Effect of gamma radiation and entomopathogenic nematodes on greater wax moth, Galleria mellonella (Linnaeus) [Lep., Pyralidae]

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of M. Sc. in Entomology

By Rehab Mahmoud Sayed Ali B.Sc. Entomology, 2001

Supervised by

Prof. Dr. Prof. Dr. Mohamed Adel Hussein Soryia El-Tantawy Hafez Professor of Entomology Professor of Entomology Entomology Department, Faculty of Entomology Department, Faculty of Science, Ain Shams University Science, Ain Shams University

Prof. Dr . Hedayat-Allah Mahmoud Mohamed Salem Professor of Entomology Natural Products Department, National Center for Radiation Sciences & Technology

Department of Entomology Faculty of Science Ain Shams University

2008

Faculty of Science

Approval Sheet M. Sc. Thesis Name: Rehab Mahmoud Sayed Ali Title: Effect of gamma radiation and entomopathogenic nematodes on the greater wax moth, Galleria mellonella (Linnaeus) [Lep., Pyralidae]

This Thesis for M. Sc. Degree in Entomology has been approved by:

Prof. Dr. Hedayat-Allah Mahmoud Mohamed Salem Professor of Entomology, Natural Products Department, National Center for Radiation Sciences & Technology.

Prof. Dr. Mohamed Adel Hussein Professor of Entomology, Entomology Department, Faculty of Science, Ain Shams University.

Prof. Dr. Mohamed Salem Abd EL-Wahed Professor of Entomology, Faculty of Agriculture, Ain Shams University.

Prof. Dr . Mahmoud Mohamed Saleh Professor of Nematodes, Pests and Plant Protection Department, National research centre.

Date: / /

Ain Shams University Faculty of Science Entomology Department

Name: Rehab Mahmoud Sayed Ali.

Degree: M. Sc. in Entomology.

Graduation: B. Sc. Entomology, 2001.

Faculty : Faculty of Science.

University : Ain Shams University.

ACKNOWLEDGEMENT

“FIRST & FORMOST, THANKS ARE DUE TO GOD, THE MOST BENEFICENT & MERCIFUL OF ALL ”

I would like to express my utmost gratitude to my professor, Mohamed Adel Hussein , professor of Entomology, Entomology Department, Faculty of Science, Ain Shams University, for supervising this work and for reading and correcting the manuscript.

I am particularly indebted to Prof. Dr. Soryia El- Tantawy Hafez professor of Entomology, Entomology Department, Faculty of Science, Ain Shams University, for her sincere help and guidance. She is both gracious and generous with her time, valuable advice as well as her discussion and critical reading of the manuscript.

I can’t by any means express my sincere thanks, appreciation and everlasting gratitude to Prof. Dr. Hedayat-Allah Mahmoud Mohamed Salem, Professor of Entomology, Natural Products Department, National Center for Radiation Research and Technology, for suggesting the subject of study, practical guidance. Truly I’m honored to be one of her sincere students.

I wish also to thank Dr. Salwa Abdo Rizk Assistant Professor of Entomology, Natural Products Department, National Center for Radiation Research and Technology, for

her kind care, continuous encouragement, practical guidance and providing knowledge to complete this work.

My deepest appreciation and sincere thanks to Dr. Mona Ahmed Hussein , Assistant Professor of Nematology, Pests and Plant Protection Department, National Research Center, for her valuable efforts for facilitating performance of this work.

Special thanks to Prof. Dr. El-Sayed Ahmed Abdel Aziz Hegazy, Chairman of the National Center of Radiation Research &Technology, for his kind care and his contribution in achieving the present work.

Finally, I would like to express my deep gratitude to my beloved parents, husband and my son for their encouragement, patience and sincere inspiration.

Contents

Title Page No. List of Tables List of Figures 1- Introduction ………………………………….. 1 2- Review of Literature
Biological effects of gamma irradiation on certain lepidopterous insects. ………………….. 5
Effect of entomopathogenic nematodes on Lepidopterous insects. ………………………….. 11
The combined effects of gamma radiation and entomopathogenic nematodes on some biological aspects of insects. ………………...... 17
Effect of gamma radiation on total and differential haemocytes count. ………………… 20 3- Materials and Methods • Insect. ……………………………………………. 23 • Entomopathogenic nematodes. . ……………… 24 • Radiation. ……………………………………….. 24 • Bioassay Experiments. ………………………... 25 4. Results
Biology of Galleria mellonella . …………… 30 A. Biological effects of gamma radiation on Galleria mellonella . I. Irradiated female mated with normal male. 32 II. Irradiated male mated with normal female. 37

Contents (Continued)

Title Page No. III. Irradiated male mated with irradiated female. ………………………………………… 41 B. Effect of Entomopathogenic nematodes and radiation on the larvae of the greater wax moth, Galleria mellonella . I. Susceptibility of wax moth larvae to Heterorhabditis bacteriophora BA1. ……………. 45 II. Susceptibility of wax moth larvae to Steinernema carpocapsae BA2. …………………. 48 III. Reproductive rate of dauer juveniles:

1. Reproductive rate of Heterorhabditis bacteriophora BA1 juveniles in G. mellonella larvae. …………………… 50 2. Reproductive rate of Steinernema carpocapsae BA2 juveniles in G. mellonella larvae. ………………………….. 53 C. Examination of the haemocytes of Galleria mellonella larvae: I. Description of the haemocytes of G. mellonella larvae. 1. Normal haemocytes. ………………………………….... 56 2. Morphological effects of Gamma radiation and/or entomopathogenic nematodes on Galleria mellonella haemocytes. …………………………………………………. 59

II. Determination of total haemocytes count (THCs) in normal and treated wax moth larvae. .. 75

Contents (Continued)

Title Page No. III. Determination of differential haemocytes count (DHCs).……………………….……………..

1. Effect of gamma radiation and Heterorhabditis bacteriophora BA1 on differential haemocytes counts (DHCs) in wax moth larvae. ………………………………. 77

2. Effect of gamma radiation and Steinernema carpocapsae BA2 on differential haemocytes counts (DHCs) in wax moth larvae. ………………………………. 83

5-Discussion. …………………………………….. 88

6- Summary. ……………………………………... 99

7- References. ……………………………………. 103

8-Arabic summary.

Figure No. Title Page No.

Life cycle of normal Galleria mellonella. 1 31 2 No. of eggs resulted from irradiated female mated with normal male. ….………………… 35

3 Sex ratio of F 1 generation resulted from irradiated female mated with normal male. … 36 4 No. of eggs resulted from normal female mated with irradiated male parent pupae. … 39 5 Sex ratio of F 1 generation resulted from normal female mated with irradiated male of Galleria mellonella . …………………………. 40 6 No. of eggs per irradiated female mated with irradiated male of wax moth. ……………… 43

7 Sex ratio of F 1 generation of irradiated female mated with irradiated male of wax moth. …… 44 8 Production rate of Heterorhabditis bacteriophora BA1 in non-irradiated and G. mellonella irradiated larvae at different nematode concentrations. ……….…………… 52 9 Production rate of Steinernema carpocapsae BA2 in non-irradiated and irradiated G. mellonella larvae at different nematode concentrations. ……………………………...... 55 10 Normal haemocytes of Galleria mellonella larvae. ………………………………………... 58 11 Morphological changes in prohaemocytes. ….. 60

12 Abnormalities in plasmatocytes. …………….. 62

List of figures (Continued)

Figure No. Title Page No. 13 Morphological changes in granulated cells. …. 64

14 Morphological changes in spindle cells. .……. 66

15 Morphological changes in sphere cells. ……… 68

16 Abnormalities in adipohaemocytes. …………. 70

17 Morphological changes in oenocytoids. ……... 72

18 Response of the haemocytes to the treatments. 74

Table No. Title Page No.

1 Effect of irradiation on eggs, larvae, pupae and adults resulted from irradiated females mated with normal males of Galleria mellonella . …………………………………. 34 2 Effect of irradiation on eggs, larvae, pupae and adults resulted from normal females mated with irradiated males of Galleria mellonella . ………………………………….. 38 3 Effect of irradiation on eggs, larvae, pupae and adults resulted from irradiated females mated with irradiated males of Galleria mellonella . .. 42 4 Effect of entomopathogenic nematodes; Heterorhabditis bacteriophora BA1 on percent mortality of unirradiated and irradiated larvae of Galleria mellonella at different time intervals (days). ………………. 47 5 Effect of entomopathogenic nematodes, Steinernema carpocapsae BA2 on percent mortality of unirradiated and irradiated larvae of Galleria mellonella at different time intervals (days). ……………………………… 49 6 Production rate of Heterorhabditis bacteriophora BA1 in non-irradiated and irradiated G. mellonella larvae at different

nematode concentrations. …………………… 51

Table No. Title Page No.

7 Production rate of Steinernema carpocapsae BA2 in non-irradiated and irradiated G. mellonella larvae at different nematode concentrations. ………………………………… 54 8 Total haemocytic count/mm 3 of blood from Galleria mellonella larvae treated with gamma radiation alone or combiend with entomopathogenic nematodes ( Heterorhabditis bacteriophora BA1 or Steinernema carpocapsae BA2). ……………………………. 76

9 Percentage haemocytes of F 1 Galleria mellonella larvae (irradiated parent male pupae with 40 Gy) alone or combiend with Heterorhabditis bacteriophora BA1 (20 and 40 IJs) treatments. ……………………………. 79

10 Percentage haemocytes of F 1 Galleria mellonella larvae (irradiated parent male pupae with 100 Gy) alone or combiend with Heterorhabditis bacteriophora BA1 (20 and 40 IJs) treatments. ………………………………… 82

11 Percentage haemocytes of F 1 Galleria mellonella larvae (irradiated parent male pupae with 40 Gy) alone or combiend with Steinernema carpocapsae BA2 (20 and 40 IJs) treatments. ……………………………………... 85 12 Percentage haemocytes of F 1 Galleria mellonella larvae (irradiated parent male pupae with 100 Gy) alone or combiend with Steinernema carpocapsae BA2 (20 and 40 IJs) treatments. ……………………………………... 87

Introduction

The greater wax moth, Galleria mellonella (L.), is a lepidopterous insect; its larval stage, feeds on wax and pollen stored in combs of active honey bee colonies (Milam , 1970 ). It does not attack adult bees but destructs combs of a weak colony by chewing the comb; spinning silk-lined tunnels through the cell wall and over the face of the comb, which prevent the bees to emerge by their abdomen from their cell, so they die by starvation as they unable to escape from their cell. They also eat out a place to spin their cocoons in the soft wood of the bee hive. Galleria mellonella can also destroy stored honey combs. Therefore, it is considered a major pest of the honeybee.

Damage will vary with the level of infestation and the time that has elapsed since the infestation first began. In time, stored combs may be completely destroyed and the frames and combs become filled with a mass of tough, silky web. In ideal conditions for wax moth development, a box (super) of combs may be rendered useless in about a week. Damage occurs mainly in the warm and hot months of the year when wax moths are most active. However, considerable damage can still occur during the cool part of late autumn and early spring as greater wax moth can produce a large amount of metabolic heat which can raise the immediate temperature around them by up to 25°C above the normal environment temperature. At the time of storage, combs that are apparently free of wax moth may contain eggs that will hatch later. They should be monitored

1

Introduction at frequent intervals for signs of moth infestation unless treated (Charrière and Imdorf, 2004 ).

Several pest control measures have been applied to suppress its damage; human efforts for keeping beehives clean and free of debris have been employed. Fumigation was also used to wax combs before placed in storage (Caron , 1992).

Recently many non-classic control methods are used, such as: physical and biological to control the pest in field and store. The use of irradiation technique as a physical control method is cheaper, safe and more reliable than chemical methods. However, the problem is to determine the minimum dosage of irradiation needed to control the insects while keeping the cost of treatment at an economical level and before reaching the threshold of irradiation that will produce quality changes in some commodities of the irradiated products.

Many organisms have potential as biological control agents: viruses, bacteria, fungi, protozoa, mites, insects and also nematodes. Entomopathogenic nematodes are a welcome addition to the natural-enemy arsenal. They are generally specific to insects, not environmentally hazardous, compatible with other biological and chemical agents, can seek pests in cryptic habitats and can be commercially produced in large quantities. Efforts to use nematodes for the biological control against pests did not commence until the 1930’s ( Glaser, 1931 ).

Parasitism by nematodes has variable deleterious effects on their insect hosts including sterility, reduced fecundity, reduced longevity, reduced flight activity,

2

Introduction delayed development, and other behavioral, physiological and morphological changes ( Poinar , 1979 ).

Many qualities render these nematodes excellent biocontrol agents: they have a broad host range, possess the ability to search actively for hosts, and present no hazard to mammals ( Gaugler and Boush , 1979 ). Finally, the USEPA (United States Environmental Protection Agency) has exempted the entomopathogenic Steinernematids and Heterorhabditis from registration and regulation requirements. They are already used commercially in high value crops (Georgis and Manweiler, 1994) .

Insects defend themselves against bacterial or parasite infection with cellular and humoral immune responses: Cellular defenses in contrast refer to responses like phagocytosis, encapsulation, and clotting that are directly mediated by haemocytes ( Lackie, 1988; Strand and Pech, 1995; Gillespie et al ., 1997; Irving et al ., 2005 ). Humoral defenses refer to soluble effector molecules including antimicrobial peptides (AMPs), complement like proteins, and products generated by complex proteolytic cascades such as the phenoloxidase (PO) pathway ( Blandin and Levashina, 2004; Cornelis and Soderhall, 2004; Theopold et al ., 2004; Imler and Bulet, 2005 ).

In insects, the blood fills the main body cavity (haemoceal), thus it is indirect contact with tissues and acts as an internal medium for the transportation of nutrients for growth, synthesis of hormones and coagulating materials (Charles , 1971 ). The haemocytes of insects have been reported to play some part in defense against foreign materials: by phagocytosis or encapsulation, they may be

3

Introduction concerned in the formation of connective tissue and activation of prothoracic gland before molting.

The high interest in biological means of controlling insects intensifies the need for investigating the response of insects to disease organisms. The haemolymph and haemocytes, in particular offer a readily accessible criterion of this response ( Barakat et al ., 2002 ). Total haemocyte counts (THCs) and differential haemocyte counts (DHCs) in different insects' species have also received a considerable attention. Much work has been done on changes in the haemocytes picture following injure and hemorrhage in insects attacked by parasites ( Salt , 1970 ).

The aim of this work is to study the biological effect of sub-sterilizing doses of gamma radiation on rate of infection of two entomopathogenic nematodes and to investigate the physiological effect of gamma radiation, entomopathogenic nematodes, and their combination on the greater wax moth, Galleria mellonella .

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Literature review

Biological effects of gamma irradiation on certain lepidopterous insects:

Abdel-Salam (1977 ) studied the effect of gamma radiation on Ephestia kuehniella and showed that the rate of adult emergence was gradually increased as the age of irradiated pupae increased. A dose of 20 krad induced complete sterility for females and high degree of sterility for males (treated as fully grown pupae).

Boshra (1982 ) studied the effect of gamma radiation on full grown pupae of Sitotroga cerealella and found that all doses reduced the number of eggs laid per female and percent hatchability of the resulting adults. While doses 35 and 60 krad caused complete sterility for females and males, respectively. The adult longevity was reduced at all doses used.

Salem et al . (1983 ) reported the effects of radiation on the rate of emergence, mating percentage, sperm transfer as well as fecundity and fertility on pupae of different ages of Ephestia kuehniella (Zell) when exposed to two different doses of gamma irradiation, 10 and 20 krad of radioactive (CO 60). Older pupae were more tolerant to irradiation damage, and females were more susceptible than males. Mating frequency and multiple mating percentages increased in irradiated females and decreased in irradiated males, and was not related to either age of pupae or dose

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Literature review used. Females from pupae irradiated with 20 krad were sterile and laid fewer eggs when mated with untreated males. Irradiated males showed a considerable degree of sterility.

Tsvetkov and Atanasov (1983 ) carried out experiment on the possibility of controlling Ephestia kuehniella by gamma-irradiation using dose levels 2-80 krad at 26°. They found that three-day-old pupae were highly susceptible to irradiation and 30 krad causing a 100 % lethal effect.

Ahmed et al . (1985 ) studied the effects of gamma radiation on the pupal stage of Ephestia kuehniella (Zell.). and they found that percent mortality of irradiated pupae increased gradually with increasing the dose. The dose 10 krad delivered to 1-2 day-old pupae prevented adult emergence, while the dose 60 krad gave rise to 100% mortality of the full grown pupae. There was a gradual decrease in the percent of adult exclusion as a result of pupal irradiation at both ages. Life span of adults of both sexes, irradiated in the pupal stages, was shortened as the dose increased. The doses 25 and 45 krad induced complete sterility for females and males, respectively. Male pupae were more radioresistant than females. The fecundity and fertility were decreased by increasing the dose. The greater reduction in fecundity and fertility was obtained when both sexes were treated and mated together rather than when one sex only was treated.

Hasaballa et al . (1985) reported that when the rice moth,Corcyra cephalonica was irradiated as pupae, some individual from most doses levels were able to complete development to the adult stage. The lethal dose for 2-and 7-

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Literature review day-old pupae was 100 krad. Females were more radiosensitive than males receiving the same dose. The doses 50 and 30 krad caused 100% sterility to males and females, respectively.

Haiba (1990 ) showed a highly significant positive relationship between dose and adult emergence when irradiated 5-days old pupae of Phthorimaea operculella with doses 10, 50, 100, 150, 200, 250 and 300 Gy. She found that, the higher the dose, the lower was the percentage of adult emergence. At all doses used, the males seemed to be more successful in emergence than the females, except at the intermediate levels of 150 and 200 Gy. The exposure to radiation dose levels (10-200) had no external effect on the emerged male and female adults. However, females were more sensitive than males. Irradiation had marked effect on the egg production and its hatchability. The dose levels 150 and 300 Gy were considered as the sterilizing doses for females and males, respectively.

Bughio (1992 ) noticed the emergence of normal moths, pupal periods and adult longevities remained statistically unaffected following treatment of the mature (5- 6 day-old) male and female pupae of Chilo partellus with 5 to 20 kilo-Roentgen (kR) and 5 to 10 kR, respectively. Fecundity of normal (nonirradiated) females mated with males irradiated as pupae was not significantly different from untreated controls, but the fecundity of females irradiated as pupae at 10 kR was significantly reduced when they were mated with normal males.

