EFFECT OF GAMMA IRRADIATION AND PARASITIC NEMATODES ON THE BLACK CUT-WORM AGROTIS IPSILON (HUFN.)
By Thanaa Mohammad Sileem
B.Sc. Agric. (Economic Entomology), Zagazig University, 1987 M.Sc. Agric. (Economic Entomology)Benha University, 2004
THESIS
Submitted in Partial Fulfillment of the Requirements for the Degree
of DOCTOR OF PHILOSOPHY IN Agricultural Sciences (Economic Entomology)
Department of Plant Protection Faculty of Agriculture Moshtohor Benha University,
2011 APPROVAL SHEET
EFFECT OF GAMMA IRRADIATION AND PARASITIC NEMATODES ON THE BLACK CUT-WORM, AGROTIS IPSILON (HFUN.)
By Thanaa Mohammad Sileem B.Sc. Agric. (Economic Entomology), Zagazig University, 1987 M.Sc. Agric. (Economic Entomology)Benha University, 2004
This thesis for Ph. D. Degree had been Approved by:
Prof. Dr. Shalaby, M. El-Awady ……………………………. Professor of Economic Entomology-Plant Protection Dept. Fac. of Agric. Al-Azhar Univ.
Prof. Dr. Azza, K. A. Emam ……………………………... Professor of Economic Entomology, and Head of Plant Protection Dept. Fac. of Agric. Ain Shams Univ.
Prof. Dr. Ezzat, F. El-Khayat ……………………………… Professor of Economic Entomology-Plant Protection Dept. Fac. of Agric. Moshtohor, Benha Univ.
Prof. Dr. Samira, El. M. El-Naggar ……………………… Professor of Economic Entomology, and Head of Biological Application Dept. Atomic Energy Authority. Dr. Safaa, M . Halawa ………………………… Assistance Professor of Pesticides-Plant Protection Dept. Fac. of Agric. Moshtohor, Benha Univ.
Committee in charge Date / /2011 EFFECT OF GAMMA IRRADIATION AND PARASITIC NEMATODES ON THE BLACK CUT-WORM, AGROTIS IPSILON (HFUN.)
By Thanaa Mohammad Sileem
B.Sc. Agric. (Economic Entomology), Zagazig University, 1987 M.Sc. Agric. (Economic Entomology)Benha University, 2004
Under the supervision of: Prof. Dr. Ezzat F. El-Khayat …………………..……...... Professor of Economic Entomology-Plant Protection Dept. Fac. of Agric. Moshtohor, Benha Univ.
Prof. Dr. Samira El. M. El-Naggar ……………………...... Professor of Economic Entomology, and Head of Biological Application Dept. Atomic Energy Authority.
Dr. Safaa M . Halawa ……………………………….. Assistance Professor of Pesticides-Plant Protection Dept. Fac. of Agric. Moshtohor, Benha Univ.
2011
ABSTRACT
The sterility effects were examined on the P 1 generation of the black cutworm; Agrotis ipsilon (Hufn.) after gamma sterilization with at 0, 75, 100,125, 150,175 and 200 Gy, to identify the dose of gamma irradiation that would allow for maximum production of partially sterile P 1 adults while inducing full sterility in the F 1 generation. The studied effects were included the percentage mated males with untreated females, copulation duration to format spermatophores directly in the female bursa copulatrix, number of eggs /female deposited by females mated to irradiated males and egg hatch through three sequential females. The irradiated males with tested doses as well as untreated control were tested for mating successive and starting copulation at the same period of the scotophase. The mating competitiveness calculated from the direct observation in A. ipsilon males emerged from pupae irradiated at doses 75 to 200 Gy was noticed. The effect of substerilizing dose (125Gy) on certain biological aspects and reproduction was studied among parental generation, as well as immature stages were investigated throughout two successive generations. The influence of two entomopathogenic nematodes, Steinernema carpocapsae and Steinernema riobrivae on the insect management was included. Special attention was given to combined effect of F1 progeny of partially sterile males and S. Carpocapsae (All) on A .ipsilon , the combination of tested treatments at all concentrations analyzed for synergistic effect. The parasitisation efficacy of EPNs, the morbidity and mortality induced by normal IJs (i.e., IJs derived from untreated host) and the incubation time taken by normal IJs were compared with these of IJs derived from irradiated host with 125 Gy. Key Words: Agrotis ipsilon – Gamma radiation – Mating comptativeness – Reproductive biology- Steinernema Carpocapsae (All) - Steinernema riobrivae – Combined effect .
CONTENTS
Subject Page 1. INTRODUCTION ……………………………..…….. 1 2. REVIEW OF LITERATURE ………………..………. 4 2.1.Effect of Gamma Irradiation on Mating Ability and Mating Competitiveness in Lepidopteran Insects …..………………………………………..… 4 2.2. Effects of the Substerili zing Doses on Lepidopterous Insects………………………………. …………………… 9
2.3.Radiation Induced F1 Sterility in the Black Cut worm, Agrotis ipsilon ……………..……………..……….. 19 2.4.Virulence of Entomopathogenic Nematodes to Some Lepidopterous Pests………………………………... 23 2.5. Virulence of EPNs to the Black Cut worm, Agrotis ipsilon ……………………………………………….. 33
2.6.Feasibility of Integrating Radiation Induced F 1 Sterility and EPNs for Population Suppression of the Insect Pests………………………………………….. 40 MATERIALS AND METHODS ……………..…....…… 45 3.1. Insect Rearing Technique and Source of irradiation ……………………………….…...... …… 45 3.2. Experimental Procedures ……………………...... ….. 46 3.2.1. Mating activity and mating competitiveness ...….…. 46
3.2.2. Evaluation of F 1 Sterility for Suppression of the Agrotis ipsilon ……...... 56
I
Subject Page 3.2.2.1. Effects of gamma radiation on Agrotis ipsilon female moths……………………………………… 56 3.2.2.2. Effects of gamma radiation on Agrotis ipsilon biological aspects. ………...... 56 3.2.2.3. Total competitiveness of irradiated Agrotis ipsilon males …………………………..…………………. 57 3.2.2.4. Reproductive performance and viability of irradiated moths and their progeny ………..……... 58
3.2.2.5. Sperm production among P 1 and F 1 of Agrotis ipsilon males.………… ………………………….. 59 3.2.2.6.Feasibility study on cytological sperm bundle assessment of F1 progeny of irradiated male Agrotis
ipsilon……………………………………………….. 60 3.3. Efficacy of Entomopathogenic Nematodes (EPNs) for the Control of the Agrotis ipsilon ………………... 65 3.3.1. EPNs Concentration studies………………………... 66 3.3.2. EPNs Exposure time studies……………………….. 66 3.3.3. EPNs application method studies…………………. 67 3.4. Interaction of Steinernema. carpocapsae All with
Radiation Induced F 1 Sterility in Agrotis ipsilon …. 68 3.5. Statistical Analysis ………………….……………….. 71
RESULTS ……………………………………..………. 66
II
Subject Page 4.1.Effect of Gamma Irradiation on Mating Activity and Mating Competitiveness of Agrotis ipsilon …… 72 4.1.1. Effect of gamma irradiation on mating activity of treated males. (T♂ x N♀) ……………………..….. 72 4.1.2. Effect of gamma irradiation on mating activity of treated males among three sequentially females(T♂ x
N♀ (1), T♂ x N♀ (2)and T♂xN♀ (3) ………………….. 76 4.1.3. mating aspects means of Agrotis ipsilon males moths which had been affected among three sequential females and tested doses ……………… 82 4.1.4. Effect of gamma irradiation on successively mated st of treated males (T♂ or N♂ x none, T♂ or N♂ x 1 st nd st N♀, T♂ or N♂ x 1 N♀ +2 N♀ and T♂ or N♂ x 1 N♀ +2nd N♀+ 3rd N♀)……………………………… 89 4.1.5.Effect of gamma irradiation on treated male entering st nd into copulation during scotophase (T♂ x 1 N♀ +2 rd N♀+ 3 N♀) within three parts of night …………. 90 4.1.6. Effect of gamma irradiation on mating competitiveness of treated male (N♂+T♂ x N♀ ) ………………………………………………. 96 4.2. Effect of Substerilize doses of Gamma Irradiation on Agrotis ipsilon ………………………………………...... 96 4.2.1.Effect of gamma irradiation on mating activity of Agrotis ipsilon females. (T♀ x N♂) …………….. 98
III
Subject Page 4.2.2. Effect of gamma irradiation on successively mated of treated females. (T♀ x none and T♀ x 1st N♂ and T♀ x 1st N♂ + 2nd N♂) ………...…..…...….. 104
4.2.3. Effect of gamma irradiation on treated females entering into copulation during scotophase ……... 104
4.2.4. Effect of gamma irradiation on some biological aspects of treated insects ……...... 104
4.2.5. Effect of gamma irradiation on total competitiveness of treated males ………...... 108
4.2.6. Latent effects on certain biological aspects of F 1and
F2………….…………………………..……… 110 4.2.7. Reproductive performance and viability of irradiated males and their F1 & F2 progeny …………… 114
4.2.8.Progeny produced from irradiated P1 male pupae throughout two successive generations ……… 118
4.2.9. Effect of gamma irradiation on gonads
measurements of P 1 nd F 2 males…………….. 120 4.2.10.Effect of gamma irradiation on sperm production of P1and F1males…………………………….. 120
4.2.11.Feasibility study on cytological sperm bundle assessment of F1progeny of irradiated male………. 124
4.3. Effect of Entomopathogenic Nematodes (EPNs) on Agrotis ipsilon ………………….……..……………… 127
IV
Subject Page 4.3.1.Effect of (EPNs) Steinernema carpocapsae on Agrotis ipsilon ………….………..…………….… 127
4.3.2 Effect of (EPN) Steinernema riobrave on Agrotis ipsilon ………………………………………….…… 128
4.3.3 Virulence of (EPNs) on Agrotis ipsilon …………….. 133
4.3.4. Effect of exposure period with Steinernema. carpocapsae All on Agrotis ipsilon ……………. 136
4.3.5.Delayed effect of exposure with (EPNs) Steinernema. carpocapsae All on Agrotis ipsilon 137
4.3.6.Effect of application method with (EPNs) Steinernema. carpocapsae on Agrotis ipsilon …. 143
4.4.Combined Effect Gamma Irradiation and (EPNs) of Steinernema. carpocapsae on Agrotis ipsilon 148
4.4.1.Potency of interaction between substerlizing dose and
(EPNs) Steinernema. Carpocapsae ……………… 152
4.4.2. Interaction of Steinernema. carpocapsae All
cultured in irradiated hosts, with F 1 sterility……… 152 DISCUSSION AND CONCLUSION……….………… 159
5.1. Induction of an effective sterility and reproductive 159
5.2.Effect of dose (125 Gy) used to irradiate male & female full grown pupae on biological aspects of
P1and F 1generation………………………………… 164
V
Subject Page 5.3. Effect of Entomopathogenic nematodes in Agrotis ipsilon ……………………………………………. 173 5.4. Combined effect gamma irradiation and Steinernema. carpocapsae on Agrotis ipsilon …………………….. 177 SUMMARY…………………………………………… REFERENCES………………………………………… ARABIC SUMMARY………………………………….
VI
LIST OF TABLES No. Table Page
(1) Effect of gamma irradiation on Agrotis ipsilon male moths on mating, time in copulation,spermatophore and sperm transfer 73 (2) Effect of gamma irradiation on some mating aspects of Agrotis ipsilon males moth (Experiment 2 1st female) 78 (3) Effect of gamma irradiation on some mating aspects of Agrotis ipsilon males moths (Experiment 2 2nd female) 80 (4) Effect of gamma irradiation on mating aspects of Agrotis ipsilon males moths (Experiment 2 3rd female) 84 (5) Means of mating aspects effects of Agrotis ipsilon male's moths among three sequential females and all tested doses 86 (6) Effect of irradiation on percentage of mating successive of Agrotis ipsilon male 91 (7) Percentage of Agrotis ipsilon males entering into copulation during scotophase ( 14L:10 D) for irradiated and non irradiate 93 (8) Assessment of mating competitiveness of radiation treated Agrotis ipsilon males by direct observation method (Experiment 3) 99
VII
(9) Effects of gamma irradiation on mating aspects of Agrotis ipsilon females moths 101 (10) Effect of irradiation on percentage of successively mated females 105 (11) Percentage of A. ipsilon female entering into copulation during scotophase (14L: 10 D) for irradiated and non irradiated 105 (12) Emergence, percentage of adult with deformities and longevity of Agrotis ipsilon adults which had been irradiated with 125 Gy as a full grown pupae 106 (13) Estimation of total competitiveness values (C.V.) of irradiated (125 Gy) A. ipsilon males 109 (14) Developmental profile and survival of progeny of irradiated Agrotis ipsilon males 111 (15) Effect of sub sterilizing gamma radiation dose(125 GY) on mating behavior, fertility and reproductive suppression of Agrotis ipsilon
parent and their F 1 & F 2 progeny 115 (16) Effect of irradiating full grown male pupae with
125 Gy on F 1 and F 2 progeny of Agrotis ipsilon 119 (17) Effect of irradiating full grown male pupae with125 Gy on the volume of the resulting Agrotis ipsilon male testes within successive generations 121 (18) Accumulation of eupyrene sperm bundles in the duplex of one day old and four day old unmated males, Agrotis ipsilon 122
VIII
(19) The biological effects of Steinernema Carpocapsae All treatment on 4th larval instar of Agrotis ipsilon 129
(20) The biological effects of Steinernema riobrivae rteatment on 4th larval instar of Agrotis ipsilon 130
(21) Virulence of EPNs to 4th instar larvae of Agrotis ipsilon 134
(22) The biological effects of Steinernema carpocapsae All 100 IJs treatment on Agrotis ipsilon 4th larval instar at a various exposure periods 138
(23) Delayed Effect of Agrotis ipsilon male treatment as 4th larvae with Steinernema carpocaps a All 100 IJs within various exposure periods on reproduction and mating ability of adults 139
(24) Delayed Effect of Agrotis ipsilon female treatment as 4th larvae with Steinernema carpocapsae All 100 IJ within various exposure periods on reproduction and mating ability of adults 144
(25) The deleterious effect of treating 4th larvae of Agrotis ipsilon with Steinernemacarpocapsae All100 IJs within various Exposure periods on
the reproduction of P1 adults 145
IX
(26) Effect of application methods with Steinernema carpocapsae All on 4th larval instar of Agrotis ipsilon 146 (27) Percentage mortality of Agrotis ipsilon when
F1larvae of irradiated and un irradiated male parents were treated with different concentrations of Steinernema carpocapsae 149
(28) Lethal concentration (LC 50 ) and slopes of
concentration mortality curves for F1 progeny treated with Steinernema carpocapsae All of non irradiated and irradiated of Agrotis ipsilon male parents 150 (29) Potency of interaction between substerilizing dose and Steinernema carpocapsae All concentrations on mortality rate of Agrotis ipsilon 154 (30) Infective performance of Steinernema
carpocapsae All on F1 sterile Agrotis ipsilon larvae resulting from irradiated males with sub 155 sterilizing gamma dose of (125Gy)
X
LIST OF FIGURES
No. Figure Page
(1) Black cutworm life cycle and typical cutworm damage on agricultural crops 47 (2) Dorsal view and ventral view of mating position 52 (3) (A)Internal reproductive organs of a normal female Agrotis ipsilon 54 (3) (B) spermathica in A. ipsilon female and sperms in spermathica 55 (4) (A)Internal reproductive organs of a normal male A. ipsilon 61 (4) (B) View of sperm bundles at a magnification of 40 & 100 X 62 (5A) The sperm bundles nuclei were non responsive to the staining procedurs 63 (5B) The sperm bundles nuclei were responsive to the staining procedure the nuclei have a different color from the hole bundle 64 (5C) The sperm bundles were stained with only Giemsa stain 64 (6) Assay arena of EPNs studies 66 (7) BCW insect, cadavers 68
XI
(8) Effect of gamma irradiation on % males observed in copulation 74 (9) Effect of gamma irradiation doses on copulation time 74 (10) Effect of gamma irradiation on % mated females with spermatophore and % mated females with sperm 75 (11) Effect of gamma irradiation on % males observed in copulation of sequentially three untreated female 75 (12) Effect of gamma irradiation on time in copula of sequentially three untreated females 79 (13) Effect of gamma irradiation on no. of eggs of sequentially three untreated females 79 (14) Effect of gamma irradiation on % of egg hatch of sequentially three untreated females 81 (15) Effect of gamma irradiation on % of mated females with spermatophore of sequentially three untreated females 81 (16) Effect of gamma irradiation on % un separation pairs of sequentially three untreated females 85 (17) Mean of effects on% males observed in copulation of females and doses 85 (18) Mean of effects on time of copulation in females and doses 87
XII
(19) Mean of effects on no. of eggs /mated female in females and doses 87 (20) Mean of effects on % of egg hatch in females and doses 88 (21) Mean of effects on % of mated females with spermatophore in females and doses 88 (22 & Effect of gamma irradiation on % of 23) successively mated males 92 (24) Effect of gamma irradiation on % of mated males during scotophase 94 (25) Effect of gamma irradiation on % of mated males during scotophase 94 (26) Effect of gamma irradiation on % of mated males during scotophase 97 (27) Effect of gamma irradiation doses on % of mating at different mating combination 97 (28) Effect of gamma irradiation doses on competitiveness values in direct observation method 100 (29) Effect of gamma irradiation doses % females observed in copulation 100 (30) Effect of gamma irradiation doses on time in copulation of female 102 (31) Effect of irradiated female on average no.of eggs 102
XIII
(32) Effect of irradiated female on both of hatchability % & average no.spermatophore 103 (33) Effect of gamma irradiation on % of successively mated females 103 (34) Effect of gamma irradiation on % of mated females during scotophase 107 (35) Effect of sub sterilizing dose of gamma irradiation on % of Emergence , % adult with deformities and % survival today (6) 107 (36) Effect of sub sterilizing dose of gamma
irradiation on developmental profile in F 1 &F 2 112 (37) Effect of sub sterilizing dose of gamma irradiation on pupation% & adult eclosion%
in F 1 &F 2 112 (38) Effect of sub sterilizing dose of gamma irradiation on growth index 113 (39) Effect of sub sterilizing dose of gamma irradiation on sex ratio 113 (40) Effect of sub sterilizing dose of gamma irradiation on mating success and % fertility 116 (41) Effect of sub sterilizing dose of gamma irradiation on no. of eggs 116 (42) Effect of sub sterilizing dose of gamma
irradiation on no.of mating frequency P, F 1
and F 2 generations 117
XIV
(43) Effect of sub sterilizing dose of gamma
irradiation on volume of testes P and F 1 generations 123 (44) Effect of sub sterilizing dose of gamma irradiation on accumulation of eupyrene sperm bundles in the duplex 123 (45) Sperm bundles from testis smears of normal males, a& b&c (40X) and A&B&C (100X). In all, views nuclei clusters of the eupyrene sperm bundles were homogenously stained 125 (46) sperm bundles from testis smears of the sterile
F1 males, c(20 X), a& b&d (40X) and A&B&D (100X). In all, views nuclei clusters of the eupyrene sperm bundles were heterogeneously stained 126 (47) Effect EPNs on % of total mortality of A. ipsilon 131 (48) Effect of EPNs on % of pupation of A. ipsilon 131 (49) Effect of EPNs on % of adult emergence of A. ipsilon 132 (50) Effect of EPNs on % of survival of A. ipsilon 132
(51) Virulence of (EPNs) on A. ipsilon . (LC 50) 135
(52) Virulence of (EPNs)on A. ipsilon (LT 50) 135 (53) Biological effects of S. carpocapsae 100 IJs treatment on 4th instar larvae of A. ipsilon at a various exposure periods 140
XV
(54) Delayed Effect of treatment with S. carpocapsae 100 IJs at various expoure period on reproduction 140 (55) Delayed effect of treatment with S. carpocapsae All 100 IJs at various exposure periods on fecundity 141 (56) Delayed effect of treatment with S. carpocapsae All 100 IJs at various exposure periods on egg viability 141 (57) Delayed effect of treatment with S. carpocapsae All 100 IJs at various exposure periods on no. of spermatophores 142 (58) Effect of application methods with S. carpocapsae All on 4th larval instar of A. ipsilon 147 (59) Combined effect of gamma irradiation and
(EPNs) on A. ipsilon . LC 50 151 (60) Time morbidity & time mortality of infested larvae with both type of EPNs 156 (61) Incubation time & harvest period of infested larvae with both type of EPNs 156 (62) Harvest (yield) of IJs of infested larvae with both type of EPNs 157
XVI
ACKNOWLEDGEMENT
Ultimate thanks to ALLAH, who without his help this work can never been done and to whom I dedicate the whole work may he accept it. I would like to express my deep thanks to Prof. Dr. Ezzat Farag El-Khayat, Professor of Entomology, Department of Plant Protection, Faculty of Agriculture, Benha University for his supervision. Deep appreciation and gratefulness are expressed to him for planning, his critical comments, advice, careful review of the manuscript and encouragement throughout this work. The author wishes to express her sincere gratitude to Prof. Dr. Samira El-Sayed Mostafa El-Naggar, Professor of Economic Entomology, head of Department of Biological Application, Nuclear Research Center (NRC), Atomic Energy Authority (AEA), for suggesting the research point, her supervision, extremely valuable suggestions, for her valuable advice and great efforts for making the manuscript in its final form. I am particularly indebted to Dr. Safaa Mahmoud Halawa Assistant Professor of Pesticide, Department of Plant Protection, Faculty of Agriculture, Benha University for her sincere help and guidance. She is gracious and generous with her time, valuable advice as well as her discussion and critical reading of the manuscript. I wish also to thank Dr. Magdy Mohamed Shibl Alm El- Din Assistant Professor of Entomology, Department of Biological Applications, Nuclear Research Center (NRC), Atomic Energy Authority (AEA), for his kind care, continuous encouragement, practical guidance and providing knowledge to complete this work. Thanks are also to Prof. Dr. Samir Mahmoud Ibrahim Professor of Economic Entomology . Department of Biological Applications, Nuclear Research Center (NRC), Atomic Energy Authority (AEA), for his enlightening discussion during the experimental work and for continuous help throughout this work. I’m also thankful to head and members of Plant Protection Department, Faculty of Agriculture, Moshtohor, Benha University for their support and encouragement. Deep appreciation is also due to Cotton Pests Unit, Department of Biological Applications, Nuclear Research Center for offering facilities and encouragements. Finally, I would like to express my deep gratitude to my beloved family and husband for their encouragement, patience and sincere inspiration. 1- INTRODUCTION
The black cutworm, Agrotis ipsilon (Hufnagel), has a wide host range, feeding on nearly all vegetables and many important grains. The origin of the black cutworm is uncertain, so as it is now found in many regions of the world. It is not considered to be a climbing cutworm, most of the feeding occurring at soil level. However, larvae will feed above ground until about the fourth instar. Larvae can consume over 400 sq cm of foliage during their development, but over 80% occurs during the terminal instar, and about 10% in the instar immediately preceding the last. Thus, little foliage loss occurs during the early stages of development. Once the fourth instar is attained, larvae can do considerable damage by severing young plants, and a larva may cut several plants in a single night (Capinera, 2006). Black cutworm is often difficult to control, especially when populations are epidemic in proportion. It has already developed into a life stage which is not as susceptible to insecticides as the early larval stages. The sporadic nature of cutworm populations can make preventive treatments futile in some areas. An additional problem for control is the soil- dwelling habits of the larvae, often beneath heavy foliage, making it difficult for insecticides to reach their targets (Hill, 1983). The indiscriminate use of broad-spectrum insecticides has caused major problems with pest resistance, residues in food, environmental contamination, outbreaks of secondary pests, and reductions in populations of beneficial insects. This results in increased demands for pest control methods that are both efficient and friendly to the environment.
Introduction -1-
The sterile insect technique (SIT), applied as part of an area-wide integrated pest management approach offers considerable potential and has been used with great success against major pests of agricultural importance to establish pest- free areas (eradication), areas of low pest prevalence (suppression) or to maintain areas free of the pest through containment or prevention (Tan 2000 ) and (Vreysen et al. 2006 ). (SIT) has been used for Lepidoptera but insects in this order are radio-resistant, presumably due to their holokinetic chromosomal configuration (Bauer 1967). Therefore, lepidopterans require large doses of radiation for sterilization, leading to somatic damage and reduced competitiveness in the irradiated insect.A favored alternative to using fully sterile moths in SIT is the use of F 1 sterility. F 1 survivor progeny of sub-sterile parental (P 1) male moths result when sub-sterilizing doses of radiation are applied to the P males. The resulting F 1 progeny are more sterile than the irradiated parent, and the irradiated moths are more competitive as a result of receiving a lower dose of radiation. Inherited sterility in the progeny of treated males has been shown to have potential in suppressing populations of lepidopteran pests (North & Holt 1969 ; Knipling 1970 ; North 1975 and La-Chance 1985). Previous studies of substerilizing gamma-radiation doses on the growth, bioenergetics and reproductive behavior of A. ipsilon in the F 1 progeny of treated moths indicated the potential of managing this pest by using inherited sterility (Elnagar et al . 1984; Bakr & Abdel-Fattah 1997; Abd-El-Hamid 2004 and Ali 2008). As Entomopathogenic nematodes (EPNs) (Rhabditida: Steinemematidae and Heterorhabditidae) are found naturally in the soil, they are particularly suited for control of soil-inhabiting
Introduction -2- insects (Ehlers, 1996 ). Researches already reported the successful use of EPNs against many insects under field conditions e.g., (Boselli et al. , 1997; Shapiro-Ilan et al ., 2004 and Sulistyanto et al . 1996). This mean that EPNs are important biological control agents of a great variety of insect pests (Poinar, 1986 ). In control of Black cutworm so far only few species of EPNs were tested, in these studies, high percentages of mortality were obtained, high concentrations were required (Abdel-Kawy, 1985 Capinera et al. 1988 , Azazy, 2001 and Hussein 2004). These have necessitated testing several factors that may improve the efficacy of the applied nematodes. Furthermore, the compatibility of efficient EPN species against Black cutworm with other biocontrol agents was envisaged thus. The sterilization process is important in determining the quality of the released insects and their ability to compete with the wild population .Thus, optimization of sterilization process is critical for the efficacy of SIT programs and should be taken in consideration. The present study aims to provide such information about ionizing radiation as a method of choice for inducing reproductive sterility, in addition to evaluat the reproductive performance and mating behavior of A. ipsilon in response to substerilizing dose. Also it studies the interaction of
EPNs, with F1 sterility (substerilizing radiation induced genetic control) for controlling A. ipsilon . An attempt was made to evaluate (i) Synergistic effect of combination between two control methods (ii) The parasitizing performance of EPNs against normal and F1 sterile hosts (iii) the infective potential of EPNs cultured in radio-sterilized hosts against normal and
F1sterile hosts.
Introduction -3-
2. REVIEW OF LITERATURE
2.1. Effect of Gamma Irradiation on Mating Ability and Mating Competitiveness in Lepidopteran Insects Lepidopterous insects require about ten time of radiation doses more to induce male sterility than Dipterous (LaChance et al., 1967) and (North & Holt, 1968) and varying degree of success have reported in controlling population of Lepidoptera by using the sterile male technique. La -Chance et al . (1973) irradiated less than 24 hour old male of pink boll worm adult, Pectinophora gossypiella with 2.5, 5, 7.5,10 or 12.5Krad of gamma irradiation. When the previous treated males caged with virgin untreated females; their ability to mate and transfer eupyrene sperm appeared to be unaffected, but their fertility was reduced. In addition, the sex ratio of F 1 generation was significantly skewed in favour of males except at the lowest dose. When males or females of F 1 generation were out crossed to untreated one, the F1 males mated but often failed to transfer normal quantities of eupyrene sperm.
Fertility of the F 1 males and females was significantly less than that of the control at all doses applied to the P 1 males above
2.5Krad. On the other number of mating of F 1 males was more in comparison with control at doses above 5krad. Nabors &Pless (1981) found that males of Ostrinia nubilalis (Hb.) exposed as pupae to gamma-radiation at a dose of 15Krad were as successful as unirradiated males in spermatophore transfer, when such males were crossed with unirradiated females. The eggs from crosses with F1 female
Review of Literature -4-
progeny were nonviable and only 2.9% of eggs from crosses with F 1 male progeny were viable. Carpenter (1985) reported that the irradiated males and females of Heliothis zea (Boddie) with substerilizing doses of radiation, then moths were inbreed, out crossed and observed for their ability to reproduce. He found that the dose of 10Krad induced deleterious effects, which were inherited through the F 2 generation. Females mated to normal males, and males irradiated with 10Krad had the same mating propensity and experienced the same intermating interval. a great potential exists for population suppression were recorded with substerilizing doses of radiation and inherited sterility on the reproductive ability and behavior of H. zea . Sallam & Ibrahim (1988) inherited deleterious effects on full-grown male pupae of Spodoptera littoralis (Boisd ) were exposed to four substerilizing doses of gamma radiation up to 20Krad and mated with unirradiated females. The resulting males of each generation were mated with normal females throughout three successive generations (F 1, F 2 and F 3). A detectable reduction in the percent mated females, having sperm in their spermathecae, was observed among F 1 generation at dose level 15 and 20 Krad. The number of spermatophores per mated female did not exhibit a dose response and did not significantly differ from the control. Henneberry (1993 ) reported that exposed the pink bollworm, Pectinophora gossypiella (Saumters), moths to 10 and 20 krad and observed in copula with untreated female moths during the last 6-h dark period after irradiation. Lower percentages of male
Review of Literature -5-
exposed to 20 krad were observed in copula as compared to untreated male and female moths. Irradiated male moths also spent less time in copula , but irradiation bad no effect on numbers of spermatophores per mated female or percentages of mated females with sperm in their spermathecae. Lower percentages of untreated female moths were mated when they were paired with previously mated males than when paired with virgin males. The sequence in which females were mated to irradiated male did not affect the transfer of spermatophores. However, the percentage of eggs hatched was higher from the first mated female compared to the second and third mated female. There were no significant effects of 10 or 20 krad exposures on female mating or time in copula . However, significantly lower percentages of mating of treated and untreated females occurred with sequentially provided male moths on days 2, 3, or 4 following the first observed female mating.
Proshold et al. ( 1993) found that when F 1 gypsy moths Lymantria dispar (L.), whose male parent received 6, 8 or 10 Krad of gamma irradiation were mated with untreated females, fewer sperm was transferred than that of untreated males. Further, the quantity of eupyrene sperm transferred by offspring of irradiated males was less than that of progeny of untreated males. The effect was depended upon dose. In untreated adults, fecundity as determined by weight of the egg mass was depended upon quantity of eupyrene sperm in the spermatheca. But when the male parent was irradiated, fecundity of its female progeny or of untreated females mated with its male progeny was less than that of control, regardless of the quantity of
Review of Literature -6-
eupyrene sperm. Females come from 10Krad treated males took longer time than treated females to begin copulating.. Seth & Reynolds (1993) recorded that, irradiated male pupae of the tobaco hornworm, Monduca sexta (Linnaeus), with dose of 10Krad. Examination of spermatheca of normal females mated with F 1 males whose the P 1 male received 10Krad showed that both eupyrene and a pyrene spermatozoa were successfully transferred from the spermatophore, but there was a slight reduction in number at higher doses. Sallam et al . (2000) studied the inherited sterility in the spiny boll worm, Earias insulana ( Boisd.), when they treated the adult male moths less than 24 hours were irradiated with doses of 100,150 and 200 Gy and mated with untreated virgin females. . The reduction in mating ability was significant only at 200 Gy for P 1 males and at 150 and 200 Gy for F 1 males as compared to control. Ocampo (2001 ) determined the effects of a substerilizing dose of gamma radiation (100 Gy) on the mating competitiveness of treated males and the effect on the Helicoverpa armigera (Hübner) mating propensity of females with which they mate. Mating competitiveness of treated and untreated male moths was measured at two different release ratios inside field-cages in a cabbage field.1st 1male:1female and 2nd 4males:1female ratios were used while keeping a constant density of moths per cage. The mean number of matings recorded was not significantly different at either ratio, suggested that treated males of this species are equally as competitive as their untreated counterparts. In the mating propensity studies,
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virgin female H. armigera was first mated to treated or untreated males and then re-exposed to untreated males 24 hours later. No statistical differences were found in the number of females that re-mated from either group. Thirty point eight percent of the females first mated with treated males and 29.17% of the females first mated to untreated males re-mated in this study. When both types of females were re-exposed to untreated males in the same field-cage, a higher percentage (38.3%) of females that had initially mated with a treated male re-mated than those initially mated with a untreated male (31.7%), although the differences were not significant. Bloem et al . (2003) compared the attractiveness and the longevity of fertile and irradiated (sterile) females deployed as bait in traps. Traps baited with females sterilized with gamma radiation were as effective as traps baited with unirradiated (fertile) females in detecting populations of feral Cactoblastis cactorum male moths Suckling et al. (2004) studied the sterile insect technique efficacy on the Australian painted apple moth, Teia anartoides , using males moths irradiated as pupae at 100 Gy. Sterilization of males has a fitness cost, which was assessed in terms of longevity and competitive fitness. Irradiated male painted apple moths when released in separate groups in the flight tunnel, irradiated males were less likely to reach calling females than untreated males (P<0.001). When single irradiated (100 Gy) males and untreated males were released together as a pair, irradiated males also showed lower arrival to females (P<0.005). However, once the males successfully located the females, there
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were no significant differences between the controls and the irradiated males in the total time spent for mate location, mounting attempts and mating duration. Marti & Carpenter, (2009) mentioned that no deleterious effects due to irradiation of male Cactoblastis cactorum at 200 Gy were found, and no differences in mating frequency or longevity of irradiated male moths or of female moths mated to irradiated males were detected in their laboratory tests. Although many factors may influence the overall competitiveness of sterile insects released in a SIT program, they conclude that the irradiation (200Gy) of C. cactorum males used in the current SIT program does not adversely affect mating ability or mating frequency. 2.2. Effects of the Substerilizing Doses on Lepidopterous Insects: Complete sterility in lepidopteran species requires high doses of radiation (20-40 k rad), which invariably cause severe somatic damage, resulting in reduced competitive efficiency of the released insects. This severe reduction can be minimized by the use of lower doses, which offers considerable promise sterility for the control of lepidopteran pests. Several studies on lepidopterous pests demonstrated that progeny raised from parents that had been partially sterilized with radiation were partially or fully sterile. (Proverbs, 1962, in Caepocapsa pomonella (L.) and Proshold & Bartell, 1970, in Heliothis virescens)
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Al-Taweel et al . (1990) irradiated one-day-old males of Ephestia cautella ( Walker ) with 150,200 and 250 Gy of gamma radiation and mated them in groups of 15 pairs with untreated virgin females. Results showed that radiation effects on F 1 males were greater than the effect on the P 1 males. Further more the results showed that the effect on F 1 males were greater than the effects on F 1 females. Harwalkar et al . (1991) irradiated males of Earias vittella (Fab .) with sub-sterilizing doses of 75, 100 and 125 Gy and crossed them with untreated females. Male sterility increased with increasing the dose. The survival and development of F 1 larvae was dose dependent. The F 1 male progeny from males irradiated with a 75 Gy were more sterile
(as assessed on the basis of egg hatch) than F 1 female progeny when out- crossed to normal parents. At 100 Gy treatment, F1 males were completely sterile and F 1 females were partially sterile when F 1 males and and females were crossed, together no progeny resulted. When P 1 males were treated with 125 Gy, F1 progeny of both sexes were sterile. Sallam & Ibrahim (1993) studied the fecundity and egg hatch of normal females of Spodoptera littoralis ( Boisd ) crossed with males irradiated as 7-day-old pupae with doses of, 75,100,125, 150 and 200 Gy of gamma radiation. The inherited effects of irradiation were studied until the F 3 generation. The fecundity of females was significantly reduced by increasing the dosage applied to P 1 males. The egg hatch of P 1 females was significantly reduced at 125, 150 and 200 Gy treatments as compared with the control. The F 1 generation was significantly
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more sterile than the irradiated parents. The larval and pupal mortality in the F 1 and F 2 generations were high and dose dependent. The average developmental time from egg hatch to adult emergence was not affected and also the sex ratio of resulting progeny was about normal. Bahari (1994) investigated the effects of increase radiation doses to adult of diamond back moth of Plutella xylostella (L.) on suppression of different stages of life cycle. Doses between 150 Gy and 200 Gy did not induce significant differences in suppression of the different stages. A dose of > 200 Gy not only caused suppression in frequencies of the different life cycle stages but also caused a shift in life cycle pattern of those stages. Irrespective of doses and crosses used on adults, their offspring showed a 1:1 ratio of males to females. Qureshi et al . (1995) irradiated one- day- old adults of pink bollworm, Pectinophora gossypiella ( Saunders ) with doses 50,100,150 and 200 Gy. The fecundity and fertility was dose dependent. The number eggs laid per female was reduced in all crosses as the radiation doses increased. Females were more susceptible to gamma radiation than males. The fertility was drastically reduced when either parent was irradiated at all test doses of gamma radiation. The fecundity and fertility of F 1 progeny reduced significantly with higher dose. The number of eggs and hatchability percentage was reduced drastically at 150
Gy. Therefore, this dose is considered suitable to induce F 1 sterility in adult moths. Saour & Makee (1996) studied the inherited sterility phenomena in partially sterile male’s Phthorimaea operculella
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(Zeller ) with 10, 15 and 20 krad. Sterility in F 1 progeny was higher than that in their irradiated male parents. The sex ratio of
F1 progeny was distorted in favor of the males. El-Shall et al . (1997) irradiated full grown pupae of the maize worm, Mythimna loreyi ( Dup ) with substerilizing doses and crossed the emerged resulted moths with untreated females or males. They found that the mating, insemination, fecundity and fertility were reduced in F 1 males or F 1 females resulting from male parents irradiated at 100, 150 and 200 Gy (male line).
