الفهرس 
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Abstract
VI. SUMMARY
The most important objective of research on honey bee (Apis mellifera) is to breed bees that resist or tolerate pests and pathogens. Upon immunological encounter with a pathogen, the honey bee queen could influence the immunity of direct progeny, thus increasing resistance to current infection in the colony. The present investigation has been conducted to characterize morphological and physiological changes in virgin honey bee queens challenged, in 4th larval instar, with a sub-lethal dose of Paenibacillus larvae larvae, the causative of American foulbrood (AFB) which is considered to be the most threatening bacterial disease of honey bee brood. It is very important to employ resistance management strategies to allow bee brood to develop resistance against bacteria. The current study can also be accomplished through identification of traits or genes linked to resistance to infection in individual queens. Expression levels of genes encoding disease-related proteins can help infer which proteins are involved with disease tolerance and resistance in honey bees.
The present study provides direct tests to assess the quality of treated honey bee queens in terms of their external physical characters of the body (body weight, body length, abdomen length, thorax width, fore wing length and head width) and internal body characteristics (ovary size and number of ovarioles per ovary) in comparison with normal ones. Quantitative and qualitative estimation of total body proteins along with specific ovary proteins in normal and treated queens were also studied to explore bacteria-related proteins involved with disease tolerance and resistance. This is in addition to study of humoral antibacterial activity, as an induced immune defensive reaction, against challenged bacteria (P. l. larvae) and other Gram +ve and Gram –ve bacteria. Finally, qualitative and quantitative identification of genes linked to appearance of this antibacterial activity in individual queens were also studied.
The results obtained from this study can be summarized in the following points:
1- Susceptibility of honey bee queens to bacterial pathogen:
The estimated LD50 for honey bee queens was 4.39 x103 CFU/queen. This dose reflects high resistance level of honey bee queens towards inoculated P. l. larvae. Additionally, the estimated LD20 was 1.07 x102 CFU/queen. This dose was enough to induce the immune response of queens and did not cause high mortality rate, so it can be used in the subsequent tests during the course of this study.
2. Measurements of external body characteristics:
The bacteria-treated queens didn’t undergo any significant changes in their body weight, body length, abdomen length, thorax width, fore wing length and head width than normal ones. This result reflects the difficulty of discrimination between normal and disease-tolerant queens in the field, i.e., there was no morphological bases of resistance, so the honey bee queens with less resistance quality can compete resistant queens in mating flights and laying eggs in the field.
3. Measurements of internal body characteristics:
The ovaries of treated queens showed an increase of their sizes as compared with normal queens. In contrast, the number of ovarioles per ovary didn’t show any significant changes between normal and treated queens. These results indicate that the bacterial treatment has no impact on the development of queen`s ovary. Also, the number of ovarioles/ovary is variable and shows interspecific morphological and physiological differences related to queen origin and breeding conditions.
4. Proteomic analysis:
The total body proteins of bacteria-treated queens showed a high significant increase, with a 99% confidence level, as compared with the normal queens. The same result was found with ovary proteins. This increase may be attributed to the protein crystals produced by bacterial cells which were toxic if eaten by the insect, or it may be resulted from the increase in protein concentration to face (resist) the bacterial infection.
Electrophoretic comparisons of the total body and ovary proteins of healthy, water-treated and bacteria-treated honey bee queens revealed that the treatment either with water or bacteria was capable of changing the protein profile qualitatively. The disappearance of some proteins may be attributed to their involvement in the immune reactions. On the other hand, the synthesis of new proteins may be a result of simultaneous induction of bacteria and a fast rate of transcription of immune genes that their activation lead to the synthesis of antimicrobial peptides.
5. Induction of antibacterial activity:
The total body as well as the ovary homogenates of honey bee queens showed antibacterial activity against P. l. larvae. Week activity was found with normal queens, while a high increase in activity was observed with bacteria-treated queens. This result may be due to the action of bacteria.
Additionally, the total body and ovary homogenates showed highly significant increase in the antibacterial activity of treated queens against Klebsiella pneumonia. Significant increases in activities were observed in total body homogenate against E. coli 2, and ovary extract against E. coli 1. This variation in response may be due to the difference in ovary protein composition than other body tissues.
6. Qualitative and quantitative identification of antibacterial genes:
The whole bodies of control and treated queens were subject to RNA extraction, and then reverse transcription to synthesis cDNA of target genes; abacien (Ab), defensin (Df) and vitellogenin (Vg) of honey bee queens was qualitatively detected. DNA fragments were found to be 72, 201 and 63 bp, respectively.
Real-time PCR was performed to quantify differences in Ab, Df and Vg genes expressions in normal and treated honey bee queens throughout fold changes using β-actin gene as a control expression gene. Expression analysis showed that Df is ˷4 fold and Vg is ˷20 fold up-regulated in treated queens compared to normal ones, while Ab gene have no changes in gene expression. The up-regulation of Df gene expression indicated to the enhancement of social and individual immunity in treated queens against antimicrobial agents. Also, the Vg up-regulation is linked to antioxidant functions which promote the queen longevity, more resistant to oxidative stress and is a good protein status indicator of treated queens.
7. Detection of P. l. larvae:
The presence of P. l. larvae in J-agar media as well as in honey bee queen`s ovary using specific primer (KAT1 and KAT2) resulted in 550 bp amplicon in treated queens’ DNA fragment.
The overall results can help attempts to breed immune honey bee queens that are resistant to disease; which could influence the immunity of direct progeny, where the queen as a single individual could positively influence the immunological status of the whole colony.