for the same tested compound.
5.2.2. Hence Fayoum population proved to recorded the highest resistance (%) against Chlorpyrifose, Lambda-Cyhalothrin and Silica nanoparticles. While Qalubia population recorded the highly resistance (%) for Chitosan nanoparticles and Bihera population for Aluminum oxid nanoparticles.
5.2.3. In general, all the investigated field population of P. gossypiella exhibited highly resistance percentages to Pyriban A (OP) followed by Lambda-Cyhalothrin than the other tested naturally ore and biopolymers with slight variation in its level from one to another.
5.3. Development of resistance in the 4th instar larvae of the pink bollworm, P. gossypiella:
5.3.1. It could be concluded here that Chlorpyrifos (Pyriban A) was somewhat good selected agent than Lambada-cyhalothrin (Lambada star).
5.3.2. The, Pyriban A caused 12.91-fold relative to lab – strain and 7.78-fold relative to parent field population after eight successive generation of selection, while Lambada star caused 12.06 relative to laboratory strain and relative to parent feild population. After the same selected generation.
5.4. Enzymatic activity in different strains of the pink bollworm, Pectinophora gossypiella collected from different Governorates:
5.4.1. Alkaline phosphatase activity:
5.4.1.1. The obtained results indicated that the 4th instar larvae homogenates of the different field population showed much higher levels of alkaline phosphatases activity than the baseline laboratory strain of the pink bollworm, P. gossypiella.
5.4.1.2. The field population collected from Fayoum Governorate showed the highest activity of alkaline phosphatase as compared with the other field colony strains.
5.4.2. Acetylcholin-esterase activity (A. Ch. E.):
5.4.2.1. The enzyme activity levels were much higher in the 4th larval instars of the various field population as compared with the baseline laboratory strain.
5.4.2.2. The highest enzymatic activity level in the field colony strains was occurred in Fayoum Governorate.
5.4.2.3. The lowest enzymatic activity was noticed in the 4th instar larvae collected from Kafrel-Sheikh Governorate.
5.4.2.4. The enzymatic activity of the 4th larval instars homogenates collected from field colony of Bihera and Qalubia Governorates occupied the middle situation among the two previous strains
5.4.3. Glutathion - S - transferases Activity (GSTs):
5.4.3.1. The 4th instar larvae of the field population showed in general slight higher levels of glutathione S- transferase activity than the laboratory strain.
5.4.3.2. The corresponding calculated activity ratio of glutathione-S-transferase in relative to laboratory strain were ranged between 1.05 and 1.44 for tested field population.
5.4.4. Nonspecific β esterase’s activity:
5.4.4.1. It was clear that the levels of β- esterase’s activity were much higher in all field instar larvae than the laboratory strain.
5.4.4.2. The activity ratios of the field colony strains of the Fayoum Behira, Qalubia and Kafr-Elsheikh Governorates were 1.51, 1.17, 1.06 and 1.05; respectively as compared with the baseline laboratory strain.
5.5. Genetic diversity of pink bollworm, P. gossypiella 4th instar larvae collected from different Egyptian Governorate using RAPD-PCR:
The PCR amplification, the number and size of fragments was differ between the tested strains. Identical sized fragments detected between different populations indicate genetic relatedness or similarity, as followes:
5.5.1. The RAPD analysis of the extracted DNA samples using Primer B12 revealed that Primer B12 showed the largest number of RAPD fragments where 52 bands were detected in the four field colony and laboratory populations of the pest under the study. The number of produced fragments distributed as 8, 14, 11, 14 and 5 bands in the laboratory strain, Kafr El Sheikh, Fayoum, Qalubia, and Behira populations respectively. Results indicated that polymorphism generated by the primer B12 showed 0 monomorphic, 11 polymorphic and 29 unique, 40 polymorphic with unique, and total polymorphism estimated 40 with polymorphism percentage 100% in the tissues of the 4th instar larvae of the tested pest. The analysis of the similarity index showed low similarity index values associated with the four field colony populations, which ranged from 0.00 to 0.30 compared with the baseline laboratory strain indicating highly resistance in these field strains to the conventional insecticidal applications used under field conditions. In this respect, highly resistance could be noticed in Fayoum followed by Qalubia field populations where the estimated similarity index values were 0.00 and 0.18 compared with the laboratory population; respectively.
5.5.2. The fingerprint of the analysis of extracted DNA samples using primer P13 revealed some variability between the five different strains of P. gossypiella. The RAPD-PCR pattern showed a total of 35 bands; including 6, 8, 1, 12 and 9 fragments for laboratory, Kafe El Sheikh, fayoum, Qalubia and Behira strains; respectively.The number of DNA fragments ranged from 1 to 12 bands, and fragments size ranged between 192 and 1316 bp. The similarity polymorphism generated by the primer P13. Data indicated 0 monomorphic, 3 polymorphic without unique, 27 unique and 30 polymorphic with unique, for the some field strains relatively. Similarity index were 0.00 in Fayoum field strain; while it recorded 0.29 and 0.11 and 0.27 for Kar El Sheikh, Qalubia and Behira field strains; respectively relative to the baseline laboratory strain, indicating highly resistance in the 4th instar larvae.
5.5.3. Fingerprint profiles generated by using primer C16 appeared low number of fragments, where 11 fragments were detected in the 4th instar larvae of both field and laboratory strains including two fragments each of Kafr El Sheikh, Fayoum, Qalubia and Behira field strains, whereas three fragments were detected in the baseline laboratory strain. The similarity index showed no similarity between the four field strains and the baseline laboratory strain, where the similarity index being 0.00 indicating very highly resistance levels in the 4th instar larvae.
5.5.4. The RAPD analysis of the extracted DNA samples generated by using primer C18 showed the least total number of fragments as compared with the other primers used under the study, these are 10 fragments distributed as two fragments for each strain. The amplified fragments size ranged between 433 to 284bp. The data of similarity polymorphism demonstrated 1 monomorphic, 1 polymorphic without unique, 3 unique, 4 polymorphic with unique, 5 total polymorphism estimated 80%. The similarity index values of the field populations compared with the baseline recorded values of 0.50 for Kafr El Sheikh and Qalubia. On the other hand, the similarity index values between Kafr El Sheikh and both of Fayoum and Behira as well as between Fayoum and each of Qalubia and Behira, and between Qalubia and Behira were differed greatly which is 0.00.
5.6. Insecticide-resistance gene expression quantitation using Real-time PCR:
5.6.1. After normalization, the fold change in BtR gene expression was 0.25 times the control for Kafr El-Sheikh, Qalubia and Behira and 0.0625 times the control for Fayoum. The fold-change in the Cad1 gene expression was 0.125 times the control for Kafr El-Sheikh and Behira; and 0.0625 times the control for Fayoum and Qalubia. Thus, after normalization, comparing the relative expression values among the two target genes is possible, showing a higher expression level for BtR than Cad1. As anticipated, both genes are relatively equal in expression performance, where the two genes are positively correlated, as the reduced Cadherin gene (Cad1) is a receptor protein associated with resistance to a Bt toxin in the pink bollworm.
5.6.2. Although both genes are positive indicators for the insect’s ability to process and tolerate the insecticide, the collected samples showed a lower expression than the control group (i.e., laboratory untreated insect lines). In the field, insect resistance to toxins from Bacillus thuringiensis (Bt) often lowering the fitness of the resistant individuals relative to susceptible individuals in the absence of Bt toxins. In all cases, the ability of pink bollworm to evolve resistance via both qualitative and quantitative changes in receptor proteins demonstrates their remarkable adaptability.