Inherited sterility was more pronounced when F 1 males were crossed with untreated females than when F 1 females were crossed with untreated males. In female line, fecundity and fertility of F 1 crosses were higher than corresponding male line at 100 and 150 Gy (the same- dose level) but still less than in control treatment. A substerilizing dose of 200 Gy induced complete sterility in male line. Shantharam & Rananavare (1998) irradiated newly emerged females of spotted bollworm, Earias Vittela (Fab .) with substerilizing doses of 75,100,125 and 175 Gy and crossed with untreated males. The level of sterility induced in females was found to increase with increasing dose when evaluated on the bases of egg hatch. A dose dependent effect in relation to fecundity of irradiated female was observed. Survival development of F 1 larvae also exhibited dose dependent response. Sex ratio distortion was not observed with any of the treatments. Sterility increased in F 1 males by increasing the dose.
Emerged F 1 females were more sterile than F 1 males. Fecundity
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of F 1 females was considerably reduced than control. However, desired level of inherited sterility was not observed. Bloem et al . (1999) studied the effect of substerilizing doses of radiation on male and female codling moth Cydia pomonella (L.). The fecundity of untreated females mated with treated males declined slowly with increasing doses of radiation. However, fecundity of treated females declined almost linearly dose at 22% per 100 Gy. The minimum dose at which treated females were found to be 100% sterile when mated to untreated males was 100 Gy. This dose was much lower than previously suggested. Fertility of treated males declined almost linearly to approach 0 near 400 Gy. Inherited effects from resulting irradiation of P 1 males at selected doses were recorded for the F 1 generation. As the dose of radiation increased, F 1 fecundity and fertility decreased, mortality increased and F 1 sex ratio shifted in favor of male progeny. Sallam et al . (2000) studied the inherited sterility in the spiny boll worm, Earias insulana (Boisd .) . Adult male moths less than 24 hours were irradiated with doses of 100,150 and 200
Gy and mated with untreated virgin females. The resulting F 1 males were mated with normal females in order to obtain F 2 generation of which only the males were pooled out to continue the male line for the third generation. The reduction in fecundity increased as the dose applied to P 1 males was increased. Egg hatch of the parental generation was obviously reduced at 100,150 and 200 Gy treatments as compared to control. The
Progeny of F 1 males were evidently more sterile than their irradiated male parents. The effect continued in the F 2
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population; however F 3 males almost regained their fertility. Larvae reaching the adult stage decreased in number as the irradiation dose was increased. The effect was more obvious at
F1 generation. Alm El-Din (2001) irradiated full-grown male and female pupae of cotton leaf worm; Spodoptera littoralis (Boisd.) with the doses of gamma rays 100,125and 150 Gy. The effects on reproduction were studied throughout parents P 1 and their F 1, F 2 and F 3 generations. The fecundity was decreased by increasing in dose. The reduction in egg hatchability was significant at all tested doses among P 1, F 1 and F 2 generations. Gamma radiation did not clearly affect the percentage of mated female in P 1 and their three successive filial generations. The average number of spermatophores per mated female was slightly affected among
P1, F 1, F2 and F 3 at the majority of the tested mating combinations. The percentages of larval and pupal survival were obviously affected among F 1 and F 2 generations. The percentage of pupation and adult emergence was obviously reduced among
F1. The sex ratio among the progeny of irradiated males and females was nearly 1:1 at all tested combinations. Lu Daguang et al.(2002) studied the effect large scale rearing and gamma radiation on selected life history parameters of Cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae) in China .They found that Mature Helicoverpa armigera female and male pupae were treated with different doses of gamma radiation and out-crossed with untreated males. Mating ability of both sexes was not affected by radiation. Treated females were highly sterile and laid significantly fewer
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eggs than untreated controls. Females treated with 300 Gy were completely sterile, while females treated with 250 Gy and 200 Gy still had minimal residual fertility. Mansour &Mohamad (2004) studied the radio- sensitivity of codling moth, Cydia pomonella (L.), eggs in different stages of development. Eggs ranging in age from 1-24 to 97-120 h were exposed, at 24 h intervals, to gamma radiation doses ranging from 10 to 350 Gy. The effects of gamma radiation on egg hatch, pupation and adult emergence was examined. Results showed that the radio-sensitivity of codling moth eggs decreased with increasing age. Egg hatch in 1-24 h old eggs was significantly affected at 20 Gy and 60 Gy doses, egg hatch decreased to about 1%. At the age of 25-48 h, however, egg hatch at 60 Gy dose was about 10%, and egg sensitivity to gamma irradiation decreased significantly in the 49-72 h age group; 60 Gy dose had no significant effect on egg hatch. Eggs irradiated few hours before hatch (at the blackhead stage), were the most resistant ones; 100 Gy had no significant effect on egg hatch and at 350 Gy dose over 56% of the eggs hatched. When adult emergence was used as a criterion for measuring effectiveness, however, the effect of gamma radiation was very sever. A dose of 60 Gy completely prevented adult emergence and at 100 Gy dose all resulted larvae died before pupation . Ayvaz &Tuncbilek (2006) conducted the experiments with Ephestia kuehniella Zeller to determine the effects of gamma radiation on life stages. Eggs, larvae, pupae and adults were irradiated with increasing doses of gamma radiation (seven
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dose levels between 50 and 400 Gy for eggs and larvae, six dose levels between 50 and 350 Gy for pupae and four dose levels between 250 and 550 Gy for adults. The number of adults that developed from irradiated eggs and larvae was lower than the untreated control. Doses of 200 Gy and above prevented adult emergence from irradiated eggs. Although a dose of 200 Gy was enough to prevent adult emergence from young larvae, 250 Gy should be used to prevent adult emergence completely from last instar larvae. Delayed developmental periods were observed for the treated eggs and larvae. Fecundity and egg hatchability were decreased depending on the doses applied. Decreased fecundity and egg hatchability were more prevalent when both the male and female pupae were treated compared to the treatment of female pupae only. There was no significant decrease in the fecundity of irradiated adults except 550 Gy, and no eggs hatched at doses of 300 Gy and above. Tate et al. (2007) examined the inherited sterility effects on the F1 and F2 generations of the cactus moth, Cactoblastis cactorum (Berg) after gamma sterilization ,to identify the dose of gamma radiation that would fully sterilize F 1 -generation moths and result in no viable offspring when F1 males were inbred- or out-crossed to fertile females, and that would allow maximum production of F1 sterile C. cactorum adults by irradiated males. Newly emerged adults of C. cactorum were exposed to increasing doses of gamma radiation and inbred or out-crossed to fertile counterparts. Inherited effects resulting from irradiation of males and females were expressed in the F1 generation as reduced egg hatch, increased developmental time
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for the F1 egg, and increased F1 larval to adult mortality. These effects were most pronounced when parental adults were irradiated at 200 Gy. Survival of F1 -generation offspring originating from irradiated male x fertile female crosses was greatest at 200 Gy. In addition, inbred- and out-crosses of surviving F1 adults, with 1 parent irradiated at 200 Gy, resulted in no. F2 adults. Maximum production of sterile F1 adults at 200 Gy suggests this dose is the most appropriate dose for implementing the sterile insect technique (SIT) - F1 sterility for control of C. cactorum in North America and for testing host suitability and potential geographical range in the field. Shamitha & Purushotham Rao (2008) studied the effect of various levels of gamma irradiation on the moth of irradiated cocoons of Tasar Silkmoth, Antheraea mylitta . (D.). The effect of irradiation on moth and pupal duration was also observed. The results have shown that the moths of indoor rearing are at par with that of outdoor reared ones. Irradiation of cocoons during diapause showed a dose-dependent, significant reduction with 5 Gy, 7.5 and 10 Gy in the pupal duration and increase in the duration of the moth stage. The irradiated cocoons have also shown changes in filament structure Triseleva & Safonkin (2009) studied the influence of ionizing radiation on oocyte development and male reproductive success in Archips podana . They found that the increase of the percentage of the vitellogenous oocyte and the decrease of the percentage of the chorion oocyte against the control has been shown.
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Jafari et al. (2010) examined the control of wax moth using the male sterile technique (MST) with gamma-rays and determine the safe and effective dosage of gamma-rays capable of sterilizing male pupae of the wax moth, male pupae were exposed to increasing single doses of gamma-rays (250, 300, 350 and 400 Gy). The release ratio of sterile to normal males was also studied in a similar experiment. Treatments included sterile males, normal males and virgin females at the following ratios: 1:1:1, 2:1:1, 3:1:1, 4:1:1 and 5:1:1. Possible parthenogenetic reproduction of this pest was also examined. The results showed that 350 Gy was the most effective dose capable of sterilizing the male pupae of the wax moth. The best release ratio was established at four sterile males, one normal male for each normal female (4:1:1). Also females were incapable of producing offspring without males. Abbas et al. ( 2011) studied the effect of gamma irradiation on the Indian meal moth Plodia interpunctella Hübner at different developmental stages and the doses required to prevent each of these developmental stages .From the results, required dose to prevent larval emergence from irradiated 1 to 24 h eggs was 400 Gray (Gy), and 400 Gy was required to prevent pupae from 15 days old larvae. Also, the dose of radiation required to prevent adult emergence from irradiated 5 days old pupa was 650 Gy. According to the results, dose of 650 Gy is adequate to control all immature stages of this pest. In addition, the effect of gamma ray was studied on developmental stage period of each irradiated existence stage till adult eclosion. The results revealed that there was a dose-dependent increase in the
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developmental periods, and the growth index of the adults was significantly decreased with increasing dose of radiation administered to the eggs, larvae and pupae too. It is concluded that irradiation can be used as a safe method to control stored pests.
2.3. Radiation Induced F1 Sterility in the Black Cut- worm, Agrotis ipsilon . Ibrahim (1981) studied the effect of gamma irradiation on the greasy cutworm, Agrotis ipsilon (Hufn.). Special attention was given to the sterilization studies in the hope of promoting the sterile insect technique for the pest control program. The obtained results could be summarized as follows: Three ages of eggs were irradiated as newly laid egg (6-12 h old), middle age egg (24-36 h-old) and old egg (60-72 h-old). The mortality among irradiated eggs was directly related to the increase of irradiation dose or the decrease of the age of treated eggs. The dose of irradiation required for complete mortality was found to be 0.0525 and 0.7 Gy for newly laid eggs, middle age and old eggs, respectively,. Irradiation of eggs had no effect on the incubation period. El-Kady et al. (1983) reported the effect of gamma radiation on aspects of the biology of Agrotis ipsilon (Hufn.) exposed as pupae in the laboratory in Egypt. Adult emergence was reduced, and the rate of malformation in survivors increased, as the radiation dose increased, the effect being greater in females than in males. Exposure of mature pupae to 200 Gy reduced mating in the ensuing adults and induced sterility in females, whereas 250 Gy was required for male
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sterility. Female fecundity was reduced proportionately to the treatment dose. Irradiation of females at any given dose always caused greater sterility than did irradiation of males at the same dose, but treatment of both partners of a mating pair reduced fertility more markedly than did treatment of either sex separately. Elnagar et al . (1984) irradiated full-grown pupae of Agrotis ipsilon (Hufn with 5 or 10Krad and crossed them with unirradiated females. They found that the F 1 progeny was more sterile than their parents. Percentage of mated females of F 1 was greatly reduced, while the mating frequency was increased less inseminated females were among the F 1 particularly when the female inherited the sterility. The mortality among larvae of the
F1 was high and dose dependent, and that among F 2 larvae was even higher. The sex ratio of the F 1 progeny was altered in favour of males; while that of the F 2 was normal. The dose of
10Krad for P 1 males were sufficient to inhibit egg-hatch produced by the F 1 adults. Ibrahim & Abdel –Baky(1989) irradiated males of the Agrotis ipsilon with 5, 10 and 15 krad of gamma irradiation and crossed with untreated females. They found that the contents of amino acids which determined in protein amino acids (P.A.A) fraction were in general higher than those in free amino acids
(F.A.A) in both check and F 1 newly formed pupae. Free and protein amino acid contents were reduced by increasing the irradiation dose to male parent (P 1). F 1 females seemed to be more sensitive to irradiation than F 1 males. In the control treatment, the ratio of (P.A.A) increased as the pupae become
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elder and also in adults. While in the irradiation treatments the ratio decreased as the dose increased. The ratio of P.A.A was higher in females than in males in both pupal and adult stages. El-Naggar & Ibrahim (1995) carried out histological studies on the effect of gamma radiation on of the ovary of F 1 female moths resulted from full grown male pupae of Agrotis ipsilon irradiated with 50,100 and 150Gy . The histological examination showed that abnormalities included shrinkage and vaculation in the cytoplasm of the oocyte and nurse cells, clumping of cytoplasmic contents, destruction of oocytes and mature ova, irregular of follicular epithelial cells and clumping of mature oocyte. The damage occurred in the ovary of F 1 female increased as the dose applied to parental males (P 1) was increased. Bakr & Abdel-Fattah (1997) studied the effect of gamma irradiation on the biological activities of Agrotis ipsilon (Hufn with doses of 100-150 Gy given to full-grown pupae. They found that the irradiation affected the insect fecundity, egg- hatch, mating ability, mortality of immature stages and adult malformation and the effect was dose dependent and sex combination. Ibrahim et al. (1999) irradiated Full-grown male pupae of Agrotis ipsilon (Hufn.) with 50,100 and 150 Gy of gamma radiation, and investigated the histological observation on the testes of F 1 fourth or sixth instar larvae and 1-day old adult and compared with unirradiated ones . Irradiation affected the process of spermatogenesis, where it caused gross damage to the testes of the F 1 larvae or adult. Many of the gonial cells were damaged,
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and the damage increased in proportion to the applied dose. Histological abnormalities included shrinkage of testis contents Abd El -Hamid (2004) exposed full-grown male and female pupae of black cut worm, Agrotis ipsilon (Hufn.) to three doses of gamma irradiation 50, 100 and 150 Gy. Increasing the dose of irradiation applied to the parental male gradually reduced the egg hatch. The reduction was significant at all tested doses level when compared to the control treatment . The average number of eggs did not significantly differ from untreated control at 50 and 100 Gy but it was significantly reduced at150 Gy. Also, the data indicated that the percentage of egg hatch was reduced gradually at all tested mating combination of F I in comparison with their untreated control. Full-grown female pupae were exposed to three doses of gamma irradiation, the average number of eggs and percentage of egg hatch of treated female mated with normal male decreased. However, the data indicated that the percentage of egg hatch was increased at all tested mating combination of F 1in comparison with their P I. The results lead to a conclusion that sterility could be inherited by irradiation of full grown male pupae more than irradiated full grown female pupae. Ali, (2008) tested three doses 50, 100 and 150 Gy of gamma irradiation against full – grown male and female pupae of Agrotis ipsilon (Hufn.). The results showed that fecundity of irradiated females crossed with irradiated males was decreased by increasing irradiation dose. In addition, the decrease in egg – hatchability % and increase in sterility % induced by gamma irradiation were found to be positively correlated with the dose.
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The parentage of larval and pupal mortality increased significantly (p<0.05) with the increase of doses used. amma irradiation induced a significant (p<0.05) malformation % among the resulted pupae and adults.The percentage of adult emergence reduced at 50 and 100 Gy, compared to control treatment The sex ratio among the progeny of irradiated males and females seemed to be skewed to male side especially at 100 Gy. In general, the results indicated that the biological activity of gamma irradiation against A. ipsilon larvae was more remarkable when both crossed females and males were irradiated followed by irradiated females crossed with non-irradiated males. 2.4.Virulence of Entomopathogenic Nematodes to Some Lepidopterous Pests. Entomopathogenic nematodes (EPNs) are exceptionally safe biological control agents. They are certainly more specific and are less of a threat to the environment than chemical insecti- cides (Ehlers & Peters, 1998). Since the first use of EPNs Steinernema glaseri against the white grub Popillia japonica in New Jersey (USA) (Glaser & Farrell, 1935), not even inferior damages or hazards caused by the use of EPNs to the environment have been recorded. The use of EPNs is safe for the user EPNs and their associated bacteria cause no detrimental effect to mammals or plants (Poinar et al. 1982; Bathon, 1996; Akhurst & Smith, 2002). Morris et al. ( 1990) screened nine entomopathogenic nematode-bacteria complexes for their virulence to larvae of six noctuids , a geometrid and a pyralid. LD 50 s of ( steinernematids
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and heterorhabditids) were 1-3, (1-8) infective juveniles in Galleria mellonella, 1-71, (2) in Peridroma saucia , 1-10 ,(1-3) in Mamestra configurata , 1-28 ,(3-7) in Euxoa ochrogaster , 19 in Lacanobia radix , 22-60, (4) in Pseudaletia unipuncta [Mythimna unipuncta ], 2-95, (111) in Agrotis ipsilon and 3-28 in Paleacrita vernata . The nematode Steinernema feltiae [Neoaplectana carpocapsae ] LIC, a cold-hardy strain isolated in Newfoundland, was highly virulent to G. mellonella and M. configurata , but not to P. saucia and E. ochrogaster . The number of nematodes invading larvae and the number produced were greater in G. mellonella than in the other insects tested. Nematode-bacteria complexes that showed potential for controlling noctuids included N. feltiae , S. glaseri [ N. glaseri ], S. bibionis [ N. bibionis ], Heterorhabditis bacteriophora and H. heliothidis . Tahir et al . (1995) used last instar larvae of the African bollworm, Helicoverpa (Heliothis) armigera , and the spotted bollworm of Asia , Erias vitella , to investigate the effect of increasing dosages of Steinernema riobravis , S. carpocapsae (ALL isolate) and a Heterorhabditis sp . on the establishment of the nematodes. There was a linear relationship between the dosage of nematodes applied and the number of nematodes established in the two hosts. H. armigera was more susceptible to S. riobravis than to S. carpocapsae and Heterorhabditis sp. but against E. vitella , Heterorhabditis sp . was the most effective. The use of the dosage-establishment relationships is a useful method of comparing the efficacy of nematode species and isolates against different insect host.
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Shamseldean et al (1996) tested the efficacies of four strains of EPNs in infecting and killing Spodoptera littoralis in the laboratory in relation to soil temperature, nematode dose and emergence from the insect cadavers. All the tested nematodes attained almost 100% insect mortality at 4, 10 and 25°C but at 35°C, Heterorhabditis bacteriophora (HP88) achieved the least (64%). As soil temperature increased to 35°C, the most adversely affected nematode in terms of recycling efficiency was H. bacteriophora (EASD98) followed by Steinernema riobravis , H. bacteriophora (HP88) and finally H. indicus (EAS59). Although all nematodes could infect and kill the host insects at 35°C, those of H. bacteriophora (EASD98) could not emerge from the cadavers. There were differences in the numbers of emerging infective juveniles in relation to nematode concentration and different soil temperatures; these should be considered in developing a biocontrol strategy for the management of the noctuid. Mason &Wright (1997) assessed the pathogenicity of two isolates of Steinernema spp . (M87 and SSL85), Heterorhabditis spp . and H. indicus against Plutella xylostella in the laboratory with a view to using these nematodes as biological control agents. Infection of larvae commenced within 3 hours of exposure, with maximum levels of infection occurring after 24 hours. Concentration-dependent studies showed that between 1 and 18% of the initial concentration of nematodes infected larvae of P. Xylostella Lacey &Unruh (1998) assessed the susceptibility of codling moth diapausing larvae to three EPNs species in the
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laboratory using a bioassay system that employed cocooned larvae within cardboard strips. The LC 50 values for Steinernema carpocapsae , S. riobrav e, and Heterorhabditis bacteriophora were 4.7, 4.8, and 6.0 infective juveniles/cm 2, respectively.When a discriminating concentration of 10 infective juveniles/cm 2of each of the three nematode species was evaluated at 15, 20, 25, and 30°C, S . carpocapsae was the most effective nematode with mortalities ranging from 66 to 90%. Mortalities produced by S. riobrave and H. bacteriophora at the four temperatures were 2– 94 and 25–69%, respectively. Studies were also conducted to test infectivity at 10, 35, and 40°C. No mortality was produced by any of the nematode species at 10°C. S. riobrave was the most infective nematode at 35°C producing 68% mortality which was more than twice that observed for S. carpocapsae or H. bacteriophora . Codling moth larvae treated with 10 infective juveniles /cm 2 of S. carpocapsae and kept in 95+% RH at 25°C for 0–24 h followed by incubation at 25–35% RH indicated that more than 3 h in high humidity was needed to attain 50% mortality. Trials of S. carpocapsae,S.riobrave ,and H. bacteriophora at 50 infective juveniles/cm 2against cocooned larvae on pear and apple logs resulted in reductions of codling moth adult emergence of 83, 31, and 43%, respectively, relative to control emergence.Trials of the three entomopathogenic nematodes at 50 infective juveniles/cm 2 against cocooned larvae in leaf litter resulted in 99 ( S. carpocapsae ), 80 ( S. riobrave ), and 83% ( H. bacteriophora ) mortality, respectively. Our results indicate good potential of EPNs, especially S. carpocapsae , for
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codling moth control under a variety of environmental conditions. El-Sadawy & Saleh (1999) tested six isolates of Steinernema and Heterorhabditis spp . isolated from Egyptian soil for their infectivity to Galleria mellonella larvae at 15, 25 and 35°C. For comparison, 3 imported nematode species of the two genera were tested for their infectivity to the same insect under the same temperatures. At 25°C, both Egyptian and imported nematode species were comparable to each other and were highly infective to insect larvae causing up to 100% insect mortality. The Egyptian steinernematid isolates were suitable for 15°C (>80% mortality) rather than 35°C (20% mortality). In contrast, the heterorhabditid i solates were suitable for 35°C (>80% mortality) rather than 15°C (<5% mortality). These isolates seem very suitable for use in biological pest control in Egyptian newly reclaimed lands and other semitropical lands. Saleh et al. ( 2000) found that the larvae and the pupae of the greater sugar cane borer, Sesamia cretica showed various levels of susceptibility to six EPNs species/isolates in laboratory and field tests. Generally, the insect larvae were more vulnerable to nematode infection than the pupae. The locally isolated nematode, Heterorhabditis taysearae and the imported one, Heterorhabditis bacteriophora were bio-assayed using filter paper and sand barrier techniques at 30°C. Half-lethal concentration (LC 50 ) and slope values obtained in both assessments revealed that the indigenous nematode proved much higher and faster infectivity than the imported one. In a field experiment, a single spray to the maize plants with nematode
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suspension of 1000 infective juveniles (IJs)/ml resulted in 67.86 and 40.62% larval mortality within one week for the indigenous and the imported nematodes, respectively. This gives evidence that the Egyptian nematodes could be included in the control of S. cretica in Egyptian maize fields Shilpa-Shinde et al . (2000) tested eight EPNs species/strains, Steinernema glaseri (Steiner), S. carpocapsae (Weiser), S. feltiae (Filipjev), Steinernema sp. Ecomax strain, Heterorhabditis bacteriophora (Pioner), Heterorhabditis sp . Ecomax strain, two locally isolated strains called as JFC and TFC against the final instar larvae of diamond back moth, Plutella xylostella (L.). All nematodes were found pathogenic. However, H. bacteriophora was considered the most pathogenic amongst the test nematodes on the basis of LD 50 (9.16 IJS / larva), LT 50 (43.26 hr.), Lex T 50 (3.24 hr.) and the propagation potential (average of 271.42 IJS/mg) on the host body weight Singh et al . (2002) tested the efficacy of Heterorhabditis bacteriophora against various developmental stages of Plutella xylostella (L.). The laboratory bioassay revealed that no nematode infection was observed in the egg masses. Final instar larvae were most susceptible among all the stages of the insect, with an LC 50 value of 9.16 infective juveniles (IJs) per insect.
The prepupal stage was equally susceptible, with an LC 50 value of 10.13 IJs per insect. However, LC 50 values recorded for freshly formed pupae and cocoons were 27.1 and 28.17 IJs per insect, respectively. Two-day-old pupae (cocoons) proved to be most resistant, with an LC 50 value of 86.88 IJs per insect.
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Sankaranarayanan et al. (2003 ) investigated the infectivity of 29 EPNs isolates belonging to Heterorhabditidae and Steinernematidae on Chilo infuscatellus at 18 and 27degrees°C in the laboratory. Heterorhabditis indica (LN2), H. bacteriophora (German isolate) and Steinernema glaseri (Australian isolate) caused 100% mortality of the pestin the field significantly reduced the rate of economic ear damage from 20% to 5%. Rosa & Simoes (2004 ) mentioned that the armyworm, Pseudaletia unipuncta , is the most important pest in Azorean pastures. Although this pest has some parasitoids and pathogens, additional biological control agents are needed to manage it. EPNs particularly Heterorhaditis bacteriophora , are good candidates because they have been isolated from pastures and crops in almost all islands of the Azorean Archipelago. They tested 28 Azorean isolates of H. bacteriophora in the laboratory against the 6th instar P. unipuncta to determine mortality rates and virulence of each isolate. Plot tests in the field were also conducted to evaluate the best time for application of the selected isolate. All isolates killed the larvae although important differences in the mortality rates were observed. Forty-eight h post exposure (HPE) to the nematode infective juvenile (IJ), insect mortality ranged from 0 to 92.5% and at 96 HPE, mortality ranged from 32.5 to 100%. Based on LC 50 and LT 50 , H. bacteriophora Az29 was the most pathogenic and the remaining 27 isolates were grouped in three classes of virulence. The most virulent class included four isolates with LC 50 ranging from 180 to 327 IJs/insect and LT 50 from 44 to 62.9 h. These isolates were
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obtained from three of the nine islands. High intrapopulational variability was detected on isolates in the moderately virulent class suggesting that these isolates are good candidates for genetic improvement. Field tests showed H. bacteriophora Az29 was more effective to control P. unipuncta larvae than Steinernema carpocapsae Az20 and H. bacteriophora Az32, belonging to the less virulent class. These tests also showed that applications performed during May resulted in better control than in July. Lacey et al . (2005) tested (EPNs) as a potential means of control to diapausing codling moth larvae, Cydia pomonella (L.), that could be applied at the time bins are submerged in dump tanks. Diapausing cocooned codling moth larvae in miniature fruit bins were highly susceptible to infective juveniles (IJs) of Steinernema carpocapsa e (Weiser) and Steinernema feltiae (Filipjev) in a series of experiments. Cocooned larvae are significantly more susceptible to infection than are pupae. Experimental treatment of bins in suspensions of laboratory produced S. feltiae ranging from 10 to 100 IJs/ml of water with wetting agent (Silwet L77) resulted in 51-92% mortality. The use of adjuvants to increase penetration of hibernacula and retard desiccation of S. feltiae in fruit bins resulted in improved effcacy. The combination of a wetting agent (Silwet L77) and humectant (Stockosorb) with 10 S. feltiae IJs/ml in low and high humidity resulted in 92-95% mortality of cocooned codling moth larvae versus 46-57% mortality at the same IJ concentration without adjuvant. Immersion of infested bins in suspensions of commercially produced nematodes ranging from 10 to 50 IJs/ml
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water with wetting agent in an experimental packing line resulted in mortality in cocooned codling moth larvae of 45-87 and 56-85% for S. feltiae and S. carpocapsae , respectively. Our results indicate that EPNs provide an alternative non chemical means of control that could be applied at the time bins are submerged in dump tanks at the packing house for flotation of fruit Cottrell & Shapiro-IlaN (2006) mentioned that the nematode Steinernema carpocapsae (All) strain was significantly more effective against Peachtree borer larvae Synanthedon exitiosa [Lepidoptera: Sesiidae] than Steinernema riobrave (7–12) strain in field and laboratory experiments. Eighty-eight percent controls of peach tree borer larvae was obtained with S. carpocapsae in the field trial when applied at 3x10 5 infective juveniles per tree, and 92% mortality was obtained in the lab assay using 50 infective juveniles per larva. Salem et al . (2007) stated that Pieris rapae (L.) tested larvae, appeared more susceptible than Spodoptera littoralis (Boisd) and Plutella xylostella (L.) larvae to all tested nematodes. For P. rapae larvae , Steinernema carpocapase All and Steinernema caprocapsae S2 were more virulent to the 2nd larval instar than 5th one; but Heterorhabditis indicus SAA2 and Heterorhabditis bacteriophora HP88 were the most virulent heterorhabditids to 5th larval instar than 2nd one. As for S. littorals, S. carpocapsae All and S. carpocapsae S2 were the most virulent and fastest in action especially against the younger instars larvae; while all Heterorhabditis sp . showed valuable efficiency in virulence and time required for killing the tested
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pest larvae as indicated by values of lethal mortality concentrations and lethal time required. As well, S. carpocapsae . All and S. carpocapsae S2 showed more efficiency in virulence and faster in action to the 2nd and 3rd instar larvae of P. xylostella . On the other hand, the Egyptian heterorhadbitids S1 and H. indiccus SAA2 were more effective than H. bacteriophara HP88 to the 3rd and 4th instar larvae Bruck et al. (2008) determined the susceptibility of the strawberry crown moth, Synanthedon bibionipennis (Boisduval) (Lepidoptera: Sesiidae) larvae to two species of EPNs. The EPNs , Steinernema carpocapsae (Weiser) strain Agriotos and Heterorhabditis bacteriophora (Steiner) strain Oswego were evaluated in laboratory, soil bioassays and the field. Both nematode species were highly infective in the laboratory bioassays. Last instars were extremely susceptible to nematode infection in the laboratory, even in the protected environment inside the strawberry Fragaria ananassa (Duch.) crown. Infectivity in the laboratory was 96 and 94% for S. carpocapsae and H. bacteriophora , respectively. Field applications in late fall (October) were less effective with S. carpocapsae and H. bacteriophora , resulting in 51 and 33% infection, respectively. Larval mortality in the field from both nematode treatments was significantly greater than the control, but treatments were substantially less efficacious than in the laboratory. Soil temperature after nematode applications in the field (11°C mean daily temperature) was below minimum establishment temperatures for both nematode species for a majority of the post-application period. It is clear from laboratory data that
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strawberry crown moth larvae are extremely susceptible to nematode infection. Improved control in the field is likely if nematode applications are made in late summer to early fall when larvae are present in the soil and soil temperatures are more favorable for nematode infection. Barbosa-Negrisoli et al . (2010 ) evaluated larval and pupal susceptibility of The Brazilian apple, leafroller, Bonagota salubricola ) to Heterorhabditis bacteriophora RS107 and H. bacteriophora RS57. Bioassays for isolates selection and determination of lethal concentration were performed in tubes of 1.5 ml (eppendorf), each containing one B. salubricola third instar larvae and filter paper. Field experiments were performed in commercial orchard, with application of 100 infecting juveniles (IJs)/cm 2 for each apple plant previously infected with five B. salubricola larvae covered with plastic trays containing thin cloth. Nematodes H. bacteriophora RS107 and H. bacteriophora RS57 had LC 50 of 13 and 4.5 IJs/larvae (4.3 and 1.5 IJs/cm 2), respectively. In the field, H. bacteriophora RS107 and H. bacteriophora RS57, applied with sorbitol as an adjuvant, reached 70.2 and 61.1% larval mortality, respectively. The results showed that both isolates had biological activity against B. salubricola under laboratory and field conditions. 2.5. Virulence of EPNs to the Black Cutworm, Agrotis ipsilon . Abdel-Kawy (1985 ) tested the infectivity of Neoaplectana carpocapsae when applied either to the soil surface or incorporated into the soil, against the fourth, fifith and sixth instar larvae of Agrotis ipsilon . A positive correlation was
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found between mortality percent of 4th , 5th and 6th instars and the nematode inoculum level. Also nematode application on soil surface gave high mortality percent in all instars than when incorporated into soil. Van-Sloun &Sikora (1986) conducted field experiments with species of. Steinernema and Heterorhabditis between 1983 and 1986 to control Agrotis. segetum on lettuce ( Lactuca sativa L.) and Delia brassicae on radish ( Raphanus sativus L. var. niger Kerner) and kohlrabi ( Brassica oleracea L. convar.acephala var. gongylodes L.). Damage caused by A. segetum on lettuce in 1983 was significantly reduced (p= 5%) when S. feltiae was applied on a per plant or as a surface treatment. There was a 80% and 87.5% reduction in damage when 104 nematodes / plant (equal to 1.2 x 10 5 nematodes / m 2) or 10 6 nematodes / m 2 were applied as a drench compared to a 57.5% reduction with Lindan. In 1985 the same nematode treatment significantly reduced damage 48% and 53.5%.The Parathion treatment only caused a 4% reduction in damage. Attempts to control D. brassicae with S. feltiae in 1985 at concentrations of 10 5 nematodes per meter row or 25,000 nematodes / kohlrabi plant as a drench gave results equal to the insecticides. Control in all treatments was inadequate. These results as well as field data from experiments conducted with both models in 1986 will be presented Capinera et al. (1988) assessed various species and strains of EPNs in the laboratory for their potential to infect black cutworm larvae, Agrotis ipsilo . Steinernema felriae (Neoaplectana carpocapsae ) strains Mexican and Kapow, S.
