التبصرة الخاصة بالتعليق أو التلخيص |
ملخص، الخ / Identification of different aphid species mostly depends on the morphological features of alate adults. Morphological characters alone have proven inadequate in differentiation between closely related species. Throughout the last few years, many authors used biochemical and molecular genetic techniques in insect differentiation. Biochemical and molecular genetic fingerprint was started with differentiation among ten aphid species belonging to genus Aphis in Egypt, so the current study has continued to complete aphid biochemical and molecular genetic fingerprints with other aphid species.
The current studies aimed to study the following points:-
1) Survey of some Macrosiphini and Rhapalosiphina aphid species in Egypt.
2) Identification of eighteen aphid species belonging to Tribe: Macrosiphini and Sub-tribe: Rhapalosiphina by using Biochemical and Molecular genetic markers (Isozyme, SDS-Page total protein and RAPD-PCR markers).
3) Evaluation the efficiency of the used biochemical and molecular genetic techniques in aphid species discrimination.
4) Survey of biochemical and RAPD–PCR species – specific bands to construct a molecular key to identify some of those tested species.
5) Studying the phylogenetic relationships among the eighteen tested aphid species.
1. Survey of Aphid Species Under Consideration and New Record Species in Egypt:-
Common aphid species belonging to Tribe: Macrosiphini and Tribe: Aphidini (Sub-tribe: Rhapalosiphina) were surveyed and collected from main host plants at some localities of Egypt, through out about two successive years extended from December, 2005 to June, 2007. Survey studies showed that fourteen aphis species belonging to tribe Macrosiphini i.e. Acyrthosiphon pisum, Brevicoryne brassicae, Brachcaudus helichrysi, Brachycaudus schwartzi , Capitophorus elaeagina, Chomaphis inculata, Doctynotus sonchi, Myzus persicae, Nasonovia (Hyperomyzus) lactucae, Pentalonia nigronervosa, Pleotrichophorus chrysanthemi, Macrosiphum rosae, Macrosiphum (Sitobion) avenae and Metopolophium dirhodum , and four aphid species belonging to Tribe: Aphidini (Sub-tribe: Rhapalosiphina) i.e Hyalopterus pruni, Schizaphis graminum, Rhopalosiphum maidis and Rhopalosiphum padi were surveyed.
Brachcaudus (Appelia) shwartzi (Borner) was recorded for the first time in Egypt during the present study. This species was attacking leaves of apricot, Prunus armeniaca and peaches Prunus persica with high density during May, 2006 at El-Tahrir, El- Behera Governorate. Identification procedure was confirmed at British Museum by Prof. R. Blackman. Verbal and drafting descriptions for alate viviparous female of this new recorded species was presented. Moreover a bracket key was constructed to identify different species of Genus Brachycaudus in Egypt.
2. Biochemical Genetic Characterizations:
2.1. Isozyme Electrophoresis.
The eighteen aphid species were subjected to Native – polyacrylamide gel electrophoresis (Native-PAGE) to identify isozyme variations among them. Four enzymatic systems were chosen for this purpose, which were α-esterase, β-esterase, Acid phosphatase, and Alkaline phosphatase. Isozyme electrophoresis analysis showed 96.88 % of polymorphism among the eighteen tested aphid species that twenty seven different bands were observed in the four enzymatic systems. Only one common band in β- esterase was detected among the eighteen tested aphid species to achieve the lowest polymorphism (87.5 %.) in all enzymatic systems, while the highest percent of polymorphism was 100% in the other enzymatic systems.
2.2. SDS- Protein Electrophoresis.
Total protein electrophoresis pattern analysis was also subjected among the eighteen aphid species. Thirty two protein bands were recorded along SDS-Polyacrlamide gel. Molecular weights of recorded bands ranged from 14.366 to 245.578 KDa. Two of them were recorded as monomorphic band; in addition one band was species – specific band. Total protein electrophoresis analysis showed the lowest level of polymorphism among the eighteen tested species (93.75%).
3. Molecular Genetic Characterizations:
3.1. RAPD-PCR analysis for the tested Aphid species.
The eighteen tested aphid species were subjected to RAPD-PCR fingerprint study, to analyze molecular marker and estimate polymorphism among them. So, ten arbitrary primers were used for this purpose. The RAPD analysis with the ten primers gave 101 different DNA fragment bands with wide range of molecular sizes. Four monomorphic distinct fragment bands were recorded; most of them occurred with primer B10. So, the lowest value of polymorphism (80%) was generated by it. On the other hand, the highest polymorphic bands were produced by primers C15, D2, I17, L12, L20, UBC75 and Z1 to achieve polymorphism levels reach 100%. The highest number of DNA fragment bands (fifteen bands) was observed in primer D5, while the lowest number was five bands, generated by primer L13. Twenty six DNA fragment bands were expressed as species – specific bands. Most of them occurred in primer I17, while each of primer L12 and L20 gave one species- specific bands. The complied data for the ten primers recoded 95% polymorphism among the eighteen tested aphid species.
4. Evaluation the Efficiency of the Used Biochemical and Molecular Genetic Techniques in the Aphid Species Discrimination:-
It is obvious that each of isozyme and RAPD electrophoresis analysis revealed the highest level of polymorphism, comparing with total protein electrophoresis analysis The electrophoresis study for those different molecular systems revealed that 160 different bands pattern in all used molecular system were detected, seven of them were considered as common bands in the all tested species, while thirty four bands were observed in some species as species - specific bands. The electrophoresis studies in those different molecular systems reflected 95.21% polymorphism among the tested species.