Al-Izzi et al . (1993 ) illustrated the effect of gamma radiation on irradiated five to six days old male pupae of fruit moth; Ectomyelais ceratonia at 0, 20, 25 and 30 krad.

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Literature review

They found that when irradiated males were mated with normal females there were deleterious effects on fertility without extreme effects on mating or fecundity. The percentage of embryonated eggs and progeny per female decreased to two-thirds of the untreated control by 25 and 30 krad treatment. The productivity of females mated with irradiated males was decreased and significant reduction in egg validity, egg hatch and larvae per female were observed in the F 1 generation.

Aguilar et al . (1994 ) Studied the effects of gamma radiation on pupae and adults of Corcyra cephalonica (Stainton), a pest responsible for heavy losses in stored grains in Brazil revealed that the sterilizing dose for pupae was 350 Gy. For the F 1 generation, the sterilization was induced on to their parents with the dose of 150 Gy. Irradiated adults did not become sterile with doses up to 400 Gy. But their descendants of the F 1 generation became complete sterile if their parents were irradiated with the dose of 100 Gy.

Qureshi et al . (1995 ) reported that irradiation of mature pupae of pink bollworm , Pectinophora gossypiella at 50- 200 Gy resulted in decreased adult emergence with increased gamma radiation doses, and more deformed moths were recorded at a dose of 200 Gy adult following irradiation of mature pupae when crossed with untreated males or treated individuals crossed to treated exhibited reduced fecundity and fertility with the increasing doses.

Al-Taweel et al . (1997 ) studied the effect on irradiated 5 to 6 days Ephestia bigulilella pupae at 0.15, 0.20, or 0.25 kgy of gamma radiation. The fecundity, egg hatch and mating ability of gamma irradiated female with

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Literature review irradiated male, unirradiated female with irradiated male, irradiated female with unirradiated male or unirradiated female with unirradiated male were also studied.

Aguilar and Arthur (1998 ) investigated the effects of sub-sterilizing doses of gamma radiation on pupae of Spodoptera frugiperda , and its effect on the transfer of genes in the first and second generations. Statistical analysis showed differences in the ageing effect of gamma radiation on larval stages, and larval viability ranged between 72 and 94% when irradiated (50 Gy) males or females were crossed with non-irradiated adults. At doses of 100, 125, 150 and 175 Gy and crossing irradiated males with non-irradiated females, larval viability ranged between 64 and 94% in the F1 and F 2 generations. The duration of the pupal and adult stage did not differ from the untreated controls. Egg laying ability was not affected by doses up to 150 Gy for both the sexes.

Yousef (2001 ) investigated the effect of gamma radiation on the 5-days old pupae of Plodia interpunctella at doses of 100- 300 and 500 Gy, and found that the rate of adult emergence ranged from 90% in untreated pupae to 12% when pupae treated with 500 Gy. The maximum of adult emergence resulting from treated pupae was 65% at doses of 100 Gy followed by 42 % at doses of 300 Gy.

Al-Taweel et al . (2002 ) investigated inherited sterility in laboratory strain of G. mellonella when exposing the pupa to several doses of gamma radiation (0.10, 0.15 and 0.20 kGy). The percentage of egg hatch reached 0% for F1 progenies produced from normal females mated with irradiated males and mated as follows: F 1 female x normal

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Literature review male and F 1 male x F 1 female, for doses of 0.15 and 0.20 kGy, respectively

Abdalla (2004 ) found that adult emergence was affected greatly in irradiated full grown pupae of rice moths, Corcyra cephalonica with the dose levels 350- 700- 1100 and 1400 Gy. The rate of increase in the efficacy of gamma irradiation was proportional with the first two dose levels. Completed reduction in percent emergence was obtained when the pupae irradiated with dose level of 1400 Gy.

Sawires (2005 ) exposed full-grown male and female pupae of the Mediterranean flour moth, Ephestia kuehniella (Zeller) to doses of gamma irradiation ranging from 50 to 450 Gy. Irradiated males were more radio-resistant than females. Reduction in fecundity and egg fertility was dose dependent. Irradiated males or females showed significant shorter life span than unpredicted (check). There were reductions in F 1 progeny as result of irradiation male and female parents with sub sterilizing doses, which was more apparent in irradiation of male parents. The average larval- pupal developmental period of F 1 male and female progeny was affected. The sex ratio of F 1 progeny was shifted in favor of males.

Zahran (2006 ) studied the effect of gamma- irradiation on 5-days old pupae of potato tuber moth, Phthorimaea operculella . She found that significant positive relationship between dose levels and the percentage of adult emergence were obtained; the higher the dose, the lower were the percentage of adult emergence. Males emerged more successful than females, except at dose levels 10 and 100 Gy, where the percentages for both sexes were equal and reached 95.5 and 77.7%, respectively.

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Literature review

Ayvaz et al. (2007) exposed newly emerged mediterranean flour moth, Ephestia kuehniella to 100, 200, 400 and 600 Gy. They reported that the dose of radiation had a significant effect on the time from oviposition to larval eclosion or adult emergence and the mortality increased with increasing the dose of radiation.

Effect of entomopathogenic nematodes on Lepidopterous insects:-

Luca (1977 ) studied the biological control of the bruchide, Acanthoscalides obtectus (Say) by using an aqueous suspension containing 500 infective larvae/ml of N. carpocapsae . He mentioned that treated bruchids became inactive a few hours later and died within 24-48hrs. from combined action of nematode and associated bacterium.

Ghally (1983 ) mentioned that, the production of Neoaplectana carpocapsae presented in one insect larvae and /or to one mg of its body weight was distincty higher in Galleria mellonella than Achroie grisella F. caterpillar The higher reproductive rate (116000/one insect larva and 879/mg was obtained from Galleria mellonella at 26C°).

Mullens et al. (1987) found that strain of S. carpocapsae were more effective than H. heliothidis when exposed third instar larvae of Musca domestica, Ophyra aenescens , Fannia canicularis and F. femoralis to four strains of S. carpocapsae and one of H. heliothidis on moist filter paper in petri-dishes.

Ghally et al . (1988 ) reported that, the reproductive rate of the nematode Steinernema feltiae is represented by

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Literature review the number of survival and dead second or third larval stages obtained from one caterpillar. The mean daur juveniles per cadaver was 12500,25500 and 48300 with the initial dosage of 50, 100 and 200 invasive larvae. It seems that increasing the dosage 100% the reproductive rate also increases by nearly 100%.

Glazer (1992 ) studied the invasion rate of three Steinernematids in the three larval stage of Spodoptera littoralis . The invasion rate was determined by recording the mortality levels of insect larvae exposed to infective juveniles of the various nematodes strain for 2, 4 and 6 hrs.

Henneberry et al . (1995 ) investigated the susceptibility of the second, third and last stage larvae of pink bollworm (PBW) to infection by infective juvenile nematodes, Steinernema riobravis Cabanillas, Poinar and Raulston and the Kapow nematode strain of S. carpocapsae (Weiser). Third and last stage PBW larvae were more susceptible than second stage larvae,. The LC50 (1.84 IJs) for last stage PBW larvae treated with S. riobravis was significantly less than the LC 50 (3.07 IJs) for PBW larvae with S. carpocapsae . S. riobravis induced higher percentages of larval mortality than S. carpocapsae in bioassay containers with 10, 50, or 100 nematodes per 10 PBW larvae and in each case at densities of 1.5 and 10 PBW larvae per bioassay container. Small percentages (4-23) of PBW larvae were found containing infective juvenile nematodes after 1h. exposure to either species.

Ratnasinghe and Hague (1995 ) investigated the susceptibility of Plodia xylostella to 3 species of Steinernema; S. carpocapsae all , S. riobravis and S. feltiae and one isolate of Heterorhabditis sp.. S. carpocapsae (All)

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Literature review was the most effective, giving 100% mortality after 6 hrs. exposure when assessed by invasion of infective juveniles. There was no significant difference in susceptibility between the Steinernema sp. and Heterorhabditis sp ..

Shannag and Capinera (1995) studied the pathogenicity of 5 entomopathogenic nematodes from the genera Steinernema and Heterorhabditis against Diaphania hyalinata . S. carpocapsae (Mexican strain) was the most pathogenic nematode species, followed by H. bacteriophora , S. feltiae , S. anomali and S. glaseri , resp.. The LC 50 for S. carpocapsae (Mexican) was 39.9 infective juveniles/ml. The rate of nematode invasion into insects was proportional to the overall pathogenic effect of the various nematodes. Insect mortality and infectivity (the number of nematodes invading the insect) were directly related to exposure time. First-instar larvae and pupae were significantly less susceptible to S. carpocapsae infection than older larvae and prepupae.

Tahir et al . (1995 ) investigated the effect of increasing dosages of Steinernema riobravis , S. carpocapsae (All isolate) and a Heterorhabditis sp. against the last instar larvae of the African bollworm, Helicoverpa armigera (Heliothis), and the spotted bollworm of Asia, Erias vitella . 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 S. carpocapsae and Heterorhabditis sp. but against E. vitella , Heterorhabditis sp. was the most effective.

Shamseldean et al . (1996 ) studied the efficacy of 4 strains of entomopathogenic nematodes in infecting and

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Literature review 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 64% mortality. 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

Ratnasinghe and Hague (1997 ) studied the efficacy of Steinernema carpocapsae , S. riobravis [S. riobravae ] and S. feltiae nematodes against different stages in the life cycle of Plutella xylostella . In larvae, the LT 50 was less than 3 hrs. for all 3 nematodes, but in S. carpocapsae it was the most virulent killing larvae after 6 hrs. exposure. Pre-pupae were very susceptible to nematode treatment and S. carpocapsae caused 40% mortality in immature and mature pupae. More infective juveniles of S. carpocapsae established in larvae than the other nematodes tested

Ben-Yakir et al . (1998 ) evaluated the potential of entomopathogenic nematodes in biological control of the European corn borer (ECB), Ostrinia nubilalis (Hubner). The "All" and "Mexican" strains of Sterinernema carpocapsae (Weiser)and the "PH88" strain of Heterorhabditis bacteriophora Poinar were compared in

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Literature review both dose response assays (5, 50 and 500 infective juveniles [IJ] per Petri dish containing five 5 th instar ECB eggs; 72 h. of incubation) and exposure time assays (3, 6 and 8 h. of incubation). In the dose response assays the highest rates of ECB killing results from infestation with the Mexican strain of S. Carpocapsae . In the exposure time assays there were no significant differences between the killing rates of the three nematode strains. Sweet corn plants (Zea mays var. saccharata) grown in a screenhouse, were infested with ECB neonates and 4 days later sprayed with a suspension of the Mexican strain of S. carpocapsae (50,000 IJ per plant).

Boff et al . (2000 ) investigated that increasing densities of Heterorhabditis megidis strain NLH-E 87.3 infective juveniles (IJ) affected invasion, reproduction, length and time to first emergence of the nematodes in larvae of the greater wax moth, Galleria mellonella . Although the number of nematodes that invaded the host increased with increasing dose, percentage of invasion declined. The number of progeny produced per host initially increased with dose. The highest production of IJ per cadaver was reached at a dose of 400 IJ per host, at that dose 62 ± 3.4 IJ were established per cadaver. Production decreased again significantly at higher densities. The smallest IJ were produced at a dose of 1000 IJ per host and the largest at dose of 300 IJ per host. Time to first emergence of juveniles was generally shorter when the number of IJ inoculated was large (300-3000 IJ/host).

Shinde and Singh (2000 ) studied eight entomopathogenic nematode species/strains, Steinernema glaseri (Steiner), S. carpocapsae (Weiser), S. feltiae (Filipjev), Steinernema sp . Ecomax strain, Heterorhabditis bacteriophora (Pioner), Heterorhabditis sp. Ecomax strain,

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Literature review two locally isolated strains called as JFC and TFC against the late instar larvae of diamondback moth, Plutella xylostella (L.). All nematodes were found pathogenic. However, H. bacteriophora was considered the most pathogenic tested nematodes.

Farag (2003 ) studied the biological control of Deudorix livia the most dangerous insect infesting the green pods and Pseudopachymerus lallemanti infesting the dry pods of Acacia farnesiana by entomopathogenic nematodes Steinernema feltiae and Steinarnema carpocapsae . Both insect species are susceptible to nematodes infection and the mortality rate and the infestation intensity of them increase with increasing the initial dosage of nematodes. The tested nematodes were able to complete their life cycle inside the two insect species.

Kumar et al . (2003 ) evaluated the efficacy of Heterorhabditis sp. against Spodoptera litura collected from castor bean leaves. Infective juveniles (IJs) of Heterorhabditis were released in containers with Spodoptera litura larvae at 25, 50, 75, 100, 125 or 150 IJs/100 g soil. The mortality of host larvae was evaluated every 24 hrs. up to 120 hrs. after inoculation. Significant mortality was observed within 48 hrs. of exposure except for 25 IJs/100 g soil. Mortality ranged from 16.6 to 88.8%. Fifty percent mortality was observed within 72 hrs. of exposure to 100 IJs/100 g soil. After 120 hrs., a mortality rate of 88.8% was obtained with 150 IJs/100 g soil.

Mathasoliya et al . (2004 ) studied the performance of the entomopathogenic nematode Steinernema riobrave (Indian isolate) against cutworm ( Agrotis ipsilon ) infesting potato. In the field, there were approximately 5 larvae of A.

16

Literature review ipsilon per waste heap before spraying. At the end of the experiment, the number of larvae per waste heap were reduced up to 2.33, 1.50 and 1.17 in entomopathogenic nematodes (EPN; 100 000 IJs/m2).

El-Mandarawy (2005 ) studied the efficacy of Heterorhabditis bacteriophora "HP88" and H. taysearae , "SI" and "All" strain Steinernema carpocapsae , against Phthorimaea operculella larvae and pupae in filter paper and sand barrier assay. Exposure time assays of H. bacteriophora was 3, 6, 12, 24 and 36 hrs. from inoculation to larval mortality and nematode production was studied. Results showed that Heterorhabditis bacteriophora "HP88" caused the maximum mortality, 60-100, 40-80 and 20-60% after 96 hrs. in the filter paper, sand barrier larval assays and sand barrier pupal assays, respectively. Filter paper assayed larvae showed the lowest LC 50 value by different nematode species inoculum, while sand barrier gave the highest LC 50 for the inoculated pupae. The time required to obtain 50% (LT 50 )mortality was decreased by increasing the concentration of different nematode species in each bioassay studied. Mortality of P. operculella larvae exposed to 6, 12 and 24 hrs., while the reproduction of nematode in larvae showed negative correlation with the time of exposure.

The combined effects of gamma radiation and entomopathogenic nematodes on some biological aspects of insects:

Abdel-Salam et al . (1995 ) studied the effect of gamma radiation on the pathogenicity of the entomopathogenic nematode Steinernema carpocapsae by infecting larvae of Phthorimaea operculella . Full-grown

17

Literature review larvae of the host were gamma-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. On the other hand, gamma irradiation of the nematodes with high doses lowered their efficiency towards the unirradiated host .

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.

Gouge et al. (1998 ) studied the interactions between F1 progeny of Pectinophora gossypiella adults irradiated in the pupal stage and entomopathogenic nematodes. Both sexes of pink bollworm pupae were exposed to 4, 8, 12 or 16 krad sub sterilizing radiation doses irradiated using a 60 Co source. The F 1 larvae were tested in a small bioassay for susceptibility to Steinernema riobravis Cabanillas, Poinar & Raulston, S. carpocapsae (Weiser), and 2 strains of Heterohabditis bacteriophora (Poinar). The numbers of infecting nematodes were counted after 48h.. Increasing parental radiation dose significantly increased F 1 larval susceptibility to S. riobravis and H. bacteriophora , but decreased susceptibility to S. carpocapsae .

Abd-Elwahed (2004 ) investigated the effect of entomopathogenic nematode, 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

18

Literature review irradiated parents. Also females was 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 entomopathogenic nematode 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 × 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 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

19

Literature review 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.

Effect of gamma radiation on total and differential haemocytes count:

El-Badry (1964 ) illustrated the total haemocyte count (THC) on treated potato tuber worm larvae, regardless of whether they were subjected to irradiation at any early stage of development or at an advanced stage.

El-Maasarawy and Abd El-Salam (1991 ) reported that exposure of larvae of Bombyx mori to gamma radiation (15, 500, 1000, 3000 and 5000 rad) resulted in the appearance of abnormalities in all types of haemolymph cells, with the numbers of deformed cells increasing with increased dose..

Rizk (1991) studied the effect of gamma radiation doses (50, 100 and 150 Gy) on the 4th larval instar of Spodoptera littoralis . She found that the total haemocytes count decreased gradually as the dose increased. The haemocytes types were prohaemocyte, plasmatocyte, spindle cell, granulated cell, oenocytoid, adipohaemocyte, spherule cell and cystocyte; the most abundant type was the prohaemocytes.

Ibrahim et al. (1993) examined the blood smear of F 1 Agrotis ipsilon after exposes to gamma radiation, They

20

Literature review reported that eight types of haemocytes were morphologically differentiated in the haemolymph of the larvae. Haemolymph smears of F1 larvae of released irradiated males showed development of abnormalities in the haemocytes. Among the 8 types of haemocytes of F 1 larvae, there were only 5 types (micronucleocytes, macronucleocytes, plasmatocytes, spherules and spindle cells) which could be considered as indicators for the recognition of damage in a smear test.

Souka et al . (1993 ) investigated that the haemocytes of fourth instar Spodoptera littoralis consist of eight kinds which are prohaemocytes, plasmatocytes and sphrule cells. The treatment of the cotton leaf worm larvae with gamma radiation gradually decreased the total haemocyte count as the dose increased.