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bibionis and Heterorhabditis heliothidis . Based on LD 50, values, rates of mortality, and storage considerations, S. felriae Mexican strain was chosen for additional evaluation. When incorporated into wheat-bran pellets and calcium alginate capsules, nematode dauerlarvae were able to escape and cause infection of cutworms, but wheatbran pellets allowed more rapid escape. Cutworms consumed wheat-bran pellets as readily as corn seedlings, but more readily than calcium alginate capsules. Addition of wheat bran to calcium alginate capsules did not enhance consumption by cutworms. Field studies suggested no value of wheat-bran bait formulation, relative to aqueous suspension, for delivering nematodes. Significant reduction in corn seedling damage by cutworms in plots inoculated with aqueous suspension of dauerlarvae was demonstrated. Application of nematodes at rates of about 5x10 5/ m2 provided over 50% reduction in plant damage in some treatments Georgis, et al. (1989 ) studied the potential of the entomophilic nematode Steinernema feltiae (Neoaplectana carpocapsae )All train against the gryllotalpid Scapteriscus vicinus and the noctuid Agrotis ipsilon was in the laboratory using plastic containers filled to a depth of 5 cm with soil compost. The nematodes were applied to the pots at 0, 500, 500 and 5000 nematodes per 100 cm 2 of soil surface in 5 ml of water or mixed with 10 g of bait. Bait formulations performed significantly better than liquid sprays, especially at low concentration Levine & oumi-Sadeghi (1992) applied the entomophilic nematode, Steinernema carpocapsae (All strain) at two rates (1.25
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and 2.5x 10 9 nematodes/ha) as compared with several registered insecticides for controlling black cutworm, Agrotis ipsilon larvae during the 1991 growing season in Illinois. Fonofos, permethrin and chlorpyrifos were applied at planting time, permethrin and the two rates of nematodes were applied as post-emergence rescue treatments. The nematodes generally performed as well as or better than the conventional insecticides in controlling black cutworm larval injury to corn seedling. Bioassays with black cutworm larvae showed that nematode efficacy was lost 8 days after application in the field. Entomopathogenic nematodes hold promise for controlling black cutworm in Corn, particularly for corn grown under irrigation Kaya et al . (1993) reported that combining two species of EPNs with different search strategies suppressed two pest species of soil insects when they occurred in the same habitat. The major of individuals of Steinerema carpocopsae have a sit and wait (ambusher) strategy and tend to be near the soil surface. This species was effective against larvae of the black cutworm, Agrotis ipsilon which feeds near or at the soil surface. Heterorhabditis bacteriophora has an active foraging (cruiser) strategy and occurs deeper in the soil profile. It was effective against larvae of the black vine weevil, Otioryhnchus sulcatus , which occurs near roots. Soil temperature influenced the results. At 22°C both nematode species killed their respective target insect within 1 week. At 10°C, S. carpocapsae killed black cutworm larvae, but H. bacteriophora was not effective against black vine weevil larvae 2 weeks after treatment. A few black weevil larvae were infected with H. bacteriophora at that
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temperature, and a longer exposure of the black vine weevil larvae to the nematode may have increase efficacy. In addition to search strategies, the susceptibility of the insects to the nematode species plays an important role in pest suppression. Buhler & Gibb (1994) studied the field evaluation of the EPNs Steinernema carpocapsae and S. glaseri in controlling black cutworm, Agrotis ipsilon , and larvae in creeping bent grass. Bentgrass plots were baited with black cutworms on several dates following nematode application. Mortality of black cutworm in plots treated with nematodes was significantly greater than in untreated plots at 1 day after treatment. S. carpocapsae provided slightly higher level of black cutworm control than S. glaseri . Persistence of nematodes was lost 8-days after nematode application in the field. Laboratory bioassays with black cutworm and greater wax moth, Galleria mellonella , larvae using soil cores extracted from field plots confirmed results. Shamseldean & Ismail (1997) tested the EPNs, Heterorhabditis bacteriophora Pionar (HP 88) and the bacterium Bacillus thuringiensis Berliner var. kurstaki in the laboratory against the black cutworm, a widely distributed polyphagous insect pest of vegetables and field crops. The experiment was conducted under 15.7 °C (14 to 19 °C) to simulate the average winter temperature in Egypt. The two biocontrol agents influenced Agrotis ipsilon differently where H. bacteriophora had a more promising control levels than that of B. thuringiensis . Generally, nematode concentration of ca 500 infective juveniles/insect vial was highly effective (100% mortality) within nine days post nematode infection. The highest
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concentration of the bacterium B. thuringiensis var. kurstaki (2,000 Intern. Units/mg) gave significant larval control after 12 days of bacterial infection. Combined effect of both the nematodes and the bacteria did not result in significantly greater control than that achieved by the nematodes used alone. The present results indicated that nematodes could be used successfully against the black cutworm , Agrotis ipsilon larvae during the winter which is the active season of this insect pest in Egypt . Hussaini et al. (2000) investigated the virulence of the entomophilic nematodes Steinernema bicornutum (isolates PDBC 3.1, PDBC 3.2, and PDBC 2.1), S. carpocapsae (PDBC 66.1, PDBC 13.1, and PDBC 6.11), and Heterorhabditis indica (PDBC 6.71 and PDBC 13.3) against Agrotis ipsilon and Agrotis segetum larvae and pupae in a sand column assay. The nematode isolates differed in terms of virulence between insect stages and species. S. bicornutum PDBC 3.1 and PDBC 3.2 resulted in the highest A. segetum larval mortalities of 93.3 and 100%, respectively, at 72 h after inoculation. S. bicornutum PDBC 3.2, S. carpocapsae PDBC 66.1, and H. indica PDBC 13.3 gave 100% A. segetum pupal mortality. The highest larval mortality in A. ipsilon larvae was recorded for H. indica PDBC 6.71 and PDBC 13.3 with 100 and 93.3% mortality, respectively. The highest pupal mortality in A. ipsilon was recorded for S. carpocapsae PDBC 66.1 and S. bicornutum PDBC 2.1 with 100 and 88.9% mortality, respectively. Azazy (2001) tested the efficacy of some native and introduced nematode strains against certain larval instars of
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Agrotis ipsilon, and Spodoptera littoralis (Boisd). He found that the native strains were more efficient than the imported ones. The fourth larval instar of A.ipsilon, was found to be more susceptible to all tested nematode species than the 5th and 6th instar larvae. Both 4th and 6th instars of S. littoralis (Boisd) were more susceptible than 5th larval instar. Hussaini et al. ( 2005) studied the effect of different temperatures on the infectivity and progeny production of indigenous entomopathogenic nematode isolates, Steinernema carpocapsae PDBC EN 6.11, S. abbasi PDBC EN 3.1, S. tami PDBC EN 2.1, and Heterorhabditis indica PDBC EN 6.71, PDBC EN 13.3, under laboratory conditions against Galleria mellonella and Agrotis ipsilon final instar larvae. All isolates caused absolute mortality of test insect larvae, and produced progeny at 25 and 32°C degrees. Low rate of infection or no infection was observed at lower temperatures, 15 and 8°C degrees, respectively. Time required killing host insects by all isolates showed difference at 15, 25 and 32°C degrees. Shortest time for mortality of A. ipsilon and G. mellonella larvae was observed at 32°C degrees followed by 25°C degrees. Absolute mortality of larvae was observed with all isolates after 48 and 72 hours at 25 and 32 degrees °C, whereas the same was not achieved at 15 °C degrees even after 96 hours post exposure. Progeny production of all isolates varied with reference to temperature. Irrespective of isolates, 25 degrees °C was found suitable for infection and development of nematode populations.
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2.6.Feasibility of Integrating Radiation-Induced F 1 Sterility and EPNs for Population Suppression of the Insect Pests. No single control measure is able to provide full control of a pest. Integration of techniques like the sterile insect (SIT) with biological control practices (BC) should be intensively sough for. The biological control (BC) has been defined by DeBach (1964) as the action of parasites, predators or pathogens in maintaining the population density of another organism at a lower average than would occur in their absence. In another vein, it could be define as the use of natural enemies to reduce to tolerable levels the damage caused by noxious organisms (DeBach and Rosen, 1991). Among the variants of BC proposed (classical, conservative, augmentative,etc.) the inundative biological control agents release – involving massive production and continuous release of insects – is comparable to SIT in many regards. Mohamed (1995 ) studied the effect of gamma irradiation doses (40, 80, 120 and 160 Gy) on Steinernema carpocapsae and Phthorimmaea operculella larvae. She found that the mortality percent of the host decreased with exposure to high doses of radiation and longest time were needed for irradiated S. carpocapsae to cause full mortality. Abdel Salam et al . (1996) studied the effect of gamma radiation on the pathogenicity of EPNs Steinernema carpocapsae was by infecting larvae of Phthorimaea operculella . Full-grown larvae of the host were gamma-
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irradiated with different doses ranging from 2.5 to 160 Gray (Gy) and subsequently infected with either unirradiated or irradiated infective juveniles (IJs) of S. carpocapsae using the same doses. For comparison, 2 groups of unirradiated and irradiated host larvae were left without nematode infection. Another group of larval hosts was also infected with nematodes either unexposed or exposed to the previous doses. The results revealed that mortality was rapid in the irradiated host infected with either unexposed nematodes or nematodes irradiated with different doses similar to the mortality in the nematode treatment alone. On the other hand, gamma irradiation of the nematodes with high doses lowered their efficiency towards the unirradiated host
Gouge et al. (1998) studied the interactions between F 1, progeny of Pectinophora gossypiella (Saun-ders) adults irradiated in the pupal stage and EPNs. Both sexes of pink bollworm pupae were exposed to 4, 8, 12, or 16 krad substerilizing radiation doses irradiated using a Cobalt 60 source.
The F 1, larvae were tested in a sand bioassay for susceptibility to Steinernema riobravis Cabanillas, Poinar &Raulston, S. carpocapsae (Weiser), and 2 strains of Heterorhabditis bacteriophora (Poinar). The numbers of infecting nematodes were counted after 48 h. Increasing parental radiation dose significantly increased F 1, larval susceptibility to S. riobravis and H. bacteriophora , but decreased susceptibility to S. carpocapsae . This difference in susceptibility may be caused by the sedentary nature of larvae from parents receiving higher levels of irradiation, combined with the passive ambush tactics
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used by S. carpocapsae to acquire an insect host. The need to sustain the F, population of pink bollworm for sterility promotion and subsequent population collapse suggests S. carpocapsae to be ideal EPNs to be used in conjunction with inherited sterility control method Abd-Elwahed (2004 ) investigated the effect of EPNs, Steinernema carpocapsae on percent mortality of un-irradiated males and females Phthorimmaea operculella after 6, 18 and 24 hrs. Post infection was not different from F 1 adults descending from irradiated parents. Also females were more susceptible to nematodes than males and percent mortality was increased by increasing time elapse after infection. El Mandarawy et al . ( 2006 ) investigated the comparative susceptibility and reproduction of three EPNs species in the non- irradiated and parental irradiated greater wax moth, Galleria mellonella with different doses of gamma radiation. G. mellonella pupae were exposed to 0, 50, 100 and 150 Gy sub- sterilizing gamma radiation doses from a Cobalt 60 source. The sixth larval instar resulting from the first progeny (irradiated males x non-irradiated females) were tested in a sand bioassay for susceptibility to Steinernema riobravae , Heterohabditis bacteriophora Hp88 and H. taysearae at concentrations of 50, 100 and 200 IJs. Also, the survivor juveniles of the most effective nematode species were tested for different periods after being irradiated at doses 250, 500 and 750 Gray. Results showed that the radiation dose 300 Gy was assumed to be the lowest to achieve 0 fertile progeny. Generally, the susceptibility of G. mellonella larvae to nematode infection was positively affected
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with the irradiation doses where the mortality rate increased by increasing the dose. S. riobravae and H. taysearae caused significant increase in the larval mortality parentally irradiated with 150 Gy as compared to those with 0, 50, 100 and 150 Gy. However, the mortality percentage of larvae infected with H. bacteriophora Hp88 increased insignificantly among the non- irradiated ones and those irradiated at 50, 100 and 150 Gy. Larvae parentally irradiated at 150 Gray applied at 100 IJs of H. taysearae yielded the lowest number of nematodes, while at 200 IJs S. riobravae and H. taysearae did not reproduce. The reproduction of the three nematode species from larvae irradiated with 150 Gy decreased significantly between the different treated concentrations. There was a positive correlation between the percent survival of H. bacteriophora Hp88 juveniles after different gamma irradiation doses and the longevity of the period of exposure. Ali (2008) assessed Heterorhabditis bacteriophora BA1 and Steinernema carpocapsae BA2 for their effect on 4th instar larvae of the greater wax moth, Galleria mellonella in the laboratory. Larvae were exposed to serial concentrations of 20, 40, 60and 80 infective juveniles (IJs)/ml/cup/4 larvae. When male pupae irradiated with 40 or 100 Gy and mated to normal female, the resulted F 1 larvae were more susceptible to the nematode than normal larvae and the mortality was increased by increasing the dose of radiation and the nematode concentration and the juveniles reproduction was decreased with increasing the dose of gamma radiation. The lowest rate was at F 1 larvae of irradiated parent at 100 Gy and treated with 80 IJs.
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Seth et al. (2009) studied the efficacy of the entomopathogenic nematode (EPN), Steinernema glaseri , (Steiner) cultured in radio-sterilized host, vis-a-vis radiation- induced F1 sterility on a tropical lepidopteran pest, Spodoptera litura (Fabr.). Steinernema glaseri cultured in S. litura larvae
(with either 40 or 70 Gy of gamma rays) was evaluated on F1 sterile insects (progeny of male moths treated with 100 Gy, 130 Gy). S. glaseri EPNs cultured in radio-sterilized larvae at 40 Gy, had better infective potential than those cultured in sterilized host larvae at 70 Gy. F1 sterile larval hosts (progeny from 100 or 130 Gy treated parents) were equally acceptable to the EPNs cultured in radiosterilized hosts, although the nematode harvest was reduced on F 1sterile hosts at 130 Gy. Infectivity of IJs derived from F 1 sterile host was almost similar on F1sterile larvae and normal larvae of S. litura , although their parasitisation efficacy on the F1sterile hosts was relatively less than the controls. The IJs performance was little influenced by irradiation of IJs’ parent host and current host’s irradiation history individually, but both the parameters together did not have any further negative interaction on the performance of IJs. The present results indicate that S. glaseri harvested from
F1sterile larval hosts (progeny from 100 or 130 Gy treated parents) retained a reasonably high degree of infectivity on normal and F1sterile S. litura hosts (61_83% of controls). Two highly promising operational modes of integrating S. glaseri
EPNs with F1sterility’ to suppress S. litura populations (initial releases of EPNs to strongly suppress the density of the pest followed by use of F 1sterility vs. simultaneous use of both techniques) are discussed.
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3. MATERIALS AND METHODS 3.1. Insect Rearing Technique and Source of irradiation The black cutworm (BCW) , Agrotis ipsilon culture used in this study originated from eggs obtained as a laboratory established strain from the Black cutworm Dept., Plant Protection Res. Inst., Dokki, Giza. These eggs were surface sterilized with formalin (10%) Vapor treatment as suggested by David et al. (1972). Group of eggs Fig (1) were kept in glass jar (1600cc) covered with muslin cloth under laboratory condition of 25Co and 65 ± 5 relative humidity. When enclosed eggs became dark in color; they were supplied with castor bean leaves. (Ricinus communis) to feed the young larvae. The leaves were daily replaced by fresh ones. In order to avoid cannibalism, six instar larvae Fig (1) were reared individually in plastic vials (3x7cm.) containing a layer of sawdust to absorb excess humidity. Prepupae were transferred to plastic trays (22.5 x 10.8cm with depth of 3.7cm) containing a layer of moistened sawdust to serve as a pupation site. Newly formed pupae were collected Fig (1), sexed and kept in groups of 25 pupae of each sex, in Petri dishes (each 10cm diameter) containing moist saw dust and covering with muslin. The mating of male and female moths and oviposition were done in cylinder glass jars (12cm diameter and 25cm height). The upper rim was covered with muslin that was fixed with rubber bands. Stripes of muslin were hanged from the rim of the jar to down in the jar space, in order to serve as a resting and oviposition sites.
Materials and Methods 45
For moth feeding a piece of cotton in small vial was soaked in 10% sucrose solution and provided to the glass jar. The jars were inspected daily in order to change the feeding solution with another one fresh and to collect the deposited eggs by replacing the old stripes with new ones. Newly deposited eggs (the collected stripes) were placed in clean jars until hatching. All rearing equipments including glass jars and handling instruments were routinely washed with soap and soaked in 1% formalin solution in order to avoid virus contamination and infection. The full grown male pupae of A. ipsilon (48 24 hours before emergence to adults) from the stock culture, were irradiated with gamma radiation at different doses, (75,100, 125 150, 175 and 200 Gy), Irradiation was carried out with doses of gamma radiation from Cobalt source at dose rate of 30 Gy/minute in Cyclotron Project, Experimental Physic Department, Nuclear Research Center. 3.2. Experimental Procedures: 3.2.1. Mating activity and mating competitiveness. Moths were preconditioned fewer than 14 .10 light: dark (L.D) cycles at 25±2°C for 24 h prior to initiation of the studies. The dark phase was initiated at 4 a.m. Observations of the of mating times and duration of copulation were made under low intensity red light (7 \V) in 25±2°C constant temperature glass jars during the last 6 h of the dark period. Little or no mating occurs in light periods or the first 4 h of the dark period Gemeno & Haynes (2000). All experiments remained for five days and replicated six times with ten individual moth pairs per replicate.
Materials and Methods 46
(1)Black cutworm egg (2) Black cutworm larvae
(3) Black cut worm moth (4) Black cutworm pupae
(5) (6) (7) Fig. (1) The different stages of black cut-worm Agrotis ipsilon and their infestation on Zea plants
Materials and Methods 47
1 Image courtes of Kelly A. cook et al University of Illinois Extension ( http://ipm.uiuc.edu/field crop imn/index.html . 2 Image courtes of John L. Capinera University of Florida Extension (http:// edis.ifas.ufl.edu/TOPIC 3 &4 Image courtes of ralph E. Berry & Leonard B. Coop University of Oregon State Exension (http://min.ippc.orst.edu/foli.ageinsect.htm 5 Image courtes of Daived Riley University of Georgia www.ipmimges.org 6 Image courtes of Clemsion University Extension www.ipmimges.org 7 Image courtes of Ronald B. Hammond et al University of Ohio State Extension (http:// Ohioline.osu.edu
Materials and Methods 48
Experiment 1.The effects of gamma irradiation on the black cutworm male moths on mating times, duration of copulation, spermatophore and sperm transfer were determined by exposing male moths to 75,100,125,150,175and 200 Gy as a full grown pupae, controls in all cases were used untreated moths pairs,. After irradiation, 10 male moths in each case treated with 75,100.125,150.175and 200 Gy, and 10 untreated males were placed individually in mating cages, each with a virgin of 1 to 2 day old female, in regulated photoperiod temperature in the laboratory. Time of initiation and termination of copulation was recorded to determine the effect of treatments on mating percent and duration of copulated moth pairs were in copulation, mating duration was calculated from the success of the male to insert its aedeagus into female’s genitalia until the mating pair separated from each other as shown in figure (2) . Female moths at the end of copulation were removed from the mating cages and held for 48 h. They were then dissected and examined for the presence of a spermatophore in the bursa copulatrix. The presence of sperm in the spermatheca was verified by placing spermatheca on a glass microscope slide, rupturing the spermatheca and examining under the microscope for sperm (figure 3A&B).
Materials and Methods 49
Plan of Work First: - SIT I II Mating activity mating competitiveness a) T♂ x N♀
b) T♂xN♀ (1) N♂+T♂ x N♀
T♂ x N♀ (2)
T♂ x N♀ (3) (successive of mating) c) un mated T♂ or N♂ st T♂ or N♂ x 1 N♀ st nd T♂ or N♂ x 1 N♀ +2 N♀ st nd rd T♂ or N♂ x 1 N♀ +2 N♀+ 3 N♀ (The effect of irradiation on success of mating during scotophase) st nd rd d) N♂ or T♂ x 1 N♀ +2 N♀+ 3 N♀ at begin of night st nd rd N♂ or T♂ x 1 N♀ +2 N♀+ 3 N♀ at middle of night st nd rd N♂ or T♂ x 1 N♀ +2 N♀+ 3 N♀ at end of night Second:- From step I and step II the dose (125 Gy) was chosen then was evaluated it by 1) Studying the effect on mating activity of female (T♀ x N♂) (on the successive of mating) (un mated T♀ and T♀ x 1st N♂ and T♀ x 1st N♂ + 2nd N♂)
Materials and Methods 50
(The effect of the dose on success of mating during scotophase) (N♀ or T♀ x 1st N♂ or 2nd N♂ at begin, middle and end of night ) 2) The effect on biological aspects and total competitiveness of parents
3) The effect on biological aspects of F 1 progeny
4) The effect on the reproduction of P 1 and F 1 progeny Third: - Entomopathogenic nematodes (EPNs) A) Biological studies on Steinernema carpocapsae All Steinernema riobrave at concentrations 0, 10, 25, 50,
100,200 IJs/larvae/ pot based on LC 50 < 50 choice S. carpocapsae B)Tested S.carpocapsae All at 100 IJs/larvae for exposure time(8,10,12hr) C) Tested application method of S. carpocapsae at 100 IJs/larvae/ pot via aqueous suspensions and application of 5 and 10 day BCW cadavers as a single cadavers /larvae/ pot Fourth: - Interaction of S. carpocapsae with gamma irradiation induced F1 sterility in A.ipsilon. 1) Study the combined effect of EPNs with gamma radiation on mortality of A. ipsilon .
2) Assayed bioefficacy of EPNs on F 1 sterile insect hosts by detected morbidity and mortality time, the incubation time and harvest potential . 3) Study the parasitizing performance and harvest potential of IJs
of EPNs cultured in F 1 sterile insects was ascertained against
normal and F 1 sterile A. ipsilon hosts.
Materials and Methods 51
Dorsal view Ventral view Fig. (2) mating position Experiment 2. The effect of radiation on male mating and oviposition of untreated females paired with treated males was determined in a similar manner to Experiment (1), except that after being observed in copulation, females were removed and placed individually in oviposition cages. The numbers of produced eggs and the hatching were recorded also, the removed female was replaced with a second virgin female on day 2 for one night to determine the ability of treated and untreated males to successfully inseminate female on successive nights. The process was repeated on day 3 until day 5. All females were un mated and 1 to 2 days old when placed in the cages with males. At the end of the oviposition periods, all females were dissected and examined for the presence of
Materials and Methods 52 spermatophore. Regarding to the mean effects of tested mating aspects in three sequential females, the reported data were generated by calculating the average values of the studied parameters (percent of copulated males, duration of copulation, fecundity of mated female, egg hatch, mated female with spermatophore and un separated pairs) of the same female through all tested doses . While this average was calculated for these parameters at the same dose for three sequential females in the case of the mean effects of tested mating aspects in doses. The effect of irradiation on successively mated males were conducted using the mating male with only 1st ♀ female as an once time , the mating male with 1st ♀ & 2nd ♀ females as a twice times and the mating male with 1st ♀ , 2nd ♀ and 3rd ♀ females as a three times. The scotophase was divided as stage I (beginning of scotophase) ,stage II (middle of scotophase ), and stage III, three (end of scotophase), various regimens were based upon the work of Saour & Makee ( 1999 ). At which the mating time (period from initiation to termination of copulation) was recorded to determine effect of treatments on the entering into copulation during scotophase . For assessment the irradiation effect on successively mated males and the effect of irradiation on the entering into copulation during scotophase.
Materials and Methods 53
Fig. (3) A [Internal reproductive organs of a normal female Agrotis ipsilon . Acgl=paired accessory glands; Bcpx=bursa copulatrix; Odc=common oviduct; Odl=lateral oviduct; Ovl=ovariole; Sdct=seminal duct; Spth=spermatheca;Vag=vagina [ Gemeno et al 1998]
Materials and Methods 54
spermathica in A. ipsilon female sperms in spermathica Fig. (3) B Experiment 3 Male competitiveness was assessed in a similar manner to Experiment 1, two types of males were used in the experiments males treated with 75,100.125,150.175and 200 Gy (T Male), as indicated above; and untreated males ( N Male) with a virgin 1 to 2 day old female, in order to distinguish the male “types” the wing tips of treated and untreated males were differentially colored with a felt tip marker. For the assessment of mating competition, only the first mating combinations were considered. All the observations were done at 25±2°C and 70 ±5 % RH. Six replicates were used in each dose. Each replication consists of ten pairs of insects. Finally, the competitiveness was calculated according to the formula of Nahar, et al . (2006 ): i / n C = —— S / N Where, C = The competitiveness value i = The number of mated irradiated males,
Materials and Methods 55
n = The number of mated normal males. S = Number of sterile male used in the experiment N = Number of normal male used in the experiment.
3.2.2. Evaluation of F1 Sterility for Suppression of the Agrotis ipsilon. 3.2.2.1. Effects of gamma radiation on Agrotis ipsilon female moths. Irradiation effects on mating of black cutworm female moth were determined by exposing full grown pupae to 125 Gy (chosen dose). Individual female moths ( ten individual in each replicate, six replicates were used) were placed in mating and oviposition cages and a single untreated male moth was introduced into each cage, untreated moth pairs were used in the control. Cages were placed in the regulated photoperiod and temperature in the laboratory during the dark phase of the photoperiod and mating observations was described. On each of days 1 to 5 following treatment, male moths were removed from each cage and replaced with new ones. Time of initiation and termination of copulation and numbers of mating pairs were recorded also eggs from single pair matings were counted daily and the number of egg hatch. All female moths were dissected and examined for the pre sence of spermatophores. 3.2.2.2. Effects of gamma radiation on biological aspects of Agrotis ipsilon . Full grown pupae were irradiated with a dose of 125 Gy). Percent emergence, percentage of moths with deformities and
Materials and Methods 56 longevity of irradiated versus untreated A. ipsilon adults were recorded in the laboratory. An average of 100 pupae was used in each of three replicates for the first two parameters. A subset of 50 adults in each of three replicates was used in the longevity study. 3.2.2.3. Total competitiveness of irradiated Agrotis ipsilon males. The effect of radiation on the total competitiveness of male A. ipsilon was determined by combining irradiated male (IM), unirradiated male (UM) and virgin unirradiated female (UF) in a desired ratio. Irradiated male : unirradiated male : unirradiated female : 1 : 1 1 : : 1 1 : 1 : 1 3 : 1 : 1 5 : 1 : 1 Each group was introduced into one litre card board ice cream containers, covered with muslin cloth fixed with a rubber band. Stripes of muslin were hanged from the rim of the jar to down in the cage space, in order to serve as a resting and oviposition sites. For moth feeding a piece of cotton in small vial was soaked in 10% sucrose solution and provided to the glass jar. The cages were inspected daily in order to change the feeding solution with another one fresh and to collect the deposited eggs by replacing the old stripes with new ones. The collected eggs were counted daily to determine fecundity and fertility. All tests were
Materials and Methods 57 terminated 10 days after crossing; these trails were folded 20 times. Expected egg infertility, for irradiated males was estimated following equation according to Fried (1971): N (Ha) + S (Hs) Expected egg infertility = ———————— N + S
Where Ha = % egg infertility of untreated male x untreated female. Hs = % egg infertility of treated male x untreated female. N = number of normal males. S = number of irradiated males. The competitiveness value (C.V.) was then calculated as follows: % actual infertility Competitiveness value (C.V.) = ——————————— %corrected expected infertility When competitiveness values near or more than 1.0 indicate full competitiveness and those between 0.75 and 1.0 indicate good competitiveness. 3.2.2.4. Reproductive performance and viability of irradiated moths and their progeny Various reproductive parameters were assessed by pairing treated insects (irradiated P males and F1& F2 moths derived from the treated P males crossed with normal females) with their normal (N) counterparts from the stock culture. Eggs from single pair mating were counted daily and the number of egg
Materials and Methods 58 hatch, corrected sterility and control of reproduction of insect population due to irradiation were determined according to the methods described by Seth & Reynolds (1993) .For fecundity and fertility data, an average of 3 moth pairs were used per replicate and 20 replicates were completed. The mating success of moths was assessed by dissection of females immediately after death. The presence of a spermatophore in the bursa copulatrix indicated that the female had mated; the number of spermatophores indicated the mating frequency. The viability of treated moths, and the survival and developmental pattern of
F1& F 2 progeny were determined. Diurnal observations on the insect behavior and the growth index for each treatment were calculated. For survival percent, an average of 100 larvae per replicates was used and 3 replications were used.
3.2.2.5. Sperm production among P 1 and F1 of Agrotis ipsilon males. Effect of gamma irradiation on gonads measurements of
P1and F 1 males, was studied; and in order to the testes were not completely spherical two dimensions were measured and their average was used as diameter and the volume was determined as 4 3 = /3 π r . The number of eupyrene sperm bundles in the reproductive tract of P and F 1 unmated males (1 and 4 day old after emergence ) was counted. Counting took place 6 9 hr after the onset of the light cycle at which time according to (La- Chance et al. 1977), males contain eupyrene sperm bundles in the duplex region of the ejaculatory duct, whereas the seminal vesicles and upper vas deferens are devoid of sperm bundles( fig.4 A). The duplex region was removed from the male and
Materials and Methods 59 placed in drop of saline on a gridded microscope slide previously lined with a diamond pencil. Tearing the organ to release the eupyrene sperm bundles (fig. 4 B) in saline solution, and spreading them out in the solution with a fine needle. This procedure permitted more accurate count of sperm bundles in dark field of phase contrast microscopy at a magnification of 10 X. 3.2.2.6. Feasibility study on cytological sperm bundle assessment of F1 progeny of irradiated male Agrotis ipsilon. Dissection of moths Moths were killed by immersion in 70 95% ethanol for a few seconds, rinsed in tap water in a small Petri dish and dissected. Saline solution may be used for the dissection instead of water, but, saline forms crystals on the slide, interfering with staining and requiring an extra step for removing. The posterior abdomen just above the claspers of the freshly killed moth was pulled with a pair of fine forceps until most of the simplex was exposed.
Materials and Methods 60
Fig. (4 A) Internal reproductive organs of a normal male A. ipsilon. Aed = aedeagus ; Acgl = paired accessory glands ; Ded = ductus ejaculatorius duplex ; Des = ductus ejaculatorius simplex ; Vsm = seminal vesicles ;Tes = fused testes ; Vd = vas deferens.Arrow indicates section filled with yellow pigment .[ Gemeno et al 1998]
Materials and Methods 61
I (10) X II B(40) X Fig.(4 B) View of sperm bundles at a magnification of 40 &100 X This procedure will pull the duplex, vasa deferentia , seminal vesicles, and testes to the posterior abdomen, where they can be more easily dissected free from adhering fat body and tracheal tubules. Because there is a daily rhythm in the descent of mature sperm bundles from the testes to the seminal vesicles and then to the duplex for lepidopteran species (Riemann et al . 1974; Seth et al. 2002a). Seminal vesicles, duplex, and testes can be smeared separately on the same slide. The 2 testes are enclosed within a single membrane and can be treated as a single structure. Each part of the reproductive tract can be opened with help of fine forceps and the contents agitated in a very small drop of water on the slide until the sperm bundles are freed from the surrounding secretions.They are readily visible on a stereoscope under low magnification (10 or 20x). Smears may be allowed to dry for several hours, or overnight, or for a few minutes on a hot plate (~60°C).
