الفهرس 
Traditional TaxonomyOnly 14 pages are availabe for public view
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Abstract
Taxonomical studies
Taxonomical studies of family Diaspididae were carried out throughout four successive years 2010 to 2014. These studies were conducted by using both traditional and molecular genetic procedures.
1. Traditional Taxonomy :
1. Survey of different diaspid species:
Monthly field excursions were carried out throughout four successive years 2010-2014 to collect samples of infested plants with diaspid species at different localities in some Governorates of Egypt. These species were carfully inspected. The shape and colour of adult fameles as well as the position and colour of adult females were recorded. Permanent mounted slides were prepared for adult females for each collected species. These slides were used for identification procedure using available keys.
Survey studies throughout the four years indicated that 35 diaspid species were surveyed. These species are belonging to 14 genera. Three species were recorded for the first time in Egypt during the present work. These species are: Abgrallaspis mendax , Diaspidiotus pronorum and Lindingaspis tingi . The surveyed species were arranged in Table according their taxonomic status with their scientific names. English and Arabic names of their hosts as well as plant families were also tabulated. The synonomy lists for each species were also recorded. Thirty eight host plants were recorded. These plants are belolnging to 14 plant families.
2. Pictorial Field Key:
A simple pictorial field key was constructed to facilate identification of these diaspid species in the field. This key was based on the shape and colour of the scale of adult female scale as well as position, colour of exuviae and shape and colour of adult female after removing the scale.
3. Bracket Pictorial Key:
Bracket pictorial key was constructed to facilate identification these surveyed diaspid species in laboratory based the morphological characters of adult females. This key need permanent mounted slides of adult females and used to confirm identification.
2. Molecular Genetic Taxonomy:
Modern molecular biology techniques were applied by using Polymeraes Chain Reaction (PCR) with five specific primers for family Diaspididae. These techniques were used to obtained finger printing for the 35 survyed species as well as to determine different taxa of this family and to investigate the phylogenetic relationships between these taxa.
Five specific primers for Diaspididae were used. These primer are [ITS2 (f&r)] and (ITS4,ITS5) were used as forward and reverse . In addition three specific primers (Oligonucleotides) with specified sequancy i.e.[A.acut R-1] ,[ A.agua R-2] and [P.stera R-3] were used to determine different taxa. These studies were carried at three steps:
1. Investigate special bands to investigate genus category:
Preliminary experiment was conduct by selecting two genera which contain the largest number of species out of 14 genera. These genera are Lepidosaphes which represented by seven species and Parlatoria which represented by five species. Three selected oligonucleotide primers[A.agua R-2], [A.acut R-1] and [P.Stra R-3] were chosen to detect the common bands for each genus.
Results showed stated that genus Lepidosaphes had three common bands from the three tested primers which considered as generic-specific markers for this genus. These bands with their molecular sizes are as follows: [A.acut R-1], 185bp. [A.agua R-2], (190bp.) and [P.stra R-3], (190bp.). While genus parlatoria had one common band with primer [A.agua R-2] , (165). This band could be considered as generic-specific band for genus Parlatoria.
2. Specific PCR profiles of 14 genera:-
Five specific primers were used to detect specific band(s) for 14 genera out of 35 species. Results obtained could be summarized as follows:
Primer ITS2:-
The PCR amplified 38 fragments with this primer from 12 genera out of 14 genera. It produced interspecific variations in nine genera only. The molecular sizes of these fragments were ranged 60-550bp.
Results showed that these bands could be discriminate nine genera as generic-specific bands (Abgrallaspis mendax, Aonidiella aurantii, Aulacaspis tubercularis, Fiorinia fioriniae, Hemiberlesia rapax , Lepidosaphes beckii , Lindingaspis rossi, Pseudaulacaspis cockerelli and leucaspis riccae.
Bands (420, 395, 210, 160bp) could be considered to distinguish genera belonging tribe Aspidiotini (Chrysomphalus aonidum, Diaspidiotus perniciosus , Hemiberlesia rapax, Abgrallaspis mendax and Aonidiella aurantii).
Band 240bp could be considered to distinguish genera belonging tribe Diaspidini Fiorinia fioriniae and Lepidosaphes beckii.
Primer, ITS4, ITS5:-
The PCR amplified with 26 informative bands with this primer from 12 genera out of 14 genera. But it gave interspecific variations in seven genera only. The molecular sizes of these bands were ranged from 70- 400bp.
Results showed that these bands could be discriminate seven genera by genaric-specific bands (Abgrallaspis mendax, Aulacaspis tubercularis, chrysomphalus aonidum, Diaspidiotus perniciosus, Dynaspidiotus britannicus, Fiorinia fioriniae and Pseudaulacaspis cockerelli)
Band (380 bp.) could be considered as distinguish some genera belonging to tribe Aspidiotini, Aonidiella aurantii, Hemiberlesia rapax and Lindingaspis rossi.
Primer A. acut R-1(Oligonucleotide)
This primer produced 20 informative bands from 8 genera out of the 14 genera. The molecular size of these bands ranged from 100 to500bp.
Results showed that these bands could be discriminate four genera by generic-specific bands (Abgrallaspis mendax, Aonidiella aurantii, Hemiberlesia rapax and Pseudaulacaspis cockerelli).
Bands(400bp) could be considered to distinguish genera belonging tribe Aspidiotini (Abgrallaspis mendax, Aonidiella aurantii, Hemiberlesia rapax)
Primer A. agua R-2 (Oligonucleotide)
This primer produced 18 informative bands fore nine genera out of the 14 genera. The molecular size of these bands were ranged from 100 to500bp.