El-Maasarawy et al . (1995 ) studied the haemocytes of Corcyra cephalonica Staint. Larvae morphologically, treated as egg by gamma irradiation doses, in smears stained with Giemsa's stain. Eight types were identifild, proleucocyte, granulocyte, eosinophil cells, spherulocte, spindle shaped cell, plasmatocyte, oenocytoid and adipohaemocyte, all of which had morphological characteristies similar to those of other lepidopterous larvae Exposure of eggs during embryonic development to gamma irradiation at doses of 5, 15, 25 and 30 Grays results in the appearance of different malformations in all types of haemocytes. Also, exposure to gamma irradiation reduced the immunity system (spherulocytes, spindle cells and plasmatocytes); their percentages decreased sharply to reach a minimum level with high doses. Spindle cells become absent. On the contrary, gamma irradiation caused many mitotic abnormalities in small granulocytes and

21

Literature review proleucocytes, they achieved the highest percentages with irradiated larvae.

El-Mandarawy (1997) reported eight types of haemocytes in the 6 th instar larvae of the greater sugar cane borer Sesamia cretica (Led.), as follows: Prohaemocytes; Plasmatocytes; Granular cells; Spindle cells; Adipohaemocytes; Oenocytoids; Spherule cells and Cystocytes.

22

Materials and Methods

A. Insect:

The strain of the greater wax moth, Galleria mellonella L. was obtained from the National Research center (NRC) and reared according to Hussein (2004) .

The greater wax moth Galleria mellonella larvae were reared on media developed from Wiesner (1993). This media consists of: 22% corn groats (polenta) 22% wheat-flour (full com) or brushed-grain wheat 11% milk powder (skim-milk) 11% honey 11% glycerol 5.5% yeast powder (“brewer’s yeast”, beer yeast) 17.5% bee wax

The larvae were originally obtained from bee hives and transferred to transparent plastic rearing jars (17 x17 x 27 cm), containing 250gm from the previous prepared media, closed with a lid of muslin for aeration and incubated at 28± 2°c with a photoperiod (L:D) 8:16 and relative humidity 65± 5% in the insect rearing chamber. When larvae grown to the pupal stages and then to the adult moths, a piece (15 x 15cm) of paper tissue was folded and placed in the container to promote egg laying. Eggs were laid on the lid and on paper tissue. These eggs were gently removed and transferred to other rearing jar containing 250gm media, closed tightly with double muslin layer to prevent the escape

23

Materials and Methods of neonatal larvae, and incubated. Add fresh food frequently (1-2) times per week.

B. Entomopathogenic nematodes:-

The entomopathogenic nematodes (EPN) were originally obtained from the National Research Center (NRC), Pests & plant Protection Department. Two different genus were used, Heterorhabditis bacteriophora BA1 and Steinernema carpocapsae BA2 . Both strains of EPN were isolated from the Egyptian soil and identified by Hussein and Abou El Soud, (2006). All nematodes used in this study were reared in vivo according to Glazer and Lewis (2000) . Newly emerged infective juveniles (IJ's) were o harvested and stored at 15 C for two weeks prior to the bioassay. Their virulence was tested before starting up the experiments.

C. Radiation:-

Irradiation of G. mellonella pupae was carried out using Gamma Cell Irradiation Unit (Co 60 source) located in the National Center for Radiation Research and Technology (NCRRT)). The dose rate was 6.6 kGy/h.

24

Materials and Methods

Bioassay Experiments:-

A. Biological effect of radiation on Galleria mellonella :

Six day-old pupae of G. mellonella were exposed to different gamma rays doses (40,100,160,200 and 260 Gy), to study the effect of irradiation on some biological aspects of G. mellonella . Four mating combinations were made:

Unirradiated male × Unirradiated Female (as control) Unirradiated male × Irradiated Female Irradiated male × Unirradiated Female Irradiated male × Irradiated Female

Each 5 combinations were kept in a separate jar, covered with pieces of thin cloth fixed in with a rubber band, three replicates were performed for each one; the eggs laid by females were recorded and counted daily under a binocular microscope. Egg masses were transferred to clean jar 250 c.c capacity, washed with 0.15% formalin solution to avoid any contamination. Hatched larvae were counted daily and provided with fresh diet.

Percentage sterility was calculated according to Chamber Lain's formula as mentioned by Guirguis (1979 ) that: a × b % Sterility = 100 − × 100 A × B Where: a: No. of eggs/female treatment b: % hatching/ female treatment A: No. of eggs/female control B: % hatching/ female control

25

Materials and Methods

B. Effect of entomopathogenic nematodes and radiation on the larvae of the greater wax moth, Galleria mellonella :

Heterorhabditis bacteriophora BA1 and Steinernema carpocapsae BA2 were assessed for virulence to 4 th instar larvae of the greater wax moth, Galleria mellonella in the laboratory. The experiments were designed as: 1- Group 1: normal 4 th larval instar (control) 2- Group 2: larvae resulted from irradiated male parent mated with normal female at doses 40 and 100 Gy. 3- Group 3: normal larvae and F 1 larvae (resulted from irradiated male parent as pupae at 40 and 100 Gy) and treated with Heterorhabditis bacteriophora BA1 doses (20 and 40 IJ). 4- Group 4: normal larvae and F 1 larvae (resulted from irradiated male parent as pupae at 40 and 100 Gy) and treated with Steinernema carpocapsae BA2 doses (20 and 40 IJ).

1. Effect of EPN on larval mortality:

Larvae were exposed to serial concentrations of nematodes in a bioassay technique based on Woodring and Kaya (1988 ). Four larvae were placed in 100cc plastic cups filled with filter paper and moistened with 15% water (v/w). Nematodes suspensions were prepared in serial concentrations of 20, 40, 60and 80 infective juveniles (IJ)/ml/cup. As many as 3 replicates of each treatment were conducted.

26

Materials and Methods

Experiments were held in the laboratory under 25±2 oC. Control plots received only water. Cups were observed and larval mortality was observed over 4-10 days.

2. Reproductive rate of dauer juveniles: -

After the larvae were dead, they were placed in the collecting dishes, which consist of a filter paper on the glass watch placed in a larger and deeper Petri dish in which distilled water was added to reach the edge of the filter paper. The cadavers were placed in the middle portion of the filter paper to migrate down the moist filter paper into water.

The juveniles suspension was collected daily by a special pipette and stored in Petri dish containing distilled water in a refrigerator at 5 oC

In order to determine the reproductive rate according to Dutcky and Hough (1955 ), the nematode suspensions were collected till the end of the reproduction. Then measuring the volume of the collective suspension and counting the invasive larvae in 1ml. the number of dauer juveniles/larvae was determine for each dose, this number equal to the reproductive rate per each larvae.

C. Determination of haemocytes (total & differential) count in Galleria mellonella larvae as affected by radiation or/and entomopathogenic nematodes :

Experiments were carried out to study the effect of gamma radiation doses (40 and 100 Gy), entomopathogenic nematodes ( Heterorhabditis bacteriophora BA1 and

27

Materials and Methods

Steinernema carpocapsae BA2) and the combined effect of both on total and differential haemocytes count of 4 th larval instar (resulted from irradiated male parent pupae).

The larvae were divided into 4 groups: 1- Group 1: normal 4 th larval instar (control) 2- Group 2: larvae resulted from irradiated male parent mated with normal female at doses 40 and 100 Gy. 3- Group 3: normal larvae and F 1 larvae (resulted from irradiated male parent as pupae at 40 and 100 Gy) treated with Heterorhabditis bacteriophora BA1 doses (20 and 40 IJ). 4- Group 4: normal larvae and F 1 larvae (resulted from irradiated male parent as pupae at 40 and 100 Gy) treated with Steinernema carpocapsae BA2 doses (20 and 40 IJ).

1. Preparation of blood film:

To determine the differential haemocytes and the pathological changes resulted from radiation and nematodes treatments, haemolymph smears were taken from 4th instar larva. A drop of blood was spread into a thin film by the aid of a cover slip, air dried, fixed in absolute methanol for five minutes, stained in Giemsa stain for 35 minutes and the stain was flushed off with distilled water Abdel-Rahman (1978 ).

2. Preparation of Giemsa stain: (Fadem and Yalow (l95l))

1. Weigh 7.6 grams of Giemsa powder stain. 2. Dissolve 7.6 grams of Giemsa powder stain in 500 ml of glycerol. 3. Incubate the mixture in water bath at 56 oC for l20 minutes (mix few times).

28

Materials and Methods

4. Add 500 ml of methanol. 5. Keep the mixture at room temperature for 7 days (mix several times daily). 6. Filter and store at room temperature in dark bottles.

Giemsa solution: 1. Giemsa solution (stock)...... 50 drops. 2. Distilled water...... 50.0 ml.

3. Method of counting:

The proleg on the sixth abdominal segment was cut with a fine pair of scissors, and blood was allowed to ooze on a clean, grease free, glass microscopic slide, the haemolymph was quickly drawn up to the 0.5 mark in a Thoma white blood cell dilution pipette and immediately diluted to 11 mark with Turek solution (1-2 % glacial acetic acid, slightly colored with gentian violet. The first two or three drops of this solution were discarded; the haemocytes were counted in the four corners and multiplied by a factor of 50 to get the number of cells per cubic millimeter. If the cells were clumped or were otherwise unevenly distributed in the chamber, the preparation was discarded, as described by Rizk (1991 ).

D. Statistical Analysis:

The data were statistically evaluated by analysis of variance (F) followed by Duncan's multiple range test to examine the significant differences between treatment. The 5% level of probability was used in all statistical tests. The statistical software program CoStat (1995) was used for all analyses.

29

Results

Biology of Galleria mellonella :

The wax moth undergoes complete metamorphosis: egg, larva, pupa, and adult. In nature all stages of the wax moth may be present at the same time and except during cold weather, development is continuous. The mated female moth deposits about 200 to 1,000 small, white, slightly oblong eggs in masses in cracks away from the light. Eggs hatch from five to eight days at 30°C.

Newly hatched larvae immediately begin to burrow into the wax. Although the larvae consume a large amount of wax, it is the impurities and remains of the old bee coccoons which provide them with most of their nourishment. Because of this, new comb or foundation is a poor food and is seldom attacked. Depending on the temperature, larval development ranges from twenty seven to thirty days at 30°C - the warmer the temperature, the faster the development.

Young larvae are white, about 1mm long, and very active. Mature larvae are about 25 mm long, golden-gray or brown in color, and move much slower. When ready to pupate, the larvae spin coccons. Pupation takes from eight to ten days depending on temperature .

The wax moth is about 2 cm long with a wing spread of 2.5 to 3 cm. The size of an adult varies depending on the quality of food consumed during a larva. The male is slightly smaller than the female and has scalloped-shaped wing edges (Fig. 1).

30

Results

Figure (1): Life cycle of normal Galleria mellonella

31

Results

A. Biological effects of gamma radiation on Galleria mellonella :-

These experiments were carried out to study the effect of gamma radiation doses (40, 100, 160, 200 and 260 Gy) on the life cycle of the greater wax moth.

I. Irradiated female mated with normal male:

The obtained results in Table (1) and Figures (2 & 3) show that when full grown pupae were irradiated with 40, 100, 160, 200 and 260 Gy, the number of eggs laid per female decrease with the increasing radiation doses being 522, 331.7, 57, 0 and 0, respectively. As compared to control (799.7).

The incubation period of the eggs was positively correlated to the dose increase, except at the high dose (160 Gy) the eggs failed to hatch.

The percent sterility significantly increased gradually with increase the dose until reach 100% sterility at the doses (160, 200 and 260 Gy). Data in Table (1) also showed that the F 1 larval duration in case of irradiated female mated to normal male were significantly increased with the dose increase. There were (35.3, 36.8 and 0) as compared with control (28.47).

A negative correlation was found between the percent pupation and the increase of doses till reach 0% at the doses

32

Results

160, 200 and 260 Gy, and a significant difference was found between treatments.

When full grown pupae were irradiated with the doses 40, 100, 160, 200 and 260 Gy, the percent of adult emergence and sex ratio of F1 generation were significantly affected; the lower dose, the highest was in the percentage of adult emergence.

The sex ratio was in favour of males at dose levels 100 and 160 Gy. While in control, the number of females exceeds the number of males.

33

Results

Sex ratio Sex ratio Male Male Female

% % Emergence : dults resulted from irradiated % % 77.6 77.6 88.51 53.4 46.6 99.57 99.6 46.66 49.89 50.1 61.11 83.33 16.66 nge test). nge Pupation No. No. 0.33 c 0.33 c 2.72 b 2.33 ± 2.33 ± 13.3 a 13.3 a 41.66 ± 41.66 ± 783.33 ± 783.33 ±

Galleriamellonella 0 d 0 d 0 c 0 c 0 0 0 0 0 0 0 0 0 d 0 c 0 0 0 0 0.37 c 0.37 c 1.01 a 35.3 ± 35.3 ± 36.8 ± 0.334 b 28.47 ± 28.47 ± Duration

0 100 100 100 63.4 63.4 94.2 % Sterility % No. No. 4.3 c 790 ± 790 ± 290 ± hatch hatch 13.3 a 13.3 a 39.2 b 44.6 ± 44.6 ± Eggs Eggs Larvae Pupae Adults 0 c 0 c 7.4 ± 7.4 ± 9.4 ± 9.6 ± ation ation (days) period period Incub- 0.433 a 0.361 a 0.365 b d 0 d 0 d 0 c 0 c 0 c 0 c eggs 552 ± 552 ± 11.3 a 11.3 a 54.9 c No. of of No. 17.2 b /female /female 799.7 ± 799.7 ± 331.7 ± 57 ± 7.81 7.81 57 ± females mated with normal males of withmales normal mated females

Effect of irradiation on eggs, larvae, pupae and a 40 100 160 200 260 (Gy) Doses Doses Control Radiation Letters indicate the variance between the means (Duncan's means variance the the multiplebetween (Duncan's indicate ra Letters Values represent the mean ±S.E. of 3 replicates for each each for 3 replicates group. of mean the ±S.E. represent Values Table (1):

٣٤

٣٤ Results

d with normal 0 0 Gamma Gamma ray(Gy) doses 57 331.7 522 0 40 100 160 200 260 male. male. Number Number of eggs resulted from irradiated female mate 799.7 0

900 800 700 600 500 400 300 200 100 No. eggs No. Figure (2):

٣٥

٣٥ Results

0 0 Doses of gamma ray (Gy) of gamma Doses 0 61.11 generation resulted from irradiated female mated % Adult Emergence % 1 88.51 99.6 0 40 100 160 200 260 0 80 60 40 20 100

with normal male: withmale: normal Percent of adult emergence of F Adult emergence Adult

Figure (3):

٣٦

٣٦

Results

II. Irradiated male mated with normal female:

The results recorded in Table (2) and Figures (4 & 5) reveal that there was no significant effect of the doses 40, 100, 160, 200 and 260 Gy on the number of eggs laid per female when irradiated male mated with untreated virgin female. On the other hand, the incubation period of the eggs was significantly increased as dose increases, excluding the dose of 260 Gy that they failed to hatch.

The larval longevity gradually increased with the dose increase except at the dose 260 Gy; it was zero. In contrast, the percent pupation was negatively correlated to the doses. Showing 83.5, 77.4, 68, 41.4 and 0 at doses 0, 40, 100, 160, 200 and 260 Gy, respectively, as compared with control (99.57).

Irradiation of full grown pupae significantly decreases the percent of adult emergence of F1 generation. The male ratio was increased with increasing the dose; the percents were 56.3, 56.5, 86.7 and 88.88 as compared with control (49.89). While, the female ratio was decreased with increasing the dose giving 43.7, 43.5, 13.3, 11.11 and 0% as compared with control (50.1%).

37

Results

Sex ratio Sex ratio Male Male Female

: % Emergence dults resulted from normal nge test). nge % % 68 52.8 86.7 13.3 83.5 83.5 77.4 87.38 85.5 56.3 56.5 43.7 43.5 41.4 41.4 51.5 88.88 11.11 99.57 99.6 49.89 50.1 Pupation No. No. Galleriamellonella 1.7 d 0.8 d 6.6 ± 6.6 ± 6.6 ± 6.6 ± 14.5 a 14.5 a 28.9 c 28.9 c 28.3 b 69.3 ± 69.3 ± 783.3 ± 783.3 ± 271.6 ±

0 d 0 d 0 0 0 0 0.37 c 0.37 c 0.35 a 0.15 a 0.15 a 0.74 a 0.33 b 36.3 ± 36.3 ± 38.3 ± 37.7 ± 37.7 ± 38.5 ± 28.47 ± 28.47 ± Duration

0 % % 100 25.9 25.9 65.8 34.9 34.9 83.4 Sterility

No. No. 91.2 c 91.2 c 29.7 b 17 d 17d 15.43b 585.6 ± 585.6 ± 790 ± 790 ± hatch hatch 13.3 a 13.3 a 276.33 ± 276.33 ± 513.33 ± 513.33 ± 130.33 ± 130.33 ± a a b Eggs Eggs Larvae Pupae Adults 0 d 0 e 9.5 ± 9.5 ± 9.8 ± ation ation 0.36 c 0.36 c 0.10 a 7.48 ± 7.48 ± (days) period period Incub- 0.23 ab 9.1 ± 0.1 9.1 ± 10 ± 0.22 10 ± a a 48 a 48 a eggs 16.58 a 756 ± 756 ± 794 ± 794 ± 11.3 a 11.3 a 27.1 a 19.5 a 19.5 a No. of of No. females mated with irradiated mated of females males /female /female 799.7 ± 799.7 ± 771.3 ± 751.33 ± 751.33 ± 743 ±10 743 ±10 Effect of irradiation on eggs, larvae, pupae and a

40 100 160 200 260 (Gy) Doses Doses Control Radiation Values represent the mean ±S.E. of 3 replicates for each each for 3 replicates group. of mean the ±S.E. represent Values Letters indicate the variance between the means (Duncan's means variance the the multiplebetween (Duncan's indicate ra Letters

Table (2):

٣٨

٣٨ Results

th irradiated male th irradiated parent male Doses of gamma (Gy) ray 0 40 100 160 200 260

810 800 790 780 770 760 750 740 730 720 710 pupae: pupae:

Number of Number eggs resulted from normal female mated wi No. eggs No.