Materials and Methods 62
Staining of Slides Cytological preparations were stained according to methods described by Carpenter et al . (2009). For tissue fixation, slides containing smears were placed on a staining rack over a sink and flooded with absolute methanol for 5 min. The slides were then drained of methanol and allowed to dry before staining with aceto orcein stain for 5 min. The stained slides were rinsed in running tap water for 10 s and allowed to dry. The dried slides were stained with freshly prepared Giemsa stain; slides may be stained from 30 min to 2 h to obtain good results. Slides were then rinsed in running tap water for 30 s, allowed to dry completely, rinsed for 5 s in absolute ethanol, and allowed to dry again before being mounted with Permount or other mounting medium. However the sperm nuclei in intact bundles were frequently non responsive to the staining procedure described above as shown in (fig.5 A).
Fig.(5 A) The sperm bundles nuclei were non-responsive to the staining procedure. In this case, before staining the slides with aceto orcein, they were hydrolyzed with 1 M HCl for 2 5 min at 60°C on a hot
Materials and Methods 63 plate and then stained satisfactorily. After treatment with HCl, the slides were rinsed in running tap water for 5 s and dried before staining with the aceto orcein and Giemsa stains, as described above (fig. 5 B).
Fig (5 B) The sperm bundles nuclei were responsive to the staining procedure the nuclei have a different color from the hole bundle. The differentiation between contrast of color was difficulty to rather thus the slight modifications for( PAM ) Teia anartoides by Wee et al . (2011) was used in case of Agrotis ipsilon (fig.5 C). That is, only Giemsa stain was used without orcein stain.
Fig (5 C) The sperm bundles were stained with only Giemsa stain .
Materials and Methods 64
3.3. Efficacy of Entomopathogenic Nematodes (EPNs) to Control Agrotis ipsilon Pest . Assay arena. This study was carried out on fine sand soil taken from the experimental farm of Nuclear Search Center at Anshas. Fourth instar larvae( L4 ) was placed at a plastic pot (6 cm diameter and 8 cm height), after the pot filled with 100 g of moistened soil (10 ml of water/100 g of dry soil) leading to a soil depth 3 cm and top area 28.3 cm 2 the moisture content of the soil at the time of cutworm introduction into the pots was ca 10% v/w approximately (i.e., volume of water to weight of soil) (fig.6) .The soil was autoclaved before each experiment. Nematodes were applied on the soil surface in volume 1 ml of water. These pots were covered with muslin, and provided with castor leaves as food for larvae. Thereby enabling some of the L4 to molt into next instars of larvae, prepupae, pupae and adult and thus providing a mixture of population with different developmental stages in the substrate. The nematodes were not more than1 2week old after being harvested from the Galleria mellonella larvae and were acclimatized for at least 6 h at room temperature before use. To prepare a new concentrations from nematodes in a stock culture, the quantification method described in Woodring & Kaya (1988) was used. In all experiments, a control treatment was pipetted with distilled water instead of nematode suspension. The experiments were carried out in a laboratory at 25 ± 2°C, ca 70% ± 5 rh, and L12:D12 photoperiod unless indicated otherwise. All experiments were repeated five times, with, a total of 100 larva
Materials and Methods 65 per treatment. In all replicates, EPNs efficacies in the different experiments were assessed according to the emergence of A.ipsilon adult emergence.
Fig. (6) Assay arena of EPNs studies. 3.3.1. EPNs Concentrations studies EPNs Steinernema carpocapsae (All) and Steinernema riobrave were obtained and stored at 5°C these nematodes were used within one –two weeks of receipt, or were cycled on G. mellonella larvae by the method of Dutky et al. (1964). EPNs species tested at 10, 25, 50,100 and 200 IJs/larvae/ pot in the assay arena. Mortality of the insects was recorded every 24 hr until moth emergence, dead insects were removed and set on individual emergence traps to verify the mortality was due to nematode infection . 3.3.2. EPNs Exposure-time studies To avoid potential nematode mortality caused by exposure to ultraviolet light nematode treatment were done at the evening (Buhler & Gibb , 1994). A. ipsilon larvae were exposed
Materials and Methods 66 to nematodes S. carpocapsae AII, ((100 IJs/larvae/ pot) for periods of 8 ,10 and 12hr (three photo regimes that they encounter in nature at different latitudes: 12:12 (L:D) h, 14:10 (L:D) h, and 16:8 (L:D) h) (late September to late March ), to establish the host killing rate among exposure period .At the end of this period, A. ipsilon larvae of each treatment were transferred into clean pots .Insect mortality was recorded every 24 48hr until moth emergence, dead insects were removed and set on individual emergence traps to verify the mortality was due to nematode infection. 3.3.3. EPNs application methods studies According to Welch & Briand (1960) they found that aqueous applications of S. carpocapsae (Weiser) DD136 strain, and those made using infected greater wax moth, Galleria mellonella (L.), cadavers, were equally effective at controlling cabbage root maggot, Hylemya brassicae (Bouché). Recent study compared the efficacy of S. carpocapsae against A. ipsilon larvae when applied via aqueous suspensions with those made using infected A. ipsilon cadavers. The experiment was conducted in the assay arena, treatments evaluated were a single aqueous application of 100 IJs/larvae/ pot of S. carpocapsae All and application of 5 and 10 day A. ipsilon cadavers shown in (fig.7), a single cadavers /larvae/ pot. Insect mortality was recorded every 24hr until moth emergence, dead insects were removed and set on individual emergence traps to verify the mortality was due to nematode infection.
Materials and Methods 67
Fig. (7) Agrotis ipsilon insect, cadavers 3.4. Interaction of Steinernema carpocapsae All with
Radiation Induced F1 Sterility in Agrotis ipsilon Experiment 1.To study the combined effect of EPNs with gamma radiation on mortality of A. ipsilon .Group of fully grown males pupae were irradiated at dose 125 Gy. The emerged adults were mated with untreated females, F 1 fourth instar larvae were exposed to nematodes S. carpocapsae AII, with concentrations 10, 25, 50, 100 and 200 IJs/larvae/ pot. Mortality of the insects was recorded every 24hr until moth emergence, dead insects were removed and set on individual emergence traps to verify the mortality was due to nematode infection. Analysis for additive, antagonistic, or. Synergistic interaction was made according to Faruki & Khan (2004) and
Koppenhofer (2007). The expected mortality M E for the combination of two agents can be calculated using the formula:
M E = M A+ M B (1 MA /100).
Where = MA and MB are the observed mortalities for the two agents alone. Results from a chi square test,
Materials and Methods 68
2 2 X = (MA B M E) / M E
Where = MA B is the observed mortality for the combination, and compared to the chi square table value for (df =1, p= 0.05). If the calculated X2 value less than the tabular value, mortalities for the combination were within the range expected, called additive effect. When the calculated X2 value exceeded the tabular value, mortalities in combination treatments were significantly different from the expected mortality and a non additive effect between the two agents is suspected ,but the synergistic or antagonistic effect is suspected ; if the difference MA B M E has a positive (negative) value, a significant interaction is considered synergistic (antagonistic)
Experiment 2. Bioefficacy of EPNs on F 1 sterile insect hosts Firstly, the bioefficacy of S .carpocapsae was assayed as indicated above against F1 sterile A. ipsilon larvae derived from matings in which their male parent had been treated with sub sterilizing gamma dose 125 Gy. Each assay for ascertaining IJs infective behavior was conducted in an individual mode with respect to host (comprising of single L4 larva inoculated by moderate concentration, 50 IJs per assay) due to the cannibalistic behavior of host larvae. Bioefficacy of S. carpocapsae was assessed by recording the times needed to induce morbidity and mortality, the incubation time, the percent parasitisation, development and proliferation (as computed by the harvest potential) of EPNs. The time profile for the induction of morbidity and mortality in the exposed A. ipsilon , larvae was recorded at 6 h intervals. Morbidity is an initial behavioral response to haemolymph septicemia caused by toxins released
Materials and Methods 69 by symbiotic bacteria of EPNs, followed by the host’s resultant mortality. Morbidity criteria of infected larva included their minimal response to a probe, sluggish nature, and delayed resumption of the normal posture with slight torsion in the body when turned upside down. After host death, the parasitized (host) larvae were incubated until the next generation of IJs was developed. After 7 8 days, IJs were seen wriggling at the outer surface of the insect cadaver. The incubation times of EPNs (i.e., from inoculation to emergence of next generation IJs from host cadaver) were recorded. The harvest of IJs was done using the white trap method (White 1927). For this, the cadavers having proliferating IJs inside were removed from the inoculative Petri dishes and transferred to sterilized harvest dishes (90mm diameter). The daily profiles of emergence of IJs out of host cadavers and their total emergence (harvest) period were recorded. Their harvest potential (IJs’ yield) was determined in terms of cumulative number of IJs harvested over the total harvest period per host, and the number of IJs harvested per mg fresh weight of host). For assessment of host morbidity timing and its mortality timing by EPNs, observations were conducted and each replicate represented the mean value of observations on a set of twenty individual hosts for five replicates; while the incubation time of EPNs (within host) and harvest potential of IJs, individual host observations were conducted and each replicate represented the mean value of observations on a set of ten individual hosts; whereas for the assessment of percent parasitisation, a cohort of 20 host larvae (evaluated by individual exposures) constituted each replicate.
Materials and Methods 70
Lastly, the parasitizing performance and harvest potential of IJs (indicating reproductive potential) of EPNs cultured in parasitized F1 sterile insects (progeny of irradiated male parents), was ascertained against normal and against F1 sterile A. ipsilon hosts. 3.5. Statistical Analysis: The data were statistically evaluated by analysis of variance (F) followed by Duncan's multiple range (1955) test to examine the significant differences between treatment. The 5% level of probability was used in all statistical tests. The statistical software program Steel &Torrie (1980) was used for all analyses. Mortality percentages were corrected using Abbot 's formula (1925), concentrations –mortality and time mortality lines were plotted using Ldp.LC50 , LC 90 and LT 50 were calculated .
Materials and Methods 71
4. RESULTS 4.1. Effect of Gamma Irradiation on Mating Activity and Mating Competitiveness of BCW, Agrotis ipsilon . Full grown pupae of A ipsilon were isolated from laboratory culture (48 24 hr before emergence) and irradiated at 0, 75, 100,125, 150,175 and 200 Gy, to examine sterility effects on the P 1 generation and to identify the dose of gamma radiation that would allow for maximum production of partially sterile P 1 adults while inducing full sterility in the F 1 generation. The results were exhibited as follow: 4.1.1. Effect of gamma irradiation on mating activity of treated males. (T ♂ x N♀) Table (1) show that negative correlation was noticed to exist between rate of mating and radiation doses. The results also show that the tested doses neither significantly affect. The percent of males observed in copulation nor the time of copulation( Figs.8 &9). On the other hand, the spermatophore formation percentage and the percentage of mated females with sperm in their spermatheca were significantly decreased at the treatment of the high doses 175 and 200 Gy in comparison to control,the spermatophore formation percentage was 94 and 91.84 % at 175 and 200 Gy compared to 98.21 % in control and the percentage of mated females with sperm in their spermatheca was 89.36 and 91.11 % at the same compared to 94.55% in control (fig. 10).
Results 72
Results 73
96 94 92 90 88 86 84 82 80 78 % Male observed in copulation copulation in observed Male % 76 74 Cont. 75 Gy 100 Gy 125 Gy 150 Gy 175 Gy 200 Gy Dose (Gy)
Fig (8) Effect of gamma irradiation on % copulated males.
124 123 122 121 120 Time( min) 119
118 Cont. 75 Gy 100 Gy 125 Gy 150 Gy 175 Gy 200 Gy Dose (Gy)
Fig (9) Effect of gamma irradiation doses on copulation time.
Results 74
%mated females 102 with 100 spermatophore %mated females 98 with sperm 96 94 92
andsperm 90 88 86 84
%Mated females spermatophorewith . y y Gy Gy t Gy 5 Gy Gy Gy Gy G Gy Gy G Gy Cont. 7 25 00 Con 75 00 75 100 1 150 175 2 1 125 150 1 200 Dose ( Gy ) Fig (10) Effect of gamma irradiation on % mated females with spermatophore and % mated females with sperm
120
100
80
60
40
20 % Of mated males mated % Of 0 75 75 75 100 125 150 175 200 100 125 150 175 200 100 125 150 175 200 Cont. Cont. Cont. First female Second female Third female Gamma doses & number of females
Fig (11) Effect of gamma irradiation on % males observed in copulation of sequentially three untreated female
Results 75
4.1.2. Effect of gamma irradiation on mating activity of treated males among three sequentially females (T♂
x N♀ (1), T♂ x N♀ (2) and T♂ x N♀ (3) ) When sequentially three untreated females were introduced to P1 males (Experiment 2) the indicated results of 1st female are included in the results indicated in Table 2,3&4 and Figs 11,12,13,14,15&16. The results belonged the 1st introduced female was shown in Table 2 and Fig,11 the percent males observed in copulation was significantly only differed at 100, 150, 175, 200 Gy (83.33, 80.00, 81.67 , 81.67 %) respectively when compared with the control (96.67 %). The results in Table, 2 & Fig,12 indicate also that the average time of copulation was not significantly affected with the radiation dose except at 100Gy dose(116.18 min). Table, 2&Fig,13 show that the reduction in fecundity of normal females mated with irradiated males was significant comparing with untreated control; and the egg hatch was reduced significantly by increasing the dose of radiation applied to male (Fig, 14). The greatest reduction in egg hatch occurred at 200 Gy, (19.78%) in comparison with control (79.25%). The data given in Table 2 &Fig. 15 indicate that the percent of females observed in copulation with spermatophore decreased by increasing dose level, while the percent of pairs which failed to separate not affected at any tested dose level. In case of 2nd introduced female, the percent males observed in copulation was drastically decreased from 78.33% in the control to 58.33, 53.33, 71.67, 40.00, 50.00 and 41.67 % at doses of 75, 100, 125, 150, 175 and 200 Gy (Table, 3& Fig. 11).
Results 76
On contrast as in case of 1st female, the average time in copulation was slightly increased with the increase of doses. The fecundity of sequentially 2nd normal introduced female mated with the same irradiated male, previous mated with1st female, was not significantly affected at 75 &175 Gy( 636.75 & 501.11 egg/female) comparing with the control (725.98 egg/female). on contrast at doses 100, 125 , 150 and 200 Gy the fecundity of was significantly decreased to 487.97 ,455.85 ,407.83 and 420.86 egg/female respectively comparing with control (Fig,13). The egg hatch as a shown in Fig 14 was severely affected at all doses of irradiation comparing with the control ,the egg hatch percentages reached to 36.56, 29.89, 27.18, 18.33 14.93 and 9.49% respectively at the treatments of 75 ,100, 125, 150, 175 and 200 Gy respectively. The percent of females observed in copulation with spermatophore in Fig 15 slightly affected at the doses of 75 & 125 (55 & 65%) comparing with (75%) in control. Where this the percent of females observed in copulation with spermatophore was diminished to 51.67, 33, 42 and 38% at the treatments with 100, 150, 175 and 200 Gy. The percent of un separated pairs in Fig.16 was not affected by the treatments. Table (4) & Fig (11) present the data of 3rd introduced female to irradiated male the percentages of treated males observed in copulation with 3rd female was less than those of untreated males (control) at each dose, this reduction was slight as shown in the treatment with the of doses 75 & 125 Gy while the doses of 100, 150, 175 and 200 Gy caused a notable reduction in the percent of mating (20, 18.33, 20 and 20.41 % respectively).
Results 77
Results 78
160 140 120 100 80 60 40 20
Time in copulation (min) in Time copulation 0 75 75 75 100 125 150 175 200 100 125 150 175 200 100 125 150 175 200 Cont. Cont. Cont. First female Second female Third female Gamma doses & number of female
Fig (12) Effect of gamma irradiation on time in copulation of Sequentially three untreated females
1200
1000
800
600
400
200
0 75 75 75 100 125 150 175 200 100 125 150 175 200 100 125 150 175 200 Cont. Cont. Cont. Average no.of eggs /mated female First female Second female Third female Gamma doses & number of female
Fig (13) Effect of gamma irradiation on no. of eggs of sequentially three untreated females
Results 79
Results 80
90 80 70 60 50 40 30
% Of egg hatch 20 10 0 75 75 75 100 125 150 175 200 100 125 150 175 200 100 125 150 175 200 Cont. Cont. Cont. First female Second female Third female Gamma doses & number of female
Fig (14) Effect of gamma irradiation on % egg hatch of sequentially three untreated females.
100 90 80 70 60 50 40 30
spermatophore 20
% Mated females with %females Mated 10 0 75 75 75 100 125 150 175 200 100 125 150 175 200 100 125 150 175 200 Cont. Cont. Cont. First female Second female Third female Gamma doses & number of female
Fig (15) Effect of gamma irradiation on % of mated females with spermatophore of sequentially three untreated females.
Results 81
As shown in Fig 12 the data about the average time in copulation was fluctuated, up and down from the time in the control. The shortest average time occurred at 175 Gy was 99.19 min comparing with 124.04 min in the control, while the longest average time occurred at 125Gy, 131.94 min. Also Table (4) & Fig (13) clear that no significant difference in the fecundity of females treated with doses 75, 100, 125, 150 and 200 Gy. On the other hand ,these fecundities were significantly less than the control. For instance the fecundity of the females treated with 175 Gy was 197.22 egg/ female while in the control was 566.86 egg/female. The fertility of the eggs was decreased gradually by increasing the doses ,the fertility decreased to about the 1/3 at treatment of 125 Gy (11.49%) comparing with that of the control (35.55%). According to that, the inhibition of egg hatch increased by increasing the doses until reached 94.08% at 200Gy (Fig, 14). Radiation dose did not significantly affect both the percent of females observed in copula with spermatophore and the un separated pairs percent (Figs, 15&16). 4.1.3.Mating aspects means of Agrotis ipsilon males moths which had been affected among three sequential females and tested doses. Table (5) summarizes the results of mean effects of male moths mating activities in sequential females and tested doses. The results indicate that the percentages males observed in copulation significantly decreased, where it was ranged between 23.98 and 86.90 % in 3rd female and 1st female, respectively. These percentages did not differ significantly between them in
Results 82 case of mean doses (Table, 5&Fig, 17). The data on the average time in copulation was presented in Table (5) &Fig (18) showed that this time was not significantly affected at the different treatments although the variation of mating time range was high. These ranges were 194.86: 79.57 min, 202.43:82.43 min and 173:79.71 min maximum time : minimum time in 1st female , 2nd female and 3rd female ,respectively. The ranges were 183.33: 87.67, 171.00: 91.00, 179.67:94.67, 182.33:83.67, 189.00: 88.00, 208.33: 62.67 and 183.67:56.33 minute maximum time: minimum time at the respective tested doses. Table(5)&Fig (19) exhibit that the fecundity of the other two sequent females was drastically decreased compared with that of the first one (679.05 eggs/female).The fecundities were significantly decreased from that of the control (760.98 eggs/female) only at two doses 125 and 200Gy (459.15 and 473.93 eggs/female). Data was found in (Table, 5 & Fig, 20) indicate that the egg fertility slightly reduced in 2nd female, however a significant reduction of the egg fertility than the control was recorded in the 3rd female. The egg fertility reduction was not obvious among tested doses. The results also showed no obvious reduction occurred in both of the percent of females observed in copulation with spermatophore and un separated pairs percent in relationship to doses increasing. The sequences of females had notable effect on of the percent of females observed in copulation with spermatophore, these percents were ( 82.38 ,51.48 and 21.31 %) in 1st female , 2nd female and 3rd female ,respectively Fig (21). The percent of pairs that failed in separation normally was more affected in 3rd female (41.67 %) compared to (14 and 10 %) in 1st female and 2nd female Table (5).
Results 83
Results 84
70 60 50 40 30 20 10
% Of un-separation pairs 0 75 75 75 100 125 150 175 200 100 125 150 175 200 100 125 150 175 200 Cont. Cont. Cont. First female Second female Third female Gamma doses &number of female
Fig (16) Effect of gamma irradiation on % un separated pairs of sequentially three untreated females.
F1 F2 F3 Cont. 75 Gy 100 Gy 125 Gy 150 Gy 175 100 90 80 70 60 50 40 30 20 10 0
% Males observed in copulation F1 F2 F3 Cont. 75 Gy 100 125 150 175 200 Gy Gy Gy Gy Gy Mean effect of females and doses
Fig (17) Mean of effects on% males observed in copulation of females and doses.
Results 85
Results 86
F1 F2 F3 Cont. 75 Gy 100 Gy 125 Gy 150 Gy 175 Gy 132 130 128 126 124 122 120 118 116 Time copulaof(min) 114 112 110 F1 F2 F3 Cont. 75 Gy 100 125 150 175 200 Gy Gy Gy Gy Gy Mean effect of females and doses Fig (18) Mean of effects on time of copulation in females and doses.
F1 F2 F3 Cont. 75 Gy 100 Gy 125 Gy 150 Gy 175 Gy 200 Gy 800 700 600 500 400 300 200 100 0
No.of eggs /m ated fem ale F1 F2 F3 Cont. 75 Gy 100 125 150 175 200 Gy Gy Gy Gy Gy Mean effect of females and doses
Fig (19) Mean of effects on no. of eggs /mated female in females and doses.
Results 87
F1 F2 F3 Cont. 75 Gy 100 Gy 125 Gy 150 Gy 175 Gy 200 Gy
70 60 50 40 30 20 10 % of hatchability0 F1 F2 F3 Cont.75 Gy100 125 150 175 200 Gy Gy Gy Gy Gy Mean effect of females and doses
Fig (20) Mean of effects on % egg hatch of females and doses.
F1 F2 F3 Cont. 75 Gy 100 Gy 125 Gy 150 Gy 175 Gy 200 Gy 90 80 70 60 50
40 30 spermatophore 20
% mated femaleswith 10 0 F1 F2 F3 Cont. 75 100 125 150 175 200 Gy Gy Gy Gy Gy Gy Mean effect females and doses Fig (21) Mean of effects on % mated females with spermatophore in females and doses.
Results 88
4.1.4. Effect of gamma irradiation on successively mated of st treated males (T♂ or N♂ x none, T♂ or N♂ x 1 N♀, st nd st T♂ or N♂ x 1 N♀ +2 N♀ and T♂ or N♂ x 1 N♀ +2nd N♀+ 3rd N♀) Table (6) present the data about effect of irradiation on percentage of successively mated males, that mate more than one time. The data show that the reduction in percent males which failed in responses to the calling female and complete the mating was more obvious when these males were treated with the high doses at 150 200 Gy. The reduction in un mated males percent was 20, 18.33 and 18.33 % at doses 150, 175 and 200 Gy, respectively compared to 3.33 % in the untreated control Fig, 22). Concerning the percentage of successive mating of male, the results represent in Table 6 & Fig 22 showed that the treatment with all doses did not clearly affect neither the percentage of successively mated males once nor the percentage of successively mated males twice, except at the dose 175 Gy the percentage of successively mated males twice significantly decreased to 25 % comparing the control (43.33%) (Fig, 23). On the other side the results showed significant reduction in the percentage of successively mated males three times was recorded. The reduction was 26.66, 20.00, 28.33, 11.66, 16.66 and 13.33 % at the doses 75, 100, 125, 150, 175 and 200Gy, respectively comparing with 35 % in the control treatment (Table, 6 & Fig, 23). Generally, it was noticed that the percentage of successively mated males was shifted in favour of twice and three times specially at first three doses. By comparing
Results 89 the averages number of mating times, and effect of irradiation on it (Table, 6) may exhibit that the dose 150 Gy produced the lowest average number of mating times(1.31) but it was 1.95 in 125 Gy case comparing to 2.10 in control treatment 4.1.5. Effect of gamma irradiation on treated male entering st nd into copulation during scotophase. (T♂ x 1 N♀ +2 N♀+ 3rd N♀) within three parts of night Data are given in Table, 7 clarify the effect of irradiation on the percentage of Agrotis ipsilon males entering into copulation during scotophase . In general the percentage of males entering into copulation during beginning of scotophase and middle of scotophase was higher than the percentage of males entering into copulation during end of scotophase among the sequentially three individual females at both of treated and un treated males. In the case of 1st female the irradiation did not clearly affect the percentage of males entering into copulation throughout (during beginning of scotophase and during end of scotophase), however, the percentage of males entering into copulation was significantly affected by gamma radiation applied to P1 males during the middle of scotophase. The percentage was 68.96% at the dose 125Gy where it reduced to 28.57% at the dose 175 Gy compared to 37.93% in the control (Fig, 24). The previous Table & Fig (25) show that the irradiation clearly affect the percentage of males entering into copulation throughout three scotophase period in the case 2nd female. During the beginning of scotophase ,the lowest percentage of
Results 90
Results 91
Effect of irradiation on % successively mated males 45 40 40 40 40 35 31.66 31.66 30 25 23.33 20 18.3318.33 18.33 20 16.66 15 % )Of mating 10 10 3.33 5 5 0 Cont. 75 Gy 75 Gy Cont. 100 Gy 125 Gy 150 Gy 175 Gy 200 Gy 100 Gy 125 Gy 150 Gy 175 Gy 200 Gy Irradiation effect Un-mated Mated-once Fig (22) Effect of gamma irradiation on % of successively mated males.
50 45 43.33 43.33 40 35 35 31.66 31.66 28.33 28.33 28.33 30 26.66 25 25 20 20 16.66 13.33 15 11.66 10 % Of m ating 5 0 Cont. 75 Gy 75 Gy Cont. 100 Gy 125 Gy 150 Gy 175 Gy 200 Gy 100 Gy 125 Gy 150 Gy 175 Gy 200 Gy Mated-twiceIrradiation effect Mated-three
Fig (23) Effect of gamma irradiation on % of successively mated males.
Results 92
Results 93
80 70 60 50 40 30 20 10 % Of mated males 0 75 75 75 100 125 150 175 200 100 125 150 175 200 100 125 150 175 200 Cont. Cont. Cont. Beginning Middle End
First mating First mating First mating Mting during scotophase
Fig (24) Effect of gamma irradiation on % of mated males during Scotophase.
90 80 70 60 50 40 30 20
% Of mted males 10 0 75 75 75 100 125 150 175 200 100 125 150 175 200 100 125 150 175 200 Cont. Cont. Cont. Beginning Middle End
second mating second mating second mating Mating during scotopase
Fig (25) Effect of gamma irradiation on % of mated males during scotophase.
Results 94 males entering into copulation resulted from treated males with 125 Gy (13.95%),where it was 46.93 in the control. At the same dose the percent of males entering into copulation increased to 81.39 % compared to 30.61% in the control during the middle of scotophase. The variation in the percent of males entering into copulation was very clear during the end of scotophase it was 8.57, 19.35, 4.65, 0.00, 20.00 and 32.00% respectively at the doses of 75, 100, 125, 150, 175 and 200Gy respectively in comparison with 22.44% in the control. In regard to the effect of irradiation on the percentage of males entering into copulation during scotophase among 3rd female as shown in Table, 7 & Fig, 26 the irradiation did not clearly affect the percentage of males entering into copulation during beginning of scotophase and the same trend was observed in the end of scotophase of the same female. But the percentage of males entering into copulation obviously affected as a result of treatment throughout both of the middle of scotophase. The percentage was 56.25, 66.66, 76.47, 14.28, 40.00 and 25.00% respectively, at the doses 75, 100, 125, 150, 175 and 200 Gy respectively, compared to 52.38% in control. In many insects, females often determine the acceptance or rejection of males. This concept is generally accepted because female investment in the production of gametes is greater than that of males. Female mate choice, however, is generally not well understood. Major point was focused that the males treated with 75 to 125 Gy appeared to be equally as capable as untreated males to arrival success, locatable success of female and initiated mating attempts with virgin females when placed at the same scotophase .
Results 95
4.1.6. Effect of gamma irradiation on mating competitiveness of treated male (N♂+T♂ x N♀ ) The effect of gamma irradiation doses on the mating competitiveness, which was studied by direct observation method, was presented in Table, 8 & Fig, 27.The results indicated that the 60 replicates with paired releases of irradiated and control males, the irradiated males( 56.52, 59.18, 52.00, 46.67, 46.67 and 40.91% respectively (n =26/46, 29/49, 26/50, 21/45, 21/45 and 18/44 respectively) at the doses of 75, 100, 125, 150, 175 and 200 Gy respectively, could locate and mate with the calling females. As a shown in Table, 8 & Fig, 28 mating competitiveness calculated from the direct observation in revealed that males emerged from pupae irradiated at 75 to 125 Gy are equally capable of mating as control. The competitiveness values calculated in the present study at 75 Gy was 1.30, at 100 Gy was 1.45 and that at 125 Gy was 1.08, results suggest that lower radiation doses may improve the competitiveness of sterile released insects. An important concern in sterile insect release programs is that treated males destined for release retain the ability to perceive /orient to pheromone signals from females and, as such, are able to compete with wild males in locating and mating with calling females in the field. 4.2. Effect of Substerilizing doses of Gamma Irradiation on Agrotis ipsilon . In this part of the study, full grown pupae were irradiated with substerilizing dose of 125 Gy. The effect on certain biological aspects was studied among parental generation, as well as immature stages were investigated throughout two successive generations. .
Results 96
90 80 70 60 50 40 30 20
% mated males Of 10 0 75 75 75 100 125 150 175 200 100 125 150 175 200 100 125 150 175 200 Cont. Cont. Cont. Beginning Middle End
Third mating Third mating Third mating Mating during scotophase
Fig (26) Effect of gamma irradiation on % of mated males during scotophase.
75 Gy NmxNf 100 Gy NmxNf 125 Gy NmxNf 150 Gy NmxNf 175 Gy NmxNf 200
75 70Gy TmxNf 100 Gy TmxNf 125 Gy TmxNf 150 Gy TmxNf 175 Gy TmxNf 200 60 50 40 30 20 10 0 % Of mating TmxNf TmxNf TmxNf TmxNf TmxNf TmxNf
NmxNf NmxNf NmxNf NmxNf NmxNf NmxNf 75 Gy 100 125 150 175 200 Gy 75 Gy 100 125 150 175 200 Gy Gy Gy Gy Gy Gy Gy Gy Gy Mating combinationan\ dose (Gy)
Fig (27) Effect of gamma irradiation doses on % of mating at different mating combination .
Results 97
4.2.1. Effect of gamma irradiation on mating activity of Agrotis ipsilon females. (T♀ x N♂) The effect of exposing female to 125 Gy gamma radiation on mating activity among successive males data is summarized in Table 9 The data show no effect on the percentages of treated females mating or times in copulation with untreated males by comparing with untreated females throughout three successive males(Table , 9 & Fig, 29). However, percentages of mating females over radiation doses (0 125Gy) significantly decreased from an average of 81.66% with the first male to 22.44% and 0 respectively with new virgin males provided during the second, and third mating periods . Also the time in copulation with untreated males significantly affected (Fig, 30). The same Table & Fig (32) show that the average number of sequential male matings was 1.2 ±0.63 and 1.25 ±0.51 times with untreated females or females exposed to 125 Gy, respectively. The average number of deposited eggs per female was significantly reduced by gamma radiation applied to females (Fig, 31), and the egg hatchability was significantly decreased to zero of irradiated females compared to 60.03 % of untreated control (Fig, 32).
Results 98
Results 99
1.6 1.45 1.4 1.3
1.2 1.08 1 0.75 0.75 0.8 0.692 0.6
0.4 comptativness value 0.2 0 NmxTmxNf NmxTmxNf NmxTmxNf NmxTmxNf NmxTmxNf NmxTmxNf 75 Gy 100 Gy 125 Gy 150 Gy 175 Gy 200 Gy Dose (Gy)
Fig (28)Effect of gamma irradiation doses on competitiveness values in direct observation method.
1st Male Cont. 1st Male 125 Gy 2nd Male Cont. 2nd Male Mean dose cont effect 125Gy Mean male 1st Male effect 2 90 a a a 80 70 60 50 a a 40 b b b 30 20 10 0
Gy Gy 126 1st 125 cont 2nd Male Male Cont. Cont. 125Gy %females obsarvedin copulation 1st 1st Male 2nd 2nd Male Mean effect Mean effect Male Male dose male Effect of radiation on mating of female
Fig (29) Effect of gamma irradiation doses % females observed in copulation.
Results 100
Results 101
1st Male Cont. 1st Male 125 Gy 2nd Male Cont. 2nd Male 126 Gy Mean dose cont effect 125Gy Mean male 1st Male effect 2nd Male
180 b b a b 160 a a a a 140 120 100 80 60 40
Timecopulain (min) 20 0 Gy Gy 125 126 1st cont 2nd Male Male Cont. Cont. 125Gy 1st 1st Male 2nd 2nd Male Mean effect Mean effect Male Male dose male
Effect of radiation on mating of female
Fig (30) Effect of gamma irradiation doses on time in copulation of female.
700 600 500 400 300 200 100 Ave.no.of eggs 0 Cont. 125 Gy
Number of eggs Irradiation dose
Fig (31) Effect of irradiated female on average no.of eggs.
Results 102
70 60 1.26 50 1.24 40 1.22 30 1.2 20 10 1.18 H atch ab ility % sperm atophore 0 Average1.16 no. of Cont. 125 Gy Cont. 125 Gy
% Of egg hatch Number of spermatophore Irradiation dose Irradiation dose
Fig (32) Effect of irradiated female on both of hatchability % &average no. spermatophore.
66.66 70 60 60
50
40
30 16.66 20 16.66 20 % Of m ating 20
10
0 Cont. 125 Gy Cont. 125 Gy Cont. 125 Gy
%un-mated Once Twice Irradiation dose & mating successive
Fig (33) Effect of gamma irradiation on % of successively mated females .
