الفهرس 
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Abstract
6- Summary
This work was aimed to evaluate the efficacy of Ultra Violet non
and exposed nuclear polyhedrosisvirus (NPV) against the 4th
larval instar of cotton leafworm, Spodoptera littoralis.
Spodoptera littoralis (Boisd.) is a serious pest of cotton and other
important plants in Egypt. This thesis studied the UV effect on
Spodoptera littoralis nuclear polyhedrosis virus by: evaluating it
against Spodoptera littoralis and virus RAPD PCR DNA
fingerprint. The results showed that UV exposure decreased virus
concentration, efficacy and OAR, also damaged the virus DNA.
6.1. Insecticidal activity of the stock original nuclear
polyhedrosis virus against 4thinstar larvae.
The fourth instar larvae of Spodoptera littoralis were treated with
the original stock concentrations of nuclear polyhedrosis virus
(1.1x108, 3.11x107 and 6.7x106 PIB/ml). The results indicated
that these concentrations caused (79.96, 78.84 and 61.11%
mortality), respectively. The percentage of larval mortalities
increased with the increase of nuclear polyhedrosis virus
concentration. The results were analyzed by SPSS Program and
showed significant differences between all viral concentrations
used and control.
Lethal concentration values of LC50& LC90 were 1.3x106 and
6.03x108, respectively, lethal time of LT50 and LT90 were 4.29&
27.28 days, 9.26 &17.11days and 12.32&43.46 days for 1.1x108,
3.11x107 and 6.7x106PIB/ml, respectively. This result confirmed
that as the concentration of the virus increased as the LT50
decreased. But the LT90 in case of the 2nd concentration was less
than the highest concentration and less than the lowest
concentration due to latent effect of the virus inside the infected
162
Summary
larvae and the threshold efficacy of the virus against the infected
larvae.
6.2. Effect of ultra violet on insecticidal activity of Spodoptera
littoralis Nuclear Polyhedrosis Virus concentrations
against Spodoptera littoralis 4th larval instar.
Three diluted concentrations were taken from the original stock
and the number of PIB /ml/concentration for each concentration
(1.0 ml) were 1.1 x 108, 3.11 x 107 and 6.7 x 106, respectively.
The concentrations without UV exposure caused mortality
percentages to 4th larval instar of 79.96, 78.84 and 61.11% at
1.1x108, 3.11x 107 and 6.7x 106 PIB/ml, respectively. Each
concentration was exposed to UV for 5 and 20 minutes
producing other two concentrations from each exposed
concentration as a result of UV exposure. After exposure period,
the PIB/ml were counted and tested against fourth instar larvae
feeding on castor leaf disks treated with aqueous suspensions of
occlusion bodies (OBs). The results indicated that increasing the
exposure period of virus to UV decreased the concentration of
virus compared to the original stock virus, also, the mortality
percentage of larvae decreased and the original activity
remaining (OAR) decreased.
The correlation between concentrations, time of UV exposure,
corrected mortality and OAR were significant. from these data,
There was strong and significant reversible correlation between
each of (time of exposure & concentration), and between (time of
UV exposure &corrected mortality), (time of UV exposure &
OAR) because the value of correlation factor is (-) and near to
1and has very high significance.
163
Summary
There was strong and significant irreversible correlation between
each of the following (concentration& mortality), (concentration
&OAR), (OAR & corrected mortality) because the value of
correlation factor is (+) and near to 1 and has very high
significance value.
The lethal concentrations & lethal times values of Spodoptera
littoralis Nuclear Polyhedrosis Virus that kill the tested 4th instar
of Spodoptera littoralis larvae were calculated for UV non
exposed concentrations and for each of them with its groups were
calculated.
6.3- Effect of sub lethal concentrations of nuclear
polyhedrosis virus on survival and some biological
aspects of Spodoptera littoralis 4th instar 2,4and 6 days
post feeding.
Feeding of Spodoptera littoralis 4th instar larvae on contaminated
leaves with sub lethal concentrations of nuclear polyhedrosis
virus affected on Relative Growth Rate (RGR) of this insect and
caused disturbance in its development.
The results of the three experiments were compared with each
other by statistical analysis showing that there were significant
differences between experiment of feeding on infected food for 2,
4 and 6 days . Experiment of feeding on infected food for 6 days
in larval duration, in mean wt of pupa, in pupal duration, in
Longevity of adult, in mean no. of eggs, in mean no. of hatching
and in mean no. of hatching alive. There was no significant
difference between experiment of feeding on infected food for 2
days & experiment of feeding on infected food for 4 days.