5. Survey of Biochemical and RAPD–PCR Species – Specific Marker for the Tested Aphid Species:-
It is obvious from obtained data that 26 of DNA fragment species – specific bands were generated by the ten arbitrary primers which could distinguish thirteen aphid species out of the eighteen aphid species. Each of Acyrthosiphon pisum, Brachycaudus schwartzi, Myzus persicae, Macrosiphum rosae and Metopolophium dirhodum didn’t generate any species- specific markers with the used primers. In contract, Doctynotus sonchi harbored the highest number of DNA species – specific bands with the used primer. Primer I17 was the most effective primer which generated the highest number of species - specific marker. So it could distinguish five aphid species [Brevicoryne brassicae, Nasonovia (Hyperomyzus) lactucae, Macrosiphum (Sitobion) avenae, Hyalopterus pruni and Schizaphis graminum]. While each of primers L12 and L20 gave one species – specific marker, which can distinguish Rhopalosiphum maidis and Hyalopterus pruni, respectively.
Most of obtained isozyme markers were observed in each of β- esterase and Acid phosphatase isozyme analysis. The isozyme analysis could distinguish only the following species [Brevicoryne brassicae, Brachycaudus schwartzi, Capitophorus elaeagina, Nasonovia (Hyperomyzus) lactucae, Macrosiphum rosae, Rhopalosiphum padi and Hyalopterus pruni]. On the other hand, Total protein analysis could distinguish only Capitophorus elaeagina by protein band with molecular weight 23.789 Kda.
The previous results declared that the used biochemical and RAPD – PCR analysis techniques could distinguish fifteen aphid species out of eighteen; however any species – specific marker weren’t observed in each of Acyrthosiphon pisum, Myzus persicae and Metopolophium dirhodum.
The present study revealed that the fifteen species, with species - specific markers, can be differentiated well by applying electrophoresis analysis program based on only using β- esterase and Acid phosphatase isozyme analyses in addition to application of RAPD-PCR analysis with primers B10, I17, L12, L13, UBC75 and Z1 which can save time, material and efforts with the fifteen aphid differentiated species. So it scored all species – specific makers, which can differentiate among them.
On the other hand, a branching molecular key was constructed to identify thirteen aphid species out of the eighteen aphid species based on 26 DNA species – specific bands.
6. Phylogenetic relationships:-
Studying the genetic similarities and phylogenetic relationships among the eighteen tested aphid species was based on obtained data of morphological character analysis, isozyme, totals protein, and RAPD- PCR analysis, as well as the combined effect of those techniques.
The proximity matrix analysis of the eighteen aphid species based on morphological characters reflected that the highest similarity value 93.8% was noticed between Brachcaudus helichrysi and B. schwartzi, while the lowest similarity value in morphological character was recorded between Acyrthosiphon pisum and Chomaphis inculata, which was 42.9%. It declared that those aphid species can be separated into three main groups depending on their morphological characters. The first main group divided into two sub groups, the first included Brachcaudus helichrysi, B. schwartzi and Chomaphis inculata, and the second sub group included Doctynotus sonchi, Macrosiphum rosae, Nasonovia (Hyperomyzus) lactucae, Rhopalosiphum padi, Macrosiphum (Sitobion) avenae and Myzus persicae. The second main group contains Rhopalosiphum maidis, Hyalopterus pruni and Brevicoryne brassicae, while the third main group divided also into two sub groups, the first sub group included Capitophorus elaeagina, Pleotrichophorus chrysanthemi and Pentalonia nigronervosa. While the second included Metopolophium dirhodum, Schizaphis graminum and Acyrthosiphon pisum.
The proximity matrix analysis for the eighteen aphid species based on combined effect of isozyme, Total protein and RAPD-PCR analysis reflected that the highest similarity value (89.9%) was recorded between Rhopalosiphum maidis and R. padi, while the lowest similarity in all tested parameters polymorphism (43.9%) was recorded between Brachycaudus helichrysi and Pleotrichophorus chrysanthemi. Dendrogram analysis can separate the Greaminaceae host plant aphid from others. Moreover it showed clearly the gab between Tribe: Macrosiphini and Tribe: Aphidini (Sub-tribe: Rhapalosiphina). The study declared that the eighteen tested aphid species can be classify into two main groups depending on combined effect of isozyme, Total protein and RAPD analysis. The first main group divided into three sub groups, the first sub group includes the Greaminaceae host plants aphid i.e. Rhopalosiphum maidis, R. padi, Schizaphis graminum, Hyalopterus pruni, Macrosiphum (Sitobion) avenae and Metopolophium dirhodum; while the second sub group includes Acyrthosiphon pisum, Brevicoryne brassicae, Capitophorus elaeagina and Doctynotus sonchi; the third sub group contains Pleotrichophorus chrysanthemi, Macrosiphum rosae, Chomaphis inculata and Myzus persicae, Nasonovia (Hyperomyzus) lactucae and Pentalonia nigronervosa. On the other hand, the second main group includes two closely related species Brachycaudus helichrysi and B. schwartzi. |