Results 103
4.2.2. Effect of gamma irradiation on successively mated treated females. (T♀ x none and T♀ x 1st N♂ and T♀ x 1st N♂ + 2nd N♂) The data given in Table (10) & Fig. (33) present the effect of irradiated females with 125 Gy on mating successive with virgin males . The results indicate that the percent females successively mated for once or for twice were almost a bout normal ,where the average number of mating times reduced from 1.016 ± 0.07 in control to 1± 0.05 in treated females Table (10); which means that female was treated with this dose has a little effect on mating successive of concerning to female. 4.2.3. Effect of gamma irradiation on treated females entering into copulation during scotophase. Data in Table (11) &Fig (34) show that percentage of treated mated females among three regimes of scotophase period was not clearly affect when compared with untreated mated females. In general the females tend to mate at the first half of the night either these were untreated or treated with 125 Gy .The same trend was noticed on the second virgin male . 4.2.4. Effect of gamma irradiation on some biological aspects of treated insects. The data was presented in Table (12) & Fig. (35) cleared out that pupae treatment with 125 Gy affected slightly on percent emergence at both of treated male and treated female, the reduction was about 3.01and 0.01% from control in male and female respectively .However the data show that percent of malformation increased from 6.85% in untreated control males to reach to 17.25% in irradiating males, also it increased from 19.68 % in untreated females to reach 26.36 % in treated females.
Results 104
Table (10): Effect of irradiation on percentage of successively mated females.
Dose to % Female successively Ave. number % Un mated mated: female of mating female times (Gy) Once Twice
Cont. 16.66 a 66.66 a 16.66 a 1.016 ±0.07 a
125 20.00 a 60..00 a 20.00 a 1 ± 0.05 a
Table (11): Percentage of A. ipsilon female entering into copulation during scotophase (14L: 10 D) for irradiated and non irradiated.
First mating Second mating
Dose to beginning of middle of end of beginning of middle of end of female scotophase% scotophase scotophase scotophase scotophase scotophase % % % (Gy) % %
Cont. 50 a 42 a 4 a 40 a 60 a 0 *
125 91.66 a 8.33 a 0 a 75 a 25 a 0
* Student Newman Keuls Test ERROR Mean Square Error zero or unknown.
Results 105
Results 106
100 91.66 90 80 75 70 60 60 50 50 42 40 40 30 25 20 % Of mated females mated % Of 8.33 0 0 4 10 0 0 125 125 125 125 125 125 Cont. Cont. Cont. Cont. Cont. Cont. Beginning Middle End Beginning Middle End
First mating second mating No.of mating & doses & period of scotophase
Fig (34) Effect of gamma irradiation on % of mated females during scotophase.
100 90 80 70 60 50 40
Percentage 30 20 10 0 125 125 125 125 125 125 Cont. Cont. Cont. Cont. Cont. Cont. Male Female Male Female Male Female
% Of emergence % Adult with deformities % Survival to day (6) Doses & biological aspectes
Fig (35) Effect of sub sterilizing dose of gamma irradiation on % of emergence , % adult with deformities and % survival today (6).
Results 107
Generally the increasing was pronounced in thee females more than the males. On other hand the longevity of adults was not significantly (p > 0.05) affected by irradiating, where it was 11.70 days compared to 11.86 days in control in case of male and it was 10.30 days compared to 10.91 days in control in case female. 4.2.5. Effect of gamma irradiation on total competitiveness of treated males. Table (13) shows that data of total competitiveness of irradiated black cut worm male against untreated adult males for mating with normal female. Results indicate that the average number of eggs per female was not affect at ratio 1IM:0UM:1UF in comparison with 0IM:1UM:1UF, while there was clear reduction in this average at ratio 1IM:1UM:1UF, where the average number of eggs per female was 622.26 in comparison with 881 for control ratio .The results show obviously reduced at ratios 3IM:1UM:1UF and 5IM:1UM:1UF the reduction was about 52.86 and 56.92% from control. The data indicate that the percentage of observed infertile eggs significantly increased as the ratio of the irradiated males was increased in comparison with the untreated control. Increasing the ratio of irradiated males against normal males from 1:1 to 3:1 and 5:1 increased the percentage of observed infertile eggs from 53.77 to 72.7 and 78.52 at the last ratios respectively. Regarding the competitiveness value (C.V.) increased from 1.4 to 1.49 and 1.51 by increasing the ratio of irradiated males from 1 to 3 and 5 at dose level. The above results lead to
Results 108
Results 109 the conclusion that treatment black cut worm males by the low sub sterilizing dose of 125 Gy were fully competitive.
4.2.6. Latent effects on certain biological aspects of F 1and F 2 The data in Table (14) summarize the effect of irradiating male pupae of A. ipsilon with substerilizing dose 125 Gy on the resulting immature stages of the first and second generations. The results indicate that total developmental period (including egg &larval period and pupal period) was clearly increased in comparison with the untreated control in two filial generations, where it was 44.76 days in treated males compared with 41.52 in control treatment Fig. (36). Among F 2 generation, total developmental period also obviously increased, where it was
55.4 and 51.7 days in F 2 progeny descent from F 2M XUF and
F2F XUM respectively. The result show that the percent of pupation at two generations did not differ from the control. However, the percentage of adult eclosion in the treated insects decreased from control especially in F 2 progeny ,where it was 68.66 and 84.98 in F 2 progeny descent from F 2M x UF and F 2F x UM respectively in comparison with 92.8 in the control treatment (Fig,37).
Table(14) & Fig (38) show that F 1 and F 2 growth index was significantly decreased according to the radiation administered to the male parent progeny. While the sex ratio was not affected in the adults of two studied generations, where it was about 1±5.2:1.28 ± 2.3 female: male which was normally obtained in control treatment Fig (39).
Results 110
Results 111
60 55.41 51.7
50 44.76 45.48 41.52 40
30 Days
20
10
0 UM X UF IM X UF UM X UF F2M X UF F2F X UM
F1 generation F2 generation
Total developmental period
Fig (36) Effect of sub sterilizing dose of gamma irradiation
on developmental profile in F 1 &F 2.
100 90 80 70 60 50 40 30 20 10 0 UM IM X UM F2M F2F UM IM X UM F2M F2F X UF X X X X UF X X X % Of puption & emergance UF UF UF UM UF UF UF UM
F1 generation F2 generation F1 generation F2 generation
Pupation% Adulteclosion %
Fig (37) Effect of sub sterilizing dose of gamma irradiation
on pupation% & adult eclosion % in F 1 &F 2.
Results 112
2.5 1.97 2.04 2 1.69 1.65 1.5 1.24
1
0.5 Value of groth index 0 UM X UF IM X UF UM X UF F2M X UF F2F X UM
F1 generation F2 generation
Growth index Generations
Fig (38) Effect of sub sterilizing dose of gamma irradiation on growth index .
1.48 1.6 1.39 1.4 1.28 1.05 1.2 1 1 1 1 0.95 1 1 0.8 0.6 0.4 0.2 0 M ale M ale M ale M ale M ale Female Female Female Female Female UM X UF IM X UF UM X UF F2M X UF F2F X
F1 generation F2 generation Sex ratio Sex ratio
Fig (39) Effect of sub sterilizing dose of gamma irradiation on sex ratio.
Results 113
4.2.7. Reproductive performance and viability of irradiated
males and their F1& F 2 progeny The effect of irradiating parent males with substerilizing dose of 125 Gy of gamma radiation on female fecundity in the parental generation as well as the following two successive generations are given in Table (15). Data show that the radiation did not significantly affect the mating percentage, while the mating frequency was adversely affected practically in F 1and F 2 generations (Figs ,40 & 42). Results exhibit that the fecundity was affected by irradiation dose given to P 1 males. Where, fecundity was significantly less than the control when P 1 males crossed with normal females.
The reduction in fecundity was more pronounced when F 1 males and F 1 females mated with opposite sex (Fig, 41). The greatest reduction in number of eggs per female was
349.25 and 325 in F 1 male and F 1female respectively. Also, fecundity in F 2 decreased significantly in comparison with the untreated control. Concerning to the egg hatchability was significantly reduced to 44.84% when P 1 irradiated males were compared with 75.78% in the control treatment (Fig, 40). The reduction was significant at all tested mating combinations among F 1 in comparison with the untreated control, while, there were no significant differences between the two tested mating combinations. The reduction in egg hatch was more pronounced among
F1 generation than their irradiated male parents where it reduced to 16.74 % and 19.97% at the mating combinations A F 1M x UF and, B F 1F x UF respectively in comparison with 71.74% in the
Results 114
Results 115
100 90 80 70 60 50 40
percentage 30 20 10 0 IM X IM X F2AF F2AF UM X UM X UM X UM X BF1 F BF1 F F2BM F2BM F2AM AF1M F2AM AF1M F2BF X F2BF X UMxUF UMxUF P1 F1 F2 generation P1 F1 F2 generation generation generationgeneration generation
Mating success Fertility(%)
Fig (40) Effect of sub sterilizing dose of gamma irradiation on mating success and % fertility .
800 700 600 500 400 300
No .of eggs 200 100 0 UF UF UF IMX XUM F2AF XUF XUF XUF UM XUM UMX UMX BF1F AF1M F2AM F2BM F2BFX UMxUF P1 generation F1 generation F2 generation
No.of egg/female Mating combination
Fig (41) Effect of sub sterilizing dose of gamma irradiation on no. of eggs.
Results 116
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 U F U F U F IM X X U M X U F U M X U M X X U F X U F U M F 2 A F X U M F 2 A M F 2 B M A F 1 M B F 1 F F 2B F X U M x U F P1 generation F1 generation F2 generation Mating frequency no.
Fig (42) Effect of sub sterilizing dose of gamma irradiation
on no. of mating frequency P, F 1 and F 2 generations.
Results 117 control treatment. The same trend was also observed in the hatchability at all tested mating combinations among F 2 generation. Table (15) also shows radiation –induced suppression effects were examined in terms of corrected sterility and control of reproduction, especially at the AF 1M X UF cross which gave 46.36 of the total eggs laid by control (Table, 15) and resulted in 16.74% egg hatch as compared with 71.74% hatch in control. Therefore this cross exhibited 76.13 % sterility and 88.56% control of reproduction.
4.2.8. Progeny produced from irradiated P1 male pupae throughout two successive. The deleterious effect of irradiating full grown male pupae with 125 Gy on reproduction of F 1 progeny resulting from irradiated parental of A. ipsilon and their latent effect on F 2 progeny was presented in Table 16. A significant reduction was noticed in the fecundity, fertility and number of survived larvae to adult (Table, 16). The values reported in the last column of Table (16). (Production of female adults) were generated by multiplying the value of fecundity, egg hatch, survived larvae and pupae and female progeny. The production of F1 female adults resulting from irradiated P 1 males as full grown pupae and crossed with P 1 normal females was sharply decreased to 39.69% from untreated control at dose level of 125 Gy. The production of adult females in F 2 was greatly reduced due to radiation effect i.e. it reduced to
7.46% and 11.71% at AF 1M X UF and BF 1 F X UM respectively.
Results 118
Results 119
4.2.9. Effect of gamma irradiation on gonads measurements
of P1and F1males. Because the testes were not completely spherical two dimensions were measured and their average was used as diameter (Table, 17 & Fig, 43)show that gamma irradiation significantly affected the testes in P 1volume,where it decreased to 1.11mm 3 in comparison with 2.07mm 3 in un irradiated control
.While the testes not clearly affected in F 1about its control ,however the volume of these testes 1.59 mm 3 differed significantly from its irradiated parents 1.11 mm 3. 4.2.10. Effect of gamma irradiation on sperm production of
P1and F 1males Generally speaking, in the treatment as well as the control, eupyrene sperm bundles were not found in the duplex region of the newly emerged males dissected immediately after emergence in several species of Lepidoptera, the release of sperm from testes has been shown to be controlled by circadian rhythms with the clock and photoreceptor located in the testis vas deferens complex ( Giebultowicz et al . 1989). Table (18) &Fig (44) present the average number of eupyrene sperm bundles found in the duplex of P 1and F 1 male one or four –day old.Statistical analysis showed that sperm production not affected during the first dark light cycle of sperm descendence. On the 4th day of emergence, the average number of eupyrene sperm bundles accumulated in the duplex of unmated F 1male significantly decreased from 613.71in the control to 488.58 bundles in F 1male descendent from irradiated parents.
Results 120
Results 121
Results 122
2.5 2.07 1.75 2 1.59 1.59 1.5 1.44 1.53 1.46 1.42 1.26 1.3 1.5 1.11 1 0.5 0 cont. cont. cont. cont. cont. cont. 125Gy 125Gy 125Gy 125Gy 125Gy 125Gy P1 F1 generation P1 F1 generation P1 F1 generation generation generation generation
Diameter A Diameter B Volume of testes mm
Fig (43) Effect of sub sterilizing dose of gamma irradiation
on volume of testes P and F 1 generations .
700 600 500 400 300 200 100 0 cont. cont. cont. cont. Ave.of eupyrene sperm bundels 125Gy 125Gy 125Gy 125Gy P1 generation F1 generation P1 generation F1 generation
One-day-old Four-day-old Day of counting
Fig (44)Effect of sub sterilizing dose of gamma irradiation on accumulation of eupyrene sperm bundles in the duplex.
Results 123
4.2.11.Feasibility study on cytological sperm bundle
assessment of F 1 progeny of irradiated male. In the attempt to count sperm bundles in the live moth samples, when seminal vesicles or duplex tissues were ruptured during dissection, milky coloured seminal fluid instantly oozed out along with the aggregated but easily separated strands of silvery, shiny and curly sperm bundles. Staining results showed that the nucleic cluster of the eupyrene sperm bundles stained by Giemsa. In the seminal vesicles, consistent and obvious differences were observed in individual sperm nuclei of eupyrene sperm bundles between the control (fertile) and
F1(sterile) males. In control males we examined, the sperm nuclei in the eupyrene sperm bundles are usually clustered and arranged in an orderly manner with the anterior ends of individual sperm usually equidistant from the anterior end of the sperm bundle Fig (45). The nuclei cluster of the eupyrene sperm bundles of normal males was stained homogeneously, indicating that equal amounts of chromatin material were present in each sperm. In contrast, individual sperm nuclei in eupyrene sperm bundles from F 1 sterile moths were irregularly arranged and irregularly stained patterns were consistently observed, often with only a few or a low fraction of sperm nuclei in the bundle being stained (Fig, 46). This heterogeneously stained nuclei cluster revealed that the amount of chromatin material in each sperm nucleus of F 1sterile males was variable and disorganized compared with control. Variations in the nuclear staining patterns were often great because some sperm within a bundle had no visible evidence of chromatin material while other sperm within the same bundle exhibited abnormally high levels of chromatin material.
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a A
b B
c C Fig (45) sperm bundles from testis smears of normal males, a& b&c (40X) and A&B&C (100X). In all, views nuclei clusters of the eupyrene sperm bundles were homogenously stained
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a A
b B
d D
Fig (46) sperm bundles from testis smears of the sterile F 1 males, a& b&d (40X) and A&B&D (100X). In all, views nuclei clusters of the eupyrene sperm bundles were heterogeneously stained.
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4.3. Effect of Entomopathogenic Nematodes (EPNs) on Agrotis ipsilon . EPNs is one of the most important method that influence the insect management .In this part of study the effect of two aforementioned EPNs species on some biological activities of A. ipsilon were studied. 4.3.1. Effect of (EPNs) Steinernema carpocapsae on Agrotis ipsilon . Data in Table (19) show the biological effects of (EPNs ) S. carpocapsae on the 4th instar larvae of A. ipsilon . Results revealed that the highest number of dead larvae (100) resulted from the larvae treated with 200 IJ/larvae concentrations of S. carpocapsae and the lowest record was 40 dead larvae in case of treatment with 10 IJ/larvae concentrations of S. carpocapsae . Data given in Table 19 &Fig 47 indicate that the percentage of total mortality until adult stage was increased with the increase of concentration comparing with the untreated control. It was increased at 200 IJ/larvae concentration to 100 % in , but the lowest mortality percentage (49%) resulted with the application of 10 IJs /larvae concentration. The results in the same table indicate that all the treatments with S. carpocapsae produced percentages of pupation lower than control (Fig, 48). The data given in Table 19 & Fig 49 revealed that the increase in adult emergence related to the decrease of concentrations, the same trend was observed in percent of final surviving individual (Fig, 50). The results also indicate that the sex ratio was fluctuated, sometime it was shifted in favor of female (as observed in case of 25 IJs /larvae). This ratio was 0.82 ±0.37 :
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1±0.58 male : female . While the sex ratio was shifted in favor of male as observed in case of 10 IJs /larvae concentration where it was1.21 ±0.68: 1± 0.40 male : female. Generally the sex ratio was significantly differ about 0.80 ±0.58: 1±0.58 as was obtained in control treatment. 4.3.2Effect of (EPN) Steinernema riobrave on Agrotis ipsilon . Data in Table (20) revealed the biological effects of (EPN) S. riobrave on 4th larval instar of A. ipsilon. Results showed that all the concentrations of S. riobrave have effect on larvae of A. ipsilon compared with the untreated control. The highest total mortality percentage (100%) was obtained with 200 IJs /larvae concentration (Fig, 47).All concentrations of, while the lowest percentage of total mortality was recorded with 10 IJs / larvae concentration which gave 37%. The data in the same table showed that pupation percentages were obviously affected all tested concentrations in comparison with untreated control. By comparing means of pupation percentages of all concentrations (Table, 20 &Fig, 48) it may be concluded that 100 IJs /larvae produced the lowest mean of pupation 8 % but in the case of 10 IJs / larvae it was 60 % comparing with 95 % in control treatment. In regard to the percentage of emergence as shown in Table (20) & Fig (49) the lowest percentage of adult emergence resulted from the treatment of 100 IJs /larvae (25 %).While the highest percentage resulted with the application with 10 IJs / larvae concentration (85%). As shown in Table (20) & Fig (50) treatment with S. riobrave at all concentrations gave higher
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Results 130
St. 120 carpocapsae St.riobrivae 100
80
60 40
% Total mortality 20
0 cont. 200I J 100I J 50I J 25I J 10I J cont 200I J 100I J 50I J 25I J 10I J
Nematodes species & concentration
Fig. (47) Effect EPNs on % of total mortality of A. ipsilon
100 90 St.carpocapsa e 80 St. riobrivae 70 60 50 40 30
pupation Of ) % 20 10 0 cont. 200I 100I 50I J 25I J 10I J cont 200I 100I 50I J 25I J 10I J J J J J
Nematodes species & concentration
Fig. (48) Effect of EPNs on % of pupation of A. ipsilon
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120 100 80 60 40 20
Adult emergance % 0 50IJ 25IJ 10IJ 50IJ 25IJ 10IJ cont cont. 200IJ 100IJ 200IJ 100IJ St.carpocapsa St. riobrivae EPNs, species & concentrations
Fig. (49) Effect of EPNs on % of adult emergence of A. ipsilon .
100 92 92 90 80 70 63 60 51 48 50 44 40 23 % Of survival % 30 20 20 10 10 0 0 0 0 cont. 200IJ 100IJ 50IJ 25IJ 10IJ cont. 200IJ 100IJ 50IJ 25IJ 10IJ St.carpocapsa St. riobrivae Nematodes species & concentration
Fig. (50) Effect of EPNs on % of survival of A. ipsilon .
Results 132 reduction in percentages of individual surviving more compared with the control treatment. The variation between means of sex ratio at all treatments was very clear in comparison with untreated control. 4.3.3 Virulence of (EPNs) on Agrotis ipsilon . From Table (21) mortality in 4th instar larvae of A. ipsilon due to S. Carpocapsae . Was between 8 and 60 % using concentrations between 10 and 200 IJs / larvae, within 48 hr. 10 days post treatment mortality increased between 36.84 and 100 % using the same concentrations.
Values of LC 50 and LC 90 as shown in Table (21) & Fig
(51) were 11.37 and 47.39 IJs / larvae, respectively. And LT 50 as shown in Fig (52) were 3.62 day due to concentration 200 IJs / larvae . R 2 Values showed strong correlation between mortality values Vs concentrations more than Vs time. Table (21) comparing susceptibility of 4th instar larvae of A. ipsilon to infection with S. riobravae . Mortality values ranged 1 32 % in first two days and 17.9 87.37 % after ten days post treatment at the concentrations 10 and 200 IJs / larvae. LC 50 and LC 90 values were 30.44 and 487.9 respectively, and LT 50 was 10.44 days.
Values of LC 50 and LT 50 against larvae of A. ipsilon indicated that S. carpocapsae looked more virulent and faster in effect than S. riobravae .
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Results 134
Fig (51) Virulence of (EPNs) on A. ipsilon . (LC 50)
Fig (52) Virulence of (EPNs)on A. ipsilon ( LT 50)
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4.3.4. Effect of exposure period with Steinernema carpocapsae All on Agrotis ipsilon . Table (22) &Fig (53) shows the effect of several of exposure period (8, 10 and 12hr) with (EPN) S. carpocapsae at concentration 100 IJs/ larvae. The percentage of total mortality was increased by increasing the time of exposed in comparison with the control, where it was 58, 66 and 66 %, at the periods levels of 8hr,10hr and 12hr respectively, compared with 12 % in control treatment. Larval and pupal durations were slightly affected among all tested in comparison with the control treatment. The data in Table (22) &Fig (53) indicated that the percentage of pupation in P 1 generation was obviously reduced. The reduction was more pronounced among levels of 10hr and 12hr. Where it was 56and 42% compared with 94% in the control. The data indicated that the percentage of adult emergence of P 1 generation was sharply reduced among 8hr, 10hr and 12hr . The reduction was 63.33, 60.71 and 80.95% at the respective periods in comparison with the control (92.55%). Also, the sex ratio of P 1 generation was affected by the treatments. The sex ratio was altered in favour of male in a dose dependent manner 1.37: 1 to 1.61: 1 male/female at the treatments in comparison with control where it was 1.04:1 male /female. The percentage of malformed adults of P 1 generation was increased by increasing time of exposed to Steinernema carpocapsae at concentration 100 IJs/larvae where it was 13.16, 14.70 and 8.82 % at the respective levels in comparison with 2.29 % in the control (Fig, 53).
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4.3.5. Delayed effect of exposure with (EPNs) Steinernema carpocapsae All on Agrotis ipsilon .
The delayed effect of treating fourth instar larvae of P 1 male A. ipsilon with various periods levels of 8hr,10hr and 12hr on reproduction and mating ability of P 1 was shown in Table (23) & Fig (54).The results show that the mating ability was 100% when moths developed from untreated immature stage. But it reduced to 66.67 % if the larvae had been treated by 100 IJs/larvae concentration of S. carpocapsae , number of spermatopheres /adult was not almost affected at the period's level (Fig, 57). As shown in Table (23) &Fig (55) reduction in average no. of eggs/mated female was more obvious, the average number of laying eggs was 524.7, 489.5 and 519.5 egg at 8, 10 and 12hr, respectively compared with 847.92 egg in the untreated control. Concerning fertility the results represented in Table (23) showed that adult fertility was also adversely affected as a result of treatments (Fig, 56).The data presented in Table (24) clarified the effects on the fecundity, the percent egg hatch and the mating when females' moths resulting from 4th larval instar treatment at different periods levels with both with 100 IJs/larvae of S. carpocapsae . The resulting females were mated with untreated males, data of the previous mating show that the fecundity in all treatment is significantly different when it compared with the untreated control groups. But the results indicate that no clear reduction occurred in mating frequency and sperm transfer at all
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Results 139
0.75 cont. 0.75 8Hr 0.75 10Hr 0.75 !2Hr
100 90 80 70 60 50 40 30
The percentage 20 10 0 %Total mortality % Pupatin %Adult emergence %Adult malformation Different biological aspects
Fig. (53) Biological effects of S. carpocapsae 100 IJs treatment on 4th instar larvae of A. ipsilon at a various exposure periods.
cont. 8Hr 10Hr !2Hr 120
cont. cont. cont. cont. 100 !2Hr 8Hr 10Hr 8Hr 10Hr 8Hr 10Hr !2Hr 80 8Hr !2Hr 10Hr !2Hr 60
40
20 Matingaspects% 0 %Mating ability %Mated female %Mating ability %Mated female resived sperm resived sperm Treated male X Normal female Treated female X Normal male Mating combination & mating aspecrs Fig. (54) Delayed Effect of treatment with S. carpocapsae 100 IJs at various expoure period on reproduction .
Results 140
cont. 8Hr 10Hr !2Hr
900 800 700 600 500
400 300 No. of eggs 200 100 0 Fecundity Fecundity Treated male X Normal female Treated female X Normal male Mating combination & exposure period
Fig.(55) Delayed effect of treatment with S. carpocapsae 100 IJs at various exposure periods on fecundity.
cont. 8Hr 10Hr !2Hr
80 72.47 72.47 70 60 46.68 50 44.18 44.35 42.63 38.74 37.24 40 30
Eggviability % 20 10 0 Mating combination & expousre period
Fig. (56) Delayed effect of treatment with S. carpocapsae 100 IJs at various exposure periods on egg viability.
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cont. 8Hr 10Hr !2Hr 1.6 1.48 1.48 1.3 1.38 1.33 1.36 1.4 1.2 1.2 1.2 1 0.8 0.6 0.4 sperm atophore A verage N0.2 o. of 0 Ave.No.of Ave.No.of spermatophore spermatophore
Treated male X Normal female Treated female X Normal male
Mating combination & expousre period
Fig.(57) Delayed effect of treatment with S. carpocapsae 100 IJs at various exposure periods on no. of spermatophore
Results 142
treatment (Figs, 54& 57). Fertility of deposited eggs was also affected by S. carpocapsae treatments the data in Table (24)& Fig (56) show that the longest time 12hr was the most effective in the reduction of hatchability producing only 37.24 % ,in general, fertility was highly reduced in adult emerging from treated immature stages in comparison with untreated ones. The deleterious effect of treating fourth instar larvae of A. ipsilon with 100 IJs/larvae concentration of S. carpocapsae ,on the reproduction of P 1 adults was shown in Table (25) .When parental females treated during fourth instar larvae at one of different exposure periods and crossed with normal males, significant reductions in fecundity, fertility and survival of larvae and pupae were recorded (Table, 25 ).The production of female adults resulting from treated P 1 females during the fourth instar larvae and crossed with normal females was drastically decline to 13.22, 12.56 and 7.92% (% of control treatment) at the treatments for 8,10 and 12hr respectively. 4.3.6.Effect of application method with (EPNs) Steinernema carpocapsae on Agrotis ipsilon . This experiment was carried out to the efficiency of methods of application, comparing between two methods and one concentration of nematodes suspension 100 IJs/pot/larvae were tried in controlling A. ipsilon . Data in Table (26) & Fig (58) showed that aqueous suspensions of S. carpocapsa e nematodes were as effective as applications of cadavers infected with S. carpocapsae for controlling A. ipsilon the percentage of cumulative mortality were 98, 92 and 100% in aqueous
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Results 146
control Aqueos tr. 5daycada. 10day cad.
120 98 100 97.82 100 100 92 92 91.3
80
60
40 The percentage 20 8 8 2 0 0 0 Cumulativ mortality % Final individual surviving% Reduction in progeny % Biological aspects
Fig. (58) Effect of application methods with S. carpocapsae on 4th larval instar of A. ipsilon .
Results 147 suspension, 5 day BCW cadavers and 10 day BCW cadavers respectively compared with 8% in untreated control. Percents of reduction in progeny from control were considerably higher in nematode treated pots than in non treated, that due to highly reduced in percentages of final individual surviving in all treated pots about no treated pots as shown in Table (26) & Fig (58). 4.4.Combined Effect Gamma Irradiation and (EPNs), Steinernema carpocapsae All on Agrotis ipsilon .
In this part of study, the 4 instar larvae of F1 progeny, resulted from irradiated full grown pupae were treated with 10, 25, 50,100 and 200 IJs/larvae concentrations to compare the responses of F1 progeny of partially sterile male parents to S. carpocapsae with those of F1 progeny of nonirradiated male parents.Results concerning the effect of radiation of 125Gy and S. carpocapsae concentrations separately or together are tabulated in Tables (27 and 28) & Fig (59)data indicated that the percentages mortality of F1 progeny of nonirradiated and irradiated parents were significantly increased when compared with the control at all concentrations , also the percentages mortality was significantly increased by increasing S. carpocapsae concentrations.
The mortality of F1 progeny of both parents was not significantly different, except at 10 and 25 IJs/ larvae concentrations. Table (28) present the LC 50 (concentration that killed 50 % of F1 progeny) and slope of concentration mortality regression. LC 50 of F1 progeny of irradiated parents was clearly lower than that of nonirradiated parents with a resistance factor of 2.08.
Results 148
Results 149
Results 150
Fig (59) Combined effect of gamma irradiation and
(EPNs) on A. ipsilon . LC 50
Results 151
4.4.1. Potency of interaction between substerlizing dose 125Gy and EPNs, Steinernema carpocapsae The data concluded in Table (29) clarified the combined effect in F 1 progeny resulting from parental males and treated with S. carpocapsae concentrations. The results show the combined effect of it radiation and S. carpocapsae was higher than when using each of them alone this may be attributed to latent effect of irradiation which can be accumulated in the larval and pupal stages which increasing the mortality rate . Data show that the percentage of expected mortality increased by increasing the concentration with dose level .Where, it was 55.27, 76.87, 83.83 89.4 and 90.8 at concentrations 10, 25, 50, 100 and 200 IJs/ larvae with 125 Gy respectively. An additive response was observed in the combinations. There was no synergistic or antagonistic response in any combinations. 4.4.2. Interaction of Steinernema carpocapsae All cultured in
irradiated hosts, with F 1 sterility The bioefficacy of normal EPNs (i.e., cultured in un irradiated insect host) was ascertained against F1 sterile A .ipsilon larvae produced in matings of untreated females with substerilized male moths, irradiated 125 Gy, in order to understand the degree of acceptability and suitability of F1 progeny as potential hosts to EPNs. And the parasitising performance and harvest potential of IJs (indicating reproductive potential) of the EPNs cultured in parasitised F1 sterile insects was ascertained against normal and against F1 sterile A .ipsilon hosts, the results were conclusion in Table (30).
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Data clear that ANOVA performed on data pertaining to bioefficacy of EPNs on F1sterile insects indicated that there was no significant influence of induced sterility of the host (F1 sterile insects) on both of the parasitisation efficacy of EPNs, and on the mortality induction process. The onset of morbidity and mortality induced by normal IJs (i.e., IJs derived from untreated host) was not different in
F1sterile hosts and un irradiated controls (Fig, 60) (i.e., untreated host) (P>0.05). The incubation time taken by IJs on F1 sterile hosts was significantly prolonged, in F1hosts derived from 125 Gy treated male parent (P< 0.05) (Fig, 61). Parasitisation response was reduced in F1 sterile hosts, with a significant impact on F1larvae derived from fathers that had been treated with 125 Gy (P<0.05). The IJs’ harvest was found significantly reduced on F1 hosts, the number of IJs harvested from F1 sterile host was reduced by about 32 – 35 % at 125 Gy with respect to controls. The harvest period was also slightly affected by the radiation dose applied to the host in the previous generation (Fig, 61). Regarding to the infective performance of IJs cultured in parasitized F1 sterile A .ipsilon larvae was ascertained on normal
(unirradiated) hosts and on F1 sterile host progeny of irradiated male parents, so as to understand the persistence of infective viability of IJs harvested from F1 sterile hosts (Table, 30 & Fig, 62). It is worth noting that no evident interaction was noticed between the irradiation background of IJs parent host, i.e., F1 sterile larvae (in which the IJs used had been cultured) and the nature (quality) of the current host (i.e., F1 sterile host) on parasitisation behavior of EPNs.
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Results 154
Results 155
40 35 30 25 20 15 10 5 0 Time hour Time F1 Ag F1 Ag F1 lAg F1 lAg F1 lAg F1 lAg NormalAg NormalAg NormalAg NormalAg NormalAg normal St F1 St normal St F1 St Morbidity time Mortality time
Fig (60) Time morbidity & time mortality of infested larvae with both type of EPNs.
18 16 14 12 10 8 6 Time Time /day 4 2 0 F1 Ag Ag F1 F1 lAg F1 lAg F1 lAg F1 NormalAg NormalAg NormalAg NormalAg NormalAg normal St F1 St normal St F1 St
Incubatio time /day Harvest period /day
Fig (61) Incubation time & harvest period of infested larvae with both type of EPNs.
Results 156
400 IJs /mg 345.87 body 350 weight normal St 300 NormalAg 250 233.19 IJs /mg body 188.86 200 weight 138.1 normal St 150 F1 lAg IJs /mg
ave.number100 of IJs body 50 weight F1 St NormalAg 0 NormalAg F1 lAg NormalAg F1 lAg IJs /mg body normal St F1 St weight F1 St F1 lAg IJs /mg body weight
Fig (62) Harvest (yield) of IJs of infested larvae with both type of EPNs.
Results 157
Incubation time taken by IJs that had been cultured
F1sterile hosts was significantly affected on F1sterile host larvae with respect to the control, whereas it was 8.5 and 9.75 days in normal hosts and F1 sterile hosts respectively, compared to 7 days in the untreated control (Table, 30 &Fig, 61). It means the incubation time taken by IJs from radio sterilized hosts was prolonged on normal as well as F1 hosts. The reduction in harvest potential of IJs depended upon the gamma dose administered to male parent of F1 insects (as hosts). The IJs’ harvest was reduced by 45 38 % when IJs that had been cultured in F1 sterile hosts parasitized normal hosts, with respect to controls. Table (30) & Fig (62) Further, a harvest reduction of 60 46% was recorded in case of infection by IJs that had been cultured in F1 sterile hosts parasitized F1sterile hosts. The harvest period of IJs in normal hosts and F 1sterile hosts occurred at a similar time, where it was 13.75 days
Results 158
DISCUSSION AND CONCLUSION
For the sterile insect technique (SIT, ionizing radiation is the method of choice for inducing reproductive sterility. The sterilization process is important in determining the quality of the released insects and their ability to compete with the wild population. Thus, optimization of the sterilization process is critical for the efficacy of SIT programs and should be given due consideration Kinpling (1992)) suggested that the integration of SIT or inherited insect technique IST with natural enemies may lead to a major reduction in the insect population. The effectiveness of IST as a genetic control method could be increased in combination with the application of biological control agent EPNs. Present study includes two biological control methods to management Agrotis ipsilon , in the following text unless otherwise stated the discussion points include both methods under present investigation. 1. Induction of an effective sterility and reproductive:- Copulation duration and frequency varies among Lepidoptera. Spermatozoa, or mature sperm, are present at eclosion in adult males that are short lived, suggesting that spermatogenesis occurs in the pupal stage (Gillott 1995). Spermatophores containing sperm are formed directly in the female bursa copulatrix during copulation when the aedeagus contacts the opening of the female bursa copulatrix. Spermatophores, which are composed of amino acids and
Discussion And Conclusion 159 proteins, often contain enough sperm to fertilize numerous eggs. They are produced from male accessory gland and simplex (ejaculatory duct in Lepidoptera) secretions, filled with sperm from the duplex (seminal vesicle), and deposited into the bursa copulatrix of the female at the time of mating. The duration of mating is determined by the time required for spermatophore formation. Osanai et al. (1987) reported that the spermatophore of Bombyx mori was formed entirely within the female bursa copulatrix during copulation. Seth et al. (2002a) observed spermatophore formation and deposition of sperm and secretions in S. litura over an average 75 min mating period. Sperm are released from the spermatophore within the female as a result of abrasive movements of spines within the bursa copulatrix. After being released from the spermatophore and released into the female reproductive tract, spermatozoa are transported to the spermatheca for storage, although the manner of transport is not known Pritchard (2004). Irradiated males tended to copulate for longer times than did untreated controls in gamma irradiated Trichoplusia ni (Holt & North 1970). In this study the irradiated males not only spent as mush offer in attempting to mate with untreated females, but their mating duration was also not significantly different to that of the untreated males. However, the significant differences in spermatophore formation occurred and percentage of females with sperm in spermatheca was observed at highly doses. This suggests that the mating aspects of irradiated males were not affect at lower treatments. In general sterilizing doses not alter the mating ability fitness (Hennebery 1993; Suckling 2004).