6.4) Effect of non exposed and exposed nuclear polyhedrosis
virus to UV on 4th instar larvae of Spodoptera littoralis.
164
Summary
Symptoms and malformations of infected larvae and
Prepupae with Sp li Nucleopolyhedro Virus:
1) Skin redness in the body bottom with girdle formation around
infected larva body causing disability of the larvae to get rid of
the molting skin leading to difficulty in molting
2) Skin redness in the body bottom and pale swollen bodies with
softness and liquefaction of the body of infected larvae with Sp
li Nucleopolyhedro Virus
3) Darkness of infected larvae color and shrinkage& dwarf
(decrease in size of infected larvae with Sp li Nucleopolyhedro
Virus.)
Symptoms of Infected Pupal stage with Sp li Nucleopolyhedro
Virus:
Shrinkage&dwarf (decrease in size of infected pupa with Sp li
Nucleopolyhedro Virus), softness and girdle formation around
infected pupa causing difficulty in molting
Symptoms of Infected adult stage with Sp li Nucleopolyhedro Virus:
1) Development of one side wings and failure in development of
the other side wings.
2) Disability of infected adult with Sp li Nucleopolyhedro Virus
to develop its wings (Abnormal adults emerged with nonwings).
3) Failure of infected adult to molt completely from infected pupa
with Sp li Nucleopolyhedro Virus.
4) Pleat of wings of infected adults with Sp li Nucleopolyhedro
Virus leading to decrease in its spreading
6.5- Identification of NPVs.
Viral DNA isolation was carried out using the method described
in QIAamp® MinElute® Virus Spin kit manual. DNA of all the
samples isolates was amplified using two RAPD primers, Operon
A20& B17 (Operon Technologies, Inc., Alameda, CA, USA).
165
Summary
In this study RAPD analysis of two arbitrary oligonucleotide
primers generated 100% polymorphism. A total of 57 bands (19
bands rows) were obtained, ranging in size from 151 to 668 base
pairs.
In this study RAPD analysis of two arbitrary oligonucleotide
primers generated 100% polymorphism.
The data presented in Figs. (17&18) & Table (38) resulted from
RAPD PCR for NPV DNA with Operon B17, showed eleven
bands characterized for Control ranged from 100-700bp with
molecular weights (650, 582, 506, 458, 388, 350, 300, 234, 214,
185&154 bp) in comparison with Operon A20 which produced
four bands only (Figs.15, 16& Table, 35) ranged from 200to 600
bp (535, 379, 275 and 231 bp). Lane 2 with R1.1 (5 min
UVexposed 1st conc. of Spodoptera littoralis nuclear
polyhedrosis virus)had 2 bands between 400-506 bp (458& 506
bp) in comparison with Operon A20 (seven bands) ranged from
200to 700 bp (668, 535, 474 ,379, 341, 275&231 bp).No bands
were found in lane 3 for R1.2 (20 min UVexposed1st conc. of
NPV virus)while with Operon A20 revealed six bands ranged
between 200 to 700 bp (668, 535, 474, 379, 275& 231 bp). Ten
bands were appeared at lane 4 for R2.1 (5 min UV exposed 2nd
conc. of NPV virus) between 100-600 bp (582, 506, 458,
388,,350, 300, 234, 214, 185&154 bp .) while with Operon A20
produced no bands to be recorded. In lane 5, 5 bands appeared
for R2.2 (20 min UV exposed 2nd conc. of NPV virus) between
100 - 600 bp (582, 506, 350, 234, 185 bp) while with Operon
A20 produced four bands between200 to 400 bp at (379, 341,
275&207 bp.) There were no bands found with lanes 6&7for
R3.1 (5 min UV exposed 3rd conc. of NPV virus) &R3.2 (20 min
166
Summary
UV exposed 3rd conc. of NPV virus) while with Operon A20
formed two lanes with four bands for each of R3.1 (5 min UV
exposed 3rd conc. of NPV virus) produced 4 bands between 200
to 400 bp at (379, 275, 231&207 bp). R3.2 (20 min UV exposed
3rd conc. of NPV virus) produced 4 bands between 200 to 400 bp
(379, 275, 231&207 bp.)