Figure Figure (4):

٣٩

٣٩ Results

0 51.5 : Doses of gammaDoses ray (Gy) 52.8 generation resulted from normal female 1 85.5 % Adult Emergence % Galleriamellonella 87.38 99.6 0 40 100 160 200 260 0

90 80 70 60 50 40 30 20 10

100 Adult Emergence Adult Percentage of adult emergence of F mated with irradiated mated of male

Figure (5):

٤٠

٤٠

Results

III. Irradiated male mated with irradiated female:

Data in table (3) and Figures (6 & 7) indicated that there was a highly significant reduction in the number of eggs per female with the increase of the doses when irradiated male mated with irradiated female. The numbers of resulted eggs per female were 545, 314, 55.6, 0 and 0 at 40, 100, 160, 200 and 260 Gy, respectively, as compared with control (799.7).

The incubation period of the eggs was in correspondence to the doses increased, it was 10 days at 100 Gy and 0 days at the dose 160 Gy as compared with 7.5 days in the control. Similarly, the sterility was in accordance to the doses increased, showing the 100% sterility at the doses 160, 200 and 260 Gy.

Larvae showed a significant decline in longevity at the doses 100 Gy.

The percent of pupation was inversely correlated to the dose increase. The percentages pupation were 42.03, 0, 0, 0 and 0 at, 40, 100, 160, 200 and 260 Gy, respectively, as compared with 99.6 in the control. Adult emergence and sex ratio were unmatched with the dose increasing as they were zero at 100, 160, 200 and 260 Gy.

41

Results

: Sex ratio Sex ratio Male Male Female dults resulted from % % Emergence Galleriamellonella nge test). nge % % 99.57 42.03 99.6 55.555 49.8 83.3 50.1 16.7 Pupation No. No. 14.5 a 14.5 a 0.33 b 2.33 ± 2.33 ± 783.33 ± 783.33 ±

0 c 0 c 0 b 0 c 0 b 0 c 0 0 b 0 b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 39 ± 39 ± 1.15 a 1.15 a 0.37 b 28.47 ± 28.47 ± Duration

0 % % 100 100 100 98.5 98.5 93.5 93.5 Sterility No. No. 52 ± 52 ± 1.7 c 790 ± 790 ± hatch hatch 13.3 a 13.3 a 11.6 b 12.66 ± 12.66 ± Eggs Eggs Larvae Pupae Adults 0 c 0 c 10 ± 10 ± 7.4 ± 7.4 ± 9.7 ± ation ation 0.11 a 0.11 a 0.15 a 0.36 b (days) period period Incub- irradiatedwith irradiated mated of females males Effect of irradiation on eggs, larvae, pupae and a 0 e 0 e 0 c 0 c 0 c 0 c eggs 1.4 d 545 ± 545 ± 314 ± 11.3 a 11.3 a No. of of No. 13.7 c 13.7 b 55.6 ± 55.6 ± /female /female 799.7 ± 799.7 ±

40 100 160 200 260 (Gy) Doses Doses Letters indicate the variance between the means (Duncan's means variance the the multiplebetween (Duncan's indicate ra Letters Values represent the mean ±S.E. of 3 replicates for each each for 3 replicates group. of mean the ±S.E. represent Values Control Radiation Table (3):

٤٢

٤٢ Results

adiated male ofadiatedmoth: wax male Dosesgamma(Gy) ray of 0 40 100 160 200 260 Number of eggs withperNumber irradiated irr mated female 0

900 800 700 600 500 400 300 200 100 No. of eggs of No. Figure(6):

٤٣

٤٣ Results ted 0 Doses of gamma Dosesof (Gy) ray 0 0 % Adult Emergence Adult % 0 generation of irradiated withmated female irradia 1 55.55

99.6 0 40 100 160 200 260 0 80 60 40 20 100

male ofmoth: wax male

Adult emergence ofF Adult Emergence Adult Figure(7):

٤٤

٤٤

Results

B. The combiend effect of Entomopathogenic nematodes and radiation on the larvae of the greater wax moth, Galleria mellonella:

Results as shown in Tables (4 to 7) and Figures (8 &9) comparing the percentage mortality that occurred between normal larvae treated with entomopathogenic nematodes only and F 1 larvae resulted from irradiated male parent mated with normal female and then treated with entomopathogenic nematodes.

I. Susceptibility of wax moth larvae to Heterorhabditis bacteriophora BA1:-

Data in Table (4) showed that percentage mortality increased with increasing the doses of radiation at all the Heterorhabditis bacteriophora treatments within 1 – 3 days of the experiment. The means of percentage mortality for the 1 st day were 0, 41.6 and 58.3 for the doses 0, 40 Gy and 100 Gy, respectively. The percentage mortality in the 2 nd day was 25, 83.3 and 100 for the doses 0, 40 and 100 Gy, respectively. While in the 3 rd day, the mortality was 100% at all gamma radiation doses used, as comparing with control 0% at 20 IJs treatment. At 40 IJs, the percent mortality was 0, 58.3, and 66.7 for the 1st day. While in the 2 nd day, the mortality increased to 50, 100 and 100% and in the 3rd day; it reached 100% for all gamma doses applied 0, 40 and 100 Gy. When 60 IJs were used, the larval mortality at 1 st day of infection was 0, 66.7 and 75%. While in 2 nd day, they

45

Results were 75, 100 and 100% for gamma radiation doses 0, 40 and 100 Gy, respectively. At 3 rd day, the mortality was 100% for all gamma doses applied. The concentration of 80 IJs caused mortality of 0, 83.3 and 91.7% for gamma doses 0, 40 and 100 Gy; respectively, at the 1 st day. While in 2 nd and 3 rd days the mortality was 100% at all gamma radiation doses applied.

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Results

Table (4): The combiend effect of entomopathogenic nematodes; Heterorhabditis bacteriophora BA1 on percent mortality of unirradiated and irradiated larvae of Galleria mellonella at different time intervals (days).

% Mortality after Doses of Radiation No. Nematode/IJs Doses/GY Larvae 1 2 3 Day Days Days Control 0 Gy 12 0 0 0 0 Gy 12 0 25 75 40 Gy 12 41.6 83.3 100

20 IJs 100 Gy 12 58.3 100 100 0 Gy 12 0 50 100 40 Gy 12 58.3 100 100

40 IJs 100 Gy 12 66.7 100 100 0 Gy 12 0 75 100 40 Gy 12 66.7 100 100

60 IJs 100 Gy 12 75 100 100 0 Gy 12 0 100 100 40 Gy 12 83.3 100 100

80 IJs 100 Gy 12 91.7 100 100

• Values represent the mean of percentages of 3 replicates for each group.

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Results

II. Susceptibility of wax moth larvae to Steinernema carpocapsae BA2 :

The obtained results in Table (5) clearly show that there was a significant effect of gamma radiation doses on larval mortality at all Steinernema carpocapsae BA2 concentrations. At 1 st day; when Steinernema carpocapsae BA2 was applied at 20 IJ/4 larvae, the percentage mortality increased from zero at normal larvae to 82.5 in larvae resulted from irradiated male parent at 100 Gy.

The average mortality reached 100% in the 1 st day of application at concentration 40 IJs in F1 larvae resulted from irradiated male pupae of G. mellonella . While in normal larvae the mortality was 50%. At the 2 nd day, the larval mortality was 75, 100 and 100% for gamma doses 0, 40 and 100 Gy, respectively. The mortality was 100% in the 3rd day at all gamma radiation doses.

At 60 IJs, the mortality increased in the 1 st day to 75, 100 and 100% for 0, 40 and 100 Gy, respectively as compared to control (0%). For the 2 nd and 3 rd days, the mortality reached 100% for gamma doses applied.

In the application of 80 IJs, all larvae were dead at all gamma radiation doses application (0, 40 and 100 Gy), from the 1 st day of the experiment.

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Results

Table (5): The combiend effect of entomopathogenic nematodes, Steinernema carpocapsae BA2 on percent mortality of unirradiated and irradiated larvae of Galleria mellonella at different time intervals (days).

% Mortality after Doses of Radiation No. Nematode/IJs Doses/Gy Larvae 1 2 3 Day Days Days Control 0 Gy 12 0 0 0 0 Gy 12 0 75 100 40 Gy 12 66.6 100 100

20 IJs 100 Gy 12 82.5 100 100 0 Gy 12 50 75 100 40 Gy 12 100 100 100

40 IJs 100 Gy 12 100 100 100 0 Gy 12 75 100 100 40 Gy 12 100 100 100

60 IJs 100 Gy 12 100 100 100 0 Gy 12 100 100 100 40 Gy 12 100 100 100

80 IJs 100 Gy 12 100 100 100

• Values represent the mean of percentages of 3 replicates for each group.

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Results

III. Reproductive rate of dauer juveniles: -

1. Reproductive rate of Heterorhabditis bacteriophora BA1 juveniles in G. mellonella larvae: -

Data in Table (6) and Figure (8) indicated that there was a negative relationship between the average production rate of Heterorhabditis bacteriophora BA1 at different concentrations (20, 40, 60 and 80 IJs) and the gamma radiation doses applied to parents as pupae (40 and 100 Gy). This means that; the average production of nematode/larvae decreased with the increase of gamma radiation dose and the increase of nematode concentration.

The highest production were 200000 IJs/larvae for Heterorhabditis bacteriophora BA1 from non irradiated larvae infected by 20 IJs, while larvae resulted from irradiated parent at 100 Gy and infected by 80 IJs of produced 44000 IJs/Larvae.

The lowest yield was 3833.33 IJs/Larvae from F 1 larvae of irradiated parent as pupae with 100 Gy and infected by 80 IJs

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Results

ns: 0 d 80 IJs 11545 ± 207.4 a 207.4 a 11545 ± 26500 ± 275.3 b 26500 ± 3833.33 ± 88.2 c 88.2 c 3833.33 ±

BA1 in non-irradiated and 0 d group. 60 IJs

BA1 Yield / Larvae BA1 Yield / Larvae 5000 ± 26.4 c 26.4 c 5000 ± ncan'smultiple range test). 124000 ± 230.9 a 230.9 a 124000 ± 59333.3 ± 288.3 b 59333.3 ± each 0 d 40 IJs 192000 ± 642.9 a 642.9 a 192000 ± 16666.6 ± 284.8 c 284.8 c 16666.6 ± 90181.8 ± 183.7 b 90181.8 ± larvae infected different by concentrationematode

Heterorhabditis bacteriophora

Heterorhabditis bacteriophora 0 d 20 IJs G. mellonella G. 44000 ± 57.7 c 57.7 c 44000 ± 96500 ± 264.5 b 96500 ± 200000 ± 346.4 a 200000 ±

conc. irradiated Reproduction rate of Nematode Nematode 40 100 Control Control Control + Control nematode Lettersindicate the variance between (Du the means of 3 replicates Valuesrepresent for ±S.E. the mean doses (Gy) doses (Gy) Radiation (IJ)

Table (6):

٥١

٥١ Results

BA1 in non-irradiated 100Gy H.bacteriophora conc. (IJs) 40Gy larvae infected by different nematode BA1 Heterorhabditis bacteriophora

G. G. mellonella

20 40 60 80 Control 0 50000 200000 150000 100000 concentrations: and irradiated Reproduction rate of Figure (8):

٥٢

٥٢

Results

2. Reproductive rate of Steinernema carpocapsae BA2 juveniles in G. mellonella larvae: -

The obtained results in Table (7) and Figure (9) show that gamma radiation doses (40 and 100 Gy, exposed to parent male pupae) had a negatively significant effect on the rate of Steinernema carpocapsae BA2 production at the different applied concentrations (20, 40, 60 and 80 IJs).

The means of the nematodes production at 20 IJs were 109500, 32666.6 and 6545.45 IJs While they were 108000, 12666.6 and 4000 IJs; at 40 IJs BA2, for gamma radiation doses 0, 40 and 100 Gy; respectively, as compared to control (0 IJs).

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Results

14.43 a 14.43 a 71.78 b 95.46 c 95.46 c ± ± 0 d ±

80 IJs ns: 1500 2000 67200 nge test). nge Yield / Larvae Yield / Larvae 57.95 c 57.95 c in non-irradiated and irradiatednon-irradiatedand in 92.38 a 92.38 a

60.36 b ± ± 0 d ± 60 IJs BA2 BA2 4500 81000 2123.3 b 100 a 100 a ٥٧.٧٤ ± ± 23.76 c 23.76 c 0 d ± 40 IJs 4000 108000 12666.6

c

Steinernema carpocapsae Steinernema Steinernemacarpocapsae

٣٦.٠٦ 40.41 b ± 76.38 a 76.38 a ± ± 0 d 20 IJs larvae infected different by concentrationematode 109500 32666.6 6545.45

conc.

G. mellonella G. 40 Nematode Reproductionof rate 100 Control Control Letters indicate the variance between the means (Duncan's means variance the the multiplebetween (Duncan's indicate ra Letters each for 3 replicates group. of mean the ±S.E. represent Values Control + Control nematode

doses doses (Gy) Radiation (IJ)

(7): Table

٥٤

٥٤ Results

ns: IJs in non-irradiated and 100Gy

BA2 S. carpocapsaS. conc. (Ijs) 40Gy BA1 larvae infected different by concentrationematode Steinernema carpocapsae

Control 20 40 60 80 G. mellonella G. 0 80000 60000 40000 20000 120000 100000 Reproduction rate of irradiated

Figure (9):

٥٥

٥٥

Results

C. Examination of the haemocytes of Galleria mellonella larvae :

I. Description of the haemocytes of Galleria mellonella larvae:

1: Normal haemocytes:

Haemocytes were distinguished based on morphological characteristic and staining affinity. Eight types had been found in 4 th larval instar of G. mellonella (Figure 10 ): a- Prohaemocytes: were small, round to slightly ovoid cells, with undifferentiated cytoplasm. The single, round nucleus with dense, homogenous chromatin is usually enlarged in size leading to formation of macronucleus, which in turn sparing the cellular cytoplasm.

b- Plasmatocytes : characterized by their round shape. The nucleus with dark stains, round with punctuate or granular chromatin and usually central in position. c- Granulated cells : were slightly round, characterized by the undifferentiated cytoplasm and nucleus. d- Spindle cells : spindle shaped cells distinguished by the presence of very large, distinct, usually spindle nucleus (nu), mostly appearing as a solid purple structure. e- Spherule cells : were round with undistinguished nucleus. After Giemsa stain, they appeared in dark pink color.

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Results f- Adipohaemocytes : were rounds containing variable amounts of refringent fat droplets and several other non lipid inclusions. f- Oenocytoids: were slightly round characterized by round, large and approximately centric nucleus with homogenous chromatin. The cytoplasm contained numerous granular inclusions and filled with intricated canaliculi. g- Cystocytes: were extremely fragile cells with a single, small, irregular nucleus in envelop containing distinct, round, acidophilic inclusions.

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Results

Figure (10): Normal haemocytes of Galleria mellonella larvae (x= 1600)

(a): Prohaemocytes (b): Plasmatocytes (c): Granulated cells (d): Spindle cells (e): Spherule cells (f): Adipohaemocytes (g): Oenocytoids (h): Cystocytes

nu: indicates the haemocyte nucleus .

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Results

2: Morphological effects of Gamma radiation and/or entomopathogenic nematodes on Galleria mellonella haemocytes:

1-Prohaemocytes: When pupae were irradiated with doses 40 Gy and 100 Gy, the prohaemocytes of the F 1 larvae showed remarkable increase in the size with increasing the dose

Heterorhabditis bacteriophora BA1 and Steinernema carpocapsae BA2 nematodes of concentrations 20 IJs and 40 IJs treatments causing enlargement of the prohaemocytes. When they were applied to larvae of irradiated parent pupae, the most change observed in the prohaemocytes was enlarged or take elongate shape. Destruction of the cell membrane was apparent and there was also evidence of vacuoles. (Figure 11).

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Results

Figure (11): Morphological changes in prohaemocytes (x=1600)

(a) Normal: small, round to slightly ovoid prohaemocyte cell. (b) 40 Gy and (c) 100 Gy: increased the size of the cell. (d) BA1 20 IJs and (e) BA1 40 IJs: increased the size of the cell. (f) BA2 20 IJs and (g) BA2 40 IJs: increased the size of the prohaemocyte. (h) 40 Gy + BA1 20IJs and (i) 40 Gy + BA1 40IJs: increased the cell size. (j) 100 Gy + BA1 20IJs and (k) 100 Gy + BA1 40IJs: the cell takes an oval shape. (l) 40 Gy + BA2 20 IJs and (m) 40 Gy + BA2 40 IJs: prohaemocyte appear in oval shape. (n) 100 Gy + BA2 20 IJs: increased the size of the cell. (o) 100 Gy + BA2 40 IJs: Destruction of the cell membrane and presence of vacules (arrow).

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Results

2-Plasmatocytes: Morphological changes were observed in plasmatocytes in F 1 larvae after different treatments. Generally radiation with 40 Gy and 100 Gy to the parent male pupae causing the cell to be enlarged or take slightly oval shape. The nucleus lost its central position and moved to the cell wall (Fig. 12 b&c).

Entomopathogenic nematodes (Heterorhabditis bacteriophora BA1 and Steinernema carpocapsae BA2) affect plasmatocytes in the same way of gamma radiation and inducing vaculation of cytoplasm. The combined effect of nematodes and radiation would be more destructive to cells than using one of them only as the cells become irregular in shape. (Figure 12).

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Results

Figure (12): Abnormalities in plasmatocytes (x=1600) (a) Normal: round with central nucleus plasmatocytes. (b) 40 Gy and (c) 100 Gy: increased the size of the cell and the nucleus loss the central position (arrow). (d) BA1 20 IJs: increased the size of the cell. (e) BA1 40 IJs and (f) BA2 20 IJs: vaculation of cytoplasm (arrow). (g) BA2 40 IJs: The cell takes an oval shape. (h) 40 Gy + BA1 20IJs and (i) 40 Gy + BA1 40IJs: irregular shape plasmatocytes. (j) 100 Gy + BA1 20IJs; (k) 100 Gy + BA1 40IJs; (l) 40 Gy + BA2 20 IJs; (m) 40 Gy + BA2 40 IJs; (n) 100 Gy + BA2 20 IJs and (o) 100 Gy + BA2 40 IJs: The cell takes an oval shape with acentric nucleus (arrow).

nu: indicates the haemocyte nucleus

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Results

3- Granulated cells: When gamma radiation (40 and 100 Gy) applied to parent pupae significantly increased the size of granulated cells in the haemolymph of F 1 G. mellonella larvae.