Discussion And Conclusion 160
The results indicate that lower percentages of copulated males with untreated virgin females in the second and third mating than the first mating. Number of deposited eggs /female mated with irradiated males and egg hatch were significantly reduced as a result of irradiation male treatment through three females. Also Number of eggs /female deposited by females mated to irradiated males and egg hatch were significantly less in second female to 23.5 & 25.37 % and third female to 37.36 & 57.98 % compared to the first female. In another related study by (Hennebery 1993) female eggs not significantly different to that of control. The mating aspects under the study (mating ability, duration of copulation, females with spermatophore and un separation pairs) and reproduction (eggs per mated female and egg hatch) significantly different from first mated female to second and third mated female. The consequences of multiple male matings reported in the literature include a decrease in spermatophore size as mating frequency increases, and a loss in the ability to change a female’s mating behavior to “mated” status. Foster & Ayers (1995) reported on three different substances transferred from males during mating that were responsible for changes in female mating behavior in lightbrown apple moth, Epiphyas postvittana (Walker). Multiple male matings have been shown to decrease spermatophore size in European corn borer (Ostrinia nubilalis ) (Royer & McNeil 1993), Plodia interpunctella (Hubner) (Cook 1999), and turnip moth (Agrotis segetum (Schiff .) (Svensson et al .1998). The transfer of eupyrene sperm has been reported to decrease in tobacco budworm H. virescens (F) (Henneberry &
Discussion And Conclusion 161
Clayton 1984 ) and the monarch butterfly (Oberhauser 1989) as the number of male matings increased. Seth et al . (2002b) reported that the number of sperm bundles descending into the duplex in S. litura was not affected by previous matings, and concluded that the negative effects of multiple matings could be due to a delayed recovery of secretions from the simplex (ejaculatory duct), accessory gland, and vas deferens associated with spermatophore formation. On the other hand the results showed that not significant difference between tested doses was observed. It means that there were significant interactions within sequential mating females, where's, no significant interactions among doses was noticed. The majority of A. ipsilon mated several times in laboratory (present results as well as previous reports, Elnagar et al. 1984 and Ibrahim, 2004 ) present study shows some degree of reduced mating of the targeted males inevitable in return for associated irradiation doses whereas the highest average times of matings was 2.1 in control, however it remains clear whether the increased number of mating time from lowered irradiation doses will prove to enough as a trade off to untreated control. The dark period was divided into three equal parts each of 3 hr, these parts covered the beginning, middle and the end of the scotophase .Our data indicated that 38.89, 42.00, 18.96, 60.41, 46.94 and 36.73 of irradiated males with tested doses 75, 100, 125, 150, 175 and 200 Gy respectively and 48.93 of normal males entered into copula during the first part of the dark period. Only a small percentage of matings occurred in the middle part where 0.00, 16.00, 12.06, 6.25, 24.49 and 24.49 at
Discussion And Conclusion 162 aforementioned doses and 13.7 of normal males started mating during the last part of the dark period, differences recorded within one part dark period were not significant except in the middle period this observation was agree with that Toth (1985) who reported that the onset of strong calling of Phthorimaea operculella female occurred 5 hr after beginning of the scotophase. Also Saour & Makee (1999) shared with him and added that the female of Ph. operculella could start calling at any hour of the dark period. The same trend of females tends to start mating at the first half of the night was observed in the second mated female and the third. An important concern in sterile insect release programs is that treated males destined for release retain the ability to perceive/orient to pheromone signals from females and, as such, are able to compete with the wild males in locating and mating with calling females in the field. The results of present study clear that the mating competitiveness calculated from the direct observation in A. ipsilon revealed that males emerged from pupae irradiated at 75 to 125 Gy are equally capable of mating as control. The competitiveness values calculated in the present study at 75 Gy was 1.30 and that at 125 Gy was 1.08, results suggest that lower radiation doses may improve the competitiveness of sterile released insects. Katiyar & Ramirez (1969) made similar observations and reported that gamma irradiation at 6 10 krad applied to mature pupae had little or no effect on the mating ability of irradiated male Ceratitis capitata which agrees with the present observation. Ocampo (2001 ) suggested that partially sterilized males Helicoverpa armigera
Discussion And Conclusion 163 were as competitive as untreated males in seeking and securing mates, males treated with 100 Gy. Nahar et al (2006 ) reported that the relationship between radiation dose and mating competitiveness evaluated by the method of direct observation indicated that 10, 20 and 30 Gy radiation treated males of Bactrocera cucurbitae were almost as successful as untreated males. On the other hand 40 Gy and 50 Gy treated males were 2 3 times less competitive than the untreated insects, which agree with our data as shown in Table (8) where competitive value decreased with the dose increased. 2. Effect of dose (125 Gy) used to irradiate male & female full grown pupae on biological aspects of
P1and F 1generation. As the aim of the present work is to choose a radiation dose for both males and females capable to inherit the sterility sufficiently through two successive generations, the immature stages descendant of irradiated parents should be evaluated. The assessment should include effect the choice dose on the, female larval and pupal mortality and adult emergence in P1&two success generations, developmental synchrony and reproduction in P 1& two success generations. In many insects, females often determine the acceptance or rejection of males. This concept is generally accepted because female investment in the production of gametes is greater than that of males. Female mate choice, however, is generally not well understood. Greenfield (1981) suggested that low release rates of pheromone by females represent a type of female sexual selection because females are selecting for males that are strong flyers over a great distance.
Discussion And Conclusion 164
Radiation exposure of 125 Gy had no effect on percentages of treated females mating or times in copulation with untreated males (Table 9). However, percentages of females mating over all radiation doses decreased from an average of 81.66% with the first male to 22.44 and 0.00%, respectively with new virgin males provided during the second and third mating period. Similar results were obtained by ( Hennebery 1993) also time in copula significantly different at over all radiation doses within new virgin males provided. The results revealed that fecundity of the black cutworm, (BCW) A. ipsilon female was significantly affect when full grown female pupae were exposed 125 Gy and the percentage of egg hatch was significantly reduced to 0.00% compared to 60.03% in control. The average numbers of sequential male matings were 1.016 ± 0.07and 1 ± 0..05 times with untreated females or females exposed to 125 Gy respectively. For all females and treatment 16.66 to 20 % of the females did not attempt to copulate, 60.00 % were observed in copulation once and 20% twice, Table (10). Successful insemination results in changes in female behaviors, production and release of pheromone cease and females are no longer receptive to copulation and female receptivity varies in Lepidoptera specie Pritchard (2004). Of the 60 females observed in copulation 50 and 91.66 % for normal and treated females mated in the first part of dark period while, 4 and 0 % of the females observed in copulation for normal and treated females mated in the first part of dark period (Table 11). It means that the treated females with 125Gy
Discussion And Conclusion 165 had no effect on calling behavior and attracting to wild males. The same trend of females whereas they tend to start mating at the first half of the night was observed in second mating with the second male. The results of this study on the effect of 125 Gy on percent emergence, percentage of adults with deformities and longevity of BCW in(Table12) show that 82.75% of the male pupae developed as normal adult and 70.02% of these survived to the 6th day while corresponding percentages for treated female were 56.39 % 71.98. Adult longevity slightly reduced by irradiating. In general, BCW female slightly affected by irradiating more than male, this result coincided with that obtained by Nguyen Thi & Nguyen Thanh(2001) on Plutella xylostela and Hazaa, (2002) on Spodoptera littoralis . A major problem in the use of irradiated male moths to suppress natural populations has been considered to be the lack of competitiveness, due primarily to the in ability of irradiated males to transfer sperm successfully (North & Holt 1971). In order to retain vigour in Lepidoptera, researchers suggested the use of substerilizing doses of radiation to produce sexually competitive moths. Furthermore, by this method the F 1 offspring of irradiated Lepidoptera are after partially or completely sterile. In an earlier work by sallam (1969 ) reported that full grown male pupae of the cotton leaf worm S. littoralis were found to require a dose of 45 50 Krad gamma rays to produce completely sterilized adult males. The treated males were less competitive than the untreated males for mating with untreated females at different ratios, up to 10 treated males: 1 untreated male. The
Discussion And Conclusion 166 results of the present work on mating competitiveness of the black cutworm lead to the conclusion that parental males irradiated with low doses of 125 Gy were fully competitive against untreated males in mating with normal females. Increasing the ratio of treated males to the competing population from 1:1 to 5:1 increased the percentage of nonviable eggs at the three tested ratios. The same trend of effect was observed by El-Shall et al .(1997 ) on Mythimna lareyi , ; El- Naggar et al . (2000) on A. ipsilon and Alm El-Din (2001) on S. littoralis who, studied the effect of gamma radiation on the mating competitiveness of the adults produced from irradiated parental male pupae. The data indicated that the reduction in egg hatch was significantly greater at the ratio of 5:1:1 than at 1:1:1 (treated male: normal male: normal female).
The developmental rate of F1 larvae originating from the crosses between treated males and normal females of A. ipsilon was slower than that of controls, and this delay in development was greater at F 1MXUF combination. Because insect development and differentiation are controlled by hormones
Gilbert (1964), the protracted development of F1 larvae might be due to alteration in hormonal or enzymatic production caused by chromosomal rearrangements, as indicated by Proshold & Bartell (1970, 1972). While the percentage pupation was not affected among in
F1 and F 2 progeny, the results indicate that the percentage of adult emergence was slightly reduced among the first filial generation descendent of parental males irradiated with 125 Gy when compared with control; however, there was clear reduction
Discussion And Conclusion 167 among F 2 progeny descendent of mating combination F 1MxUF. These results agree with those obtained by Ahmed et al. (1985) on Ephestia kueniella and Hazaa (2002) on S. littoralis. These authors studied the effect of gamma radiation on the percentage of pupation and adult emergence which was reduced throughout the F 1 generation descendant of irradiated P 1 males compared to the control. The growth index showed a decrease as a consequence of irradiation of male parents in F 1and F2 generations this result coincided with that obtained by Seth & Sharma (2001 ) on S. litura . The sex ratio in F1 generation was skewed towards male 1.48:1 male:female as compared with a 1.28:1 male: female in the control group of insects. Sex distortion appears to be general phenomenon occurring in the progeny of irradiated male lepidopterans Proverbs (1962) and Carpenter et al. (1986), probably resulting from the expression of recessive lethal mutations on the single Z chromosome in females but not in ZZ males Marec (1990 ). As a result of the reproductive performance and somatic damage caused by the radiation, we selected dose 125 Gy for a more in depth study. The reduced reproductive performance of the treated moths resulted from the combined effects of reduced longevity, fecundity, mating success, and fertility. In this part of study the parental males and irradiated with 125 Gy were crossed with untreated opposite sex. All possible mating combinations of the resulting F 1 adults and the untreated adult moths were carried out in order to obtain F 2 generation.
Discussion And Conclusion 168
The present work indicate that low dose,125Gy, did not affect the mating success of irradiated male parents, their F 1 and
F2 generation resulting from all possible mating combinations of
F1 adults compared with control group . The mating frequency was more adversely affected in F1 females crossed with normal males as compared with the F1 males paired with normal females. This can be considered an advantage of inherited sterility over completely sterile insect techniques; such results should ensure the success of partially sterile male technique against black cutworm moth. Similar debilitating effects of irradiation on the reproduction of moths have been reported by several workers ( North 1975; Carpenter et al . 1983; 1993; Omar & Mansor 1993; and Makee & Saour 1997). Poor reproductive performance and low fertility in treated A. ipsilon could be attributed to one or more of the following reasons: (i) poor ability to mate El-Sayed & Graves (1969), (ii) failure to produce and transfer as many spermatophores as normal males Rule et al. 1965; Flint & Kressin (1969), (iii) transfer spermatophores that contain little or no sperm; (iv) abnormal sperm structure, which fails to fertilize the eggs Ashrafi & Roppel (1973 ), or (v) inheritance of special chromosome rearrangements La-Chance (1985); Anisimov et al. (1989). The number of deposited eggs per mated female and the egg, hatchability percentage P 1, F 1 and F 2 generations were significantly reduced as a result of irradiated parent with 125
Gy.The present results indicate that F1 progeny were far more affected than their irradiated P1male parents, the sterility reach to
76.13 and 70.44 % in F1 Male and F1 female, the number of
Discussion And Conclusion 169 progeny from out crossed F1 pairs, of either male or female was much lower than that in the following out crossed F2. Thus deleterious effects on F2 pairs were not as severe as on F1 pairs, but still greater than the irradiated parents. Therefore, it is possible, from a practical point of view; a single release of irradiated substerilized P1males may decrease both the size attained and growth rate of population. In the present case, the object of partially sterile male release technique is primarily to reduce the size of first generation, beside a fair reduction in subsequent generations.
Data on the ability of irradiated P 1 males to reproduce throughout successive generations indicate that significant reduction in egg hatch and larval/pupal survival were recorded among two successive generations for male line and. So the production of female adult was reduced and the greatest reduced in egg hatch and female production was observed in F2 more than that was observed in F1. The same trend of effect was observed by North (1975), Carpenter et al. (1987 ) on Heliothis Zea (Boddie). and Mohamed (2002). Male Lepidoptera transfer eupyrene (nucleated) and apyrene (anucleated) sperm, as well as accessory gland secretions in a spermatophore at the time of copulation, only eupyrene sperm are capable of fertilizing eggs. In several species of Lepidoptera, the release of sperm from testes has been shown to be controlled by circadian rhythms with the clock and photoreceptor located in the testis vas deferens complex Giebultowicz et al. (1989), sperm is stored in the duplex until
Discussion And Conclusion 170 copulation occurs and the presence of eupyrene sperm bundles in the duplex is an indication of a male’s mating readiness. The previous studies mentioned the absence of eupyrene sperm bundles in the duplex region of newly emerged males immediately after emergence, this agrees with Henneberry &Clayton (1984) and Ibrahim (1987). The present results on the average number of eupyrene sperm bundles in the duplex of A. ipsilon show that this average reduce to 483.16 compared with 511.67 in untreated males in P 1,males at four dark light cycle of sperm descendence, this reduction was associated with significant reduction in the volume of the testis. While a significant reduction which obtained only in F 1 males at four dark light cycle of sperm descendence Table (18). This reduction to about 1/3 of the control was also observed in P. gossypiella by LaChance et al . (1977 ) who concluded that spermatogenesis and spermiogenesis in the F 1 males were affected and the majority of eupyrene sperm bundles produced are abnormal at structural or functional level, these are then eliminated or resorbed before they can descend into the duplex. Ibrahim (1987) added that the sperm bundles that had severe genetic damage might not develop to full mature bundles and resorbed within the testis because at the higher doses 12.5 Krad there was a significant reduction in testis volume associated with a reduction in the first cycle. According to Reimann (1973), it could be some kind of selection against abnormal sperm bundles within the testis basilar membrane which prevent the damage or abnormal sperm bundles.
Discussion And Conclusion 171
As an overall conclusion, our observations indicate that F 1 males (sterile) from irradiated A.ipsilon fathers can be distinguished from The normal fertile males based on the cytological stained pattern of the nuclei clusters of the eupyrene sperm bundles by using Giemsa stain alone. The chromatin materials of the nuclei cluster of a normal fertile A.ipsilon male was homogeneously stained, while it was heterogeneously stained in the F 1 sterile males. These cytological characteristics, observed in all males examined, are consistent with the findings of Wee et al. (2011) on Teia anartoides . Similarly to when stained by orcein and Giemsa stains by Carpenter et al .(2009). The species used in their investigation represent five lepidopteran families varied in size from the small diamondback moth, Plutella xylostella L. to larger corn earworm, Helicoverpa zea (Boddie), similarly the mature sperm bundles of these species varied in shape, length, and staining characteristics. The use of this cytological attribute for differentiation between the F 1progeny of irradiated males and the fertile males was first demonstrated in the codling moth by using orcein and Giemsa stains Carpenter & Marti (2005). This technique will be a great asset and potentially be adaptable for all lepidopteran pests involved in future SIT programs. The inherited sterility in Lepidoptera is a very complex phenomenon that is based on the induction of a large variety of genetic alterations by irradiation. The alterations reduce fertility of F 1 individuals in two ways. Some of them directly prevent the normal course of reproduction in their carriers, i.e., cause physiological sterility. Particular chromosome rearrangements
Discussion And Conclusion 172 lead to the production of genetically unbalanced gametes, resulting in inviable progeny and thus, cause genetical sterility of their carriers. La-Chance (1985) extended the hypothesis that multiple chromosomal translocations as well as simple nonreciprocal and reciprocal translocations are most responsible for the genetical sterility of F 1 generation. 3. Effect of Entomopathogenic nematodes on Agrotis ipsilon. In previous studies the susceptibility of developmental stages of Agrotis ipsilon to selected EPN species has already been done, most of the studies were in agreement with our finding. In the experiment was carried out to evaluate the efficiency of two EPN species, Steinernema. carpocapsae (All) and S. riobrivae on A. ipsilon , the obtained results are given in tables (19 & 20). It could be mentioned the used species were effective against tested pest, statistical analysis showed that there were significant increases in percent of mortality, significant decreases in adult emergence and significant decreases in surviving from infested larva to adulthood. In general, at all concentrations, S. Carpocapsae gave higher percentages of mortality more than concentrations, S. riobrivae,.
From Table (21) LC 50 values were 11.37 and 30.44 IJs
/larvae in S. Carpocapsae and S. riobrivae respectively, the LT 50 values were 3.62 days in case of S. Carpocapsae and 10.44 days in case of S. riobrivae . Values of LC 50 and LT 50 against larvae of A. ipsilon indicated that S. Carpocapsae looked more virulent and faster in effect than S. riobrivae . R 2 values showed strong correlation between mortality values and concentrations;
Discussion And Conclusion 173 however R 2 values showed moderate correlation between mortality values and time, indicating that the former responded more than the latter. In agreement with the present results, Abdel-kawy (1985) found a positive correlation between mortality percent of 4th , 5th and 6th instars larvae of A. ipsilon and the nematode inoculum level. Our results also confirm these of Levine & Oloumi-Sadeghi (1992), who found out that the EPNs, S. Carpocapsae (All strain) generally performed as well as or better than the conventional insecticides in controlling A. ipsilon . Mogahed & Abbas (1998) studied the efficacy of S. Carpocapsae and Heterorabditis bacteriophora against A.ipsilon they found that 4th instar larvae were more susceptible to S. Carpocapsae and H. bacteriophora than 6th instar larvae of A.ipsilon . Pouring the nematode suspension on the soil surface proved to be more effective against the larvae than being incorporated with the soil; both S. Carpocapsae and H. bacteriophora were still active through the first month after field application. Table (22) depended on S. Carpocapsae exposure periods, the respective experiment was conducted to assess killing effect of S. Carpocapsae during any dark period was coincided A.ipsilon season. Mortality percentages were 58 to 66 % at periods 8 to 12 hr, significant reduces in both of percentage of pupation and percentage of adult emergence and sex ratio favors to male line. The efficacy of various nematode species or strains for controlling a particular insect pest is influenced by the rate of IJ penetration into the insect, the time it takes to release the symbiotic bacteria, and the virulence of the latter Glazer &
Discussion And Conclusion 174
Navon, (1990). In the present study, when A.ipsilon 4th instar larvae were treated with 100 IJs for various exposure periods, S. carpocapsae killed the greatest number of larvae. This means that direct relationships were found between results obtained in exposure time assays in studies of the effect of nematodes on other lepidopteran pests Glazer, (1992) and Ricci et al. (1996). Ben-Yakir et al. (1998) reported that indeed, within the exposure time assay of ECB, Ostrinia nubilalis (Hǖbner), larval death rate gradually increased and was significantly higher after 9 h of exposure than after 3 or 6 h to to S. carpocapsae and H. bacteriophora , at an infestation rate of 500 IJs per Petri dish. Concerning to the delayed effect of treatment 4th instar larvae with S. Carpocapsae on reproduction of developed male and female. Present data exhibit adverse effect on reproduction of male and female, decreased mating ability and mated female received sperm.The fecundity of females reduced by increasing of exposure period. Abd-Elwahed(2004) investigated the effect of S. carpocapsae on Ph. operculella after 6, 18 and 24 hrs. Percent mortality was increased by increasing time elapse after infection, adverse effect on reproduction of male and female was observed. The employed application methods is important as well as nematode concentrations in controlling A.ipsilon i.e., the application method had obvious effect on the percent of cumulative mortality of A.ipsilon , the percent mortality was 98 and 100 % in aqueous suspension method and 10 day A.ipsilon cadavers method it could be mentioned that the used two method
Discussion And Conclusion 175 were effective against the insect and this was the reason of increasing to percent of reduction in resulted progeny. These results in harmony with those obtained by Jansson & Lecrone (1994 ) they suggested that aqueous suspensions of H. bacteriophora 88 nematodes were as effective as applications of Galleria mellonella cadavers infected with H. bacteriophora 88 for controlling damage by sweet potato weevil, Cylas formicarius (Fabricius) to storage roots. Creighton & Fassuliotis (1985) and Jansson (1993) mentioned that entomopathogenic nematodes may be applied in infected insect cadavers and in this approach, nematode infected cadavers are disseminated and pest suppression is subsequently achieved by the progeny IJs that exit the cadavers. Field application of EPNs in infected hosts may be superior to application in aqueous suspension, in terms of infectivity, dispersal and survival Shapiro-Ilan & Glazer (1996) and Shapiro-Ilan & Lewis (1999). EPNs can survive dry or harsh conditions or desiccation for extended periods within host cadaver Brown & Gaugler (1996) and Koppenhofer et al . (1997). Improved persistence within the host cadavers Perez, et al. (2003) has been reported as compared to aqueous suspensions wherein EPNs might face osmotic stress. However, EPNs carried within infected hosts are compromised by limitations of storage and application. To an extent, this constraint can be solved by improved formulations Shapiro-Ilan et al. (2001). It is reported that EPNs have the ability to seek out and quickly kill hosts within 24 48 h Gaugler (1981).
Discussion And Conclusion 176
Therefore, after an adequate time of exposure to IJs, the host can be transported immediately to the field, but this may pose a serious limitation if some viable, non parasitized host insects escape parasitization. This problem can be overcome by radiosterilization of hosts before host exposure to IJs, and transport to the field before the hosts die and turn into cadavers. In this manner, the IJs from infected hosts can then directly interact with the ecosystem after emergence and seek new hosts (target pests) for progeny propagation. 4. Combined effect gamma irradiation and Steinernema. Carpocapsae on Agrotis ipsilon . Before combining inherited sterile technique (IST) with S carpocapsae application to manage A .ipsilon populations, the sensitivity of sterile F1progeny, produced from released irradiated insects, to S .carpocapsae should be determined. The primary objective of this study is to compare the responses of F1 progeny of partially sterile male parents to Xenorhabdus nematophelus released by S .carpocapsae with those of
F1progeny of nonirradiated male parents. When Lepidopteran species are irradiated, several physiological, biochemical and genetical changes occur and are inherited by their progeny Anisimov et al . (1989) and Tothova& Marec, (2001). In the present study, the efficiency of the integration of such technique with S. carpocapsae application so as to control A .ipsilon was determined. S. carpocapsae had a clear effect on the mortality of F1 progeny of A. ipsilon whether they were from irradiated or nonirradiated parents Tables (27&28). F1 progeny of irradiated parents were more susceptible than those of
Discussion And Conclusion 177 nonirradiated parents at a low concentration, but F1 progeny of both parents had similar susceptibility at higher concentrations S. carpocapsae was more effective against F1 progeny of irradiated parents overall. Correspondingly, Jafri & Sabiha (1974) reported that irradiated insects had higher susceptibility to many pathogenic microorganisms. Our results in agreement with the findings by Abdel Salam et al . (1995) on ph. operculella larvae infected with S. carpocapsae ; Gouge et al . (1998) on susceptibility of Pectinophora gossypiella larvae to S. riboravae and H. bacteriophora and El- Mandarawy et al . (2006)on Galleria mellonella response to S.riboravae, H. bacteriophora and H. tayserae . In this study, all combinations resulted in additive response Table (29), this coincided with the work of Faruki &
Khan (2004) they studied the combination between F 1 sterility and insecticides on Cadra cautella and the work of Mahmoud (2007) he studied the combination between some botanical insecticides and EPNs, S. feltiae on Bactrocera zonata (Saunders). These authors studied synergistic interaction between biological control methods against the pests. Regarding to interaction of (EPNs) cultured in irradiated hosts, with F 1 sterility, data in Table (30) clear that no significant influence of induced sterility of the host (F1 sterile insects) on both of the parasitisation efficacy of EPNs, and on the mortality induction process. The onset of morbidity and mortality induced by normal IJs (i.e., IJs derived from untreated host) was not different in
F1sterile hosts and un irradiated controls. The incubation time
Discussion And Conclusion 178 taken by IJs on F1 sterile hosts was significantly prolonged, in F 1 hosts derived from 125 Gy treated male. While, the IJs harvest was found significantly reduced on F1 hosts, the harvest period was also slightly affected by the radiation dose applied to the host in the previous generation. Concerning to the infective performance of IJs cultured in parasitized F1 sterile A. ipsilon ., no evident interaction was noticed between the irradiation background of IJs parent host (as cultured) and the nature of the current host (as infective) on parasitisation behavior of EPNs.
Incubation time taken by IJs that had been cultured F1sterile hosts was significantly affected. It means the incubation time taken by IJs from radio sterilized hosts was prolonged on normal. The reduction in harvest potential of IJs depended upon the gamma dose administered to male parent of F1 insects (as hosts) as well as F1 hosts. Inherited sterility induced by irradiated male parent moths has been proposed for the reproductive suppression of lepidopteran pests. Combination with certain ecologically safe strategies like biological control may further improve the control of lepidopteran pests using F1 sterility; hence, the probability of integrating S. carpocapsae EPNs with this genetic control measure was investigated in the present study on A .ipsilon . It is difficult to find complementary control strategies for synergistic use in conjunction with sterile moth release programmes. Gouge, et al. (1998) suggested that S. carpocapsae may be an ideal entomopathogenic nematode to be used in conjunction with inherited sterility for the management of the
Discussion And Conclusion 179 pink bollworm, P. gossypiella, as S. carpocapsae would more likely infect the mobile native pink bollworm larvae than the sedentary F1 larvae from irradiated parents. But in our present experiments, parasitize hosts possessing low mobility and residing within the soil profile, as suggested by Hill (1983). We have attempted to study the feasibility of integrating the use of EPNs with F1 sterility and have focused on investigations regarding the interaction with F1 sterile insects of normal EPNs, and EPNs that had been cultured in radio sterilized insects. F1insects, derived from 125Gy irradiated male parent, were acceptable and suitable hosts for EPNs, almost with the same degree as untreated insects control. Therefore, simultaneous release of EPNs with F1 insects from 125 Gy treated parents might be good proposition. Hence, release of
EPNs along with F1 sterile insects might limit or influence the effectiveness of F1 sterility for pest suppression, depending upon the gamma dose (to be used in F1 sterility) and the timing of EPNs release. Further, since the compatibility of two control measures was confirmed, and the pest population suppression was feasible by both techniques, a management strategy could be devised. In this situation, simultaneous application of both tactics, due to acceptability and suitability of F1 insects as host for EPNs reared in radio sterilized host, may show an additive effect, because these two methods are not antagonistic to each other. Provided timing and logistics are taken into consideration, synergy may be achieved in response to inoculative release of
EPNs along with F1 sterility.
Discussion And Conclusion 180
As per review of our investigations, the use of ‘genetic pest control method’ (F1sterility technique) in conjunction with EPNs could be a feasible strategic component in IPM of A .ipsilon , in which operational modality might be either (i)
‘sequential’, i.e., EPNs application preceding the use of F1 sterility so as to reduce the load of release of sub sterilized moths, or (ii) ‘simultaneous’ for some initial specific phase, because F1 insects (at 125Gy) would be equally acceptable as normal insects, and pest suppression would be operating against different stages of the life cycle, i.e., against larvae and pupae
(through EPNs) and against adults (via F1sterility). The intermittent (inundative) releases of EPNs could also be effectively pursued, alternately with F1 sterility, so as to keep the pest population below the economic threshold. Further, in a situation where F1 sterility has been successful in suppression of pest population, the inoculative releases of EPNs could be considered for biological pest management, especially as a quarantine measure along with release of partially sterile insects, in view of bio infective potential of IJs, cultured in F1 sterile insects, observed in the present study. Cautious field simulated studies are warranted to judge the operational approach with respect to pest density and timing, so as to optimally integrate the use of EPNs with F1sterility. The inundative and inoculative releases of EPNs might be possible and effective in view of acceptability and suitability of normal and irradiated A .ipsilon and their F1 sterile progeny as hosts of EPNs, according to the present investigation, where the pest was found to be responsive towards both control tactics.
Discussion And Conclusion 181
Final necessary word:- From the previously mentioned results, in addition to what me know and feel about the recent level of ecosystem destroy because of the extensive use of toxic pesticides during the foregoing time, which caused lethal dangerous diseases symptoms which had dispersed among people and domestic animals because within tissues agricultural products, that are used as food. Thus we find it necessary to call all humans all over the world to stop, instantaneously, the use of “chemical control” and replace it with the prolyphic “biological control” method. That surely will be useful for preventing environment from being polluted, man and animal from being toxicated, and finally a state “natural balance dominance”.
Discussion And Conclusion 182
SUMMARY The present work deals with the effects of gamma irradiation on Agrotis ipsilon (Hufn.), to induce inherited sterility. Special attention was given to study reproductive biology and inherited sterility throughout two successive generations, in hope of promoting the sterile insect method for the pest. This work comprised the study of effects of six low doses (75, 100, 125,150, 175 and 200Gy) of gamma radiation as well as two species of EPNs (Steinernema Carpocapsae All and Steinernema riobrivae ). Special stress was given to compare the responses of F1 progeny of partially sterile male parents to S.
Carpocapsae with those of F1progeny of nonirradiated male parents. The obtained results can be summerized as follows: 1. Effect of Gamma Irradiation on Reprodutivity: When full grown male pupae were irradiated with the doses of, 75 100, 125,150, 175 and 200Gy and crossed with untreated females 1. Both the percentage mated males with untreated females and copulation duration to format spermatophores directly in the female bursa copulatrix were not affect with the doses used. 2. The percent of mated female with spermatophores and percent of mated female with sperm in spermatheca were not evidently different from the control except on the high doses 175 &200 Gy.
Summary -183-
3. Lower percentages of males were observed in copulation with untreated virgin females in the second and third mating than the first mating . 4. Number of eggs /female deposited by females mated to irradiated males and egg hatch were significantly reduced as a result of irradiation male treatment through three females. 5. Also Number of eggs /female deposited by females mated to irradiated males and egg hatch were significantly less in second female and third female compared to the first . 6. Significant differences in mating aspects and reproductive parameters within three sequential mated females, however significant differences was not be observed within the doses. 7. Slight reduction in the average number of matings of the treated males was observed due to radiation doses. 8. The irradiated males with tested doses as well as untreated control could be responsing to calling females and start copulation at the same period of the scotophase. 9. The mating competitiveness calculated from the direct observation in A. ipsilon revealed that males emerged from pupae irradiated at 75 to 125 Gy are equally capable of mating as control. While, the competitive value decreased with the dose increased after that.
Summary -184-
2. Effect of dose 125 Gy used to irradiated males& females full grown pupae on biological aspects of
P1and F1generations A. Effect of dose 125 Gy used to irradiated female 1. Radiation exposure of 125 Gy had no effect on percentages of treated females mating or times in copulation with untreated males. 2. Percentages of females mating over all radiation doses decreased with new virgin males provided during the second and third mating period the first male more than the first male. 3. The fecundity of the black cutworm, female was not significantly affect when full grown female pupae were exposed 125 Gy while, the percentage of egg hatch was significantly reduced 4. The average numbers of sequential male matings not affect 5. The treated females with 125Gy had no effect on calling behavior and attracting to wild males. B- Effect of dose 125 Gy used to irradiated male on biological aspects of P1and F1generation . 1. The percent emergence not affect, the percentage of malformed adults increased in treated insect compared to untreated insect and adult longevity slightly reduced by irradiating. In general, BCW female slightly affected by irradiating more than male 2. The mating competitiveness of the black cutworm parental irradiated males with low doses of 125 Gy were fully
Summary -185-
competitive against untreated males in mating with normal females in all tested ratios. 3. The average developmental period from egg hatch to adult emergence was obviously increased in the progeny of two filial generations.
4. The percentage of pupation was not affected among in F 1 and
F2 progeny, the percentage of adult emergence was slightly reduced among the first filial generation descendent of parental irradiated males, however this reduction was clear
in F 2 progeny. 5. The growth index showed a decrease as a consequence of
irradiation of male parents in F 1and F2 generations.