By using Operon A20 the relationship between Control&R1.1 in
sharing bands was 0.73 in bands having (535&379&275&231 bp
m.wt), while Control&R1.2 in sharing bands was 0.8 in bands
having (535&379&275&231 bp m.wt). While Relationship
between R1.1&R1.2 in sharing bands was 0.92in bands having
(668&535&474&379&275 &231 bp m.wt) while the relationship
between Control &R2.2 was 0.5 in bands having (379&275 bp
m.wt) on other hand the relationships between (Control&R2.1,
R2.1, R2.2) in sharing bands were zero, while Control&R3.1 in
sharing bands was0.75 in bands having(379&275&231 bp m.wt)
but Relationship between Control&R3.2 was 0.75 in bands
having( 379&275&231 bp m.wt) on other hand R3.1&R3.2 in
sharing bands was1 in bands having(379&275&231&207 bp
m.wt). This was due to the effect of UV on the DNA of exposed
samples. UV exposure also affected the degradation rate of DNA
such as number of produced bands of UV exposed virus more
than UV non exposed virus that indicated that there was
degradation leading to more fragments for UV exposed virus
when we used Operon A20 more than non-exposed virus and the
%reduction in produced bands was negative but when appearing
of smear in UV exposed virus indicated there was completely
reduction (100%) as in R2.1 (5 min UV exposed 2nd conc. of
NPV virus).
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Summary
The reduction % in bands of UV exposed virus was calculated
and was (-ve) such as in R1.1 and R1.2, reduction was -75% and
-50% respectively. But there were 100% reduction as in R2.1due
to the effect of UV exposure caused very high degradation level
leading to smear on the electrophoresis gel but there were
0%reduction in R2.2, R3.1 and R3.2. All these results were due
to the effect of exposure of virus to UV.
While by using Operon B17 the relationship between
Control&R1.1 in sharing bands was 0.31 in bands having
506&458 bp m.wt. While between Control& R2.1 in sharing
bands was 0.95 in bands having (582 &506 &458 &388 &350&
300&234 & 214 &185&154 bp m.wt) ,on other hand control
&R2.2 in sharing bands was 0.625 in bands having(582& 506&
350& 234&185 bp m.wt), while the relationship between
R2.1&R2.2 in sharing bands was 0.67in bands having (582&
506& 350& 234&185 bp m.wt) while the relationship between
(Control &R1.2; R1.1&R1.2 ; Control&R3.1; Control&R3.2;
R3.1&R3.2) in sharing bands were zero. This was due to the
effect of UV on the DNA of exposed samples. UV exposure also
affected the degradation rate of DNA by the reduction in bands
of UV exposed virus such as there were 100% reduction as in
(R1.2 ,R3.1&R3.2 appeared as smears and had very high
degradation level in DNA) but there were reduction% in R2.1
was 9.1, R2.2 was 54.55%. All these results were due to the
effect of exposure of virus to UV.
Data were scored for computer analysis on the basis of the
presence or absence of the amplified products. If a product was
present in NPV virus sample, it was designated as „1‟, and if
absent it was designated as „0‟. Specific bands were studied (that
band which was present or absent only in a specific NPV virus
168
Summary
sample) for two used RAPD primers. The data generated were
analyzed with software Past version 3. Data were analyzed with
the (similarity and distance indices) function using Euclidean
distance and Jaccard‟s similarity coefficient to determine how
UV exposure affected the presence or absence of RAPD PCR
product bands for each virus sample by using these two primers.
A dendrogram was constructed using (multivar – cluster
analysis) in the same program (Past) Cluster analysis was
performed based on Euclidean distance (coefficient matrices
calculated from RAPD markers to generate a dendrogram of
Spodoptera littoralis NPV samples and coordinate graphs &
Jacquard’s similarity (coefficient matrices calculated from
RAPD markers to generate a dendrogram of Spodoptera littoralis
NPV samples. Each Dendrogram was resulted from RAPD
analysis of 2 primers used.
Pooled RAPD analysis of all 2 arbitrary oligonucleotide
primers generated 100% polymorphism, the most similar
Lanes were Lane 6 ( R3.1(5 minutes UV exposed 3rd
concentration of nuclear polyhedrosis virus)) and Lane 7(
R3.2(20 minutes UV exposed 3rd concentration of nuclear
polyhedrosis virus)) (0 as measured in Euclidean distance for
each of them and (1 as measured in Jacquard similarity for each
of them for two primers totally, this indicated that the effect of
UV exposure strength on these two samples leading to equality
of them in numbers of appeared bands with degradation in DNA
of each of them as by using Operon A20 and appeared smears
with strong degradation in DNA of each of them by using
Operon B17 .