Granulated cells enlarged or may take oval shape as a result of Heterorhabditis bacteriophora BA1 and Steinernema carpocapsae BA2 treatment with the concentrations 20 IJs and 40 IJs alone Figure 13 (d-h).

The combined effect of gamma radiation (40 Gy and 100 Gy) with Heterorhabditis bacteriophora BA1 (20 IJs) causing the same effect of EPN alone (Figure 13).

The other combinations of gamma radiation (40 and 100 Gy) with Heterorhabditis bacteriophora BA1 (40 IJs) and Steinernema carpocapsae BA2 (20 and 40 IJs) lead to disintegration and disappearance of granulated cells.

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Results

Figure (13): Morphological changes in granulated cells (x=1600)

(a) Normal granulocytes: slightly round, with undifferentiated cytoplasm and nucleus granulocyte. (b) treated granulocyte with 40 Gy; (c) 100 Gy and (d) BA1 20 IJs: showing increase in the cell size. (e) BA1 40 IJs: increase in the cell size with slight cytoplasm vaculation (arrow). (f) BA2 20 IJs and (g) BA2 40 IJs treatments showing granulocyte in oval shape with cytoplasm vaculation. (h) 40 Gy + BA1 20 IJs: round granulocyte with cytoplasm vaculation (arrow). (i) 100 Gy + BA1 20 IJs: oval granulocyte with cytoplasm vaculation (arrow).

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Results

4- Spindle cells:

Gamma radiation affected the shape of the spindle cells significantly. The nucleus may become slightly round Figure 14 (b&c ).

Spindle cells showed notable changes in their shape when Heterorhabditis bacteriophora BA1 and Steinernema carpocapsae BA2 were applied alone to G. mellonella larvae. When larvae of irradiated parent pupae with 40 Gy were treated with Heterorhabditis bacteriophora BA1 and Steinernema carpocapsae BA2 of concentrate 20 IJs, the spindle cells became compressed in size with approximately round nucleus (Fig. 14).

The combined effect of 40 Gy with 40 IJs of Heterorhabditis bacteriophora BA1 or Steinernema carpocapsae BA2 and 100 Gy with 20 or 40 IJs of EPN led to disappearance of the spindle cells.

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Results

Figure (14): Morphological changes in spindle cells (x=1600)

(a) Normal: spindle shaped cells with spindle nucleus (arrow). (b) irradiation with 40 Gy and (c) 100 Gy: increased the cell size. (d) BA1 20 IJs; (e) BA1 40 IJs; (f) BA2 20 IJs; (g) BA2 40 IJs; (h) 40 Gy + BA1 20 IJs and (i) 40 Gy + BA2 20 IJs: the spindle cell takes irregular shape approximately round nucleus (arrow).

nu: indicates the haemocyte nucleus

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Results

5- Spherule cells:

Irradiation of parent pupae with 40 Gy or 100 Gy showed a significant effect in the shape of the spherule cells of F 1 G. mellonella larvae.

Heterorhabditis bacteriophora BA1 (20 IJs and 40 IJs) treatment caused appearance of vacuoles in the cell. When Steinernema carpocapsae BA2 was applied, they lose their round shape Figure 15 (d-g).

Applications of Heterorhabditis bacteriophora BA1 (20 and 40 IJs) and Steinernema carpocapsae BA2 (20 and 40 IJs) to F 1 larvae of irradiated parent pupae with 40 Gy caused vacuoles formation in the cells Figure 15 (h-j).

The other combinations of gamma radiation (40 and 100 Gy) and EPN (20 and 40 IJs) disappeared spherule cells.

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Results

Figure (15): Morphological changes in spherule cells (x=1600)

(a) Normal cells: round with undistinguished nucleus (b) 40 Gy and (c) 100 Gy: increased the cell size. (d) BA1 20 IJs: irregular sphere cell. (e) BA1 40 IJs induced cytoplasmic vaculation (arrow). (f) BA2 20 IJs (arrow) and (g) BA2 40 IJs: irregular shape of the sphere cell. (h) 40 Gy + BA1 20 IJs; (i) 40 Gy + BA2 20 IJs and (j) 40 Gy + BA2 40 IJs: increased the cell size with cytoplasm vaculation (arrow).

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Results

6- Adipohaemocytes:

Gamma radiation of pupae with 40 and 100 Gy increased the size of adipohaemocytes significantly in F 1 larvae of irradiated male pupae mated with normal female. Application of Heterorhabditis bacteriophora BA1 (20 IJs) or Steinernema carpocapsae BA2 (20 IJs) also caused increase in the size (Figure 16).

Adipohaemocytes vanished from the haemolymph of G. mellonella larvae under the effect of Heterorhabditis bacteriophora BA1 or Steinernema carpocapsae BA2 (40 IJs) and under the combined effect of gamma radiation (40 and 100 Gy) and EPN (20 and 40 IJs).

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Results

Figure (16): Abnormalities in adipohaemocytes (x=1600)

(a) Normal: round containing variable amounts of refringent fat droplets (arrow).

(b) 40 Gy and (c) 100 Gy; (d) BA1 20 IJs and (e) BA2 20 IJs: increased the cell size than in control.

nu: indicates the haemocyte nucleus.

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Results

7- Oenocytoids:

Exposure of G. mellonella pupae to 40 Gy caused enlargement of the cell size in the haemolymph of F1 larvae. When the pupae exposed to 100 Gy, oenocytoids cell enlarged and the cytoplasm degranulated (Figure 17).

The treatment of Heterorhabditis bacteriophora BA1 or Steinernema carpocapsae BA2 alone or in F 1 Galleria mellonella larvae lysed oenocytoids; except when Heterorhabditis bacteriophora BA1 (20 IJs) was applied. The cells were enlarged and degranulated.

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Results

Figure (17): Morphological changes in oenocytoids (x= 1600)

(a) Normal: oenocytoid is slightly round with round, large and approximately centric nucleus (arrow). (b) 40 Gy: increase in the cell size. (c) 100 Gy and (d) BA1 20 IJs: oenocytoids cell enlarged and the cytoplasm degranulated (arrow).

nu: indicates the haemocyte nucleus

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Results

8- Cystocytes:

Treatment with Gamma radiation to parent pupae with 40 and 100 Gy or larvae treatment with Heterorhabditis bacteriophora BA1 or Steinernema carpocapsae BA2 in different concentrations (20 and 40 IJs) caused completely disappearance of cystocytes from the haemolymph of F 1 G. mellonella larvae.

In case of nematodes treatments, there is a heamocytes response appeared as immunocomponenet cells. This response is by phagocytosis (Figure 18 ‘a&b’) or by encapsulation of foreign body (Figure 18 ‘e&f’) or by increasing the mitotic division (Figure 18 ‘c&d’) .

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Results

Figure (18): Response of the haemocytes to the nematodes treatments: (a & b) phagocytosis, (c & d) mitotic division and (e & f) encapsulation of foreign body. (x= 1600)

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Results

II. Determination of total haemocytes count (THCs) in normal and treated wax moth larvae:

The larvae were divided into 3 groups, Group A which represents larvae resultsed from irradiated male parent mated with normal female, Group B represents normal larvae and F 1 larvae treated with H. bacteriophora BA1 doses (20 and 40 IJs) and Group C represents normal larvae and F 1 larvae treated with S. carpocapsae BA2 doses (20 and 40 IJs).

Data in Table (8) showed that in group (A) the total number of haemocytes of G. mellonella larvae decreased gradually by increasing the dose of radiation which applied to parent male pupae as compared by control being 32.08, 21.75 and 20.56 for 0, 40 and 100Gy, respectively.

In group (B) treatment with H. bacteriophora (20IJs and 40 IJs) caused significant decrease in the total haemocytes counts and more decrease occurred when H. bacteriophora applied to F1 larvae of irradiated parent male pupae (40 and 100 Gy). The THC decreased with increasing the dose of gamma radiation (0, 40 and 100 Gy) and increasing the concentrations of the nematodes (20IJs and 40 IJs).

In group (C) with S. carpocapsae treatment the THC of G. mellonella larvae was significantly decreased. When S. Carpocapsae was used against larvae resulted from irradiated male parent as pupae, THC revealed high reduction with increasing the radiation doses (0, 40 and 100 Gy) as compared with control. In treated G. mellonella larvae with 20 IJs, the THCs were 13.13, 10.4 and 8.73 while in 40 IJs treatment; THCs were 10.91, 9.2 and 5.61 for 0, 40 and 100 Gy respectively.

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Results

± ± 3 ± ±

3 3 0.75 a a 0.75 0.2 c 0.2 c BA2 0.57 b 0.57 b

± ± 10.91×10 9.2×10 BA2): BA2): 5.61×10 enic enic nematodes larvae treated with ± ± ± ± ±

3 3 3 Group C Group c c 21 0.34 a 0.34 a nge test). nge 0.32 b 0.32 b 0. S. carpocapsae 10.4×10 8.73×10 13.13×10

3 3 3 3 0.26 c c 0.26 BA1 0.57 a a 0.57 0.47 b b 0.47 ± ± ± ± ± ± Galleria mellonella 20.76×10 13.03×10 10.11×10 THC/mm Steinernema carpocapsae Steinernema ± ± ± ± ± ±

3 3 3 Group B Group 20 IJ 40 IJ 20 IJ 40 IJ 0.08 c 0.08 c 0.37 a 0.37 a 0.06 b 0.06 b BA1 or BA1 21.5×10 H. bacteriophora 15.03×10 13.45×10 of blood from 3 ± ± ± ± ±

3 3 3 b b 29 rays rays 0.54 a 0.54 a 0.22 b 0.22 b 0. Gamma Gamma Group AGroup 32.08×10 21.75×10 20.56×10

40 100 Control Control Heterorhabditis bacteriophora Heterorhabditis gamma gamma radiation ( alone or combined with entomopathog Total haemocytic count/mm Letters indicate the variance between the means (Duncan's means variance the the multiplebetween (Duncan's indicate ra Letters Values represent the mean ±S.E. of 3 replicates for each each for 3 replicates group. of mean the ±S.E. represent Values Types of treatments Radiation Radiation Doses (Gy)

Table Table (8):

٧٦

٧٦

Results

III. Determination of differential haemocytes count (DHCs):

1. Effect of gamma radiation and Heterorhabditis bacteriophora BA1 on differential haemocytes count (DHCs) in wax moth larvae :

(A): Male pupae of` the greater wax moth were irradiated with dose 40 Gy and mated with normal female, the resulted F 1 larvae were treated with Heterorhabditis bacteriophora BA1 (20 and 40 IJs).

Results in Table (9) showed that gamma radiation had slight effect on prohaemocytes number by increasing their percent. While with H. bacteriophora BA1 treatment alone or in F 1 larvae their percent was significantly increased. The percentages of prohaemocytes were 49.59, 65.21, 77.94, 83.41 and 91.98 as compared to control 49.56 in treatment with 40 Gy, 20IJ, 40IJ, 40 Gy+ 20 IJ and 40 Gy+ 40 IJ, respectively.

It is also noticed that the percentage of plasmatocytes was 23.16 in control sample, and when Galleria mellonella was irradiated with 40 Gy; a slight increase in percentage plasmatocytes was occurred that reach 23.69. While in the treatment of H. bacteriophora BA1 to normal or F 1 G. mellonella larvae, the percent plasmatocytes decreased with increasing H. bacteriophora BA1 conc. (20-40IJ). They were 23.69, 16.71, 13.48, 9.37 and 8.02 as compared to control 23.16 by the treatment of 40 Gy, control + 20IJ, control + 40IJ, 40 Gy+ 20 IJ and 40 Gy+ 40 IJ, respectively.

Granulocytes: Their percent was decreased in larvae resulted from irradiated parent pupae, larvae treated with

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Results

(20 and 40 IJ) H. bacteriophora BA1 and in their combinations. In control sample, the percentage of granulocytes was 5.62 and decreased to 0 at 40 Gy + 40IJ.

Spindle cell: 40 Gy of gamma radiation applied to parent as pupae had no effect on the percentage of spindle cells in F 1 larvae. When H. bacteriophora BA1 was applied to G. mellonella larvae, the percentage of spindle cells decreased from 7.9 to 6.44 for 20 IJs and 5 for 40 IJs, respectively. More decrease was obtained when H. bacteriophora BA1 was applied to larvae of irradiated parent as it became 4.43% and 0% for 40 Gy + 20 IJs and 40 Gy + 40 IJs, respectively.

Spherule cells: the percent of sphere cells was decreased gradually by gamma radiation 40 Gy, H. bacteriophora BA1 treatment (20 IJs and 40 IJs) and their combinations. In control, the percent of sphere cells was 3.05 and it decreased till 0 at the treatment of 40 Gy + 40 IJs.

The percent of adipohaemocytes and Oenocytoids in G. mellonella F1 larvae were slightly decreased by 40 Gy applied to parent as pupae. When BA1 was applied to normal or F1 larvae. Their percents were significantly decreased to 0% in the treatments of 40 IJs, 40 Gy + 20 IJs and 40 Gy+ 40 IJs as compared with control of 7.15% and 3.27% for adipohaemocytes and Oenocytoids, respectively.

Cystocytes had a percent of 0.28 in control. On the other hand, in larvae treated with BAI (20IJ and 40 IJs), larvae resulted from irradiated parent at 40 Gy, or in their combinations, cystocytes were completely disappeared.

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Results

BA1 40 IJs 40 Gy + group.

20 IJs 40 Gy + catesfor each larvae (irradiated parent male

Heterorhabditis Heterorhabditis bacteriophora Galleria Galleria mellonella

1 7.9 7.9 6.44 5 4.43 0 5.62 3.05 5.55 7.15 3.27 3.02 2.55 0.28 7.06 3.19 2.15 1.52 0 5.38 1.57 2.06 1.04 0 0 0 1.74 0 0 0 0 0 0 0 0 0 49.56 23.16 49.59 23.69 65.21 16.71 77.94 13.48 83.41 9.37 91.98 8.02 Control 40 Gy 20 IJs 40 IJs Cystocytes Oenocytoid Spindle cells Granulocytes Spherule cells Plasmatocytes Prohaemocytes Valuesrepresent of percentages the mean of 3 repli pupae with 40 Gy) alone or combined with treatments: IJs) 40 and (20 Treatments

Adipohaemocytes • Percentage haemocytes of F Cell Type

Table Table (9):

٧٩

٧٩

Results

(B): Experiments carried out in male pupae of the greater wax moth were irradiated with dose 100 Gy and mated with normal female, the resulted larvae were treated with Heterorhabditis bacteriophora BA1 (20 and 40 IJs) as F1 larvae had been shown in table (10).

Data from Table (10) represented that the percent of prohaemocytes was gradually increase in the haemolymph of 4 th instar larvae of G. mellonella as a result of male parent irradiation with 100 Gy alone or in combination with H. bacteriophora BA1 (20 IJs and 40 IJs). They were 50.29, 65.21, 77.94, 92.44 and 97.88 as compared to control 49.56.

Plasmatocytes in the control, the percentage number was 23.16. Th treatment with 100 Gy to parent showed a slight increase effect, while H. bacteriophora BA1 treatment induced a reduction in plasmatocytes number. Treatment of H. bacteriophora BA1 (20IJs and 40 IJs) to larvae resulted from irradiated parent at 100 Gy was significantly reduce their percent from 23.16 in control to 7.55 and 2.11 at 20 IJs + 100 Gy and 40 IJs + 100 Gy, respectively.

Granulocytes: Their percent was decreased in larvae resulted from irradiated parent pupae, larvae treated with (20 and 40 IJ) H. bacteriophora BA1 and in their combinations. In control it was 5.62% and decreased to 0% at 100 Gy + 40IJ.

The percent of both spindle cells and sphere cells were gradually decreased when 100 Gy was applied to parent pupae and when larvae was treated with H. bacteriophora

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Results

BA1 (20 IJ and 40 IJ) alone. While, in case of F 1 larvae of irradiated parent treated with H. bacteriophora BA1 (20IJs and 40 IJs) the percents became zero for both spindle cells and sphere cells. Larval treatment with 40 IJs of H. bacteriophora BA1 alone or after irradiation of parent pupae with 100 Gy, led to the disappearance of both adipohaemocytes and Oenocytoids . When 100 Gy or 20 IJs alone was applied to G. mellonella larvae the percent numbers were gradually decreased as compared with control of 7.15 and 3.27 for adipohaemocytes and oenocytoids, respectively.

The percentage of adipohaemocytes were 6.39 and 5.38 for 100 Gy and 20 IJs of H. bacteriophora BA1, respectively. While for Oenocytoids they were 3.17 and 1.57.

Cystocytes had a percent of 0.28 in normal larvae. Treatment with BA1 (20 and 40 IJs), gamma radiation (100 Gy) or in their combinations; cystocytes were completely disappeared.

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Results

BA1 40 IJs 100 Gy + group.

20 IJs 100 Gy + larvae larvae (irradiated parent male

catesfor each Heterorhabditis Heterorhabditis bacteriophora Galleria Galleria mellonella

1 7.9 7.8 6.44 5 0 0 5.62 3.05 4.81 7.15 3.27 2.37 2.55 0.28 6.39 3.17 2.15 1.52 0 5.38 1.57 2.06 0.77 0 0 0 0 0 0 0 0 0 0 0 0 0 49.56 23.16 50.29 25.12 65.21 16.71 77.94 13.48 92.44 7.557 97.88 2.112 Control 100 Gy 20 IJs 40 IJs

pupae pupae with 100 Gy) alone or combined with (20 and 40 IJs) treatments: treatments: IJs) 40 and (20 Valuesrepresent of percentages the mean of 3 repli

Percentage haemocytes of F • Cystocytes Oenocytoid Spindle cells Granulocytes Spherule cells Plasmatocytes Prohaemocytes Treatments Adipohaemocytes Cell Type Table (10):

٨٢

٨٢

Results

2. Effect of gamma radiation and Steinernema carpocapsae BA2 on differential heamocytes counts (DHCs) in wax moth larvae:

(A): Male pupae of the greater wax moth were irradiated with dose 40 Gy and mated with normal female, the resulted F 1 larvae were treated with Steinernema carpocapsae BA2 (20 and 40 IJs).