Results showed that these bands could be discriminate four genera by generic-specific band Abgrallaspis mendax, Aonidiella aurantii, Diaspidiotus perniciosus, Lepidosaphes beckii, Parlatoria ziziphi(.
Primer P. stra R-3 (oligonucleotide)
This primer produced 12 informative bands from six genera only out of 14 genera. The molecular sizes of these bands were ranged from 90- 400bp.
Results showed that there bands could be discriminate four genera with generic-specific bands (Abgrallaspis mendax, Aonidiella aurantii, Hemiberlesia rapax and Lepidosaphes beckii)
from these results it could be stated that the five tested primers could be determine agroup of bands as specific group for each genus.
3. Species PCR profiles of 35 species
Four specific primers [2singl+2mix] were used to investigate 35 diaspid species. Results obtained could be summarized as follows:
Primer [ITS2]
The PCR amplified with this primers 99 bands from 27 species out of 35 species. The molecular sizes of these bands were ranged between 50 – 500bp.
Results showed that this primer produced species-specific bands to discriminate ten species by species-specific band(s) (Aonidia lauri [325,190], chrysomphalus bifasciculatus [145], Dynaspidiotus britannicus [480,215], Hemiberlesia lataniae [360], Hemiberlesia rapax [370], Aulacaspis tubercularis [380], Lepidosaphes ficus [460], Lepidosaphes pinnaeformis [295], Lindingaspis rossi [90], Lindingaspis tingi [315], Parlatoria blanchardi [280], Parlatoria ziziphi [330])
Distinguish species belonging to two genera particularly Lepidosaphes (390 pb.), Parlatoria (350 pb.) by generic- specific bands.
Distinguish species belonging to tribe, Diaspidini (200 pb.), Lepidosaphes ulmi, Lepidosaphes pinnaeformis and Aulacaspis tubercularis.
Primer [ITS4, ITS5]
The PCR amplified 78 bands with this primer from 28 species out of 35 species. The molecular sizes of these bands were ranged between 90 – 550bp.
Results showed that this primer produced species-specific bands to discriminate ten species by species-specific band(s) (Chrysomphalus aonidum [330], chrysomphalus dictyospermi [240,180], Dynaspidiotus britannicus [420,230,120], Hemiberlesia rapax [90] , Fiorinia linderae [125], Lepidosaphes beckii [540,460,345,225], Lindingaspis rossi [440,245], Lindingaspis floridana [510], Parlatoria proteus [520], Parlatoria ziziphi [380,315] )
Distinguish species belonging to two genera, particularly Lepidosaphes (360 pb.), Parlatoria (500 pb.) by generic- specific band. Band (415bp.) could be considered to distinguish two species belonging to tribe Diaspidini, (Aulacaspis tubercularis and Lepidosaphes beckii). Band (310bp.) could be considered to distinguish two species belonging to tribe Diaspidini, (Lepidosaphes tapleyi and Aulacaspis tubercularis).
Mix primer [A. acut R-1 (r)] + [ITS2(f)]
The PCR amplified 80 bands with these mix primers produced from 30 species out of 35 species under investigation. The molecular sizes of these band(s) were ranged between 70 – 545bp.
Results showed that these mix primers produced species-specific bands used to discriminate ten species by species-specific bands (Aonidia lauri 155, Aonidiella citrine [215,290,325,460], chrysomphalus aonidum 220, chrysomphalus bifasciculatus [140], Aulacaspis tubercularis [260,500], Lepidosaphes beckii [165, 90], Lepidosaphes tapleyi [270], Lepidosaphes ficus [105], Parlatoria blanchardi [330], Parlatoria pergandii [185,140], Parlatoria proteus [375,295], Parlatoria ziziphi [340,145])
Mix primer [P. stra R-3 (r)] + [ITS2 (f)]
The PCR amplified 52 bands with these primers from 28 species out of 35 species under investigation. The molecular sizes of these bands were ranged between 60 – 330.
Results showed that these mix primers produced species-specific bands used to discriminate ten species by species-specific bands (Chrysomphalus aonidum [300], Diaspidiotus pronorum [125], Dynaspidiotus britannicus [110,280], Hemiberlesia rapax [230], Fiorinia linderae [60,140], Lepidosaphes pallidula [100], Pseudaulacaspis cockerelli [120], Parlatoria ziziphi [240])
Distinguish species belonging to two genera particularly Lepidosaphes [265], Parlatoria [170], Lindingaspis[205], Hemiberlesia [290] ) by generic- specific bands.
from these results it could be stated that these four specific primers produced species- specific bands for 24 species out of 35 species under investigation. Also, these primers found to produced groups of bands which considered as a group specific band for nine species. Therefore, these four primers could be used to determine 33 out of 35 species.
4. Proposal Molecular Branching key for Identification of thirty five species:
During the present work of molecular genetic a branching key was constructed. This key was based on the specific bands produced from the four specific primers with the thirty five species. These specific – bands produced for both genus and species categories with their molecular sizes were cited in this key. This key could be facilate determination 33 diaspid species out 35 species.
5. Phylogenetic Relationships:
Genetic similarities and phylogenetic relationships between the thirty five species were based on three criteria; morphological characters of female, molecular sizes of females species-specific band(s) detected from the four tested primers and combination of morphological and molecular characters. These phylogenetic criteria are represented different approaches could be used to determine genetic similarity between these diaspid species.
Results showed that using of both morphology (as a morphological expression) and DNA analysis (as a genetic material) criteria could be provided more conclusive results regarding phylogenetic relationships among different taxa of Diaspdidae.