The arrangement of the clades tells us which leaves (NPV virus
samples) are the most similar to each other in the effect strength
169
Summary
of UV exposure. The height of the branch points indicates similar
or different among each other: when the height increased, the
difference increased as Euclidean distance Dendrogram and
coordinate graphs.
The other dendrogram and coordinate graph separated the
NPV virus samples by Euclidean distance for Operon A20
only based on as the more value as the more difference between
the NPV virus samples in the clades in the strength of UV
exposure degradation effect and as the low number of bands
produced. Euclidean distance ranged between 0(the most similar
NPV virus samples (lane 6 (R3.1), lane7 (R3.2)) and 2.6458 (the
most dissimilar NPV virus sample and the most UV affected
sample (R2.1) appeared as smear meaning that it has the most
degradation level in this region compared to (R1.1) and has 2 as
Euclidean distance compared to Control), while (lane2( R1.1) ,
lane3( R1.2)) were near to each other so that they were in one
group as two clades, also (lane 5(R2.2) ) was near to (lane 6
(R3.1) and lane 7(R3.2) which were considered as one clade) so
that they were in one group as two clades, the nearness here in
their degradation effect by UV exposure and number of produced
bands.
Other dendrogram and coordinate graph separated the NPV
virus samples by Euclidean distance for Operon B17 only
based on as the more value as the more difference between the
NPV virus samples in the clades in the strength of UV exposure
degradation effect and as the low number of bands produced.
Euclidean distance ranged between 0(the most similar NPV virus
samples (lane 3(R1.2), lane 6 (R3.1), lane7 (R3.2)) and 3.32 (the
most dissimilar NPV virus sample and the most UV affected
sample compared to Control(lane 3(R1.2),lane 6 (R3.1), lane7
170
Summary
(R3.2)) appeared as smear meaning that it has the most
degradation level in this region).While(Lane4( R2.1))was near to
the Control and has 1 as Euclidean distance compared to Control.
Other dendrogram separated the NPV virus samples by
Jacquard’s similarity coefficient as the high value of
Jacquard‟s similarity coefficient as the high similarity between
samples. Jacquard‟s similarity coefficient was between zero (the
most dissimilar NPV virus sample (R2.1 compared to Control)
and 1(the most similar NPV virus samples (R3.1, R3.2).
from all results it was shown that the differences between these
samples of entomopathogenic Spodoptera littoralis nuclear
polyhedrosis virus were caused by the strength of the effect of
UV exposure on virus. This strength depended on the
concentration of the exposed virus and the time of UV exposure.
These effects of UV made degradation in DNA of this virus
leading to reduction in some bands resulted by used primers in
some studied samples of virus and also leading to increasing in
some bands resulted by used primers in other studied samples of
virus as a result of degradation in DNA into many fragments
facilitating primer binding with DNA.
there were 100% polymorphism between NPV virus samples in
bands produced by Operon A20 and Operon B17 and there are
0% monomorphic band for Operon A20 and for Operon B17 and
There is one positive marker band in raw5 having 650 bp m.wt
for Control( UV-non exposed Spodoptera littoralis nuclear
polyhedrosis virus)by operonB17 and there are two negative
marker bands in raw 10&raw 13 having 0 bp m.wt for R2.1(5
min UV exposed 2nd concentration of Spodoptera littoralis
nuclear polyhedrosis virus) by Operon A20.
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Summary
6.6- Investigation of UV non exposed Nuclear polyhedrosis
virus and UV exposed Nuclear polyhedrosis virus under
Light and electron microscopes.
photos for UV non exposed virus by different magnification
powers showed complete normal structural shapes of UV non
exposed nuclear polyhedrosis virus(virion containing DNA
surrounded by capsule surrounded by complete polyhedron),but
photos for UV exposed nuclear polyhedrosis virus for 5.0
minutes by different magnification powers showed malformed
incomplete leading to releasing of capsules without polyhedra
and also photos for UV exposed virus for 20.0 minutes by
different magnification powers showed malformed destroyed
structural shape of the virus polyhedra with released free
destroyed capsules and destroyed virions and incomplete virus
particle without polyhedra nor capsule