Data from Table (11) showed that the percent of prohaemocytes number was increased with all the treatments applied to G. mellonella larvae. The percents were 49.56, 49.59, 74.41, 82.81, 85.71 and 92.49 for control, 40 Gy, BA2 20 IJs, 40 IJs, 40 Gy+20 IJs and 40 Gy + 40 IJs, respectively.

Plasmatocytes: the irradiation of wax moth male pupae with 40 Gy caused a slight increase in percent of plasmatocytes to reach 23.69, when S. carpocapsae BA2 was applied to normal or F 1 G. mellonella larvae. The percent plasmatocytes decreased with increasing S. carpocapsae BA2 conc. (20-40 IJs) .

In treatment of Gamma radiation (40Gy) to parent pupae or with S. carpocapsae BA2 (20IJs and 40 IJs) alone, the percent of granulocytes reduced in the haemolymph of 4th instar of G mellonella from 5.62 in control to 5.55, 2.64 and 1.55 for 40 Gy, 20 IJs and 40 IJs, respectively. While in the combination of 40 Gy to parent and S. carpocapsae BA2 (20IJs and 40 IJs), granulocytes disappeared.

When 40 Gy of gamma radiation was applied to parent pupae, the percentage of spindle cells in the haemolymph of

83

Results

4th instar G. mellonella larvae were not affected. S. carpocapsae BA2 (20IJs and 40 IJs) alone or 20 IJs + 40 Gy treatments decreased the percent than in control 7.9. In case of 40 Gy + 40 IJs S. carpocapsae BA2, the spindle cells percent became zero.

Spherule cells percent was significantly affected by all the treatments. The percents were 3.05, 3.02, 2.2, 1.53, 1.59 and 1 for control, 40 Gy, 20 IJs, 40 IJs, 40 Gy + 20 IJs and 40 Gy + 40 IJs, respectively.

Adipohaemocytes percent was slightly decreased from 7.15 at control to 7.06 when 40 Gy was applied to parent pupae of G. mellonella . In S. carpocapsae BA2 (20IJs) treatment the percent was sharply decreased to 1.32, while in case of 40 IJs alone or BA2 (20IJs and 40 IJs) combined with 40 Gy adipohaemocytes percent was zero.

S. carpocapsae BA2 (20IJs and 40 IJs) alone or combined with Gamma radiation (40 Gy to percent) treatment lead to disappearance of Oenocytoids , except at the dose 40 Gy applied alone to parent pupae; the percent showed slightly reduction than in control.

Cystocytes had a percent of 0.28 in control. When G. mellonella larvae treated with S. carpocapsae BA2 (20IJ and 40 IJs), larvae resulted from irradiated parent at 40 Gy, or in their combinations, cystocytes were completely disappeared.

84

Results

BA2 (20 and 40 IJs 40 Gy + group.

20 IJs 40 Gy + larvae larvae (irradiated parent male

cates for each for cates Steinernema Steinernema carpocapsae Galleria Galleria mellonella

1 7.9 7.9 5.588 3.817 4.46 0 5.62 3.05 5.55 7.15 3.27 3.02 2.64 0.28 7.06 3.19 2.2 1.55 0 1.32 0 1.53 0 0 0 1.59 0 0 0 0 1 0 0 0 0 0 49.56 23.16 49.59 23.69 74.41 13.84 82.81 10.29 85.71 8.23 92.49 7.51 Control 40 Gy 20 IJs 40 IJs

Values represent the mean of percentages of3 repli of percentages mean the represent Values pupae pupae with 40 Gy) alone or combined with treatments: 40IJs) Percentage haemocytes of F

• Cystocytes Oenocytoid Spindle cells Granulocytes Spherule cells Plasmatocytes Prohaemocytes Treatments Adipohaemocytes Cell Type

Table Table (11):

٨٥

٨٥

Results

(B): Male pupae of the greater wax moth were irradiated with dose 100 Gy and mated with normal female, the resulted F 1 larvae were treated with Steinernema carpocapsae BA2 (20 and 40 IJs).

Results in Table (12) illustrated that the percentage of prohaemocytes increased in the haemolymph of F 1 larvae under the effect of 100 Gy applied to parent pupae, S. carpocapsae BA2 (20IJ and 40 IJs) and their combinations. In contrast, the percentage plasmatocytes was gradually decreased by the treatment applied.

The combined effect of gamma radiation (100 Gy) applied to parent as pupae and S. carpocapsae BA2 (20IJs and 40 IJs) resulted in the absence of granulocytes , spindle cells and sphere cells . When gamma radiation or S. carpocapsae BA2 was applied separately, their percent were reduced gradually. In control, the percents were 5.62, 7.9 and 3.05 for granulocytes, spindle cells and spherule cells, respectively.

Percent oenocytoids was 3.27 in control and reduced to 3.17 in larvae resulted from irradiated parent at 100 Gy treatment of S. carpocapsae BA2 (20IJ + 40 IJ) alone or combined with 100 Gy when applied to parent led to the disappearance of oenocytoids.

Cystocytes had a percent of 0.28 in control. The treatment of gamma radiation and nematodes or their combinations caused a complete disappearance of cystocytes.

86

Results

BA2 (20

40 IJs 100 Gy + group.

20 IJs 100 Gy + larvae (irradiated parent male cates for each for cates

Steinernema Steinernema carpocapsae Galleria Galleria mellonella

1 7.9 7.8 5.588 3.817 0 0 5.62 3.05 4.81 3.27 2.37 2.64 0.28 3.17 2.2 1.55 0 0 1.53 0 0 0 0 0 0 0 0 0 0 0 7.15 6.39 1.32 0 0 0 49.56 23.16 50.29 25.12 74.41 13.84 82.81 10.29 95.27 4.73 96.55 3.45 Control 100 Gy 20 IJs 40 IJs

Values represent the mean of percentages of3 repli of percentages mean the represent Values pupae with 100 Gy) alone or combined with treatments: IJs) 40 and

Percentage haemocytes of F • Cystocytes Oenocytoid Spindle cells Granulocytes Spherule cells Plasmatocytes Prohaemocytes Adipohaemocytes Treatments Cell Type

Table (12):

٨٧

٨٧

Discussion

Biological effects of irradiation on Galleria mellonella :-

Irradiation studies on several insects species indicated that, the pupal stage was generally the most effective stage for treatment as it was easier to handle and leads to a reduction in reproductive potential Proverbs and Newton (1962 a&b) Lepidopterous insects require high doses of radiation to achieve fully sterilized adults, these doses often render them less competitiveness than unpredicted , therefore, using sub sterilizing doses of radiation are increased competitiveness of released insects and possible integration with other non – polluting methods to control insects pests North & Holt (1968a&b) . The present study showed that the dose of radiation was inversely proportional to the fecundity and fertility when both sexes of G. mellonella were exposed as pupae to all tested doses of gamma radiation. Similarly, Bloem et al . (1999) on male Cydia pomonella (L.); Bloem et al. (2003) on female Cryptophlebia leucotreta; Mohamed (2004) on male Agrotis ipsilon ; Allinghi et al. (2007) on female Anastrepha fraterculus , all reported that the reduction in both fecundity and egg viability were increased by increasing the radiation doses applied and Aye et al. (2008) who stated that fecundity and hatchability of irradiated Plodia interpunctella were reduced when pupae were



Discussion irradiated at 0.1 kGy and completely inhibited at 0.25 kGy and higher doses.

The reduction in both fecundity and egg hatchability was more significant when both irradiated parents mated together than one irradiated parent mated to the opposite normal sex. This is in accordance with El-Kady et al. (1983) whom said that treatment of both parent pupae of Agrotis ipsilon reduced fertility more markedly than did treatment of either sex separately . Also Milcheva (2005) found that the cross of 2 irradiated parents pupae of Galleria Mellonella results in sterility at 150 Gy and Hofemeyr et al. (2005) stated that fecundity of treated female pupae of Cryptophlebia Leucotreta mated to treated males pupae declined as the dose of radiation increased.

Female G. Mellonella was more sensitive to radiation doses rather than male. The reasons of fecundity and fertility reduction may relate to dominant lethal mutation as reported by Marec et al. (2001) on Ephestia Kuehniella and Milcheva (2005) on Galleria mellonella or lack of eupyrene sperm transferred by irradiated male during mating as suggested by Bloem et al. (1999) ; Alm El- Din (2001) on Spodoptera littoralis . Haiba (1990) reported that sever effect of gamma irradiation on the germanium and vitellarium of the overioles in the resulted females of Ph. Operculella . Sawires (2005) said that thinness in the follicular epithelium and disrupted nurse cells in female E. Kuehniella ovaries.



Discussion

Also the loss of sperm bundles, degeneration and malformation in the resulting sperm, which were too short and clumped in damaged testis were the way to explain the deleterious effects occurred in the fecundity and fertility of treated male pupae of Ephestia Kuehniella (Sawires, 2005) .

On the other hand, larval duration of larvae resulted from irradiated parents pupae, was increased in accordance to the dose increased at all different combinations this result agree with Gharieb (1989) on Trogoderma granarium who said that the effect of gamma irradiation on growth rate seemed to be dose-dependent, the higher the dose to the parental males and females, the slower the development of the F 1 generation.

The effect of radiation given to male parent pupae increased the mean number of days for F 1 larval development, reported by Bloem et al . (2003) on false codling moth; Cryptophelebia leucotreta and Sawires (2005) on the mediterranean flour moth; Ephestia kuehniella .

Our results indicated that the percent pupation and percent of adult emergence in F 1 progeny were suppressed by increasing the dose of gamma radiation given to the parental males or females or both. Similar results obtained by El-nagar et al . (1984) on Agrotis ipsilon ; Sallam & Ibrahim (1993) on Spodoptera littoralis and Sallam et al . (2000) on Earias insulana . They reported that the rate of



Discussion pupation and percent adult eclosion were decreased and correlated with irradiation dose used. Male bases of sex ratio had been obtained in the first generation of all matings at the different doses used (40 – 260 Gy), this result goes parallel to that of Hoe and Tien (2001) on Plutella xylostella ; Abd-Elwahed (2004) on Phthorimaea operculella and Sawires (2005) on Ephestia kuehniella .

Effect of entomopathogenic nematodes and gamma radiation on the larvae of the greater wax moth, Galleria mellonella :

The nematodes attack a wide spectrum of insects in the laboratory, where host contact is assured, environmental conditions are optimal, and no ecological or behavioral barriers to infection exist (Gaugler, 1988; Kaya and Gaugler, 1993).

Infectivity of steinernematid and heterorhabditid nematodes varied greatly due to some environmental factors or requirements. These environmental requirements could be Physical or biotic parameters (Woodring and Kaya, 1988).

Some other important factors affect the infectivity for a great extent such as nematode species/strain, exposure period, nematode concentration and period of nematode storage. The degree of infectivity of each of the nematode



Discussion species/strain for different hosts varied considerably and any species of nematode was not the most infective for all insect species (Bedding et al., 1983).

The present study showed that increasing parental radiation dose increased larval susceptibility (F1) to both genus of nematodes. The radiation debilitated G. mellonella larvae, which made the penetration of nematodes easier so the mortality increased at higher doses of radiation.

Increasing irradiation of parents may increase carbon dioxide release from F 1 larvae and establishing chemical and heat gradients to which entomopathogenic nematodes are known to cue in and use in host finding ( Lewis et al ., 1993 and Grewal et al ., 1993 ).

There were two extreme modes of behavior used when entomopathogenic nematodes are attempting to locate the insect host; which can be categorized as cruising Heterorhabditis bacteriophora, which are highly mobile and responsive to volatiles (carbon dioxide) released by the host and ambushing Steinernema carpocapsae, which tend to stand on their tails in a rigid position allowing them to attack pests (Grewal et al ., 1994 ).

In the preset study increasing the parental radiation doses (40- 100 Gy) increased F 1 larval susceptibility to Heterorhabditis bacteriophora BA1 and Steinernema carpocapsa e BA2 in different concentrations. This agree with results of Abdel Salam et al . (1995) on phthorimaea



Discussion 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 .

From the results, it was noticed that Steinernema carpocapsa BA 2 was faster in Killing Galleria mellonella larvae than Heterorhabditis bacteriophora BA1. This result was in accordance with those obtained by Stark and Lacey (1999) on Rhagoletis indifferens mortality to S. carpocapsae , S. feltiae , S. riboravae and H. bacteriophora and Zawrah (2008) who reported that S. carpocapsae was faster than S. feltiae , H. bacteriophora (1-3) and H. bacteriophora in killing C. vittata .

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 used by S. carpocapsae to acquire an insect host. The need to sustain the F 1 population of Galleria mellonella for sterility promotion and subsequent population collapse suggests S. carpocapsae to be an ideal entomopathogenic nematode to be used in conjugation with inherited sterility control method.

Our results indicated that the average production of nematodes decreased with increasing the concentration applied to the host and the radiation dose increasing. This



Discussion result coincided with those obtained by Saleh & Alheji (2003) and El- Mandarawy et al . (2006). The reason of reduction was explained by Kaya and Koppenhofer (1996) who reported that when the number of IJ's penetrating into a host exceeds an optimal level, exploitative interaspecific competition occurs among the developing nematodes, which reduces the total number of progeny emerging from the cadaver.

Similarly, Rizk (1991) reported that the blood volume of Spodoptera littoralis larvae decreased with increasing gamma radiation doses. This may be the reasone for reduction of EPN yield, according to thier habitate of releasing the symbiotic bacteria into the insect haemocoel to multiply and create the conditions necessary for the nematodes to mature, mate and reproduce . Also the anatomical structure of separete sexs of Steinernema spp. and hermaphroditic Heterorhabditis was the reason of higher generation production of Heterorhabditis bacteriophora BA1 than Steinernema carpocapsae BA2 (Poinar, 1975 ).

Determination of haemocytes count in Galleria mellonella larvae affected by gamma radiation and EPN:

Insect haemocytes were identified by a combination of morphological, antigenic, and functional characteristics (Gupta, 1985; Brehelin and Zachary, 1986; Willott et al .,



Discussion

1994; Gardiner and Strand, 1999; Lanot et al ., 2001; Jung et al ., 2005 ). Although haemocytes have similar functions in immunity across all insects, the naming of different haemocytes types varies somewhat among species and taxa Strand (2008) .

The most common types of haemocytes reported are prohemocytes, granular cells (granulocytes), plasmatocytes, spherule cells (spherulocytes), and oenocytoids. These haemocytes have been described from species in diverse orders including Lepidoptera, Diptera, Orthoptera, Blattaria, Coleoptera, Hymenoptera, Hemiptera, and Collembola (Ahmed, 1988; Azambuja et al ., 1991; Ahmed, 1992; Luckhart et al ., 1992; Sonawane and More, 1993; Joshi and Lambdin, 1996 ). The other haemocytes described from Lepidoptera are non adhesive spherule cells, oenocytoids and prohemocytes. Spherule cells have been suggested to transport cuticular components ( Sass et al ., 1994 ), while oenocytoids contain cytoplasmic phenoloxidase precursors that likely play a role in melanization of haemolymph (Ashida and Dohke, 1980; Iwama and Ashida, 1986; Jiang et al ., 1997 ). Prohemocytes are hypothesized to be stem cells that differentiate into one or more of the aforementioned haemocytes types (Lavine and Strand, 2002 ).

th From the results, THC of normal 4 instar larvae or F 1 G. mellonella larvae from irradiated male parent pupae with doses 40 and 100 Gy mated with normal female then treated with Heterorhabditis bacteriophora BA1 and Steinernema carpocapsa BA2 at conc. 20 IJs and 40 IJs, showed



Discussion remarkable decrease with increasing the dose of radiation and the EPN conc. applied. The results were in agreement with those obtained by Souka et al . ( 1993 ) who studied the effect of gamma radiation on Spodoptera littoralis ; El- Mandarawy (1997 ) on Sesamia cretica parasitized by Bracon brevicornis ; Ayaad et al . ( 2001 ) on Paresarcophaga surcoufi injected by Heterorhabdities bacteriophore HP 88 and Barakat et al . (2002 ) on Schistocerca gregaria injected with Bacillus thuringiensis.

Once the symbiotic bacteria appeared in the haemolymph, they adhere to the haemocyte in order to reproduce within it and in parallel with their re-emergence into the haemolymph; the bacteria induce damage to the haemocytes of Lepidoptera ( Dunphy and Thurston , 1990 ; Dunphy , 1994 & 1995 ). Under condition of nematode infections the haemocytoes seems to be under stress of inhibitory toxin which is released from the outer membrane of the nematode – symbiotic bacteria. Therefore, the decrease in the THC of 4 th instar G. mellonella larvae may be considered as a result of this stress of both the infective nematodes H. bacteriophora and S. carpocapsae and their symbiotic bacterium Xenorhabdus luminescens and Photorhabdus sp ., respectively (Dunphy and Webster , 1988 a & b ).

Eight types of haemocytes were observed in the haemolymph of 4 th instar G. mellonella larvae: prohaemocytes (PRs), plasmatocytes (PLs), granular cells (GRs), spindle cells (SPs), adipohaemocytes (Ads), Oenocytoids (OEs) and spherule cells (SHs).



Discussion

The prohaemocytes were the predominant type and the cystocytes were the rare type. The present study on DHCs of normal G. mellonella larvae treated with different radiation doses or entomopathogenic nematodes (BA1 or BA2) showed that the percent of prohaemocytes increased. While the other types decreased with increasing the treatments. Our results were in accordance with those of Rizk (1991) on Spodoptera littoralis .

Due to nematodes infections stimulatory factor stimulates the haemopietic organs to form new haemocyte generations and/or mitosis of the circulating haemocytes (Feir, 1979 and Lackie, 1988 ).

Therefore, the increase in prohaemocytes is a result of mitosis in response to nematode treatment. As prohemocytes hypothesized to be stem cells that differentiate into one or more of the aforementioned haemocytes types (Lavine and Strand, 2002 ), this is simlar to that obtained by Ayaad (2001) on P. Surcoufi injected by H. bacteriophora . Abu El- Magd & El-Kifl (1993) found that H. heliothidis nematodes decreased the plasmatocytes and granulocytes of Spodoptera littoralis larvae after injection. El-Maasarawy et al. (1995) reported that Corcyra cephalonica exposure to gamma radiation reduced the immunity system (spherulocytes, spindle cell and plasmatocytes) and Matha et al. (1990) who said that the plasmatocytes considered as immunocompetent cells in the greater wax moth. Therefore; the decrease in plasmatocytes, granulocytes, spindle cells and sphere cells in this study may reflect their importance in phagocytosis and encapsulation.