6. The sex ratio, among the progeny descend of P 1 generation,
and the progeny descend of F1 generation did not differ from the ratio observed in the control treatment. 7. The percentage of mating success was not affected among in
P1 generation, F 1 and F 2 progeny, the percentage of mating
frequency was slightly reduced among the F1 and F 2 progeny . 8. The number of deposited eggs per mated female and the egg
hatchability percentage P 1, F 1 and F 2 generations were significantly reduced as a result of irradiated parent with 125 Gy
9. The present results indicate that F1 progeny were far more
affected than their irradiated P1male parents, and the
deleterious effects on F2 pairs were not as severe as on F1 pairs, but still greater than the irradiated parents
Summary -186-
10. The production of female adults resulting from P 1 irradiated
males and F 1 generation decreased as result of irradiation
treatment, and the greatest reduction in F 2 progeny. 11. Although the number of eupyrene sperm bundles descended to duplex was not affect during the first dark-light cycle of sperm descendence, the volume of testes significantly
reduced specially in P 1male parents.
12. Accumulation of eupyrene sperm bundles of unmated F 1 males was significantly reduced. 3. Effect of EPNs on Agrotis ipsilon. 1. The used species Steinernema Carpocapsae (All) and Steinernema riobrivae were effective against tested pest . 2. There were significant increases in percent mortality, significant decreases in both percent adult emergence and percent surviving from infested larva to adulthood as a result of increasing of concentrations 3. R2 value showed strong correlation between mortality values and concentrations
4. The LT 50 values showing that the S. carpocapsae was faster to be infested A. ipsilon. than S. riobriva 5. Tabulated data revealed collinear relationship between the exposure periods to 100 IJs/larvae of S. carpocapsae and the total morality percent and the percentage of adult malformation. 6. On the contrary, treating larvae with S. carpocapsae at concentration 100 IJs /larvae showed significant decreasing
Summary -187-
to the percentage of pupation and the percentage of adult emergence. 7. The reduction in mating ability percentage and percentage of mated females received sperm increased almost gradually with the increase in exposure period. The highest effect occurred when treated males were descended from treating larvae with S. carpocapsae at concentration 100 IJs /larvae . 8. It was observed that the number of deposited eggs per mated female and the eggs hatchability percentages were significantly reduced at the most mating combinations through the both tested adult lines . 9. It could be mentioned that the employed application methods were effective against and had obvious effect on the percent of cumulative mortality and percent of reduction in progeny of A.ipsilon . 4. Combined effect gamma irradiation and (EPNs) S. carpocapsae on Agrotis ipsilon .
1. F 1 progeny of A .ipsilon irradiated parents with 125 Gy were more susceptible S. carpocapsae than those of nonirradiated parents at a low concentration.
2. But F1 progeny of both parents had similar susceptibility at higher concentrations S. carpocapsae . 3. In this study, the combination of tested treatments at all concentrations resulted in additive effect. 4. Data clear that no significant influence of induced sterility
of the host (F1 sterile insects) on both of the parasitisation efficacy of EPNs, and on the mortality induction process.
Summary -188-
The onset of morbidity and mortality induced by normal IJs (i.e., IJs derived from untreated host) was not different in
F1sterile hosts and un-irradiated controls.
5. The incubation time taken by IJs on F1 sterile hosts was significantly prolonged, in F1 hosts derived from 125 Gy treated male parent (P< 0.05).
6. The IJs harvest was found significantly reduced on F1 hosts. The harvest period was also slightly affected by the radiation dose applied to the host in the previous generation. 7. Regarding to the infective performance of IJs cultured in
parasitized F1 sterile A .ipsilon ., no evident interaction was noticed between the irradiation background of IJ’s parent host (as cultured) and the nature of the current host (as infective) on parasitisation behavior of EPNs.
8. Incubation time taken by IJs that had been cultured F1sterile hosts was significantly affected. It means the incubation time taken by IJs from radio-sterilized hosts was prolonged on normal 9. The reduction in harvest potential of IJs depended upon the
gamma dose administered to male parent of F1 insects (as
hosts)as well as F1 hosts
Summary -189-
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ﺒﺴﻡ ﺍﷲ ﺍﻝﺭﺤﻤﻥ ﺍﻝﺤﻴﻡ ﺍﻝﻤﻠﺨﺹ ﺍﻝﻌﺭﺒﻰ ﺎﺘﺘﻨ ﻭل ﺍ ﻝﺩﺭﺍﺴﺔ ﺍﻝﺤﺎﻝﻴﺔ ﺘﺄﺜﻴﺭ ﺃﺸﻌﺔ ﺠﺎﻤﺎ ﻋﻠﻰ ﺍﻝﺩﻭﺩﺓ ﺍﻝﻘﺎﺭﻀﺔ ﺤﻴﺙ ﺃﻭﻝﻴـﺕ ﺍﻝﺩﺭﺍﺴﺎﺕ ﺍﻝﺨﺎﺼﺔ ﺒﺒﻴﻭﻝﻭﺠﻴﺎ ﺍﻝﺘﻨﺎﺴل ﻭﺴﻠﻭﻙ ﺍﻝﺘﺯﺍﻭﺝ ﺍﻫﺘﻤﺎﻡ ﺨﺎﺹ ﺒﻬﺩﻑ ﺍﺴـﺘﺨﺩﺍﻡ ﻫﺫﻩ ﺍﻝﻤﻌﻠﻭﻤﺎﺕ ﻓﻰ ﺘﺤﺴﻴﻥ ﺍﺴﺘﺨﺩﺍﻡ ﺍﻹﺸﻌﺎﻉ ﻭﺍﻝﺤﺸﺭﺍﺕ ﺍﻝﻌﻘﻴﻤﺔ ﻝﻤﻜﺎﻓﺤﺔ ﺍﻷﻓـﺎﺕ ٠ ٠ ﻜﻤﺎ ﺘﺘﻨ ﺎﻭل ﺍﻝﺩﺭﺍﺴﺔ ﺘﺄﺜﻴﺭ ﺃﺸﻌﺔ ﺠﺎﻤﺎ ﻭﺒﻌﺽ ﺍﻝﻨﻴﻤﺎﺘﻭﺩﺍ ﺍﻝﻤﻤﺭﻀﺔ ﻝﻠﺤﺸـﺭﺍﺕ ﻜـل ﻋﻠﻰ ﺤﺩﻩ ﺃﻭ ﻤﺠﺘﻤﻌﺔ ﻋﻠﻰ ﺒﻌﺽ ﺍﻝﻨﻭﺍﺤﻰ ﺍﻝﺒﻴﻭﻝﻭﺠﻴﺔ . ﻭﻗﺩ ﺘﻨﺎﻭﻝﺕ ﺍﻝﺩﺭﺍﺴﺔ ﺘـﺄﺜﻴﺭ ﺍﻝﺠﺭﻋﺎﺕ ( ٧٥ ، ١٠٠، ١٢٥، ١٥٠، ١٧٥، ٢٠٠ ﺠﺭﺍﻯ ) ﻭﻜﺫﻝﻙ ﺨﻤﺴﺔ ﺘﺭﻜﻴﺯﺍﺕ ﻤﻥ ﻨﻭﻋﻴﻥ ﺘﺎﺒﻌﻴﻥ ﻝ ﻠﻨﻴﻤـﺎﺘﻭﺩﺍ ﺍﻝﻤﻤﺭﻀـﺔ ( Steinernema carpocapsae All (، Steinernema riobrave ) ﺍﻫﺘﻤﺕ ﺍﻝﺩﺭﺍﺴﺔ ﺒﺼﻔﺔ ﺨﺎﺼﺔ ﺒﺎﻝﺘﺄﺜﻴﺭ ﻋﻠﻰ ﺍﻝﺘﻔﺎﻋـل ﺍﻝﻤﺸﺘﺭﻙ ﺒﻴﻥ ﺍﻝﻌﻘﻡ ﺍﻝﻤﻭﺭﻭﺙ ﻭ ﺍﻝﻨﻴﻤﺎﺘﻭﺩﺍ ﺍﻝﻤﻤﺭﻀﺔ ﻝﻠﺤﺸﺭﺍﺕ ٠ ﻭﻴﻤﻜﻥ ﺘﻠﺨﻴﺹ ﺍﻝﻨﺘﺎﺌﺞ ﺍﻝﻤﺘﺤﺼل ﻋﻠﻴﻬﺎ ﻓﻰ ﺍﻵﺘﻰ -: ﺃﻭﻻ : ﺘﺄﺜﻴﺭ ﺃﺸﻌﺔ ﺠﺎﻤﺎ ﻋﻠﻰ ﺍﻝﻨﻭﺍﺤﻲ ﺍﻝﺘﻜﺎﺜﺭﻴﺔ ﻭﺴﻠﻭﻙ ﺍﻝﺘﺯﺍﻭﺝ -: ﻋﻨﺩ ﺘﻌﺭﻴﺽ ﺍﻝﻌﺫﺍﺭﻯ ﺍﻝ ﻤﻜﺘﻤﻠـﺔ ﺍﻝﻨﻤـﻭ ﻝﻠـﺫﻜﻭﺭ ﻝﻠﺠﺭﻋـﺎﺕ ( ،٧٥ ١٠٠ ، ، ١٢٥، ١٥٠، ١٧٥، ٢٠٠ ﺠﺭﺍﻯ ) ﺜﻡ ﺘﻘﺩﻴﻤﻬﺎ ﻝﻺﻨﺎﺙ ﺍﻝﻐﻴﺭ ﻤﻌﺎﻤﻠﺔ ﻝﻜﻰ ﻴـﺘﻡ ﺍﻝﺘـﺯﺍﻭﺝ ﺒﻴﻨﻬﻡ ﻭﺠﺩ. ١- ﻝﻡ ﻴﺘﻡ ﺘﺄﺜﺭ ﻜل ﻤﻥ ﻨﺴﺒﺔ ﺍﻝﺫﻜﻭﺭ ﺍﻝﻤﺘﺯﺍﻭﺠﺔ ﻤﻊ ﺍﻹﻨﺎﺙ ﻭﻻ ﺍﻝﻤﺩﺓ ﺍﻝﻤﺴﺘﻐﺭﻗﺔ ﻓﻰ ﺍﻝﺘﺯﺍﻭﺝ ﻭﺘﻜﻭﻴﻥ ﺍﻝﺤﺎﻤل ﺍﻝﻤﻨﻭﻱ . . ٢- ﻝﻡ ﺘﺘﺄﺜﺭ ﻨﺴﺒﺔ ﺍﻹﻨﺎﺙ ﺍﻝﻤﺘﺯﺍﻭﺠﺔ ﻭﺍ ﻝﺤﺎﻭﻴﺔ ﻋﻠﻰ ﺍﻝﺤﺎﻤـل ﺍﻝﻤﻨـﻭﻯ ﻭﻻ ﻨﺴـﺒﺔ ﺍﻹﻨﺎﺙ ﺍﻝﻤﺘﺯﺍﻭﺠﺔ ﻭﺍﻝﺤﺎﻭﻴﺔ ﻋﻠﻰ ﺤﻴﻭﺍﻨﺎﺕ ﻤﻨﻭﻴﺔ ﻓﻰ ﺍﻝﻘﺎﺒﻠـﺔ ﺍﻝﻤﻨﻭﻴـﺔ ﺇﻻﻋﻨـﺩ ﺍﻝﺠﺭﻋﺎﺕ ﺍﻝﻤﺭﺘﻔﻌﺔ ( ١٧٥ ، ٢٠٠ ﺠﺭﺍﻯ .) ٣- ﺍﻨﺨﻔﺎﺽ ﻨﺴﺒﺔ ﺍﻝﺫﻜﻭﺭ ﺍﻝﻤﺘﺯﺍﻭﺠﺔ ﻤﻊ ﺍﻹﻨﺎﺙ ﺒﺎﻝﻨﺴﺒﺔ ﻝﻸﻨﺜﻰ ﺍﻝﺜﺎﻨﻴﺔ ﻭﺍﻝﺜﺎﻝﺜﺔ ﻋﻨﻬﺎ ﺒﺎﻝﻨﺴﺒﺔ ﻝﻸﻨﺜﻰ ﺍﻷﻭﻝﻰ . .
ﺍﻝﻤﻠﺨﺹ ﺍﻝﻌﺭﺒﻲ -١- ٤- ﺍﻨﺨﻔﺎﺽ ﻤﻌﺩل ﻭﻀﻊ ﺍﻝﺒ ﻴﺽ ﻭﻜﺫﻝﻙ ﻨﺴﺒﺔ ﻓﻘﺱ ﻫﺫﺍ ﺍﻝﺒﻴﺽ ﺍﻨﺨﻔـﺎﺽ ﻤﻌﻨـﻭﻱ ﻨﺘﻴﺠﺔ ﻝﻠﻤﻌﺎﻤﻠﺔ ﺒﺎﻝﺠﺭﻋﺎﺕ ﻭﺫﻝﻙ ﺒﺎﻝﻨﺴﺒﺔ ﻝﻜل ﺍﻨﺜﻰ ﻋﻠﻰ ﺤﺩﺓ . . ٥- ﺍﻨﺨﻔﺎﺽ ﻤﻌﺩل ﻭﻀﻊ ﺍﻝﺒﻴﺽ ﻭﻜﺫﻝﻙ ﻨﺴﺒﺔ ﻓﻘﺱ ﻫﺫﺍ ﺍﻝﺒﻴﺽ ﺍﻨﺨﻔـﺎﺽ ﻤﻌﻨـﻭﻱ ﺒﺎﻝﻨﺴﺒﺔ ﻝﻸﻨﺜﻰ ﺍﻝﺜﺎﻨﻴﺔ ﻭﺍﻝﺜﺎﻝﺜﺔ ﺒﺎﻝﻤﻘﺎﺭﻨﺔ ﻤﻊ ﺍﻷﻨﺜﻰ ﺍﻷﻭﻝﻰ. ٦- ﻝﻭﺤﻅ ﻭﺠﻭﺩ ﺍﺨﺘﻼﻑ ﻤﻌﻨﻭﻱ ﻓﻲ ﺍﻝﻨﻭﺍﺤﻲ ﺍﻝﺘﻜﺎ ﺜﺭﻴﺔ ﻭﺴﻠﻭﻙ ﺍﻝﺘﺯﺍﻭﺝ ﺒﻴﻥ ﺍﻹﻨﺎﺙ ﺍﻝﻤﺘﺘﺎﻝﻴﺔ ، ﻓﻲ ﺤﻴﻥ ﻝﻡ ﻴﻜﻥ ﻫﺫﺍ ﺍﻻﺨﺘﻼﻑ ﻤﻌﻨﻭﻴﺎ ﺨﻼل ﺍﻝﺠﺭﻋﺎﺕ ﺍﻝﻤﺨﺘﺒﺭﺓ . . ٧- ﻭﺠﻭﺩ ﺍﻨﺨﻔﺎﺽ ﺨﻔﻴﻑ ﻓﻲ ﻤﺘﻭﺴﻁ ﻋﺩﺩ ﻤﺭﺍﺕ ﺘﺯﺍﻭﺝ ﺍﻝﺫﻜﻭﺭ ﺍﻝﻤﻌﺎﻤﻠـﺔ ﻨﺘﻴﺠـﺔ ﺍﻹﺸﻌﺎﻉ . . ٨- ﻜﺎﻨﺕ ﺍﺴﺘﺠﺎﺒﺔ ﺍﻝﺫﻜﻭﺭ ﺍﻝﻤﺸﻌﻌﺔ ﻻﺴﺘﺩﻋﺎﺀ ﺍﻹﻨﺎﺙ ﻭﺒﺩﺀ ﺍﻝﺘﺯﺍﻭﺝ ﻤﺴﺎﻭﻱ ﻝﻠﺫﻜﻭﺭ ﺍﻝﻁﺒﻴﻌﻴﺔ ﺨﻼل ﻨﻔﺱ ﺍﻝﻔﺘﺭﺓ ﻤﻥ ﺍﻝﻠﻴل ( ﻭﻫﻭ ﻭﻗﺕ ﻨﺸﺎﻁ ﺍﻝﻔﺭﺍﺸﺎﺕ ). ). ٩- ﺃﺸﺎﺭﺕ ﻗﻴﻤﺔ ﺍﻝﺘﻨﺎﻓﺱ ﺍﻝﺘﺯﺍﻭﺠﻰ ﻭﺍﻝﻤﺩﺭﻭﺴﺔ ﺒﻁﺭﻴﻘﺔ ﺍﻝﻤﻼﺤﻅـﺔ ﺍﻝﻤﺒﺎﺸـﺭﺓ ﺃﻥ ﺍﻝﺫﻜﻭﺭ ﺍﻝﻤﻌﺎﻤﻠﺔ ﺒﺎﻝﺠﺭﻋﺎﺕ ﻤﻥ ٧٥ ﺤﺘﻰ ١٢٥ ﺠﺭﺍﻯ ﻜﺎﻨﺕ ﻜﺎﻤﻠﺔ ﺍﻝﺘﻨﺎﻓﺱ ﻤﻊ ﺍﻝﺫﻜﻭﺭ ﺍﻝﻐﻴﺭ ﻤﻌﺎﻤﻠﺔ ﻓﻲ ﺍﻨﺨﻔﻀﺕ ﻫﺫﻩ ﺍﻝﻘﻴﻤﺔ ﺒﺯﻴﺎﺩﺓ ﺍﻝﺠﺭﻋﺔ ﻋﻥ ﺫﻝﻙ . . ﺜﺎﻨﻴﺎﹰ : ﺘﺄﺜﻴﺭ ﺍﻝ ﺠﺭﻋﺔ ﺘﺤﺕ ﺍﻝﻤﻌﻘﻤﺔ ١٢٥ ﺠﺭﺍﻯ ﺍﻝﻤﺴـﺘﺨﺩﻤﺔ ﻓـﻰ ﻤﻌﺎﻤﻠـﺔ ﺍﻝﺫﻜﻭﺭ ﻭ ﺍﻹﻨﺎﺙ ﻋﻠﻰ ﺍﻝﻨﻭﺍﺤﻰ ﺍﻝﺒﻴﻭﻝﻭﺠﻴﺔ ﺨﻼل ﺠﻴﻠﻲ ﺍﻷﺒﺎﺀ ﻭﺍﻷﺒﻨﺎﺀ . . ﺃ – ﺘﺄﺜﻴﺭ ﺍﻝﺠﺭﻋﺔ ﺘﺤﺕ ﺍﻝﻤﻌﻘﻤﺔ ١٢٥ ﺠ ﺭﺍﻯ ﻭﺍﻝﻤﺴﺘﺨﺩﻤﺔ ﻓﻰ ﻤﻌﺎﻤﻠﺔ ﺍﻹﻨﺎﺙ . . ١- ﻝﻡ ﺘﺘﺄﺜﺭ ﻨﺴﺒﺔ ﺘﺯﺍﻭﺝ ﺍﻹﻨﺎﺙ ﺍﻝﻤﻌﺎﻤﻠﺔ ﺒﺎﻝﺠﺭﻋﺔ ١٢٥ ﺠـﺭﺍﻯ ﻤـﻊ ﺍﻝـﺫﻜﻭﺭ ﺍﻝﻁﺒﻴﻌﻴﺔ ﻤﻘﺎ ﺭﻨﺔ ﺒﺎﻹﻨﺎﺙ ﺍﻝﻐﺒﺭ ﻤﻌﺎﻤﻠﺔ ﻭﻜﺫﻝﻙ ﻝﻡ ﻴﺘﺄﺜﺭ ﺍﻝﻭﻗﺕ ﺍﻝﻤﺴﺘﻐﺭﻕ ﻓـﻰ ﺘﻜﻭﻴﻥ ﺍﻝﺤﺎﻤل ﺍﻝﻤﻨﻭﻯ . . ٢- ﺍﻨﺨﻔﻀﺕ ﻨﺴﺒﺔ ﺘﺯﺍﻭﺝ ﺍﻹﻨﺎﺙ ﻤﻊ ﺍﻝﺫﻜﺭ ﺍﻝﺜﺎﻨﻰ ﻭﺍﻝﺜﺎﻝﺙ ﺒﺸﻜل ﻤﻌﻨـﻭﻯ ﻋـﻥ ﻨﺴﺒﺔ ﺘﺯﺍﻭﺝ ﺍﻹﻨﺎﺙ ﻤﻊ ﺍﻝﺫﻜﺭ ﺍﻷﻭل.
ﺍﻝﻤﻠﺨﺹ ﺍﻝﻌﺭﺒﻲ -٢- ٣- ﻝﻡ ﻴﺘﺄﺜﺭ ﻤﻌﺩل ﻭﻀﻊ ﺍﻝﺒﻴﺽ ﻤﻌﻨﻭﻴـﺎ ﻓـﻰ ﺍﻹﻨـﺎﺙ ﺍﻝﻤﻌﺎﻤﻠـﺔ ﺒﺎﻝﺠﺭﻋـﺔ ١٢٥ ﺠﺭﺍﻯ ﻓﻰ ﺤﻴﻥ ﺍﻨﺨﻔ ﻀﺕ ﻨﺴﺒﺔ ﻓﻘﺱ ﺍﻝﺒﻴﺽ ﺒﺸـﻜل ﻤﻌﻨـﻭﻯ ﻨﺘﻴﺠـﺔ ﺍﻹﺸﻌﺎﻉ. ٤- ﻴﻝﻡ ﺘﺄﺜﺭ ﻤﺘﻭﺴﻁ ﻋﺩﺩ ﻤﺭﺍﺕ ﺘﺯﺍﻭﺝ ﺍﻹﻨﺎﺙ ﺍﻝﻤﻌﺎﻤﻠﺔ ﺒﺎﻝﺠﺭﻋـﺔ ١٢٥ ﺠـﺭﺍﻯ ﻤﻘﺎﺭﻨﺔ ﺒﺎﻹﻨﺎﺙ ﺍﻝﻁﺒﻴﻌﻴﺔ. ٥- ﺘﻤﻜﻨﺕ ﺍﻹﻨﺎﺙ ﺍﻝﻤﻌﺎﻤﻠﺔ ﺒﺎﻝﺠﺭﻋﺔ ١٢٥ ﺠﺭﺍﻯ ﻤﻥ ﺠﺫﺏ ﺍﻝﺫﻜﻭﺭ ﻭﺒﺩﺀ ﺍﻝﺘﺯﺍﻭﺝ ﻤﻌﻬﺎ ﺨﻼل ﻨﻔﺱ ﺍﻝﻔﺘﺭﺓ ﻤﻥ ﺍﻝﻠﻴل ﺍﻝﺘﻲ ﺘﻨﻁﻠﻕ ﻓﻴﻬﺎ ﺍﻹﻨﺎﺙ ﺍﻝﻐﻴﺭ ﻤ ﻌﺎﻤﻠﺔ . . ﺏ - ﺘﺄﺜﻴﺭ ﺍﻝﺠﺭﻋﺔ ﺘﺤﺕ ﺍﻝﻤﻌﻘﻤـﺔ ١٢٥ ﺠـﺭﺍﻯ ﻋﻠـﻰ ﺒﻌـﺽ ﺍﻝﻨـﻭﺍﺤﻰ ﺍﻝﺒﻴﻭﻝﻭﺠﻴﺔ ﻓﻰ ﺠﻴﻠﻲ ﺍﻷﺒﺎﺀ ﻭﺍﻷﺒﻨﺎﺀ . . ﺃﻭﻀﺤﺕ ﺍﻝﻨﺘﺎﺌﺞ ﻤﺎ ﻴﻠﻰ -: ١- ﻝﻡ ﻴﺅﺜﺭ ﺍﻻﺸﻌﺎﻉ ﺒﻭﻀﻭﺡ ﻋﻠﻰ ﺨﺭﻭﺝ ﺍﻝﻔﺭﺍﺸﺎﺕ ﻭﺯﺍﺩﺕ ﻨﺴﺒﺔ ﺍﻝﺘﺸﻭﻩ ﻓ ﻴﻬـﺎ ﻤﻘﺎﺭﻨﺔ ﺒﺎﻝﻜﻨﺘﺭﻭل ﺒﻴﻨﻤﺎ ﺍﻨﺨﻔﻀﺕ ﻤﺩﺓ ﺤﻴﺎﺓ ﺍﻝﻔﺭﺍﺸـﺎﺕ ﺍﻨﺨﻔـﺎﺽ ﺒﺴـﻴﻁ ٠ ٠ ﻭﻝﻭﺤﻅ ﺍﻥ ﺍﻹﻨﺎ ﺙ ﺃﻜﺜﺭ ﺤﺴﺎﺴﻴﺔ ﻝﻺﺸﻌﺎﻉ ﻤﻥ ﺍﻝﺫﻜﻭﺭ. ٢- ﻋﻨﺩ ﻤﻌﺎﻤﻠﺔ ﺍﻝﺫﻜﻭﺭ ﺒﺎﻝﺠﺭﻋﺔ ١٢٥ ﺠﺭﺍﻯ ﻭﺘﺭﻜﻬﺎ ﻝﻠﺘﻨﺎﻓﺱ ﻋﻠﻰ ﺍﻝﺘﺯﺍﻭﺝ ﻤـﻊ ﺫﻜﻭﺭ ﻏﻴﺭ ﻤﻌﺎﻤﻠﺔ ﺃﻨﻬﺎ ﻜﺎﻨﺕ ﻜﺎﻤﻠﺔ ﺍﻝﺘﻨﺎﻓﺱ ﻋﻨﺩ ﻜل ﻨﺴﺏ ﺍﻹﻁﻼﻕ . . ٣- ﺃﺩﺕ ﺍﻝﻤﻌﺎﻤﻠﺔ ﺒﺎﻝﺠﺭﻋﺔ ١٢٥ ﺠﺭﺍﻯ ﺍﻝﻰ ﺍﻁﺎﻝﺔ ﻤﺩﺓ ﺍﻝﺘﻁﻭﺭ ﺒﺸـﻜل ﻤﻌﻨـﻭﻯ ﺨﺼﻭﺼﺎﹰ ﺨﻼل ﺍﻝﺠﻴل ﺍﻷﻭل ﻭﺍﻝﺠﻴل ﺍﻝﺜﺎﻨﻰ . . ٤- ﺃﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﺯﻴﺎﺩﺓ ﻋﺩﻡ ﺘﺄﺜﺭ ﻨﺴﺒﺔ ﺍﻝﺘﻌﺫﻴﺭ ﻓﻰ ﺍﻝﺠﻴل ﺍﻷﻭل ﻭﺍﻝﺠﻴل ﺍﻝﺜـﺎﻨﻰ ﻭﻗﻠﺕ ﻨﺴﺒﺔ ﺨﺭﻭﺝ ﺍﻝﻔﺭﺍﺸﺎﺕ ﻓﻰ ﺍﻝﺠﻴل ﺍﻷﻭل ﻭﻗﺩ ﺯﺍﺩ ﻫﺫﺍ ﺍﻻﻨﺨﻔـﺎﺽ ﻓـﻰ ﺍﻝﺠﻴل ﺍﻝﺜﺎﻨﻰ . . ٥- ﻝﻭﺤﻅ ﺍﻨﺨﻔﺎﺽ ﺩﻝﻴل ﺍﻝﻨﻤﻭ ﻓﻰ ﺤﺸﺭﺍﺕ ﺍﻝﺠﻴل ﺍﻷﻭل ﻭﺤﺸ ﺭﺍﺕ ﺍﻝﺠﻴل ﺍﻝﺜـﺎﻨﻰ ﻨﺘﻴﺠﺔ ﺍﻝﻤﻌﺎﻤﻠﺔ.
ﺍﻝﻤﻠﺨﺹ ﺍﻝﻌﺭﺒﻲ -٣- ٦- ﻝﻡ ﺘﺘﺄﺜﺭ ﺍﻝﻨﺴﺒﺔ ﺍﻝﺠﻨﺴﻴﺔ ﺤﻴﺙ ﺒﻘﻴﺕ ﺸﺒﻪ ﻋﺎﺩﻴﺔ ﻓﻰ ﺤﺸـﺭﺍﺕ ﺍﻝﺠﻴـل ﺍﻷﻭل ﻭﺤﺸﺭﺍﺕ ﺍﻝﺠﻴل ﺍﻝﺜﺎﻨﻰ . . ٧- ﻝﻡ ﺘﺘﺄﺜﺭ ﻨﺴﺒﺔ ﻨﺠﺎﺡ ﺍﻝﺫﻜﻭﺭ ﻓﻰ ﺍﻝﺘﺯﺍﻭﺝ ﻤﻊ ﺍﻹﻨﺎﺙ ﺨﻼل ﺍﻷﺠﻴﺎل ﺍﻝﺜﻼﺜﺔ ﻭﻗﺩ ﺍﻨﺨﻔﻀﺕ ﻨﺴﺒﺔ ﺘﻜﺭﺍﺭ ﺘﺯﺍﻭﺝ ﻫﺫﻩ ﺍﻝﺫﻜ ﻭﺭ ﻓﻰ ﺍﻝﺠﻴل ﺍﻷﻭل ﻭﺍﻝﺠﻴل ﺍﻝﺜﺎﻨﻰ. ٨- ﺃﻅﻬﺭﺕ ﺍﻝﻤﻌﺎﻤﻠﺔ ﺒﺎﻝﺠﺭﻋﺔ ١٢٥ ﺠﺭﺍﻯ ﻝﻠﺫ ﻜﻭﺭ ﺍﻻﺒﺎﺀ ﺍﻨﺨﻔﺎﺽ ﻤﻌﻨﻭﻯ ﻝﻜـل ﻤﻥ ﻤﻌﺩل ﻭﻀﻊ ﺍﻝﺒﻴﺽ ﻭﻜﺫﻝﻙ ﻨﺴﺒﺔ ﻓﻘﺱ ﺍﻝﺒﻴﺽ ﺨﻼل ﺍﻷﺠﻴﺎل ﺍﻝﺜﻼﺜﺔ. ٩- ﺃﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﺍﻥ ﺍﻝﺘﺄﺜﻴﺭ ﻓﻰ ﺍﻝﺠﻴل ﺍﻷﻭل ﻜﺎﻥ ﻤﻌﻨﻭﻴـﺎ ﻤﻘﺎﺭﻨـﺔ ﺒﺎﻝﺠﻴـل ﺍﻝﺜﺎﻨﻰ ﻭﺇﻥ ﻅل ﺍﻝﺘﺄﺜﻴﺭ ﻓﻰ ﺍﻝﺠﻴل ﺍﻝﺜﺎ ﻨﻰ ﺍﻜﺒﺭ ﻤﻥ ﺍﻻﺒﺎﺀ ﺍﻝﻤﺸﻌﻌﺔ ﻨﻔﺴﻬﺎ. ١٠- ﺃﻨﺨﻔﺽ ﺇﻨﺘﺎﺝ ﺍﻹﻨﺎﺙ ﺍﻝﺒﺎﻝﻐﺔ ﻭﺍﻝﻨ ﺎﺘﺠﺔ ﻤﻥ ﺍﻻﺒﺎﺀ ﺍﻝﻤﻌﺎﻤﻠﺔ ﻭﻝﻜﻥ ﺇﻨﺨﻔـﺎﺽ ﺍﻹﻨﺎﺙ ﺍﻝﺒﺎﻝﻐﺔ ﻭﺍﻝﻨﺎﺘﺠﺔ ﻤﻥ ﺍﻝﺠﻴل ﺍﻻﻭل ﺍﻜﺜﺭ ﻭﻀﻭﺤﺎ. ١١- ﻋﻠﻰ ﺍﻝﺭﻏﻡ ﻤﻥ ﻋﺩﻡ ﺘﺄﺜﺭ ﻋﺩﺩ ﺤﺯﻡ ﺍﻝﺤﻴﻭﺍﻨـﺎﺕ ﺍﻝﻤﻨﻭﻴـﺔ ﺍﻝﻤﺘﻭﺍﺠـﺩﺓ ﻓـﻰ ﺍﻝﻘﻨﻭﺍﺕ ﺍﻝﺘﻨﺎﺴﻠﻴﺔ ﻝﻠﺫﻜﺭ ﻓﻰ ﺍﻝﻠﻴﻠﺔ ﺍﻻﻭﻝﻰ ﻤﻥ ﺍﻝﺨـﺭﻭﺝ ﺇﻻ ﺍﻥ ﺤﺠـﻡ ﺍﻝﻐـﺩﺩ ﺍﻝﺘﻨﺎﺴﻠﻴﺔ ﺍﻨﺨﻔﺽ ﺒﺸﻜل ﻤﻌﻨﻭﻯ ﺨﺎﺼﺔ ﻓﻰ ﺍﻷﺒﺎ ﺀ ﺍﻝﻤﻌﺎﻤﻠﺔ . ١٢- ﺍﻨﺨﻔﺽ ﻋﺩﺩ ﺤﺯﻡ ﺍﻝﺤﻴﻭﺍﻨﺎﺕ ﺍﻝﻤﻨﻭﻴﺔ ﺍﻝﻤﺘﺭﺍﻜﻤﺔ ﻓﻰ ﺍﻝﻘﻨﻭﺍﺕ ﺍﻝﺘﻨﺎﺴﻠﻴﺔ ﻝﻠﺫﻜﺭ ﻓﻰ ﺍﻝﻠﻴﻠﺔ ﺍﻝﺭﺍﺒﻌﺔ ﻤﻥ ﺍﻝﺨﺭﻭﺝ ﻓﻰ ﺍﻝﺠﻴل ﺍﻷﻭل ﺒﺸﻜل ﻤﻌﻨﻭﻯ . ﺜﺎﻝﺜﺎ: ﺘﺄﺜﻴﺭ ﺍﻝﻨﻴﻤﺎﺘﻭﺩﺍ ﺍﻝﻤﻤﺭﻀﺔ ﻝﻠﺤﺸﺭﺍﺕ ﻋﻠﻰ ﺍﻝﺩﻭﺩﺓ ﺍﻝﻘﺎﺭﻀﺔ ١- ﺃﻅﻬﺭ ﻜـل ﻤـﻥ ﺍﻝﻨـﻭﻉ Steinernema carpocapsae All ﻭﺍﻝﻨـﻭﻉ Steinernema riobrave ﻜﻔﺎﺀﺓ ﻀﺩ ﺍﻝﺤﺸﺭﺓ. ٢- ﺃﺩﺕ ﺍﻝﺯﻴﺎﺩﺓ ﻓﻰ ﺍﻝﺘﺭﻜﻴﺯﺍﺕ ﺍﻝﻤﺴﺘﺨﺩﻤﺔ ﺇﻝﻰ ﺯﻴﺎﺩﺓ ﻨﺴﺏ ﺍﻝﻤﻭ ﺕ ﺍﻝﻜﻠﻴﺔ ﺒﺸـﻜل ﻤﻌﻨﻭﻯ ﻭﺇﻝﻰ ﺇﻨﺨﻔﺎﺽ ﻜل ﻤﻥ ﻨﺴﺒﺔ ﺨﺭﻭﺝ ﺍﻝﻔﺭﺍﺸﺎﺕ ﻓﻰ ﺍﻝﺤﺸﺭﺍﺕ ﺍﻝﻤﻌﺎﻤﻠﺔ ﻭﻨﺴﺒﺔ ﺒﻘﺎﺀ ﺍﻝﻴﺭﻗﺎﺕ ﺍﻝﻤﻌﺎﻤﻠﺔ ﺤﺘﻰ ﺍﻝﻁﻭﺭ ﺍﻝﺒﺎﻝﻎ . . ٣- ﺃﻅﻬﺭﺕ ﻗﻴﻤﺔ ﻤﻌﺎﻤل ﺍﻹﺭﺘﺒﺎﻁ ﻭﺠﻭﺩ ﻋﻼﻗﺔ ﻗﻭﻴﺔ ﺒﻴﻥ ﻨﺴﺏ ﺍﻝﻤـﻭﺕ ﺍﻝﻨﺎﺘﺠـﺔ ﻭﺍﻝﺘﺭﻜﻴﺯﺍﺕ ﺍﻝﻤﺴﺘﺨﺩﻤﺔ . .
