الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
Abstract
Date palm (Phoenix dactylifera L.) family; Arecaceae, (Angiosperms,
monocotyledon) is one of the most important and oldest trees in the arid
and semi-arid regions, in which it was cultivated for its edible sweet
fruits. It is considered as an economic crop in the Arab land, and Egypt is
the first county in date palm cultivation and dates production in the world.
This economic tree faces many problems, the biggest one is the Red Palm
Weevil (RPW) which known of its rapid expansion and destruction
during the last three decades. The knowledge of the red palm weevil gut
microbiota composition is of importance, as it could lead to establish new
and innovative approaches for red palm weevil management or at least it
could help in improving classic management approaches. This study was
applied as a first exploration of the gut bacterial community of healthy
field caught larvae of Red Palm Weevil (Rhynchophorus ferrugineus), in
Egypt, by using culture-independent techniques based on 16S RNA
genes.
1. Sample collection
Three groups of 20-22 healthy field caught Rhynchophorus
ferrugineus larvae – dominated by the late instars – were collected from
three different date palm trees from two different places; El-Badrashein
and Agriculture Research Center (ARC) in El-Giza governorate, Egypt.
The sampling palm trees have a known history clear of any insecticidal or
biological control treatment.
2. Larvae dissection and metagenomic DNA extraction
Field caught Larvae transferred within 24 hours to the laboratory
and undergone dissection and gut content evacuation under aseptic
conditions. After dissection process, we have got three pools of larvae gut
content in three 50ml tube. Metagenomic DNA extracted from the three
gut content pools by using DNA isolation kit (MO-BIO Laboratories).
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SUMMARY
Bahaa El-Din M. A. Osman, (2018), M. Sc., Fac. Agri., Ain Shams Univ.
3. PCR amplification of 16S rRNA genes
Bacterial 16S rRNA genes of the bulk DNA of the gut content
pools were amplified by using Touch-up and Touch-Down PCR
programs, with 341F-GC as a forward primer and 907R as a reverse
primer. All of the three pools were amplified positively and produced a
16s rRNA amplicons with an approximate size 586 bp
4. Denaturing Gradient Gel Electrophoreses (DGGE) of 16S rRNA
amplicons
16S rRNA amplicons were run on 6% polyacrylamide gel in
DCodeTM Mutation Detection System (Bio-Rad,UK). The
electrophoresis was conducted at 120 V and 35 Amp under a constant
temperature of 60C for 17 hours followed by staining with ethidium
bromide for 20 min, washed in double distilled water for 15 min ,
visualized and photographed under UV gel documentation system; Gel
DocTM XR+ imager (Bio-Rad Laboratories inc,UK)
5. DGGE Bands profiling analysis
DGGE bands pattern showed 26 distinguishable bands with an
average of 8–9 bands per sample. 9 bands were excised, eluted, and
undergone Re-PCR amplification with the same primers (without the GC
clamp)
6. Sequencing and construction of phylogenetic tree
The Bands that shown PCR positive results were purified and
sequenced. The sequences were analyzed by nucleotide-nucleotide
BLAST, Sequence alignments and Phylogenetic trees were constructed by
bioinformatics programs.
7. Results of DGGE profiling and its sequence analysis
DGGE bands pattern showed relatively low complexity of the gut
microbiota. DGGE bands sequences analysis have shown the relatively
prevalence of Lactic Acid Bacteria; Leuconostoc lactis, Lactococcus
46
SUMMARY
Bahaa El-Din M. A. Osman, (2018), M. Sc., Fac. Agri., Ain Shams Univ.
lactis and Lactobacillus nagelli. The presence of Cellulomonas cellasea ,
Bifidobacterium minimum and Gordonibacter urolithinfaciens as a
common bacterial phylotype among all three samples was recorded. The
DGGE results led to the assumption of RPW larvae does not depend on
the digestion of cellulose, hemicellulose and xylane as a primary nutrition
function, while it is primarily dependent on readily sugars and
carbohydrates in palm sap. LAB and bifidobacterium might have a key
role on insect nutrition and antimicrobial activity that need more
investigations.