Discussion

The sharply decrease in adipohaemocytes, Oenocytoids and cystocytes may be due to the toxin released by the symbiotic bacteria which lyses them ( Dunphy and Webster , 1988 a & b ).

The remarkable decrease in THCs and the changes in DHCs of 4 th instar Galleria mellonella larvae increased with gamma irradiation doses (40-100 Gy), this is in agreement with that of Rizk (1991) on Spodoptera littoralis and this may be due to the effect of gamma radiation on the physiological condition of the body ( Stoltz & Guzo (1986) and Davies et al ., 1987 ).

The observed pathological conditions in the infected haemocytes, characterized as (1) enlargement or elongate the cell (2) vacuolization and degeneration of the cytoplasm (3) lyses of the cell membrane. The changes were a response of the actions of gamma radiation and the entomopathogenic nematodes.

Similar results were previously indicated by Rizk (1991) on effect of gamma radiation on Spodoptera littoralis ; El-Kattam (1995) on Plodia interpunctella infected by B.t and Barakat et al . (2002) on Schistocera gregaria injected with Bacillus thuringiensis.



Summary

Summary

The present work was carried out to determine the biological effect of gamma irradiation, entomopathogenic nematodes and their combination on the greater wax moth, Galleria mellonella .

The obtained results can be summarized as follows:

A. Biological effect of gamma irradiation on full grown pupae:

1. A negative relationship between radiation doses and number of eggs deposited by treated females mated with normal or treated males. When unirradiated females, mated with irradiated males, laid less number of eggs comparing with the control, however there was no significant reduction between treatments.

2. The incubation period of eggs had a positive correlation with the applied doses to both male and female irradiated as full grown pupae; increasing the dose lead to increasing period. In contrast, there was adverse relationship between the applied radiation doses and the percentage of egg hatchability.

3. The radiation dose levels 160 and 260 Gy were considered as the complete sterilizing doses for females and males, respectively.

Summary

4. The larval duration of F 1 generation resulted from irradiated males and/or female increased as the irradiated dose increased as compared with the control.

5. Percent of pupation of F1was reduced as the radiation dose increased rather than male and / or female was treated. While, the pupal duration was increased with increasing the dose.

6. Adults emergence showed adverse reduction to the dose increase; the higher the dose, the lower was in the percentage of adult emergence in F 1 generation of irradiated males and/or females.

7. The sex ratio among the progeny of irradiated parent (male and/or female ) was in favor of males.

Effect of gamma radiation and entomopathogenic nematodes on the larvae of the greater wax moth, Galleria mellonella :

1. Effect of EPN on larval mortality:

Heterorhabditis bacteriophora BA1 and Steinernema carpocapsae BA2 were assessed 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.

Summary

The results showed that the larval mortality incresed with increasing the nematode concentration and increasing in the time of infection. When male pupae irradiated with 40 or 100 Gy and mated to normal female, the resulted F 1 larvae were more succeptable to the nematode than normal larvae and the mortality was increased by increasing the dose of radiation and the nematode concentration.

2. Reproductive rate of dauer juveniles: -

The reproductive rate of the juveniles was decreased with increasing the nematode concentrations (20, 40, 60 and 80 IJs). In F 1 larvae resulted from male pupae irradiated with 40 or 100 Gy and mated to normal female, the juveniles reproduction was decreased with incresing the dose of gamma radiation. The lowest rate was at F 1 larvae of irradiated parent at 100 Gy and treated with 80 IJs.

The yield of Heterorhabditis bacteriophora BA1 was more than Steinernema carpocapsae BA2.

Determination of haemocytes count in Galleria mellonella larvae affected by gamma radiation and EPN :

I. Description of 4 th instar larvae of Galleria mellonella haemocytes:

Eight types of haemocytes were described in the haemolymph of the larvae. They are Prohaemocytes; Plasmatocytes; Spindle cells; Granulated cells; Spherule cells; Oenocytoids; Adipohaemocytes and Cystocytes.

Summary

II. Total haemocytes count (THC):

The total haemocyte count of F 1 larvae of irradiated male parent as pupae with gamma radiation doses 40 and 100 Gy was gradually decreased as the dose increased. Also larvae treated with 20 IJs of Heterorhabditis bacteriophora BA1 or Steinernema carpocapsae BA2. The total haemocyte count significantly decreased, and more decrease occurred when Heterorhabditis bacteriophora BA1 or Steinernema carpocapsae BA2 were treated in F1 Larvae of irradiated male parent pupae mated with normal female.

III. Differential haemocytes count (DHC):

The prohaemocytes were the predominant type and the cystocytes were the rare type. The present study on DHC showed that in the treatment of normal G. mellonella larvae or F 1 (from irradiated male parent pupae with 40 and 100 Gy and mated to normal female) with BA1 or BA2 at concentrations 20 and 40 IJs, the percentage of prohaemocytes increased and the other types decreased with increasing the treatments.

References

References

• Abdalla, R.S. (2004): Effect of gamma irradiation and plant substances on certain stored product insects. M.Sc.Thesis. Fac. Agric. Ain Shams Univ. • Abdel Salam, K.A. (1977): The effect of gamma radiation on certain stored product insects. M.Sc. Thesis, Fac. Agric., Cairo University. • Abdel-Rahman, A.M. (1978 ): The haemocytes of Aedes caspius . Palls. Bull. Fac. Sci. Cairo University, 51(1): 95 – 100. • Abdel Salam, K.A.; Gally, S.E.; Kamel, E.G. and Mohamed, S.A. (1995): Effect of gamma irradiated entomopathogenic nematode Steinernema carpocapsa (Fil) on the larval of the potato tuber moth, Phthorimaea operculella (Zell). Anzeiger Fur schall lingskunde, Pflanzenschat, Umweltschutz. 68(3): 51 – 54. • Abd-Elwahed, S.A.M. (2004) : Biological and histological studies on the effects of gamma irradiated parents to entomopathogenic nematodes (Rhabditidae) and some plant extraction potato tuber moth, Phthorimaea operculella , (Zeller). Ph. D. Thesis, Girls College for Arts, Science and Education, Ain Shams Univ. • Abu EI-Magd, A.A. and El- Kifl, T.A.H. (1993): Cellular and humoral immune reactions of Spodoptera littoralis and Agrotis ipsilon larvae against the nematode Heterorhabditis heliothidis . J. Egypt. Ger; Soc. Zool., 12 (A): 475-488. • Aguilar, J.A.D. and Arthur, V. (1998): Effect of sub sterilizing doses of gamma radiation on Spodoptera frugiiperda (Smith) pupae. J. Nuclear Agriculture and Biology., 27(2) : B2 – B8.



References

• Aguilar, J.A.D.; Arthur, V. and Wiendl, F.M. (1994): Effects of gamma radiation on pupae, adults and first generation of Corcyra cephalonica. J. Nuclear Agriculture and Biology., 23(2): 98 – 101. • Ahmed, A. (1988 ): Free haemocytes in adult Polistes hebroeus Fabr. (Hymenoptera: Vespidae). J. Entomol. Res., 12: 28–35. • Ahmed, A. (1992): Study of haemocytes of two coleopterous insects, Aulacophora foveicollis Lucas (Chrysomelidae) and Mylabris pustulata Thunberg (Cantharidae). J. Animal Morphol. Physiol., 39: 19–26. • Ahmed, M.Y.Y.; Abdel-Baky, S.M.; EI-Bamby, M.A. and Salem, Y.S. (1985): Gamma radiation effect on the pupal stage of Ephestia kuehniella Z. Egypt. J. Rad. Sci. and Appl., 2 (l): 69-77. • Al-Izzi, M. A. J; AL-Maliky, S. K. and Khalaf, M. Z. (1993 ): Effects of gamma irradiation on sterility of pomegranate fruit moth, Ectomyelois ceratoniae zeller. Insect Sci. Appl.14 (5): 675-679. • Allinghi, A.; Gramajo, C.; Willink, E. and Vilardi, J. (2007): Induction of sterility in Anastrepha fraterculus (Diptera: Tephritidae) by gamma radiation. Florida Entomologist, 90(1): 96 – 102. • Alm-El-Din, M.M.S. (2001): Studies on the sterilization of Egyptian cotton leaf worm, Spodoptera littoralis (Boisd.) in Egypt. Ph. D. Thesis, Faculty of Agricultural, AL-Azhar University. • Al-Taweel, A.A.; Ahmed, M.S.; Wakeed, A.K. and Nasser, M. (1997): Inherited sterility in progeny of gamma irradiated of raisin moth , Ephestia bigulilella (Grey) (Lepidoptera: Pyralidae). Dirasta. Agric. Sci. 24:128-134.



References

• Al-Taweel, A.A.; Al-Tememi, N.K. and Al-Ali, A.M. (2002): Inherited sterility in the greater wax moth adults, Galleria mellonella . J. Nat. and App. Sci., 6 (2): 287-296. • Ashida, M. and Dohke, K. (1980) Activation of prophenoloxidase by the activating enzyme of the silkworm Bombyx mori . Insect Biochem., 10: 37–47. • Ayaad, T.H.; Darrah, M.A.; Shaurub, E.H. and EI- Sadawy, H.A. (2001): Effects of the entomopathogenic nematode, Heterorhabditis bacteriophora HP 88 and Azadirachtin on the immune defense response and prophenoloxidase of Parasarcophaga Surcoufi larvae (Diptera: Sarcophagidae). J. Egypt. Soc. Parasitol., 31(1): 295 - 325. • Aye, T.T.; Shin, J.K.; Ha, D.M.; Kwon, Y.J.; Kwon, J.H. and Lee, K.Y. (2008): Effects of gamma irradiation on the development and reproduction of Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). J. Sto. Prod. Res., 44: 77 – 81. • Ayvaz, A.; Albayrak, S. and Tunçbilek, A.S. (2007): Inherited sterility in mediterranean flour moth Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae): Effect of gamma radiation on insect fecundity, fertility and developmental period. J. Sto. Prod. Res., 43(3): 234-239. • Azambuja, P.D.; Garcia, E.S. and Ratcliffe, N.A. (1991): Aspects of classification of Hemiptera hemocytes from six triatomine species. Mem. de Insitit. Oswaldo Cruz, 86: 1–10. • Barakat, E.M.S.; Meshrif, W.S. and Shehta, M.G. (2002): Changes in the haemolymph of the desert locust, Schistocerca gregaria after injection with Bacillus thurigiensis . J. Egypt. Acad. Soc. Environ. Develep., 2, (1) : 95 – 115.



References

• Bedding, R.A.; Molyneux, A.S. and Akhurst, R.J. (1983): Heterorhabditis spp..; Neoaplectana spp. and Steinernem kraussei : Interspecific and intraspecific differences in infectivity for insects. Exp. Parasitol., 55: 249-257. • Ben-Yakir, D.; Efron, D.; Chen, M. and Glazer, I. (1998): Evaluation of entomopathogenic nematodes for biocontrol of the European corn borer, Ostrinia nubilalis, on sweet corn in Israel. Phytoparasitica, 26 (2): 1-8. • Blandin, S. and Levashina, E.A. (2004): Thioester- containing proteins and insect immunity. Mol. Immunol ., 40 : 903–908. • Bloem, S.; Bloem, K.A.; Carpenter, J.E. and Calkins, C.O. (1999): Inherited sterility in codling moth (Lepidoptera: Tortricidae): Effect of substerilizing doses of radiation on field competitvness. Environ. Entomol., 28: 660 – 674. • Bloem, S.; Carpenter, J.E. and Hofmeyr, J.H. (2003): Radiation biology and inherited sterility in false codling moth (Lepidoptera: Tortricidae). J. Econ. Entomol., 96(6):1724 – 31. • Boff, M.I.C.; Wiegers, G.L.; Gerritsen, L.J.M. and Smits, P.H. (2000): Development of the entomopathogenic nematode Heterorhabditis megidis strain NLH-E 87. 3 in Galleria mellonella . Nematology, 2 (3): 303 – 308. • Boshra, S.A. (1982): Radiological studies on the angoumois moth, Sitotroga cerealella . M. Sc. Thesis, Fac. Agric., Ain Shams University . • Brehelin, M. and Zachary, D. (1986): Insect haemocytes, a new classification to rule out controversy. In Immunity in Invertebrates (M. Brehelin, Ed.), pp. 36–48. Springer Verlag, Berlin.



References

• Bughio, A.R. (1992): Effects of gamma radiation on mature pupae of maire borer, Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae). Insect Science and its Application, 13 (3): 363 – 368. • Caron, D.M. (1992): Wax Moth. American Bee Journal , 132 (10): 647-49. • Charles, J. (1971): Haemolymph arthroapoda. Chemical Zoology, 6: 64-118. • Charrière, J.D. and Imdorf, A. (2004): Protection of honey combs from moth damage , Communication nr. 24, Swiss Bee Research Centre. Bern. • Cornelis, L. and Soderhall, K. (2004) The prophenoloxidse-activating system in invertebrates. Immunol. Rev., 198 : 116–126. • CoStat (1995): CoStat, user's manual. CoHortSoftware, Minneapolis, MN. • Davies, D.H.; Strand, M.R. and Vinson, S.B. (1987): Changes in differential haemocytes content and in vitro behaviour of plasmatocytes from host Heliothis virescens caused by Campoletis sonorensis polydnavirus. J. Insect Physiol., 33 (3): 143 - 153. • Dunphy , G.B. (1994): Interaction of mutants of Xenorhabdus nematophilus (Enterobacteriaceae) with antibacterial system of Galleria mellonella larvae (Insecta: Pyralidae). Canadian Journal of Microbiology, 40: 161 – 168. • Dunphy , G.B. (1995): Physiochemical properties and surface components of Photorhabdus luminescens influencing bacterial interaction with non-self response system of nonimmune Galleria mellonella larvae. J. Inver. Pathol., 65: 25 – 34.



References

• Dunphy, G.B. and Thurston, G.S. (1990): Insect immunity. In Gaugler, R. and Kaya, H.K. (eds) Entomopathogenic Nematodes in Biological Control. CRC Press, Boca Raton. Florida, pp.:301 – 323. • Dunphy, G.B. and Webster, J.M. (1988 a): Virulence mechanisms of Heterorhabditis heliothidis and its bacteria associated Xenorhabdus luminescens in non-immune larvae of the greater wax moth, Galleria mellonella . J. Pathol., 18:729-737. • Dunphy, G.B. and Webster, J.M. (1988 b): Lipopolysaccharides of Xenorhabdus nematophilus (Enterobacteriaceae) and their haematocytes toxicity in non- immune Galleria mellonella . J. Ger. Microb., 134: 1017- 1028. • Dutcky, S.R. and Hough, W.S. (1955): Note on a parasitic nematode from codling moth larvae, Carpocapsa pomonella (Lepidoptera, Olethreutidae). Proceedings of the Entomological Society of Washington, 57(5): 244. • El Badry, E.A. (1964): The effect of gamma irradiation on the haemocyte counts of larvae of the potato tuber worm Gnoriomoschema Operculella (Zeller.) J. Insec. Path., 6: 327 – 330. • El Kady, E.A.; Salem, Y.S. and Hekal, A.M. (1983): Effect of gamma irradiation on pupae of the greasy cutworm, Agrotis ipsilon (Hufn) (Lepidoptera: Noctuidae). Mededelingen Van de Faculteit Landbouwwetensckappen, Rijksuniversiteit –Gent. 48(2): 385- 392. • EI Kattam, N.A.1. (1995): Physiological studies on the Indian meal moth. Plodia interpunctella HB. (Pyralidae: Lepidoptera) infected with microbial entomopathogen. Ph.D. Thesis, Ain Shams Univ.



References

• El-Maasarawy, S.A.S.; Abd El-Baki, S.M. and Salama, R.A.K. (1995): Abnormalities of larval haemocytes of Corcyra cephalonica (staint.) after exposure of egg to Gamma irradiation. Bull. Ent. Soc. Egypt. 73: 107 – 119. • El-Maasarawy, S.A.S. and Abd El-Salam, K.A.A. (1991 ): The effect of irradiation on the haemocytes picture in Bombyx mori L. larvae. Bull. Ent. Soc. Egypt. Econ. Ser., 17: 63- 76. • El Mandarawy, M.B.R. (1997): Effects of insect diapause and parasitization of a braconid, Bracon brevicornis wesm. on the haemolymph of its host Sesamia cretica (Led.). J. Egypt. Soc. Parasitol., 27(3): 1997: 805- 815. • El Mandarawy, M.B.R. (2005): Efficacy of entomopathogenic nematodes against potato tuber moth Phthorimaea operculella (Zeller.) (Lepidoptera: Gelechiidae) J. Egypt. Ger. Soc. Zool., 46 (E): 93-104. • EL Mandarawy, M.B.R.; Rizk, S.A.; and Abdcl Samea, S.A. (2006): Effect of some entomopathogenic nematodes and gamma irradiation on the greater wax moth, Galleria mellonella (L.) (Lepidoptera: Pyralidae).J. Egypt. Grer. Soe. Zool. 49 (4):29-40. • El-nagar, S.; Megahed, M.M.; Sallam, H.A. and Ibrahim, S.M. (1984): Inherited sterility among Agrotis ipsilon laboratory population exposed to gamma irradiation. Insect Sci. Appl., 6:501 – 503. • Fadem, R.S. and Yalow, R. (l95l): uniformity of cell counts in smears of bone marrow particles. American Journal of Clinical Pathology, 27: 54l.