ﺍﻝﻤﻠﺨﺹ ﺍﻝﻌﺭﺒﻲ -٤- ٤- ﺃﻭﻀﺤﺕ ﻗﻴﻡ LT 50 ﺍﻥ ﺍﻝﻨﻭﻉ ﺸﺘﻴﻨﺭﻨﻴﻤﺎ ﻜﺎﺭﺒﻭﻜﺒﺴﺎ ﺃﺴـﺭﻉ ﻓـﻲ ﺇﺤـﺩﺍﺙ ﺍﻝﻌﺩﻭﻯ ﻭﺍﻝﻤﻭﺕ ﻝﻠﺤﺸﺭﺓ ﻤﻥ ﻭﺍﻝﻨﻭﻉ ﺸﺘﻴﻨﺭﻨﻴﻤﺎ ﺭﺍﻴﻭﺒﺭﻴﻔﺎ. ٥- ﺃﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﻭﺠﻭﺩ ﻋﻼﻗﺔ ﺒﻴﻥ ﺯﻴﺎﺩﺓ ﻨﺴﺏ ﺍﻝﻤﻭﺕ ﻝﻠﺤﺸﺭﺓ ﻭﺯﻴـﺎﺩﺓ ﻨﺴـﺏ ﺍﻝﺘﺸﻭﻫﺎﺕ ﺍﻝﻨﺎﺘﺠﺔ ﻭﻁﻭل ﺍﻝﻤﺩﺓ ﺍﻝﺘﻰ ﺘﻌ ﺭﺽ ﻝﻬﺎ ﺍﻝﻴﺭﻗﺎﺕ ﻝﻠﻨﻴﻤﺎﺘﻭﺩﺍ ﺸﺘﻴﻨﺭﻨﻴﻤﺎ ﻜﺎﺭﺒﻭﻜﺒﺴﺎ. ٦- ﺃﺩﻯ ﻁﻭل ﺍﻝﻤﺩﺓ ﺍﻝﺘﻰ ﺘﻌﺭﺽ ﻝﻬﺎ ﺍﻝﻴﺭﻗﺎﺕ ﻝﻠﻨﻴﻤﺎﺘﻭﺩﺍ ﺸـﺘﻴﻨﺭﻨﻴﻤﺎ ﻜﺎﺭﺒﻭﻜﺒﺴـﺎ ﺇﻝﻰ ﺇﻨﺨﻔﺎﺽ ﻜل ﻤﻥ ﻨﺴﺒﺔ ﺘﻌﻴﺭ ﻫﺫﻩ ﺍﻝﻴﺭﻗﺎﺕ ﻭﻨﺴﺒﺔ ﺨﺭﻭﺝ ﺍﻝﻔﺭﺍﺸﺎﺕ. ٧- ﻗﻠﺕ ﻗﺩﺭﺓ ﺍﻝﻔﺭﺍﺸﺎﺕ ﺍﻝﻨﺎﺘﺠﺔ ﻤﻥ ﻴﺭﻗـﺎﺕ ﻤﻌﺭﻀـﺔ ﻝﻠﻨﻴﻤـﺎﺘﻭﺩﺍ ﺸـﺘﻴﻨﺭﻨﻴﻤﺎ ﻜﺎﺭﺒﻭﻜﺒﺴﺎ ﻋﻠﻰ ﺍﻝﺘﺯﺍ ﻭﺝ ﻭﻜﺫﻝﻙ ﻨﺴﺒﺔ ﺍﻝﻔﺭﺍﺸﺎﺕ ﺍﻝﻤﺘﺯﺍﻭﺠﺔ ﻭﺍﻝﺤﺎﻭﻴﺔ ﻋﻠﻰ ﺃﻯ ﻤﻥ ﺍﻝﺤﺎﻤل ﺍﻝﻤﻨﻭﻯ ﺍﻭ ﺍﻝﺤﻴﻭﺍﻨﺎﺕ ﺍﻝﻤﻨﻭﻴﺔ ٠ ﻭﻜﺎﻥ ﺍﻝﺘﺄﺜﺭ ﻓﻰ ﺍﻝﻔﺭﺍﺸﺎﺕ ﺍﻝﺫﻜﻭﺭ ﺃﻭﻀﺢ ﻤﻥ ﺍﻝﻔﺭﺍﺸﺎﺕ ﺍﻹﻨﺎﺙ. ٨- ﺃﺩﺕ ﺍﻝﻤﻌﺎﻤﻠﺔ ﺇﻝﻰ ﺍﻨﺨﻔﺎﺽ ﻜل ﻤﻥ ﻤﻌﺩل ﻭﻀﻊ ﺍﻝﺒﻴﺽ ﻭﻜﺫﻝﻙ ﻨﺴـﺒﺔ ﻓﻘـﺱ ﺍﻝﺒﻴﺽ ﺍﻝﻔﺭﺍﺸﺎﺕ ﺒﺸﻜل ﻤﻌﻨﻭﻱ. ٩- ﺃﺜﺭﺕ ﻁﺭﻴﻘﺘﻰ ﺍﻝﻤﻌﺎﻤﻠﺔ ﺒﺎﻝ ﻨﻴﻤﺎﺘﻭﺩﺍ ﺸﺘﻴﻨﺭﻨﻴﻤﺎ ﻜﺎﺭﺒﻭﻜﺒﺴﺎ ﺘﺄﺜﻴﺭﺍ ﻭﺍﻀﺤﺎ ﻋﻠـﻰ ﻨﺴﺒﺔ ﺍﻝﻤﻭﺕ ﺍﻝﻜﻠﻴﺔ ﻓﻰ ﺍﻝﻴﺭﻗﺎﺕ ﻤﻤﺎ ﺃﺩﻯ ﻹﺭﺘﻔﺎﻉ ﺍﻝﻨﻘﺹ ﻓﻰ ﺍﻝﺠﻴل ﺍﻝﺘﺎﻝﻰ. ﺭﺍﺒﻌﺎ : ﺍﻝ ﺘﺄﺜﻴﺭ ﺍﻝﻤﺸﺘﺭﻙ ﻝﻜل ﻤﻥ ﺍﻝﻤﻌﺎﻤﻠﺔ ﺒﺎﻝﺠﺭﻋﺔ ﺘﺤﺕ ﺍﻝﻤﻌﻘﻤﺔ ﻭﻨﻴﻤـﺎﺘﻭﺩﺍ ﺸﺘﻴﻨﺭﻨﻴﻤﺎ ﻜﺎﺭﺒﻭﻜﺒﺴﺎ . . ﺃﻭﻀﺤﺕ ﺍﻝﻨﺘﺎﺌﺞ ١- ﺃﻥ ﺍﻝﻨﺴل ﺍﻝﻨﺎﺘﺞ ﻤﻥ ﺘﺯﺍﻭﺝ ﺃﺒ ﺎﺀ ﻤﻌﺎﻤﻠﺔ ﺒﺎﻝﺠﺭﻋﺔ ١٢٥ ﺠﺭﺍﻯ ﻭﺇﻨـﺎﺙ ﻏﻴـﺭ ﻤﻌﺎﻤﻠﺔ ﺃﻜﺜﺭ ﺤﺴﺎﺴﻴﺔ ﻝﻺﺼﺎﺒﺔ ﺒﺎﻝﻨﻴﻤﺎﺘﻭﺩﺍ ﺸﺘﻴﻨﺭﻨﻴﻤﺎ ﻜﺎﺭﺒﻭﻜﺒﺴﺎ ﻤﻥ ﺍﻝﻨﺴـل ﺍﻝﻨﺎﺘﺞ ﻤﻥ ﺫﻜﻭﺭ ﻏﻴﺭ ﻤﻌﺎ ﻤﻠﺔ ﻭﺫﻝﻙ ﻋﻨﺩ ﺍﻝﺘﺭﻜﻴﺯﺍﺕ ﺍﻝﻤﻨﺨﻔﻀﺔ . . ٢- ﺃﻥ ﺍﻝﺘﺭﻜﻴﺯﺍﺕ ﺍﻝﻤﺭﺘﻔﻌﺔ ﻜﺎﻥ ﻝﻬﻤﺎ ﻨ ﻔﺱ ﺍﻝﺤﺴﺎﺴﻴﺔ ﺍﻝﻌﺎﻝﻴﺔ ﻝﻠﻨﻴﻤﺎﺘﻭﺩﺍ . .
ﺍﻝﻤﻠﺨﺹ ﺍﻝﻌﺭﺒﻲ -٥- ٣- ﺃﻥ ﺍﻝﺘﺄﺜﻴﺭ ﺇﻀﺎﻓﻲ ﻻﺸﺘﺭﺍﻙ ﺍﻝﻤﻌﺎﻤ ﻠﺘﻴﻥ ﻋﻠﻰ ﺍﻝﺤﺸﺭﺓ ﻭﻝـﻡ ﻴﻼﺤـﻅ ﺍﻝﺘـﺄﺜﻴﺭ ﺍﻝﻤﻨﺸﻁ ﺃﻭ ﺍﻝﻤﺘﻀﺎﺩ . . ٤- ﺃ ﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﻋﺩﻡ ﻭﺠﻭﺩ ﺘﺄﺜﺭ ﻤﻌﻨﻭﻱ ﻤﻥ ﻗﺒل ﻴﺭﻗﺎﺕ ﺍﻝﻨﺴل ﺍﻝﻨـﺎﺘﺞ ﻤـﻥ ﺘﺯﺍﻭﺝ ﺃﺒﺎﺀ ﻤﻌﺎﻤﻠﺔ ﺒﺎﻝﺠﺭﻋﺔ ١٢٥ ﺠﺭﺍﻯ ﻭﺇﻨﺎﺙ ﻏﻴﺭ ﻤﻌﺎﻤﻠﺔ ﺘﺠـﺎﻩ ﺍﻝﻌـﺩﻭﻯ ﺒﺎﻝﻨﻴﻤﺎﺘﻭﺩﺍ ﺤﻴﺙ ﻝﻡ ﺘﺨﺘﻠﻑ ﻨﺴﺒﺔ ﺍﻝﺘﻁﻔل ﻭﺍﻝﻤﻭﺕ ( ﺍﻝﻭﻗـﺕ ﺍﻝـﻼﺯﻡ ﻝﻅﻬـﻭﺭ ﺍﻷﻋﺭﺍﺽ ﻭﺍﻝﻭﻗ ﺕ ﺍﻝﻼﺯﻡ ﻝﻤﻭﺕ ﺍﻝﻴﺭﻗﺎﺕ ﺍﻝﻤﺼﺎﺒﺔ ) ﻋﻨﻬﺎ ﻓـﻰ ﻨﺴـل ﺍﻷﺒـﺎﺀ ﺍﻝﻁﺒﻴﻌﻴﺔ . ٥- ﺃﻥ ﻭﻗﺕ ﺤﻀﺎﻨﺔ ﺍﻝﻁﻔﻴل ﺩﺍﺨل ﺍﻝﻴﺭﻗﺎﺕ ﺍﻝﻨﺎﺘﺠﺔ ﻤﻥ ﺃﺒﺎﺀ ﻤﺸﻌﻌﺔ ﺯﺍﺩﺕ ﺒﺸـﻜل ﻤﻌﻨﻭﻯ ﻋﻨﻬﺎ ﻓﻰ ﺍﻝﻴﺭﻗﺎﺕ ﺍﻝﻁﺒﻴﻌﻴﺔ . ٦- ﺃﻥ ﻴﺭﻗﺎﺕ ﺍﻝﻁﻔﻴل ﺍﻝﻤﺘﺤﺼل ﻋﻠﻴﻬﺎ ( ﺍﻝﺤﺼﺎﺩ ) ﻗل ﺒﺸﻜل ﻤﻌﻨﻭﻯ ﻓﻰ ﻴﺭﻗـﺎﺕ ﺍﻝﻨﺴل ﺍﻝﻨﺎﺘﺞ ﻤﻥ ﺘﺯﺍﻭﺝ ﺃﺒﺎﺀ ﻤﻌﺎﻤﻠﺔ ﺒﺎﻝﺠﺭﻋﺔ ١٢٥ ﺠﺭﺍﻯ ٠ ﻭﻜﺫﻝﻙ ﺘـﺄﺜﺭﺕ ﻓﺘﺭﺓ ﺍﻝﺤﺼﺎﺩ. ٧- ﺒﺎﻝﻨﺴﺒﺔ ﺍﻝﻰ ﻴﺭﻗﺎﺕ ﺍﻝﻁﻔﻴل ﺍﻝﻤﺘﺤﺼل ﻋﻠﻴﻬﺎ ( ﺍﻝﺤﺼﺎﺩ ) ﻤﻥ ﻴﺭﻗـﺎﺕ ﺍﻝـﺩﻭﺩﺓ ﺍﻝﻘﺎﺭﻀﺔ ﺍﻝﻨﺎﺘﺠﺔ ﻤﻥ ﺃﺒﺎﺀ ﻤﻌﺎﻤﻠﺔ ﺒﺎﻝﺠﺭﻋﺔ ١٢٥ ﺠﺭﺍﻯ ﻻ ﻴﻭﺠﺩ ﺩﻝﻴل ﻋﻠﻰ ﺍﻥ ﺘ ﺄﺜﻴﺭﻫﺎ ﻤﻨﺤﺎﺯ ﻝﻨﻭﻉ ﻤﻌﻴﻥ ﻤﻥ ﺍﻝﻨﺴل. ٨- ﺃﻥ ﻓﺘﺭﺓ ﺤﻀﺎﻨﺔ ﻫﺫﻩ ﺍﻝﻴﺭﻗﺎﺕ ﺩﺍﺨل ﺍﻝﻌﺎﺌل ﺍﻝﻨﺎﺘﺞ ﻤ ﻥ ﺃﺒﺎﺀ ﻤﻌﺎﻤﻠـﺔ ﺒﺎﻝﺠﺭﻋـﺔ ١٢٥ ﺠﺭﺍﻯ ﺍﻁﻭل ﻤﻨﻬﺎ ﺩﺍ ﺨل ﺍﻝﻌﺎﺌل ﺍﻝﻨﺎﺘﺞ ﻤﻥ ﺃﺒﺎﺀ ﻁﺒﻴﻌﻴﺔ. ٩- ﺃﻥ ﻜﻔﺎﺀﺓ ﺍﻝﺤﺼﺎﺩ ﺘﻌﺘﻤﺩ ﻋﻠﻰ ﻤﻌﺎﻤﻠﺔ ﺍﻻﺒﺎﺀ ﺒﺎﻻﺸﻌﺎﻉ ﺴﻭﺍﺀ ﺍﻝﻴﺭﻗـﺎﺕ ﺍﻝﺘـﻰ ﺘﻨﻤﻰ ﺒﻬﺎ ﻴﺭﻗﺎﺕ ﺍﻝﻁﻔﻴل ﺍﻭ ﺍﻝﻴﺭﻗﺎﺕ ﺍﻝﻤﺘﻌﺭﻀﺔ ﻝﻺﺼﺎﺒﺔ ﺒﻪ.
ﺍﻝﻤﻠﺨﺹ ﺍﻝﻌﺭﺒﻲ -٦- ﺍﻝﻤﺴﺘﺨﻠﺹ
ﺘﻨﺎﻭﻝﺕ ﺍﻝﺩﺭﺍﺴﺔ ﺍﻝﺤﺎﻝﻴﺔ ﺍﺨﺘﺒﺎ ﺭ ﺍﻝﻌﻘﻡ ﻓﻲ ﺠﻴل ﺍﻵﺒﺎﺀ ﺍﻝﺫﻜﻭﺭ ﻝﺤﺸﺭﺓ ﺍﻝـﺩﻭﺩﺓ ﺍﻝﻘﺎﺭﻀﺔ ﺒﻌﺩ ﺘﻌﺭﻴﺽ ﺍﻝﻌﺫﺍﺭﻯ ﻜﺎﻤﻠﺔ ﺍﻝﻨﻤـﻭ ﻝ ﻠﺠﺭﻋـﺎﺕ ( ٧٥ ، ١٠٠ ، ١٢٥ ، ١٥٠ ١٧٥، ، ٢٠٠ﻭ ﺠﺭﺍﻯ ) ﻤﻥ ﺃﺸﻌﺔ ﺠﺎﻤﺎ ﻭ ﺫﻝﻙ ﺒﻬﺩﻑ ﺍﻝﺘﻌﺭﻑ ﻋﻠﻰ ﺍﻝﺠﺭﻋـﺔ ﺍﻝﻤﺴـﺒﺒﺔ ﻷﻋﻠﻰ ﻨﺴﺒﺔ ﻋﻘﻡ ﺠﺯﺌﻲ ﻝﻶﺒﺎﺀ ﺜﻡ ﺍﻝﻭﺼﻭل ﻝﻠﻌﻘﻡ ﺍﻝﻜ ﺎﻤل ﻓﻲ ﺍﻝﻨﺴل ﺍﻝﻨﺎﺘﺞ . ﻭﺍﺸﺘﻤﻠﺕ ﺍﻝﺩﺭﺍﺴﺔ ﻋﻠﻰ ﻤﻼﺤﻅﺔ ﺍﻝﺘﺄﺜﻴﺭﺍﺕ ﻋﻠﻰ ﻨﺴﺒﺔ ﺍﻝﺫﻜﻭﺭ ﺍﻝﻤﺘﺯﺍﻭﺠﺔ ﻤﻊ ﺍﻹﻨـﺎﺙ ، ﻤـﺩﺓ ﺍﻝﺘﺯﺍﻭﺝ ﺍﻝﻼﺯﻤﺔ ﻝﺘﻜﻭﻴﻥ ﺍﻝﺤﺎﻤل ﺍﻝﻤﻨﻭﻱ ، ﻋﺩﺩ ﺍﻝﺒﻴﺽ ﻝﻺﻨﺎﺙ ﺍﻝﻤﺘﺯﺍﻭﺠﺔ ﻤـﻊ ﻫـﺫﻩ ﺍﻝﺫﻜﻭﺭ ﻭﻜﺫﻝﻙ ﻨﺴﺒﺔ ﻓﻘﺱ ﻫﺫﺍ ﺍﻝﺒﻴﺽ ﻭﺫﻝﻙ ﺨﻼل ﺍﻹﻨﺎﺙ ﺍﻝﺜﻼﺜـﺔ ﺍﻝﻤﺘﺘﺎﻝﻴـﺔ . ﻜﻤـﺎ ﺍﺨﺘﺒﺭﺕ ﺍﻝﺫ ﻜﻭﺭ ﺍﻝﻤﻌﺎﻤﻠﺔ ﺒﺎﻝﺠﺭﻋﺎﺕ ﺍﻝﻤﺨﺘﺒﺭﺓ ﻤﻥ ﺃﺠل ﺩﺭﺍﺴﺔ ﺍﻝﺘﺄﺜﻴﺭ ﻋﻠـﻰ ﺘﻌﺎﻗـﺏ ﺘﺯﺍﻭﺝ ﺍﻝﺫﻜﻭﺭ ﻭﺩﺭﺍﺴﺔ ﺍﻝﺘﺄﺜﻴﺭ ﻋﻠﻰ ﺒﺩﺀ ﺘﺯﺍﻭﺝ ﻫﺫﻩ ﺍﻝﺫﻜﻭﺭ ﺨﻼل ﻨﻔﺱ ﺍﻝﺠﺯﺀ ﻤـﻥ ﺍﻝﻠﻴﻠﺔ ﻤﻘﺎﺭﻨﺔ ﺒﺎﻝﻜﻨﺘﺭﻭل . ﻭﻗﺩ ﺸﻤﻠﺕ ﺍﻝﺩﺭﺍﺴﺔ ﺘﺄﺜﻴﺭ ﺍﻝﻤﻌﺎﻤﻼﺕ ﺍﻝﻤﺨﺘﻠﻔﺔ ﻋﻠﻰ ﺍﻝﺘﻨﺎﻓﺱ ﺍﻝﺘﺯﺍﻭﺠﻰ ﻓﻰ ﺍﻝﺫﻜﻭﺭ ﺍﻝﻤﻌﺎﻤﻠﺔ . ﻭﺘﻀﻤﻨﺕ ﺍﻝﺩﺭﺍﺴﺔ ﺍﺨﺘﺒﺎﺭ ﺍﻝﺠﺭﻋﺔ ﺘﺤﺕ ﻤﻌﻘﻤﺔ ١٢٥ ﺠﺭﺍﻯ ﻋﻠﻰ ﺍﻝﻨﻭﺍﺤﻰ ﺍﻝﺒﻴﻭﻝﻭﺠﻴﺔ ﻭﺍﻝﺘﻜﺎﺜﺭ ﻓﻰ ﺠﻴل ﺍﻵﺒﺎﺀ ﻭﺍﻝﺠﻴﻠﻴـﻴﻥ ﺍﻝﺘـﺎﻝﻴﻴﻥ ﻝـﻪ . ﻭﺍﺤﺘــﻭﺕ ﺍﻝﺩﺭﺍﺴــﺔ ﻋﻠــﻰ ﺩﺭﺍﺴــﺔ ﺩﻭﺭ ﻨــﻭﻋﻴﻥ ﻤــﻥ ﺍﻝﻨﻴﻤــﺎﺘﻭﺩﺍ ﺍﻝﻤﻤﺭﻀــﺔ ﻝﻠﺤﺸﺭﺍﺕ( ﺸﺘﻴﻨﺭﻨﻴﻤﺎ ﻜﺭﺒﻭﻜﺎﺒﺴﺎ ﻭﺸﺘﻴﻨﺭﻨﻴﻤﺎ ﺭﺍﻴﻭﺒﺭﻴﻔﺎ ﻓﻰ ﻤﻜﺎﻓﺤﺔ ﺍﻻﻓـﺔ . ﻭﺃﻫﺘﻤـﺕ ﺍﻝﺩﺭﺍﺴﺔ ﺒﺼﻔﻪ ﺨﺎﺼﺔ ﺒﺎﻝﺘﺄﺜﻴﺭ ﺍﻝﻤﺸﺘﺭﻙ ﻝ ﻠﻌﻘﻡ ﺍﻝﺠﺯﺌﻰ ﻭﺸﺘﻴﻨﺭﻨﻴﻤﺎ ﻜﺭﺒﻭﻜﺎﺒﺴﺎ ﻋﻠـﻰ ﺍﻝﺩﻭﺩﺓ ﺍﻝﻘﺎ ﺭﻀﺔ ﻤﻊ ﺘﺤﻠﻴل ﺍﻝﺘﺄﺜﻴﺭ ﺍﻝﻤﻨﺸﻁ ﺍﻝﻤﻌﺎﻤﻠﺘﻴﻥ ﻤﻌﺎ ﻋﻨﺩ ﻜل ﺍﻝﺘﺭﻜﻴﺯﺍﺕ . ﻭﺘﻤﺕ ﻤﻘﺎﺭﻨﺔ ﺍﻝﻘﻴﺎﺴﺎﺕ ( ﻜﻔﺎﺀﺓ ﺍﻝﺘﻁﻔل ، ﺍﻝﻭﻗﺕ ﺍﻝﻼﺯﻡ ﻝﻺﺤﺘﻀﺎﺭ ﻭﺍﻝﻤﻭﺕ ﻭﻭ ﻗﺕ ﺤﻀـﺎﻨﺔ ﺍﻝﻁﻔﻴل ﺩﺍﺨل ﺍﻝﻌﺎﺌل ) ﻝﻠﻨﻴﻤﺎﺘﻭﺩﺍ ﺍﻝﺘﻰ ﺘﻡ ﺤﺼ ﺎﺩﻫﺎ ﻤﻥ ﻋﺎﺌل ﻨﺎﺘﺞ ﻤﻥ ﺍﺒﺎﺀ ﻤﺸﻌﻌﺔ ﻤﻊ ﺘﻠﻙ ﺍﻝﻘﻴﺎﺴﺎﺕ ﻨﻔﺴﻬﺎ ﻝﻠﻨﻴﻤﺎﺘﻭﺩﺍ ﺍﻝﺘﻰ ﺘﻡ ﺤﺼﺎﺩﻫﺎ ﻤﻥ ﻋﺎﺌل ﻨﺎﺘﺞ ﻤﻥ ﺍﺒﺎﺀ ﻏﻴﺭ ﻤﻌﺎﻤﻠﺔ. أ وا دا ا ا ودة ا ر
ﺩﺭﺍﺴﺔ ﻤﻘﺩﻤﺔ ﻤﻥ ء ﺒﻜﺎﻝﻭﺭﻴﻭﺱ ﻋﻠﻭﻡ ﺯﺭﺍﻋﻴﺔ ( ﺤﺸﺭﺍﺕ ﺍﻗﺘﺼﺎﺩﻴﺔ ) ﺠﺎﻤﻌﺔ ﺍﻝﺯﻗﺎﺯﻴﻕ ( ١٩٨٧ ) ) ﻤﺎﺠﻴﺴﺘﻴﺭﻓﻰ ﺍﻝﻌﻠﻭﻡ ﺍﻝﺯﺭﺍﻋﻴﺔ ( ﺤﺸﺭﺍﺕ ﺇﻗﺘﺼﺎﺩﻴﺔ ) ﺠﺎﻤﻌﺔ ﺒﻨﻬﺎ ( ٢٠٠٤ ) )
ﻝﻠﺤﺼﻭل ﻋﻠﻰ ﺩﺭﺠﺔ ﺩﻜﺘﻭﺭﺍﺓ ﺍﻝﻔﻠﺴﻔﺔ ﻓﻰ ﺍﻝﻌﻠﻭﻡ ﺍﻝﺯﺭﺍﻋﻴﺔ ( ﺤﺸﺭﺍﺕ ﺍﻗﺘﺼﺎﺩﻴﺔ) ﻗﺴﻡ ﻭﻗﺎﻴﺔ ﺍﻝﻨ ﺒﺎﺕ ﻜﻠﻴﺔ ﺍﻝﺯﺭﺍﻋﺔ- ﻤﺸﺘﻬﺭ / ﺠﺎﻤﻌﺔ ﺒﻨﻬﺎ
א א :
د.أ ./ ت ج ا ط …………………… ...... ﺃﺴﺘﺎﺫ ﺍﻝﺤﺸﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻴﺔ- ﻗﺴﻡ ﻭﻗﺎﻴﺔ ﺍﻝﻨﺒﺎﺕ- ﻜﻠﻴﺔ ﺯﺭﺍﻋﺔ ﻤﺸﺘﻬﺭ - ﺠﺎﻤﻌﺔ ﻬﺎﺒﻨ ﻬﺎﺒﻨ
د.أ ./ ا ا ر …………………… ......
ﺃﺴﺘﺎﺫ ﺍﻝﺤﺸﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻴﺔ - ﻭﺭﺌﻴﺱ ﻗﺴﻡ ﺍﻝﺘﻁﺒﻴﻘﺎﺕ ﺍﻝﺒﻴﻭﻝﻭﺠﻴﺔ - ﻫﻴﺌﺔ ﺍﻝﻁﺎﻗﺔ ﺍﻝﺫﺭﻴﺔ . .
د ./ ء د وة …………………… ......
ﺃﺴﺘﺎﺫ ﻤﺴﺎﻋﺩ ﺍﻝﻤﺒﻴﺩﺍﺕ - ﻗﺴﻡ ﻭﻗﺎﻴﺔ ﺍﻝﻨﺒﺎﺕ- ﻜﻠﻴﺔ ﺯﺭﺍﻋﺔ ﻤﺸﺘﻬﺭ - ﺠﺎﻤﻌﺔ ﺒﻨﻬﺎ
٢٠١١ أ و ا دا ا ا ودة ا ر
ﺩﺭﺍﺴﺔ ﻤﻘﺩﻤﺔ ﻤﻥ ء ﺒﻜﺎﻝﻭﺭﻴﻭﺱ ﻋﻠﻭﻡ ﺯﺭﺍﻋﻴﺔ ( ﺤﺸﺭﺍﺕ ﺍﻗﺘﺼﺎﺩﻴﺔ ) ﺠﺎﻤﻌﺔ ﺍﻝﺯﻗﺎﺯﻴﻕ ( ١٩٨٧ ) ) ﻤﺎﺠ ﺴﺘﻴﺭ ﻓﻰ ﺍﻝﻌﻠﻭﻡ ﺍﻝﺯﺭﺍﻋﻴﺔ ( ﺤﺸﺭﺍﺕ ﺍﻗﺘﺼﺎﺩﻴﺔ ) ﺠﺎﻤﻌﺔ ﺒﻨﻬﺎ ( ٢٠٠٤ ) )
ﻝﺠﻨﺔ ﺍﻝﻤﻨﺎﻗﺸﺔ:
د.أ ./ ا …………………… ...... ﺃﺴﺘﺎﺫ ﺍﻝﺤﺸﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻴﺔ- ﻗﺴﻡ ﻭﻗﺎﻴﺔ ﺍﻝﻨﺒﺎﺕ- ﻜﻠﻴﺔ ﺍﻝﺯﺭﺍﻋﺔ- ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ . .
د.أ ./ ة آ ل ا إ م …………………… ...... ﺃﺴﺘﺎﺫ ﺍﻝﺤﺸﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻴﺔ - ﻭﺭﺌﻴﺱ ﻗﺴﻡ ﻭﻗﺎﻴﺔ ﺍﻝﻨﺒﺎﺕ- ﻜﻠﻴﺔ ﺍﻝ ﺯﺭﺍﻋﺔ - ﺠﺎﻤﻌﺔ ﻋﻴﻥ ﺸﻤﺱ . .
د.أ ./ ت ج ا ط …………………… ......
ﺃﺴﺘﺎﺫ ﺍﻝﺤﺸﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻴﺔ- ﻗﺴﻡ ﻭﻗﺎﻴﺔ ﺍﻝﻨﺒﺎﺕ- ﻜﻠﻴﺔ ﺯﺭﺍﻋﺔ ﻤﺸﺘﻬﺭ - ﺠﺎﻤﻌﺔ ﺒﻨﻬﺎ
د.أ ./ ا ا ر …………………… ......
ﺃﺴﺘﺎﺫ ﺍﻝﺤﺸﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻴﺔ - ﻭﺭﺌﻴﺱ ﻗﺴﻡ ﺍﻝﺘﻁﺒﻴﻘﺎﺕ ﺍﻝﺒﻴﻭﻝﻭﺠﻴﺔ - ﻫﻴﺌﺔ ﺍﻝﻁﺎﻗﺔ ﺍﻝﺫﺭﻴﺔ . .
د ./ ء د وة …………………… ......
ﺃﺴﺘﺎﺫ ﻤﺴﺎﻋﺩ ﺍﻝﻤﺒﻴﺩﺍﺕ - ﻗﺴﻡ ﻭﻗﺎﻴﺔ ﺍﻝﻨﺒﺎﺕ- ﻜﻠﻴﺔ ﺯﺭﺍﻋﺔ ﻤﺸﺘﻬﺭ - ﺠﺎﻤﻌﺔ ﺒﻨﻬﺎ ﺍﻝﺘﺎﺭﻴﺦ / / ٢٠١١ أ وا دا ا ا ودة ا ر
ﺭﺴﺎﻝﺔ ﻤﻘﺩﻤﺔ ﻤﻥ ء ﺒﻜﺎﻝﻭﺭﻴﻭﺱ ﻋﻠﻭﻡ ﺯﺭﺍﻋﻴﺔ ( ﺤﺸﺭﺍﺕ ﺍﻗﺘﺼﺎﺩﻴﺔ ) ﺠﺎﻤﻌﺔ ﺍﻝﺯﻗﺎﺯﻴﻕ ( ١٩٨٧ ) ) ﻤﺎﺠﻴﺴﺘﻴﺭﻓﻰ ﺍﻝﻌﻠﻭﻡ ﺍﻝﺯﺭﺍﻋﻴﺔ ( ﺤﺸﺭﺍﺕ ﺇﻗﺘﺼﺎﺩﻴﺔ ) ﺠﺎﻤﻌﺔ ﺒﻨ ﻬﺎ (٢٠٠٤ ) )
ﻝﻠﺤﺼﻭل ﻋﻠﻰ ﺩﺭﺠﺔ ﺩﻜﺘﻭﺭﺍﻩ ﺍﻝﻔﻠﺴﻔﺔ ﻓﻰ ﺍﻝﻌﻠﻭﻡ ﺍﻝ ﺯﺭﺍﻋﻴﺔ ( ﺤﺸﺭﺍﺕ ﺍﻗﺘﺼﺎﺩﻴﺔ ) )
ﻤﻥ و א א א −
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