References

• Farag, S.R.M. (2003) : Relationship between entomopathogenic nematodes and insect pests associated with acacia trees in Egypt. M SC. Thesis, Girl College for Arts, Science and Education, Ain shams Univ. PP: 219. • Feir, D. (1979): Cellular and humoral responses to toxic substances: In: Insect Haemocytes (Ed. Gupta, A. P.), Cambridge University Press, Cambridge. • Gardiner, E.M.M. and Strand, M.R. (1999) : Monoclonal antibodies bind distinct classes of haemocytes in the moth Pseudoplusia includens. J. Insect Physiol. , 45 : 113–126. • Gaugler, R. (1988) : Ecological consideration on the biological control of soil inhibiting insect pests with entomopathogenic nematodes. Agric. Ecosys. Environ., 24: 351-360. • Gaugler, R. and Boush, G.M. (1979): Nonsusceptibility of rats to the entomopathogenic nematode Neoaplectana carpocapsae . Environmental Entomology, 8: 658 - 660. • Georgis, R. and Manweiler, S.A. (1994): Entomopathogenic nematodes: a developing biological control technology. In: Evans, K. (ed.) Agricultural Zoology Reviews. Intercept, Andover, UK, pp. 63 – 94. • Ghally, S.E. (1983): Relationships between nematodes of Neoaplectana carpocapsae (Weiser) and Heterorhabditis bacteriophora (Poinar) and their hosts under different conditions. Ph.D. Thesis, Acad, Sci., Insti-Zoo-Warsaw, Poland P: 151. • Ghally, S.E.; Kamel, E.G. and Nasr, N.M. (1988): Study on the influence of entomophilous nematodes on Spodoptera littoralis (Boisd.). J. Egypt. Soc. Parasitol., 18 (1): 119-127.



References

• Gharieb, O.H. (1989 ): Effectiveness of gamma irradiation on Trogoderma granarium (Everts). Ph. D. Thesis, Fac. Agric. Ain Shams University. • Gillespie, J.P; Kanost, M.R. and Trenczek, T. (1997): Biological mediators of insect immunity. Annu. Rev. Entomol. , 42 : 611–643. • Glaser, R.W. (1931): The cultivation of a nematode parasite of an insect. Science 73: 614-615. • Glazer, I. (1992): Invasion rate as a measure of infectivity of Steinernematid nematodes Spodoptera Littoralis . J. Invert. Pathol., 59 : 90-94. • Glazer, I. and Lewis, E.E. (2000): Bioassays for Entomopathogenic Nematodes. In Bioassays for Entomopathogens and Nematodes. Navon A. [Ed.] Kluver Academic Publisher, Holland. • Gouge. D.H.; Lee, L.L.; Bartlett, A. and Henneberry, T.J. (1998): Pectinophora (Lepidoptera: Gelechiidae): Susceptibility of F 1 larvae from irradiated parents to entomopathogenic nematodes (Rhabditida: Steinernematidae, Heterorhabditidae). J. Econ. Entomol., 91 (4) : 869 – 874. • Grewal, P.S.; Gaugler, R. and Selvan, S. (1993): Host recognition by entomopathogenic nematodes: behavioral response to contact with host feces. J. Chem. Ecol., 19: 1219 – 1231. • Grewal, P.S.; Lewis, E.E.; Gaugler, R. and Campbell, J.F. (1994): Host finding behaviour as a predictor of foraging strategy in entomopathogenic nematodes. Parasitology, 108: 207-215.



References

• Guirguis, A.A. (1979 ): The use of some physical and chemical mutagens for the induction of sterility in Spodoptera littoralis (Boisd). M.Sc. Thesis. Fac. Agric., Zagazig University. • Gupta, A.P. (1985) : Cellular elements in haemolymph. In: Comprehensive Insect Physiology, Biochemistry, and Pharmacology (G.A. Kerkut, L.I. Gilbert, Eds.), 3: 401–451. Pergamon Press, Oxford. • Haiba, I.M. (1990): Gamma irradiation effects on the potato tuber worm, Phthorimaea operculella (Zeller). Ph. D. Thesis, Fac. Sci., Cairo University. • Hasaballa, Z.A.; Ahmed, M.Y.Y and Rizk, M.M.A. (1985): Effect of gamma radiation on the immature stages of Corcyra cephalonica . Assuit J. Agric. Sci., 16: 291-298. • Henneberry T.J.; Lindegren, J.E.; Forlow Jech, L.J. and Burke, R.A. (1995): Pink bollworm (Lepidoptera: Gelechiidae): Effect of steinernematid nematodes on larval mortality. Southwestern Entomologist, 20(1): 25 - 32. • Hoe, N.T.Q. and Tien, N.T.T. (2001): Radiation induced F 1 sterility in Plutella xylostella (Lepidoptera: Plutellidae): Potential for population suppression in the field. Florida Entomologist, 84(2): 199 – 208. • Hofemeyr, J.H.; Bloem, S. and Carpenter, J.E. (2005): Radiation biology and inherited sterility in false codling moth (Lepidoptera: Tortricidae). International Atomic Energy Agency. Vienna (Austria); Food and Agriculture Organization of the United Nations, Rome (1ta1y) FAO/IAEA International conference on area-wide control of insect pests: Integrating the steri1c insect and related nuclear and other techniques. Book of extended synopses 386, p.p.: 187.



References

• Hussein, M.A. (2004): Utilization of entomopathogenic nematodes for the biological control of some lepidopterous pest Entomology (BioControl). Ph. D. Thesis. Faculty of Science, Ain Shams University, Egypt Pp.203 • Hussein M.A. and Abou El-Soud A.B. (2006): Isolation and characterization of two Heterorhabditids and one Steinernematid nematodes from Egypt. International J. Nematol., 16(1):7-12. • Ibrahim, S.M; El-Maasarawy, S.A.S. and El-Sheikh, M.A.K. (1993): Haemocyte pattern in Agrotis ipsilon F1 progeny of gamma-irradiated pupae. Bulletin-of-the- Entomological-Society-of-Egypt,-Economic-Series, 20:141- 150. • Imler, J.L. and Bulet, P. (2005): Antimicrobial peptides in Drosophila , structures, activities and gene regulation. In Mechanisms of Epithelial Defense (D. Kabelitz and J. M. Schroder, Eds.), 86: 1–21. • Irving, P.; Ubeda, J.; Doucet, D.; Troxler, L.; Lagueux, M.; Zachary, D.; Hoffmann, J.; Hetru, C. and Meister, M. (2005): New insights into Drosophila larval haemocyte functions through genome wide analysis. Cell. Microbiol. , 7: 335–350. • Iwama, R. and Ashida, M. (1986) Biosynthesis of prophenoloxidase in hemocytes of larval hemolymph of the silkworm, Bombyx mori . Insect Biochem., 16: 547–555. • Jiang, H.; Wang, Y.; Ma, C. and Kanost, M.R. (1997 ): Subunit composition of pro-phenoloxidase from Manduca sexta : molecular cloning of subunit proPO–P1. Insect Biochem. Mol. Biol., 27: 835–850. • Joshi, P.A. and Lambdin, P.L. (1996 ): The ultrastructure of hemocytes in Dactylopius confusus (Cockerell), and the role of granulocytes in the synthesis of cochineal dye. Protoplasma, 192: 199–216.



References

• Jung, S.H.; Evans, C.J.; Uemura, C. and Banerjee, U. (2005): The Drosophila lymph gland as a developmental model of hematopoiesis. Development, 132 : 2521 2533. • Kaya, H.K. and Gaugler, R. (1993): Entomopathogenic nematodes. Ann. Rev. Entomol., 38: 181-206. • Kaya, H.K. and Koppenhofer, A.M. (1996) : Effects of microbial and other antagonistic organism and competition on entomopathogenic nematodes. Biocontrol Sci. Technol., 6: 357-371. • Kumar, M.R.; Porihor, A. and Sibbiqui, A.U. (2003): Effects of entomopathogenic nematode, Heterorhabditis sp ., on Spodoptera litura . Annals of Plant Protection Sciences, 11(2): 406 – 407. • Lackie, A.M. (1988): Haemocyte behaviour. Adv. Insect Physiol., 21: 85-178. • Lanot, R.; Zachary, D.; Holder, F. and Meister, M. (2001): Postembryonic hematopoiesis in Drosophila. Dev. Biol., 230 : 243–257. • Lavine, M.D. and Strand, M.R. (2002 ): Insect hemocytes and their role in immunity. Insect Biochemistry and Molecular Biology, 32: 1295–1309 • Lewis, E.E.; Gaugler, R. and Harrison, R. (1993): Response of cruiser and ambusher entomopathogenic nematodes (Steinernematidae) to host volatile cues. Can. J. Zool., 71: 765-769. • Luca, Y.D. (1977): Destruction of the adult form of Acanthoscalids obtecus (Say) by Neoaplectana carpocapsa (weiser). Vegetale, 7: 127-131. • Luckhart, S.; Cupp, M.S. and Cupp, E.W. (1992 ) Morphological and functional classification of the hemocytes of adult female Simulium vittatum (Diptera: Simuliidae). J. Med. Entomol., 29: 457–466.



References

• Marec, F.; Tothova, A.; Sahara, K. and Traut, W. (2001): Meiotic pairing of sex chromosome fragments and its relation to a typical transmission of a sex-linked marker in Ephestia Kuehniella (Insecta: Lepidoptera). Heredity, 87: 659 - 671. • Matha, V.; Grubhoffer, L.; Weyda, F. and Herliianova, L. (1990): Detection of B-1,3 glucan-specific lectin on the surface of plasmatocytes, immunocompetent cells of great wax moth, Galleria mellonella L. Cytobios., 64 (256): 35 - 42. • Mathasoliya, J.B.; Maghodia, A.B. and Vyas, R.V. (2004): Efficacy of Steinernema riobrave against Agrotis ipsilon Hufnagel (Lep: Noctuidae) on potato. Indian Journal of Nematology, 34(2): 177 – 179. • Milam, V.G. (1970): Moth pests of honeybee combs. Glean. Bee Culture 68: 824 – 428. • Milcheva, R.Y. (2005): Radiobiological studies on the greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae) II Radiation induced sterility. Bulgarian Journal of Agricultural Science. 11(4):423 – 430. • Mohamed, S.A. (1995): Interaction of gamma irradiation and parasitic nematodes on potatoes tuber worm, Phthorimaea operculella . M. SC. Thesis, College for Girls. Ain Shams Univ. Egypt. • Mohamed, H.F. (2004): Inherited sterility induced by gamma irradiation and or barnoof plant extract on reproductive potential and mating ability of the black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae). Isotopes and Radiation Research, 36(4): 713– 726.



References

• Mullens, B.A.; Meyer, J.A. and Georgis, R. (1987): Field tests of insect-parasitic nematode (Rhabditidae: Steinernematidae, Heterorhabditidae) against larvae of manure-breeding flies (Diptera: Muscidae) on caged layer poultry facilities. J. Econ. Entomol., 80: 438-442. • North, D.T. and Holt, G.G. (1968 a): Inherited sterility in progeny of irradiated male cabbage loopers. J. Econ. Entomol., 61:928 – 931. • North, D.T. and Holt, G.G. (1968 b): Genetic and cytogenetic basis of radiation-induced sterility in the adult male cobbage looper, Trichoplusia ni . Isotopes and Radi. In Entomol., (LAEA- SM- 102/39): 391 – 520. • Poinar, G.O. Jr. (1975): Description and biology of a new parasitic rhabditoid, Heterorhabditis bacteriophora n. gen., n. sp. Nematologica, 21: 463-470. • Poinar, G.O. Jr. (1979): Nematodes for the Biological Control of Insects. CRC press, Boca Raton, 93-174. • Proverbs, M.D. and Newton, J.R. (1962 a): Influence of gamma radiation on the development and fertility of the codling moth, Carpocapsa pomonella. Canad. J. Zool., 40: 401-420. • Proverbs, M.D. and Newton, J.R. (1962 b): Some effects of gamma radiation on the reproductive potential of the codling moth, Carpocapsa pomonella . Cand. Entomol., 94 :1162-1170. • Qureshi, Z.A.; Almad, N.; Hussain, T. and Ali, S.S. (1995): Effects of gamma radiation on one-day adults of pink bollworm and their F 1 progeny. Pak. 3. of Zool., 27(l): 21-26. • Ratnasinghe, G. and Hague, N.G.M. (1995): The Susceptibility of the diamond black moth Plutella xylostella (Lepidoptera: Plutellidae) to entomopathogenic nematodes. Afro. Assion. Journal of Nematology, 5(1): 20 – 23.



References

• Ratnasinghe, G. and Hague, N.G.M. (1997): Efficacy of entomopathogenic nematodes against the diamondback moth, Plutella xylostella. Pakistan Journal of Nematology., 15 (½): 45 – 53. • Rizk, S.A. (1991) : Effect of gamma radiation and some insecticides on the cotton leaf worm, Spodoptera littoralis (Boisd.). M.Sc. Thesis, Faculty of Science, Cairo University. • Saleh, M.M.E. and Alheji, M.A. (2003): Biological control of red palm weevil with entomopathogenic nematodes in eastern kingdom of Saudi Arabia. Egypt. J. Biol. Pest Control, 13(1&2):12 – 25. • Salem, M.S.; Eid, M.A. and Abdel-Salam, K.A. (1983): Effects of gamma irradiation on the pupae of the flour moth Ephestia kuehniella Zell. Bull. Fac. Agric. Cairo Univ., 34: 641-658. • Sallam, H.A.; El-Shall, S.S.A. and Mohamed, H.F. (2000): Inherited sterility in progeny of gamma irradiated spiny bollworm, Earias insulana (Boisd). Arab J. Nucl. Sci. and Appl., 33(1): 263 – 270. • Sallam, H.A. and Ibrahim, S.M. (1993): Inherited sterility in progeny of gamma irradiated mate cotton leaf worm, Spodoptera littoralis . Liv Radiation induced F 1 sterility in Lepidoptera area-wide control. (Proc. Final, research Co-ordination meating Phoenix, Az. 9 – 13 Sept. 1991 IAEA STI/PUB/929: 81 – 100). • Salt, G. (1970 ): The celluar defense reaction of insects. Cambridge Monograph in Experimental Biology, no.16 • Sass, M., Kiss, A. and Locke, M. (1994): Integument and hemocyte peptides. J. Insect Physiol. 40, 407–421.



References

• Sawires, S.G.N. (2005): Biological and biochemical effects of gamma irradiation on the Mediterranean flour moth, Ephestia kuehniella (Zell). M.Sc. Thesis, Fac. Agric. Ain Shams Univ. • Shamseldean, M.M.; Abd-Elgawad, M.M. and Atwa A.A. (1996): Evaluation of four entomopathogenic nematodes against Spodoptera littoralis (Lepid.: Noctuidae) larvae under different temperatures. Anzeiger fur schadlingskund, pflanzenschutz. U mweltschutz, 69(5): 11 – 113. • Shannag, H.K. and Capinera, I.L. (1995): Evaluation of entomopathogenic nematode Species for the control of melon worm (Lepidoptera: Pyralidae). Environmental Entomology, 24(1): 143 – 138. • Shinde, S. and Singh, N. P. (2000): Susceptibility of diamond black moth, Plutella xylostella (L) to entomopathogenic nematodes. Indian Journal of Experimental Biology 38 (9): 956 – 959. • Sonawane, Y.S. and More, N.K. (1993 ): The circulating hemocytes of the bed bug, Cimex rotundatus (Sign.) (Heteroptera: Cimicidae). J. Animal Morphol. Physiol., 40: 79–86. • Souka, S.; Morsi, A.; Abdel- Rahman, A. and Rizk, S. (1993): The effect of gamma radiation and certain insecticides on the total hemocytes counts and its different types in larvae of the cotton leaf worm Spodoptera littoralis (Boisd.). Arab J. Nucl. Sc. Appl., 26(2): 99-113. • Stark, J.E.P. and Lacey, L.A (1999): Susceptibility of western cherry fruit fly (Diptera: Tephritidae) to five species of entomopathogenic nematodes in laboratory studies. J. Invertebr. Pathol., 74(2): 206-208.



References

• Stoltz, D.B. and Guzo, D. (1986): Apparent haemocytic transformations associated with parasitoid-induced inhibition of immunity In Mala cosoma disstria larvae. J. Insect Physiol., 32(4): 377 - 388. • Strand, M.R. (2008): Insect haemocytes and their role in immunity. Pg. 25-48. In: Insect Immunity (Beckage, N.E. ed). Academic Press, San Diego, CA. • Strand, M.R. and Pech, L.L. (1995): Immunological basis for compatibility in parasitoid–host relationships. Annu. Rev. Entomol. , 40 : 31–56. • Tahir, H.I.; Oho, A.A. and Hague, N.G.M. (1995): The susceptibility of Helicoverpa armigera (Heliothis) and Erias vitella larvae to entomopathogenic nematodes. Afro. Asian. Journal of Nematology., 5(2): 161 – 165. • Theopold, U.; Schmidt, O.; Soderhall, K. and Dushay, M.S. (2004): Coagulation in arthropods, defence, wound closure and healing. Trends Immunol., 25 : 289–294. • Tsvetkov, D. and Atanasov, K.H. (1983): Possibilities of using radiation for the control of the Mediterranean flour moth, Ephestia kuehniella Zeller. Rasteniev" dni Nauki, 20(5): 51-59. • Wiesner, A. (1993): Die Induktion der Immunabwehr eines Insekts ( Galleria mellonella , Lepidoptera). Durch Synthetische Materialien und Arteigene Haemolymphfaktoren, Berlin. • Willott, E.; Trenczek, T.; Thrower, L.W. and Kanost, M.R. (1994): Immunochemical identification of insect hemocyte populations: Monoclonal antibodies distinguish four major hemocyte types in Manduca sexta . Eur. J. Cell Biol. , 65 : 417–423.



References

• Woodring, J.L. and Kaya, H.K. (1988): Steinernematid and Heterorhabditid nematodes: A handbook of echniques. Arkansas agricultural Experiment Station Southern Cooperative Bulletin 331, 30p. • Yousef, W.M.A. (2001): Efficiency of radiation and storage methods on Gibbium psylloides (zemp) and Plodia interpunctella (Hbn.) Ph.D. Thesis, Cairo University, 161, pp. • Zahran, N.F.M. (2006): Combined effect of gamma irradiation with certain biocides on potato tuber moth, Phthorimaea operculella Zeller. M.Sc. Thesis Fac. Agric., Ain Shams University. • Zawrah, M.F.M. (2008): Biological control of some sugar beet insect pests using entomopathogenic nematodes. Ph.D. Thesis, Faculty of Agriculture (Damanhour), Alexandria University.